ABSTRACT BOOK

of the 3rd meeting on the

 

Molecular Mechanisms of Neurodegeneration

Milan, Italy. May 19-21, 2007

 

 

 

 

 

 

Satellite Symposium on the

Molecular Mechanisms of Neurodevelopment

Neuronal migration and related diseases

 

(A1-A4)

 

A1   

 

NEURONAL MIGRATION: A VERY COMPLEX DEVELOPMENTAL MOVEMENT

 

John G. Parnavelas

 

Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK

 

The molecular mechanisms that guide the migration of the GABA-containing interneurons from their origin in the ganglionic eminence in the ventral telencephalon into the cortex are the subject of intensive investigations at present. We have investigated the role of the LIM-homeodomain gene, Lhx6, which has been localized in neurons of the medial ganglionic eminence (MGE), including those destined for the developing cortex.  We performed loss of function studies for Lhx6 in mouse brain slices and dissociated MGE neuronal cultures using Lhx6-targeted siRNA. We found that silencing Lhx6 impeded the migration of interneurons into the cortex, although it did not obstruct their dispersion within the ganglionic eminence. Blocking Lhx6 expression in dissociated neurons taken from the MGE did not interfere with the production of GABA in these cells. These results indicate that Lhx6 does not specify the neurochemical identity of interneurons, but regulates their migration to the cortex.

Previous studies have suggested that cortical interneurons express neuropilin (Npn) receptors that enable them to respond to chemorepulsion produced by class 3 semaphorins in the striatal mantle. These studies further suggested that the repulsive activity of semaphorins in the developing striatum creates an exclusion zone for migrating interneurons and channels them into adjacent paths, leading to the formation of their migratory routes into the cortex. We have been investigating the role of Slit signalling in interneuron migration by examining the forebrains of Robo1 and Robo2 knockout mice generated by targeted deletion. We have found that Robo1 is required to keep the cells originating in the MGE clear of the striatum on their way to the cortex. However, it has been reported that neurons avoid the striatal area in Slit1/Slit2 double mutant mice, indicating that this may be a Slit independent event. Taken together, these observations suggest that both Npn/Sema and Robo1 signalling are required to steer interneurons around the striatum and into the cortex. Our analysis has also shown that more interneurons migrate into the cortex of Robo1 null mice. Our recent proliferation studies in MGE dissociated cultures have shown that the increase in interneuron numbers in the cortex is due, at least in part, to increased proliferation in the MGE. I shall also present results of ongoing studies on the role of Robo3 in cortical interneuron migration.

A2   

 

SPECIFICATION AND INTEGRATION OF NEW NEURONS IN THE CEREBELLAR NETWORK

 

Rossi F

 

Department of Neuroscience, University of Turin, Corso Raffaello 30, I-10125 Turin (Italy)

tel. +39-011-6708165; fax +39-011-6708174; E-mail: ferdinando.rossi@unito.it

 

The different neuronal phenotypes that populate the cerebellar cortex are generated according to a precise spatio-temporal schedule, in which projection neurons precede local interneurons. Glutamatergic neurons develop from the rhombic lip, whereas GABAergic types originate from the ventricular neuroepithelium. Progenitors in these germinal layers are committed towards specific phenotypes already at early ontogenetic stages. Transplantation experiments show that postnatally proliferating precursors exposed to the heterochronic environment of the embryonic cerebellar primordium are unable to adopt the identities of projection neurons (Purkinje cells or deep nuclei neurons), suggesting that the sequence of phenotype generation results from the progressive restriction of progenitor cell developmental potential. GABAergic interneurons derive from a subset of ventricular zone cells, which migrate in the white matter and proliferate up to postnatal life. During this period, different interneuron categories are produced according to an inside-out sequence, from the deep nuclei to the molecular layer. Progenitors for these interneurons heterochronically transplanted to embryonic or postnatal cerebella achieve a high degree of integration in the recipient cortex and deep nuclei, and acquire GABAergic interneuron phenotypes appropriate for the host age and engraftment site. Therefore, contrary to other cerebellar types, which derive from fate-restricted precursors, GABAergic interneurons are produced by a common pool of progenitors, which maintain their full developmental potentialities up to late ontogenetic stages and adopt mature identities in response to local instructive cues. In this way, the numbers and types of inhibitory interneurons can be set by spatio-temporally patterned signals in order to match the functional requirements of developing cerebellar circuits.

 

A3   

 

MECHANISMS CONTROLLING THE MIGRATION OF GNRH NEURONS: THE KALLMANN'S DISEASE

 

CARIBONI A (1),(2)

 

(1) Institute of Endocrinology, University of Milan, Via Balzaretti,9 - 20133 Milan, ITALY tel. +39-02-50318216; fax +39 02 50318204; e-mail:anna.cariboni@unimi.it

(2) Anatomy Department, University College London, Gower street, WC1E 6BT, London, UK

 

Gonadotropin-releasing hormone (GnRH) neurons, a small number of cells scattered in the hypothalamus, play an important role in reproduction. During development, GnRH-neurons are born in the olfactory placode and migrate along olfactory nerves (vomeronasal and terminal) in the nasal compartment (NC) to gain access into the forebrain (FB) and reach the hypothalamus (HYP). All these steps are under control of several cues and defects in this process might represent the cause of disorders, as Kallmannâs syndrome (KS) and hypogonadotropic hypogonadisms (HH). Mutations in several genes have been identified in patients with HH, including KAL1, FGFR1, DAX1, GPR54, EBF2, NELF and PKR2. However, these known mutations do not account for all cases of HH, implying that other unknown genes must be crucial for GnRH development. Studies of GnRH neurons were initially hampered by their small number and dispersed distribution in the HYP. However, new technologies have been developed to facilitate the study of GnRH neurons, as nasal explants, immortalised cell lines (GT1-7;GN11) and transgenic mice carrying GFP-GnRH neurons. Accordingly, a number of molecules affecting directly or indirectly the migration of GnRH neurons have been identified in the last few years. Using GN11 cells as a model of migrating GnRH neurons we were the first to demonstrate the chemotactic effect of KAL-1 protein in the pathogenesis of X-linked KS. Moreover, based on some observations of impaired fertility in two mutant mice, we demonstrated the importance of reelin and Neuropilin-2 (NRP-2) in the migration of GnRH-neurons. In particular we found that reelin exerts a repulsive role on GnRH neurons when they penetrate the FB, whereas NRP-2 seems to be necessary for the fasciculation of the vomeronasal nerves and migration of GnRH neurons in the NC. These findings suggest that mutant/transgenic mice showing defects in reproductive functions can be useful tools to identify new candidate genes causing HH.

 

A4   

 

DEFECT OF NEURONAL MIGRATION IN A MOUSE MODEL OF ZELLWEGER SYNDROME

 

Baes M

 

Laboratory of Cell Metabolism, Department of Pharmaceutical Sciences, KUleuven, 3000 Leuven

 

Defects in the formation of the cerebral cortex and of the cerebellum are prominent features of Zellweger syndrome, a peroxisome biogenesis disorder and of MFP-2 deficiency, a peroxisomal -oxidation disorder. In order to study the molecular mechanisms underlying these pathologies, generalized and conditional knockout mouse models were generated for these diseases by respectively targeting the Pex5 and the MFP-2 gene.

Importantly, in Pex5 knockout mice at birth, cytoarchitectonic abnormalities were present in the cortex which were proven to be due to migration defects using BrdU birthdating experiments. Quite surprisingly and at variance with the patients, no cortical migration defects were found in MFP-2 knockout, neither in double MFP1/MFP2 knockout mice. Since very long chain fatty acid levels (VLCFA) were elevated to the same extent in these -oxidation deficient mice as in the Pex5 knockout mice, we concluded from these studies that VLCFA are not the only causative factor for the migration defects.

By generating mice with brain- or liver selective elimination of Pex5, we addressed the question whether brain malformations in peroxisome deficient mice are caused by the local absence of peroxisomes in the brain or by extraneural deletion of peroxisomal metabolic activity.  We found that absence of peroxisomal function from liver has a more severe and persistent impact on cortical neuronal migration than absence from brain. The longer survival of these conditional knockout mice allowed to evaluate cerebellar development which was also more affected in the liver selective than in the brain selective knockout mice.

In summary, these investigations were important to prove that peroxisome deficiency leads to cortical and cerebellar malformations in another species besides humans. A number of metabolites were excluded as single causative factors but there is still no clear view on the molecular mechanism underlying the migration defects.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

III Meeting on the

MOLECULAR MECHANISMS OF NEURODEGENERATION

 

Plenaries

PL1

 

RECENT PROGRESS IN PRION BIOLOGY

Aguzzi A

 

Institute of Neuropathology, Department of Pathology, University Hospital of Zurich, CH-8091 Zurich (Switzerland), tel +41-44-255 2107, fax +41-44-255 4402, e-mail: adriano.aguzzi@usz.ch

 

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative diseases of humans and animals. The underlying infectious agent, the prion, accumulates not only in the central nervous system (CNS) but also in secondary lymphoid organs. I will revisit the role of the immune system in peripheral prion pathogenesis, while focusing on the mechanisms by which extraneural and extralymphatic prion infectivity develops. Interestingly, the same pro-inflammatory cytokines and homeostatic chemokines that are involved in lymphoid neogenesis and compartmentalization of immune cells appear to represent the crucial molecular switches responsible for the establishment of extraneural prion reservoirs. 

 

PL2        

 

NEURODEGENERATION IN LYSOSOMAL STORAGE DISEASES IS ASSOCIATED WITH IMPAIRMENT OF AUTOPHAGY

 

Settembre C (1), Fraldi A (1), Jahreiss L (2), Spampanato C (1), de Pablo R (1), Medina DL (1), Lombardi A (1), Venturi C (3), Tacchetti C (3), Rubinsztein DC (2) and Ballabio A (1,4)

 

(1) TIGEM (Telethon Institute of Genetics and Medicine), Via Pietro Castellino, 111 - 80131 Naples (Italy) tel +39-081-6132297; fax +39-081-5790919; E-mail: ballabio@tigem.it

(2) Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, United Kingdom;

(3) Department of Experimental Medicine and MicroSCoBiO Research Center and IFOM Center of Cell Oncology and Ultrastructure, University of Genoa 16126 Genoa, Italy;

(4) Medical Genetics, Department of Pediatrics, Federico II University, 80131 Naples, Italy

 

Autophagy is the mechanism responsible for the turn-over of intracellular organelles and digestion of protein aggregates which are sequestered by autophagosomes and degraded upon the fusion of the autophagosome with the lysosome. Autophagy is involved in the pathogenesis of several neurodegenerative disorders, such as Alzheimer, Parkinson and Huntington diseases. Furthermore, knock-out of autophagy genes in transgenic mice is associated with neurodegeneration. We have analyzed the autophagic pathway in two different murine models of lysosomal storage disorders (LSDs), Multiple Sulfatase Deficiency (MSD) and Mucopolysaccharidosis type IIIA (MPS-IIIA). Western blotting, immunofluorescence and immunohistochemical analyses using anti-LC3 antibodies demonstrated a significant intracellular accumulation of autophagic (LC3-positive) vacuoles in MEFs as well as in several brain regions of both MSD and MPS-IIIA mice. Accumulation of autophagosomes was also confirmed by ultrastructural analysis. Co-staining of MEFs using both anti-LC3 and anti-LAMP2 antibodies demonstrated that autophagosomes do not co-localize with lysosomes, suggesting the presence of a fusion defect. As a consequence of an impairment of autophagy, a massive intracellular accumulation of ubiquitin-positive aggregates and an increased number of mitochondria with altered membrane potential were detected in the brain of both MSD and MPS-IIIA mice. Interestingly, the build-up of polyubiquitinated proteins and dysfunctional mitochondria has been associated with neuronal cell death in neurodegenerative diseases. Taken together our data indicate that accumulation of storage material, due to the lysosomal enzyme deficiency, causes a lysosomal dysfunction which affects the autophagic pathway, and more specifically the formation of autophagolysosomes. We postulate that neurodegeneration in LSDs is caused by secondary storage of toxic protein aggregates due to an impairment of autophagy. This identifies lysosomal storage diseases as autophagy disorders, representing a paradigm shift for these long-known inborn errors of metabolism.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

III Meeting on the

MOLECULAR MECHANISMS OF NEURODEGENERATION

 

Symposia

 

S1            

 

THE SEQUENCE AND STRUCTURAL DETERMINANTS OF PROTEIN AGGREGATION

 

Fabrizio Chiti

 

Dipartimento di Scienze Biochimiche, Universitˆ di Firenze

Viale Morgagni 50, I-50134 Firenze, Italy

 

Formation of insoluble fibrillar aggregates is a process that represents an essential feature of the chemistry of proteins and plays a central role in human pathology, including neurodegenerative diseases. The study of the effects of mutations on a number of protein systems has allowed the identification of the physico-chemical factors governing aggregation. Such an elucidation has allowed the development of algorithms able to determine and predict (i) the effect of mutations of the aggregation of unstructured proteins, (ii) the absolute aggregation rate of unfolded systems and (iii) the regions of the sequence determining aggregation and forming the core of the fibrils.

Examples will be shown on the good agreement between regions of the sequence forming the beta-core of the fibrils and determined experimentally and the regions predicted to promote aggregation. An agreement particularly good is found for alpha-synuclein, the amyloid-beta-peptide, tau, and may other systems.

In case of proteins having both unfolded portions and well-defined folded domains, such as the mammalian prion protein and other systems, structural factors also become important determinants in aggregation that need to be considered in such predictive tools. When the aggregation propensity profile of the mammalian prion protein is edited using the structural factors into account, the profile can identify the regions of the sequence involved in aggregation.

 

 

S2   

 

PREVENTION OF AMYLOIDOGENESIS BY CONVERTING alpha-SYNUCLEIN AND AMYLOID-beta POLYPEPTIDES INTO NONTOXIC OLIGOMERS OR AMORPHOUS AGGREGATES

 

Dagmar E. Ehrnhoefer, Jan Bieschke,  Annett Boeddrich and Erich E. Wanker

 

Max Delbrueck Center for Molecular Medicine (MDC), Department of Neuroproteomics, Robert-Roessle-Stra§e 10, 13125 Berlin, Germany

 

Amyloid diseases are a large group of protein misfolding diseases, which include Alzheimer's, Parkinson's, Huntington's disease or forms of ataxia. They are characterized by the accumulation of amyloid inclusions in tissues or the extra-cellular matrix and the detrimental consequences of this process. Amyloid formation involves misfolding of polypeptide chains, formation of transient, soluble oligomers and protofibrils as well as their self-assembly into large insoluble fibrils. Several studies indicate that natural polyphenols such as green tea epigallocatechin-3-gallate (EGCG) may have anti-amyloidogenic activities. However, their inhibitory mechanism of action is largely unclear. We demonstrate that EGCG very efficiently inhibits fibrillogenesis of the unstructured, non-homologous polypeptides alpha-synuclein (alphaS) and amyloid-beta (Abeta). It directly binds to the aggregation-prone polypeptide chains and converts them into highly stable, spherical oligomers consisting of about 30 molecules. The EGCG-generated oligomers are non-toxic, seeding-incompetent and do not self-assemble into fibrillar structures, indicating that they are off-pathway, end stage aggregation products which differ structurally from toxic amyloid oligomers and protofibrils previously described. Continued investigation revealed that EGCG efficiently remodels preformed alphaS and Abeta beta-sheet-rich fibrils leading to the formation of non-toxic amorphous protein aggregates. Together these data demonstrate that EGCG is a highly potent modulator of protein misfolding and aggregation, converting toxic amyloidogenic structures into benign aggregation products. Our studies have revealed a novel pharmacological property of polyphenols and provide experimental evidence that such compounds may have therapeutic potential in combating various protein misfolding diseases such as Alzheimer's and Parkinson's disease.

 

 

S3  

 

EXOGENOUS INDUCTION OF Abeta-AMYLOIDOGENESIS IS GOVERNED BY INTRINSIC PROPERTIES OF AGENT AND HOST

 

Mathias Jucker(1) and Lary Walker(2)

 

(1) Department of Cellular Neurology, Hertie Institue of Clinical Brain Research, D-72076 Tuebingen (Germany); (2) Division of Neuroscience, Yerkes National Primate Research Center, Emory University, Atlanta, GA, USA. E-mail: mathias.jucker@uni-tuebingen.de

 

The misfolding and aggregation of specific proteins is well-established in the pathogenesis of Alzheimer's disease, but little is known about how protein aggregation is initiated in vivo. We show that intracerebral injection of highly dilute, amyloid-beta (Abeta)-containing human or APP23 transgenic (tg) mouse brain extract can induce cerebral beta-amyloidosis and associated pathology in APP23 tg mice in a time- and concentration-dependent manner. By injecting extracts from APPPS1 tg mice into APP23 hosts and vice versa, our results suggest the occurrence of polymorphic Abeta species with varying biological activities, reminiscent of prion strains. Formic acid treatment, but not boiling, abolished the Abeta-inducing activity of the extract. Moreover, beta-amyloid-induction was effectively blocked when brain extracts were Abeta-immunodepleted, or by Abeta-immunization of the host. Notably, intracerebral injections of synthetic Abeta preparations in concentrations similar to brain extract level, as well as cell culture-derived Abeta did not yield significant seeding activity, suggesting that amyloid-induction is dependent on a conformation of Abeta that is generated in the in vivo environment. Our results demonstrate that cerebral Abeta-amyloidosis can be induced by exogenous, Abeta-rich brain extract, and that induction is governed by intrinsic properties of both agent and host.

 

 

S4   

 

MEMBRANE PERMEABILIZATION: A COMMON MECHANISMS IN PROTEIN MISFOLDING DISEASES

 

Hilal A. Lashuel (Switzerland)

              

 

Abstract not received

 

S5   

 

PROTEOLYSIS OF HUNTINGTIN AND ITS ROLE IN THE PATHOGENESIS OF HUNTINGTON DISEASE

 

Rona K. Graham, Simon Warby and Michael R. Hayden

 

Centre for Molecular Medicine and Therapeutics, University of British Columbia, Vancouver, British Columbia, Canada. V5Z 4H4

 

Cleavage of huntingtin (htt) has been characterized in vitro and accumulation of caspase cleavage fragments represents an early pathological change in brains of Huntington disease (HD) patients. However, the relationship between htt proteolysis and the pathogenesis of HD is unknown. To determine whether caspase cleavage of htt is a key event in the neuronal dysfunction and selective neurodegeneration in HD, we generated YAC mice expressing caspase 3- and caspase 6-resistant mutant htt. Mice expressing mutant htt, resistant to cleavage by caspase-6 but not caspase-3, maintain normal neuronal function and do not develop striatal neurodegeneration. Furthermore, caspase-6 resistant mutant htt mice are protected against neurotoxicity induced by multiple stressors including NMDA, quinolinic acid and staurosporine. These results are consistent with proteolysis of htt at the caspase-6 cleavage site as being an important event in mediating neuronal dysfunction and neurodegeneration and highlight the significant role of htt proteolysis and excitotoxicity in HD. Factors influencing proteolysis include posttranslational modification, in particular phosphorylation of htt. These findings provide links between posttranslational modification of htt and proteolysis.

Grant support: Huntington Disease Society of America, Canadian Institutes of Health Research, Hereditary Disease Foundation, Merck-Frosst, Michael Smith Foundation for Health Research.

 

 

S6            

 

PARKIN LINKED TO MITOCHONDRIA: IDENTIFICATION OF A NOVEL SUBSTRATE

 

Traver S (1), Ardila-Osorio H (1), PŽriquet M (1), Hampe C (1), Brice A (1) (2), Corti O (1)

 

(1)INSERM U679, Neurologie et ThŽrapeutique expŽrimentale, H™pital de la PitiŽ-Salptrire, 47 Boulevard de l'H™pital - 75013 Paris (France)

(2) DŽpartement de GŽnetique,CytogŽnetique et Embryologie, H™pital de la PitiŽ-Salptrire, 47 Boulevard de l'H™pital - 75013 Paris (France)

 

Mutations in the parkin gene, encoding an E3 ubiquitin-protein ligase, are a frequent cause of autosomal recessive parkinsonism with early onset. Loss of Parkin function is thought to compromise the polyubiquitylation and proteasomal degradation of specific substrates, leading to their deleterious accumulation and to neurodegeneration. However, we and others have shown that polyubiquitylation may not be the preferential mode of action of Parkin, which rather promotes the attachment of single ubiquitin molecules in vitro. Therefore, depending on the cellular environment and protein context, Parkin may mediate proteasome-dependent polyubiquitylation or monoubiquitylation, a posttranslational modification involved in an increasing variety of non-proteolytic processes. By analysing the brain proteome of parkin knock-out mice and wild-type controls comparatively, we recently identified the multifunctional mitochondrial hydroxysteroid dehydrogenase ERAB/ABAD as being specifically up-regulated in the absence of Parkin. Here, we demonstrate that Parkin interacts physically with ERAB/ABAD by GST pull-down, and that it promotes its ubiquitylation in cell models. When Parkin was co-produced with ERAB/ABAD in transfected cells, we observed massive clustering of mitochondria in the perinuclear region, indicating physical interaction between the two proteins at the mitochondrial surface. Consistent with this idea, co-localization studies by double immunofluorescence showed recruitment of Parkin to assembled mitochondria containing exogenous ERAB/ABAD. Clustering of mitochondria was significantly enhanced upon concomitant overproduction of ubiquitin or a lysine-less ubiquitin derivative able to promote monubiquitylation but not polyubiquitylation. Based on previous studies suggesting that ubiquitylation processes may be involved in mitochondrial protein import, our current efforts aim at exploring whether and how Parkin plays a role in targeting ERAB/ABAD to mitochondria.

 

 

S7   

 

INHIBITORS OF APOPTOSIS (IAPS), UBIQUITINATION AND PROTEOSOME DEGRADATION

 

Robert G. Korneluk (Canada)

 

Abstract not received

S8   

 

HUNTINGTIN IS A CASPASE-3 INHIBITOR

 

Robert M. Friedlander (USA)

 

Abstract not received

S9   

 

TREATMENT OF MOTOR NEURON DEGENERATION BY VEGF

 

Peter Carmeliet  (Belgium)

 

Abstract not received

S10

 

CALCIUM-REGULATED GENE EXPRESSION DURING NEURODEGENERATION

 

Jose Naranjo (Spain)

 

Abstract not received

S11  

 

PRESENILINS FUNCTION AS ENDOPLASMIC RETICULUM CALCIUM LEAK CHANNELS - IMPLICATIONS FOR ALZHEIMER'S DISEASE

 

Ilya Bezprozvanny

 

UT Southwestern Medical Center at Dallas

 

Mutations in presenilin 1 (PS1) and presenilin 2 (PS2) are responsible for approximately 40% of all early onset familial Alzheimerâs disease (FAD) cases in which a genetic cause has been identified. In addition, a number of mutations in PS1 have been associated with the occurrence of frontal temporal dementia (FTD) although a formal proof of their causal involvement has not been provided.  Presenilins are highly conserved transmembrane proteins that support cleavage of the amyloid precursor protein by gamma-secretase.  By using planar lipid bilayer reconstitution assays and Ca2+ imaging experiments with PS-null mouse embryonic fibroblasts (MEF) we discovered that presenilins also function as passive endoplasmic reticulum calcium (Ca2+) leak channels.  Interestingly, gamma-secretase activity was not essential for Ca2+ channel function of presenilins in our experiments.

In additional experiments we investigated functional effects of FAD and FTD mutations in presenilins on ER Ca2+ leak function.  We discovered that majority of FAD-linked mutations act as "loss of function" mutations for PS-supported ER Ca2+ leak pathway.  In contrast, FTD-associated mutations had no effect on ER Ca2+ leak function of PS1.  These results suggest that the observed effects are disease-specific. Our observations are consistent with the potential role of disturbed Ca2+ homeostasis in AD pathogenesis.

References:

[1]   Tu, H. et al. (2006) Presenilins form ER calcium leak channels, a function disrupted by mutations linked to familial Alzheimer's disease. Cell 126, 981-993.

[2]   Nelson, O., Tu, H., Lei, T., Bentahir, M., de Strooper, B. and Bezprozvanny, I. (2007) Familial Alzheimer's disease-linked mutations specifically disrupt calcium leak function of presenilin 1. J Clin Invest in press.

 

 

S12  

 

CELLULAR AND MITOCHONDRIAL CALCIUM SIGNALS TO THE NEURONAL DEATH 

 

Pierluigi Nicotera (UK)

 

Abstract not received

S13  

 

RELATIONSHIP OF ALPHA-SYNUCLEIN TO PROTEIN DEGRADATION PATHWAYS

 

Vekrellis K, Emmanouilidou E, Vogiatzi T, Xylouri M, Stefanis L

 

Laboratory of Neurodegenerative Diseases, Division of Basic Neurosciences

 

Genetic, biochemical and pathological data indicate that alpha-synuclein (ASYN) is a key player in Parkinsonâs Disease (PD) pathogenesis, and that maintenance of normal cellular ASYN levels is of paramount importance.  The manner in which ASYN leads to neurodegeneration and the mechanisms through which it is degraded in cells are unclear.   We and others have proposed that ASYN is degraded through the lysosomal system, and that it may impact both lysosomal and proteasomal protein degradation pathways. In the current work, we have first further characterized the effects of A53T mutant ASYN on the proteasomal system.  We find that this mutant inhibits 26S proteasomal activity in stably transfected PC12 cells.  Furthermore, select, specific soluble oligomeric species of ASYN co-elute with the 26S proteasome upon gel filtration.  Inhibition of fibrillization with Congo Red in vitro restores proteasomal activity and removes oligomeric ASYN species from the proteasomal fractions. Furthermore, pharmacological proteasomal inhibitors dramatically increase these specific ASYN species.  These results suggest that specific soluble oligomeric ASYN species, a very small fraction of total ASYN, are degraded normally by the proteasome, but, due to their aberrant conformation, they partially inhibit its activity.  These data are consistent with the 'clogging' theory of proteasomal dysfunction. 

Purified monomeric ASYN can be degraded by isolated lysosomes through the mechanism of Chaperone Mediated Autophagy (CMA).  To examine whether this mechanism could account for ASYN degradation in neuronal cells, we have created mutant ASYN that is not recognized and degraded by this system.  We find that this form is degraded more slowly than the WT form in PC12 cells. Furthermore, cells expressing double ASYN mutant with the A53T substitution and an additional mutation that prevents CMA recognition show significantly less lysosomal dysfunction compared to cells expressing the A53T mutant alone.  These results indicate that ASYN is, at least in part, degraded by CMA in a neuronal cell context and that the aberrant effects of the A53T mutant on lysosomal function are largely due its effects on CMA.  In conjunction, these data highlight the relationship between ASYN and the major intracellular protein degradation pathways, which may be important in PD pathogenesis.        


S14  

 

INTRACYTOSOLIC AGGREGATE-PRONE PROTEINS ARE AUTOPHAGY SUBSTRATES ö THERAPEUTIC IMPLICATIONS

 

Rubinsztein DC

 

Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Addenbrookeâs Hospital, Hills Road, Cambridge, CB2 0XY, UK;tel. +44-1223-762608; fax. +44-1223-331206; email: dcr1000@hermes.cam.ac.uk

 

Intracellular protein misfolding/aggregation are features of many late-onset neurodegenerative diseases, called proteinopathies. These include Alzheimer's disease, Parkinson's disease, tauopathies, and polyglutamine expansion diseases (like Huntington's   disease (HD) and various spinocerebellar ataxias (SCAs), like SCA3).  Currently, there are no effective strategies to slow or prevent the neurodegeneration resulting from these diseases in humans. The mutations causing many proteinopathies (e.g. polyglutamine diseases and  tauopathies) confer  novel toxic functions on the specific protein, and disease severity frequently correlates with the expression levels of the protein. Thus, the factors regulating the synthesis and clearance of these aggregate-prone proteins are putative therapeutic targets.  The proteasome and autophagy-lysosomal pathways are the major routes for mutant huntingtin fragment clearance. While the narrow proteasome barrel precludes entry of oligomers/aggregates of mutant huntingtin (or other aggregate-prone intracellular proteins), such substrates can be degraded by macroautophagy (which I will call autophagy). We showed that the autophagy inducer rapamycin reduced the levels of soluble and aggregated huntingtin and attenuated its toxicity in cells, and in transgenic Drosophila and mouse models. Recently, we extended the range of intracellular proteinopathy substrates that are cleared by autophagy to a wide range of other targets, including proteins mutated in certain spinocerebellar ataxias, forms of alpha-synuclein mutated in familial forms of Parkinson's disease, and tau mutants that cause fronto-temporal dementia/tauopathy. I will consider the therapeutic potential of autophagy upregulation for various proteinopathies, and describe how this strategy may act both by removing the primary toxin (the misfolded/aggregate-prone protein) and by reducing susceptibility to apoptotic insults. I will also describe novel mTOR-independent pathways which regulate autophagy and compounds that induce additive effects along with rapamycin.

S15  

 

MOLECULAR AND CELLULAR MECHANISMS OF AXON DEGENERATION

 

Coleman MP (1), Adalbert R (1), Babetto E (1), Beirowski B (1), Bridge K (1), Conforti L (1), Gilley J (1), Janeckova L (1), Morreale, G (1), Wilbrey A (1)

 

(1) Laboratory of Neuronal Development and Survival, The Babraham Institute, Babraham, Cambridge CB2 4AT United Kingdom  Tel. +44-1223-496315;  Fax +44-1223-496348; E-mail: michael.coleman@bbsrc.ac.uk

 

Recent developments to further our understanding of axon degeneration mechanisms in disease include the identification of the slow Wallerian degeneration gene (WldS), the demonstration that WldS delays axon degeneration in some diseases as well as after injury, improved longitudinal imaging of degenerating axons using YFP-H transgenic mice, and live imaging of axonal transport that is now moving towards applications in vivo.  This talk will demonstrate how these genetic tools are being used and combined to identify similarities and differences between axon degeneration in a range of disorders.  In particular, examples will be shown from amyotrophic lateral sclerosis and Alzheimer's disease.  By moving towards a molecular understanding of how the ubiquitin proteasome system and/or NAD metabolism may help regulate Wallerian degeneration we also aim to shed light on molecular events in such disorders and on how axon degeneration may be approached therapeutically. 

 

 

S16  

 

PATHOGENESIS IN POLYQ-EXPANSION DISEASES AND FAST AXONAL TRANSPORT

 

Brady S and Morfini G

 

Dept of Anatomy and Cell Biology, Univ. of Illinois at Chicago, Chicago, IL USA 60612; Email: stbrady@uic.edu

 

Polyglutamine (polyQ) expansion diseases are adult-onset, dominantly inherited neurodegenerative diseases with unknown pathogenic mechanisms. Recent experiments showed reduced fast axonal transport (FAT) in several different polyQ-expansion disease models, including Huntington Disease and Spinal Bulbar Muscular Atrophy, but specific targets and the molecular basis underlying this inhibition had not been identified. Here we report specific alterations in the phosphorylation of the anterograde molecular motor kinesin induced by polyQ proteins. Pharmacological and biochemical studies in squid axoplasm showed that the inhibition of FAT by two unrelated polyQ expanded proteins, Androgen Receptor and Huntingtin, is mediated by increased stress-activated protein kinase (SAPK) activity, suggesting a common pathway for their toxic effects. SAPK inhibitors blocked effects of pathogenic polyQ proteins on FAT and reversed polyQ-expansion mediated reductions in neurite outgrowth in culture. Our results suggest SAPK as a mediator of polyQ-expansion protein inhibition of kinesin-based motility.

 

 

S17  

 

THE AXONAL-SMN (a-SMN) PROTEIN AND MOTOR NEURON DEGENERATION IN SPINAL MUSCULAR ATROPHY

 

Battaglia G (1), Setola V (1), Locatelli D (1), Finardi A (1), D'Errico P (1), Terao M (2), and Garattini E (2)

 

(1) Molecular Neuroanatomy Lab, Exp. Neurophysiology and Epileptology Unit, Neurological Institute "C. Besta", via Celoria 11, 20133 Milano (Italy) tel. ++ 39 02-23942606; fax ++ 39 02-23942619; E-mail: battaglia@istituto-besta.it

(2) Molecular Biology Lab, Centro Catullo e Daniela Borgomainerio, "Mario Negri" Institute of Pharmacological Research, via Eritrea 62, 20157 Milano (Italy)

 

The survival motor neuron (SMN1) gene (Lefebvre et al, Cell 1995) is the determining gene of spinal muscular atrophy (SMA), a lethal autosomal recessive disease of childhood characterized by selective motor neuron death. SMA is the most frequent hereditary disease involving motor neurons and no effective treatment for affected patients is available. The main protein product of the SMN1 gene, the full-length SMN protein (FL-SMN), has its primary and more important role in spliceosomal assembly and pre-mRNAs maturation (Pellizzoni et al, Cell 1998). Therefore, the FL-SMN housekeeping cell functions do not explain how reduced FL-SMN levels lead to selective degeneration of motor neurons in SMA.

We have recently identified a novel splicing variant of the rat SMN protein, a-SMN (axonal SMN), with selective expression in the axons of spinal cord motor neurons (Setola et al, PNAS 2007). The possible involvement of this novel SMN isoform in the pathogenesis of SMA is suggested by several lines of evidence: i) first, a-SMN is preferentially encoded by the SMN1 gene, the disease gene for SMA; ii) second, it is abundant in motor neurons and down-regulated during ontogenesis, as expected for a protein of key relevance for motor neuron development and survival; iii) third, it has a striking effect on axon growth in both NSC34 cells and primary cultured cortical neurons, and its reduced expression would be consistent with the decreased ability to sprout of motor neuron axons in type I SMA patients. The fact that specific SMA-derived SMN1 point mutations impair the axonogenic properties of a-SMN represents a further indication of the relevance of a-SMN for SMA. At the applied level, the axonogenic properties of a-SMN may serve to design viral-vector-based strategies for the future development of gene therapy approaches to treat the human disease.

S18  

 

MITOCHONDRIAL ENERGY METABOLISM IN HUNTINGTON DISEASE

 

Marcy MacDonald (USA)

 

Abstract not received

S19  

 

THE ROLE OF THE MITOCHONDRIAL M-AAA PROTEASE IN NEURODEGENERATION

 

Bernacchia A (1), Martinelli P (1), Pirozzi M (2), Ferreirinha F. (2), Flore G. (2), Auricchio A. (2), Langer T (3), Quattrini A. (4), Rugarli EI (1,5)

 

(1) Istituto Neurologico \"C. Besta\" via Temolo 4, 20126 Milano (Italy). tel. +390223942614; fax +390223942619; E-mail: rugarli@istituto-besta.it

(2) TIGEM, Napoli (Italy)

(3) University of Cologne, Cologne (Germany)

(4) Ospedale San Raffaele, Milano (Italy)

(5) Dipartimento di Neuroscienze e tecnologie biomediche, Universitˆ Milano-Bicocca, Milano

 

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous condition, characterized by weakness and spasticity of the lower limbs and due to axonal degeneration of the corticospinal tracts and fasciculus gracilis. The gene SPG7 is responsible for an autosomal recessive complicated form of HSP and encodes paraplegin. Paraplegin assembles with the highly homologous Afg3L2 protein to form the m-AAA protease, a large mitochondrial ATPase with proteolytic activity in the inner membrane, involved in protein quality control and in maturation of specific substrates. We previously knocked out the Spg7 gene in the mouse. Spg7 -/- mice are affected by a late-onset retrograde degeneration of long axons of the spinal cord, of the peripheral nerves, and of the optic nerves. In all cases, axonal degeneration is preceded by axonal swellings due to accumulation of abnormal enlarged mitochondria and neurofilaments, as a consequence of impaired axonal transport. Paraplegin-deficient mice begin to show an impaired motor performance on the rotarod apparatus at 4 months of age, well before the first signs of pathology that are only detected 3 months later. We designed a gene therapy strategy to rescue the phenotype of a subset of the axons affected in paraplegin-deficient mice, those of the spinal motoneurons. These cells projects long axons in the peripheral nerves that degenerate during the course of the disease, and can be targeted by adeno-associated viral (AAV2/2) vectors after intramuscular delivery. Recombinant AAV2/2 viral vector encoding for paraplegin was administered intramuscularly to Spg7 -/- mice in different experimental settings. Intramuscular delivery of Spg7 cDNA by AAV vectors was effective in both preventing the motor phenotype of paraplegin-deficient mice and in slowing down the progression of neuropathological changes in the peripheral nerves, representing a proof of principle that restoration of paraplegin is sufficient to rescue axonal degeneration.

 

S20  

 

IN VIVO IMAGING OF BRAIN PATHOLOGY

 

Martin Kerschensteiner

 

Institute of Clinical Neuroimmunology, Ludwig-Maximilians University Munich, Marchionistr. 17, 81377 Munich (Germany), tel.++49-89-2180 78282, fax ++49-89-2180 78285, E-mail:Martin.Kerschensteiner@med.uni-muenchen.de

 

Each neurological disease has its unique clinical course. Underlying the clinical pattern are disturbances in neuronal and glia function that are often fast, asynchronous and involve complex cellular interactions. In vivo imaging provides a new approach to visualize disease dynamics and reveal the cellular mechanisms that cause pathology.

In this presentation I want to introduce a recently developed imaging approach that allows us to follow cellular interactions with high temporal and spatial resolution in the spinal cord. The approach is based on transgenic mice in which neuronal, glial or immune cells types are stably labelled with fluorescent proteins. Cellular dynamics can then be visualized in the central nervous system using either two-photon or widefield in vivo microscopy. I will introduce this approach in two models of neurological disease. First, in spinal cord injury where we study how axons are lost after injury and why they fail to regenerate. Second, in experimental autoimmune encephalomyelitis "an animal model of multiple sclerosis" where we try to reveal how immune cells damage axons.

In summary I will present an introduction to the in vivo imaging approach and try to outline how in vivo imaging can help us to better understand neurological disease.

 

 

S21  

 

NEURAL STEM CELLS DIFFERENTIATION TO FUNCTIONAL MATURE NEURONS IN VITRO AND IN VIVO

 

Conti L

 

Department of Pharmacological Sciences and Centre for Stem Cell Research, University of Milano, Via Balzaretti 9, 20133 Milano (Italy). tel. +39 0250318349/403; fax +39 0250318284; E-mail: Luciano.Conti@unimi.it

 

Defining protocols for neural stem cells (NSCs) isolation, expansion and conversion to desired mature neuronal phenotypes are crucial steps for dissecting the molecular mechanisms regulating neuronal maturation and for successful development of cell therapy approaches for neurodegenerative diseases.

We have recently developed an innovative neural stem cell system that we named NS cells (Conti, Pollard et al, PLoS Biol. 2005). NS cells are homogeneous and long-term stable and can be generated from embryonic stem cells, fetal nervous tissues and adult subventricular zone. These cells have radial glia-like features and can be maintained ad infinitum in vitro as cell lines while retaining their neurogenic potential.

Importantly, NS cells retain neurogenic potential after extensive in vitro expansion, being able to generate both neurons and glial cells. Here I will present in vitro results demonstrating that these cells can efficiently generate neurons with remarkably uniform morphological and biochemical. Furthermore, these neuronal population exhibit hallmarks of functional mature neurons in terms of electrophysiological and neurochemical properties.

Remarkably, grafting experiments performed with in vitro primed EGFP-positive NS cells showed that these cells are able to incorporate, survive and produce mature cells types into the different regions of the host CNS. Donor cells acquired site-specific neuronal identities in many regions, including cortex, hippocampus and striatum. Notably, no glial phenotypes and no tumor formation were observed at any stage.

On the whole, our data indicate that this NS line could be useful for exploring the potential of NSCs as a valuable experimental tool to study the regulatory role of intrinsic and extrinsic factors in NSC biology and serve as system exploitable for genetic and chemical screenings. Additionally, NS can be exploited in the future as a potential cellular resource to replace dead or damaged neuronal cells in acute and chronic neurodegenerative diseases.

 

 

 

S22  

 

THE THERAPEUTIC POTENTIAL OF NEURAL STEM CELLS

 

Martino G.

 

Neuroimmunology Unit - DIBIT, San Raffaele Scientific Institute, via Olgettina 58, 20132, Milano (Italy) tel. +39-02-2643 4958; fax +39-02-26434855; E-mail: g.martino@hsr.it

 

Recent evidence consistently challenges the sole and limited view that neural stem/precursor cells (NPCs) may protect the central nervous system (CNS) from inflammatory damage leading to neurodegeneration exclusively throughout cell replacement. As a matter of fact, NPC transplantation may also promote CNS repair via intrinsic neuroprotective bystander capacities, mainly exerted by undifferentiated stem cells releasing, at the site of tissue damage, a milieu of neuroprotective molecules whose in situ release is temporally and spatially orchestrated by environmental needs. This milieu contains molecules (e.g. immunomodulatory substances, neurotrophic growth factors and stem cell regulators), which are constitutively expressed by NPCs for maintaining tissue homeostasis either both during development and adult life. The intrinsic nature (pleiotropism and redundancy) of these molecules as well as their ÔconstitutiveÕ characteristics, may also reconcile data showing that other sources of somatic stem cells (e.g. mesenchymal stem cells), with very low capabilities of neural (trans) differentiation, may efficiently promote CNS repair. Thus, cell plasticity can also be viewed as the capacity of somatic stem cells to adapt their fate and function(s) to specific environmental needs occurring as a result of different pathological conditions (therapeutic plasticity). The challenging ability of transplanted NPCs to protect the brain from several types of injuries using different and/or articulated bystander strategies is of pivotal importance for the future of stem cell based therapeutic approaches.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

III Meeting on the

MOLECULAR MECHANISMS OF NEURODEGENERATION

 

Oral Communications

OC1

 

ANDROGEN RECEPTOR PHOSPHORYLATION AT AKT CONSENSUS SITES IMPAIRS LIGAND BINDING AND BLOCKS TOXICITY IN SBMA CELL MODELS.

 

Palazzolo I (1),  Burnett BG (1), Brenne PL (1), Fischbeck KH (1),  Howell BW (1), Pennuto M (1)

 

(1) Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA;

 

Spinal and bulbar muscular atrophy is a neurodegenerative disease caused by a polyglutamine expansion in the androgen receptor (AR). The disorder is ligand dependent, as only males are affected, but the mechanisms that regulate ligand binding have not been completely delineated. Post-translational modifications, such as phosphorylation, can modulate protein activity. We investigated the role of AR phosphorylation by Akt. AR has two residues that are predicted to be phosphorylated by Akt: S215 and S792. We found that polyglutamine AR and Akt interact in vitro. Phosphorylation at S215 is increased in the presence of a constitutively active mutant Akt, and decreased by inhibition of PI3K/Akt pathway. Substitution of S215 and S792 with aspartic acid, which mimics constitutive phosphorylation, reduced ligand-dependent protein stabilization, nuclear translocation, and transcriptional activity of the AR. Further, phosphomimetic mutants showed impaired ligand binding and rescued ligand-dependent toxicity. Co-expression of a constitutively active mutant Akt reduced transcriptional activity and ligand binding of AR65Q, and the effect was greatly reduced with substitution of S215 and S792 by alanine. Akt is known to be activated by insulin-like growth factor-1 (IGF-1) through activation of PI3K. We found that expression of AR65Q in motor neuron-derived MN-1 cells results in caspase 3 activation and cell toxicity. IGF-1 treatment rescued cell viability, and the effect was dependent on the PI3K/Akt pathway but not on MEK-ERK or mTOR. IGF-1 activity was partially lost in alanine substituted AR, indicating that the effect is due at least in part to direct phosphorylation of the AR by Akt. These results establish a pathway for regulation of AR ligand binding and highlight potential targets for therapeutic intervention in spinal and bulbar muscular atrophy.

OC2

 

INCREASED LONGEVITY AND REFRACTORINESS TO CA2+-DEPENDENT NEURODEGENERATION IN SURF1 KNOCKOUT MICE

 

Carlotta DellâAgnello (1), Sara Leo (2), Alessandro Agostino (1), Gyorgy Szabadkai (2), Cecilia Tiveron (3), Alessandra Zulian (1), Alessandro Prelle (4), Pierre Roubertoux (5), Rosario Rizzuto (2),

and Massimo Zeviani (1)

 

(1) Unit of Molecular Neurogenetics, Pierfranco and Luisa Mariani Center for the Study of Childrenâs Mitochondrial Disorders, National Neurological Institute ÎC. Bestaâ, Milano, Italy, tel: 02-23942618; fax 02-23942619; e-mail: dellagnello@istituto-besta.it;

(2) Department of Experimental and Diagnostic Medicine, Section of General Pathology, Interdisciplinary Center for the Study of Inflammation (ICSI) and ER-GenTech, University of Ferrara, Ferrara, Italy,

(3) Foundation EBRI Rita Levi-Montalcini Disease Modelling Facility,Rome, Italy,

(4) Centro Dino Ferrari, UO Neurologia, Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, IRCCS, Milano

(5) Universite« Marseille-2, CNRS-Universite« de la Mediterrane« e, Marseille, France

 

Leigh syndrome associated with cytochrome c oxidase (COX) deficiency is a mitochondrial disorder usually caused by mutations of SURF1, a gene encoding a putative COX assembly factor. We present here a Surf1-/- recombinant mouse obtained by inserting a loxP sequence in the open reading frame of the gene. The frequency of -/-, +/+ and +/- genotypes in newborn mice followed a mendelian distribution, indicating that the ablation of Surf1 is compatible with postnatal survival. The biochemical and assemblyCOX defect was present in Surf1loxP-/- mice, but milder than in humans. Surprisingly, not only these animals failed to show spontaneous neurodegeneration at any age, but they also displayed markedly prolonged lifespan, and complete protection from Ca2+-dependent neurotoxicity induced by kainic acid.

Experiments on primary neuronal cultures showed markedly reduced rise of cytosolic and mitochondrial Ca2+ in Surf1loxP-/- neurons, and reduced mortality, compared to controls. The mitochondrial membrane potential was unchanged in KO versus wild-type neurons, suggesting that the effects of the ablation of Surf1 on Ca2+ homeostasis, and possibly on longevity, may be independent, at least in part, from those on COX assembly and mitochondrial bioenergetics.

OC3

 

THE INTERPLAY BETWEEN POLYQ AND PROTEIN CONTEXT DELAYS AGGREGATION THROUGH THE FORMATION OF A PROTOFIBRILS RESERVOIR

 

Masino L* (1), Bulone D (2), San Biagio PL (2), Thomas DJ (3), and Pastore A (1)

 

(1) National Institute for Medical Research, The Ridgeway, London NW7 1AA (U.K.) Tel. +44-20-88162629;  Fax +44-20-89064477; e-mail: lmasino@nimr.mrc.ac.uk

(2)  CNR, Istituto di Biofisica di Palermo, Palermo (Italy)

(3)  Scientific Software Solutions, Paisley, (U.K.)

 

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by expansion of CAG repeats coding for polyQ in the corresponding gene products. These diseases are associated with the presence of amyloid-like protein aggregates, induced by the polyQ expansion. However, it has been suggested that the toxic species responsible for pathogenesis might be soluble oligomers and protofibrils rather than mature fibres. It has also been recently observed that the aggregation properties of polyQ stretches can be strongly modulated by the protein domains surrounding the polyQ region.

      To assess the importance of protein context in polyQ aggregation, we have investigated the misfolding pathway and the aggregation kinetics of polyQ tracts of lengths above (Q41) and below (Q22) the pathological threshold, fused to the protein carrier glutathione S-transferase (GST). This protein, chosen as a model system, is per se able to misfold and aggregate irreversibly, and thus mimicks the behaviour of domains of naturally occurring polyQ proteins. Using light scattering and optical spectroscopy methods, we prove that, while it is generally accepted that aggregation kinetics of polyQ depend on its length and are faster for longer polyQ tracts, the presence of GST alters the polyQ aggregation pathway and reverses this trend. Aggregation occurs through formation of a reservoir of soluble intermediates whose population and kinetic stability increase with polyQ length. Our results provide a new model that explains the toxicity of expanded polyQ proteins, in which the interplay between polyQ regions and other aggregation-prone domains plays a key role in determining the aggregation pathway. 


OC4

 

HSPB8 AND BAG3: A NEW CHAPERONE COMPLEX STIMULATING MISFOLDED PROTEINS DEGRADATION BY MACROAUTOPHAGY

 

Carra S * (1), Seguin S (1), Vinet J (2), Lambert H (1), Sik A (2) and Landry J (1)

 

(1) Centre de recherche en cancŽrologie de l'UniversitŽ Laval, L'H™tel-Dieu de QuŽbec, 9 rue McMahon, QuŽbec, Canada G1R 2J6; serena_carra@yahoo.ca; tel. : +01(418) 6915281; fax : +01(418) 691-5439

(2) Centre de recherche UniversitŽ Laval Robert-Giffard, UnitŽ de neurobiologie cellulaire 2601 Chemin de la Canardire, QuŽbec, Canada G1J 2G3

 

HspB8 is a member of the small heat shock proteins or HspB family of molecular chaperones, which comprises ten members in mammals (HspB1-10). We previously demonstrated that, in cultured cells, overexpression of HspB8, but not of HspB1 or HspB5, totally blocked the insolubilization and accumulation of a pathogenic aggregation-prone form of Huntingtin (Htt43Q). Here we report the identification of a new chaperone complex comprising the small heat shock protein HspB8 and the co-chaperone Bag3. By siRNA technique we show that HspB8 interaction with Bag3 is essential for both its structural stability and chaperone function. Moreover, we demonstrate in cultured cells that, like HspB8, Bag3 facilitates Htt43Q degradation. The chaperone activity of the HspB8/Bag3 complex was blocked by specific macroautophagy inhibitors, thus suggesting the implication of the macroautophagy process in the HspB8/Bag3 complex mechanism of action. These is further supported by the finding that HspB8 and Bag3 both increase the number of cells containing LC3 positive-vacuoles and stimulate the lipidation of LC3, a step which is necessary for LC3 incorporation into the autophagosomes. By joining the ability of recognizing endogenous misfolded proteins and of stimulating the macroautophagic vacuole formation, the new HspB8/Bag3 chaperone complex may play a crucial role in the protein quality control system responsible for eliminating potentially harmful aggregating proteins.


OC5

 

EFFECT OF THE SMALL HEAT SHOCK PROTEIN HSPB8 OVEREXPRESSION IN CELLULAR MODELS OF MOTONEURONAL DISEASES

 

Crippa V* (1), Simonini F (1), Carra S (2), Rusmini P (1), Sau D (1), Poletti A (1)

 

(1) Institute of Endocrinology, Centre of Excellence on Neurodegenerative Diseases of the University of Milan and InterUniversity Centre on Neurodegenerative Diseases (University of Florence, Rome and Milan)

Via Balzaretti 9, 20133 Milan. tel 02-5031.8215; fax 02-5031.8204; E-mail: angelo.poletti@unimi.it

(2) Centre de recherche en cancerologie de l'Universite Laval. L'Hotel-Dieu de Quebec,

9, rue McMahon, Quebec, QC, CANADA G1R  2J6. tel: (418) 691-5281/fax: (418) 691-5439

 

The small Heat Shock Proteins (HSP) family comprises 10 members in mammals where they are called the HSPB proteins (HspB1-10). They have chaperone activity, protect cells against diverse stress and may exert a neuroprotective role in conformational diseases, including polyglutamine diseases. HSPB proteins are upregulated in neurodegenerative disorders and mutations in HSPB1 and HSPB8 have been associated with peripheral neuropathies. The aim of this work was to investigate the role of HSPB8 in two different motoneuronal diseases: spinal and bulbar muscular atrophy (SBMA) and a familial form of amyotrophic lateral sclerosis (fALS). SBMA is caused by an expanded polyglutamine tract (polyQ) in the Androgen Receptor (AR); about 20% of hereditary cases of fALS are associated to mutations in Superoxide Dismutase 1 (SOD1) gene. Although AR and SOD1 do not share structural or functional domains, their mutant forms are unstable and tend to aggregate. In our studies, we used an immortalized motor neuronal cell line (NSC34) transfected with plasmids encoding for wild type and mutant forms of SOD1 (SOD1wt/G93A) or for mutant ARpolyQ (ARQ46). Both mutant proteins are characterized by: i) formation of intracellular aggregates, detectable by immunofluorescence technique; ii) the presence of PBS-insoluble materials, observed by filter retardation assay and iii) inhibition of proteasome activity, revealed by accumulation of YFPu (Yellow Fluorescent Protein with a degron signal for the proteasome system).

Overexpression of HSPB8 in NSC34 cells transfected with ARQ46 and SOD1G93A led to decrease of the levels of both mutant proteins. Both PBS-insoluble and SDS-insoluble forms of ARQ46 and SOD1G93A were found decreased in filter retardation assay and western blotting, respectively. Moreover, YFPu levels were reduced in presence of HSPB8, thus suggesting a desaturation of the proteasome system in these conditions. These results suggest that HSPB8 exerts chaperone activity towards both mutated AR and SOD1.

By immunoprecipitation analysis we found no direct interaction between HSPB8 and the two mutant proteins (AR or SOD1), indicating that HSPB8 does not need a protein-protein interaction to exert its chaperone function. Finally, the finding that treatment with proteasome inhibitor did not block the chaperone activity of HSPB8 towards mutant SOD1, suggests that HSPB8 could be a component of an alternative degradative pathway, such as the autophagy. Grants Telethon - Italy (#GGP06063),  MIUR-FIRB (#RBAU01NXFP); MIUR-Cofin (2005057598_002),  University of Milan-FIRST,  FONDAZIONE CARIPLO.


 

 

OC6

 

PROGRESSIVE MOTOR NEURONOPATHY : A CRITICAL ROLE OF THE TUBULIN CHAPERONE TBCE IN AXONAL TUBULIN ROUTING

 

SchŠfer MKE. (1), Schmalbruch H (2), Buhler E (1), Lopez C (1), Martin N(3), GuŽnet JL (3), and Haase G (1,*)

 

(1) Institut de Neurobiologie de la MŽditerranŽe, INSERM Equipe AVENIR, UniversitŽ de la MŽditerranŽe, campus de Luminy, F-13288 Marseille, France; Tel +33.4.91.82.81.27; Fax +33.4.91.82.81.01, E-mail: haase@inmed.univ-mrs.fr

(2) Panum Institute, University of Copenhagen, Denmark

(3) Institut Pasteur, Paris, France

 

Axonal degeneration represents one of the earliest pathological features in motor neuron diseases such as ALS. We here studied the underlying molecular mechanisms in progressive motor neuronopathy (pmn) mice mutated in the tubulin-specific chaperone TBCE. We demonstrate that TBCE is a peripheral membrane-associated protein that accumulates at the Golgi apparatus. In pmn mice, TBCE is destabilized and lost from the Golgi apparatus of motor neurons leading to rarefaction of microtubules in distal axons and axonal dying back. These degenerative changes are prevented in a dose-dependent manner by transgenic complementation with wildtype TBCE. In cultured motor neurons, siRNA-mediated TBCE depletion leads to defective axonal tubulin routing, an effect mimicked by Brefeldin A-mediated Golgi disruption. These data indicate that motor neurons critically depend on axonal tubulin routing from the Golgi apparatus, a process that involves TBCE and possibly other tubulin chaperones.

 

OC7

 

PROTEOME ANALYSIS OF SOLUBLE NUCLEAR PROTEINS UNRAVELS A NOVEL CELL-PROTECTIVE ROLE OF HMGB1/2 TO SUPPRESS GENOTOXIC STRESS IN THE POLYGLUTAMINE DISEASE PATHOLOGY

 

Okazawa H (1) and Qi M-L(1)

 

(1) Department of Neuropathology, Tokyo Medical and Dental University 1-5-45, Yushima, Bunkyo-ku, Tokyo, Japan tel.+81-3-5803-5847; fax. +81-3-5803-5847; E-mail: okazawa-tky@umin.ac.jp

 

Nuclear dysfunction is a key feature of the pathology of polyglutamine (polyQ) diseases. Mutant polyQ proteins are suggested to impair functions of nuclear factors by interacting with them directly in the nucleus. However, a systematic analysis of quantitative changes of soluble nuclear proteins in neurons expressing mutant polyQ proteins has not been performed. We performed a proteome analysis of soluble nuclear proteins prepared from neurons expressing huntingtin (Htt) or ataxin-1 (AT1) protein, and found that mutant AT1 and Htt similarly reduce the concentration of soluble high mobility group B1/2 (HMGB1/2) proteins. Immunoprecipitation and pull-down assays indicate an interaction of HMGBs with mutant AT1 and Htt. Immunohistochemistry with mouse models showed a reduction of these proteins from the nuclear region outside of inclusion bodies in affected neurons. Compensatory expression of HMGBs ameliorated polyQ-induced pathology in primary neurons and in Drosophila polyQ models. Furthermore, HMGBs repressed genotoxic stress signals induced by mutant Htt or transcriptional repression. Collectively, HMGBs might be critical regulators of polyglutamine disease pathology and could be targets for therapy development.

OC8

 

PML CLASTOSOMES PREVENT NUCLEAR ACCUMULATION OF MUTANT ATAXIN-7 AND OTHER POLYGLUTAMINE PROTEINS

 

Alexandre Janer*,1,2,3 Elodie Martin,1,2,3 Marie-Paule Muriel,1,2,3 Morwena Latouche,1,2,3 Hiroto Fujigasaki,6 Merle Ruberg,1,2,3 Alexis Brice,1,2,3,4 Yvon Trottier,5 and Annie Sittler1,2,3

 

1- Institut National de la SantŽ et de la Recherche MŽdicale U679, Neurologie et ThŽrapeutique ExpŽrimentale, 75651 Paris Cedex 13, France. tel:  01-42-16-22-22; fax: 01-44-24-36-58; E-mail: janer@ccr.jussieu.fr

2- H™pital de la PitiŽ-Salptrire, 75651 Paris Cedex 13, France.

3- FacultŽ de MŽdecine, UniversitŽ Pierre et Marie Curie, 75651 Paris Cedex 13, France.

4- DŽpartement de GŽnŽtique, CytogŽnŽtique et Embryologie, Groupe Hospitalier PitiŽ-Salptrire, 75651 Paris Cedex 13, France.

5- DŽpartement de Pathologie MolŽculaire, Institut de GŽnŽtique et Biologie MolŽculaire et Cellulaire, Institut National de la SantŽ et de la Recherche MŽdicale, Centre National de la Recherche Scientifi que, UniversitŽ Louis Pasteur, BP 10142, Illkirch Cedex, CU de Strasbourg, France.

6- Department of Neurology, Musashino Red Cross Hospital, Tokyo 108-8339, Japan.

 

In spinocerebellar ataxia type 7 (SCA7) and other neurodegenerative polyglutamine (polyQ) disorders, nuclear accumulation of toxic polyQ expanded proteins is associated with the disease process. It leads to the formation of insoluble nuclear inclusions (NI) in neurons, a hallmark of polyQ diseases. They could originate from a defect in protein folding, turnover or degradation. As neurons are post-mitotic and long-lived cells, failure to prevent the accumulation of toxic proteins may compromise their survival. Accordingly, molecules that prevent nuclear accumulation of polyQ proteins were shown protective against polyQ toxicity.

In the brains of patients with SCA7 and other polyQ disorders, PML (ProMyelocytic Leukaemia protein) nuclear bodies colocalized with NIs, suggesting a role of PML bodies in the polyQ protein aggregation. PML bodies are multiprotein nuclear complexes suggested to play a role in many cellular processes. However, a subset of PML bodies, called clastosomes, contain components of the ubiquitin-proteasome system (UPS) and were suggested to be sites of protein degradation in the nucleus.

We investigated how PML relates to the SCA7 pathogenesis. We studied the effect of modulating the expression of different PML isoforms. We demonstrate that PML isoform IV builds up PML bodies reminiscent of clastosomes, as they are enriched in UPS components. PML IV bodies actively recruit mutant ATXN7. As a result, PML IV leads to a loss of the fibrillar structure of mutant ATXN7 and inhibits the formation of ATXN7 aggregates, by increasing the degradation of the soluble form. Moreover, PML IV clastosomes also target other polyQ proteins for their degradation. Interestingly, beta-INF treatment, which up-regulates PML expression, mimicked the effects of PML IV overex

pression. Our study reveals that protein degradation capacity of the nucleus can be modulated by beta-INF in order to prevent the aberrant accumulation of mutant ATXN7 and other expanded polyQ proteins.

OC9

 

REDUCED HUNTINGTONâS DISEASE-LIKE STRIATAL NEURODEGENERATION IN MICE EXPRESSING A HUMAN 8-oxodGTPase

 

De Luca G (1a),  Russo MT (1a),  Degan P (2),  Tiveron C (3),  Zijno A (1), Mattei E (4),  Nakabeppu Y (5),  Crescenzi M (1),  Pzzola A (6),  Popoli P (6) and  Bignami M (1)

 

(1)Department of Environment and Primary Prevention, Experimental Carcinogenesis Division,  Istituto Superiore di Sanitˆ, Viale Regina Elena 299, 00161 Rome, Italy.

(2) Department of Translational Oncology, Istituto Nazionale per la Ricerca sul Cancro (IST-CBA), Genova, Italy.

(3) European Brain Research Institute, Rome, Italy

(4) Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy.

(5) Division of Neurofunctional Genomics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

(6)  Department of Drug Research and Evaluation, Central Nervous System Pharmacology Division Viale Regina Elena 299, 00161 Rome, Italy.

 

Several human neurodegenerative disorders are characterized by the accumulation of DNA 8-oxo-7,8-dihydroguanine (8-oxoG) in affected neurons. This can occur either through direct oxidation of DNA guanine or via incorporation from the oxidized dNTP pool during replication. Incorporation is normally prevented by the action of hydrolases that degrade oxidized purine nucleoside triphosphates. The major hydrolase in human cells is hMTH1 which degrades 8-oxodGTP and 8-oxoGTP as well as 2-hydroxy-dATP and 2-hydroxy-ATP1,15. We investigated the oxidized nucleic acid precursors as a possible source of DNA 8-oxoG by constructing a transgenic mouse (hMTH1-Tg+/+) expressing the human hMTH1 8-oxodGTPase. Steady-state and oxidant-induced levels of DNA 8-oxoG, and sensitivity to oxidant-induced killing were all lower in hMTH1-Tg+/+ embryonic fibroblasts than in wild-type cells. Paraquat treatment produced lower levels of DNA 8-oxoG in brain and in other organs of transgenic animals expressing high hMTH1 levels. Exposure of mice to 3-nitropropionic acid (3-NP) induces Huntingtonâs disease (HD)-like behavioural and neuropathological symptoms. Transgene expression conferred a dramatic protection against these, including 3-NP-induced weight loss, dystonia and gait abnormalities, death, and striatal degeneration. The findings implicate oxidized nucleic acid precursors in the neuropathological features of HD and identify striatal cell oxidized nucleoside triphosphates as a significant contributor to the pathogenesis of this disorder.


OC10

 

NEURODEGENERATION IN INHERITED ATAXIAS: FUNCTIONAL INTERACTIONS OF THE PRODUCT OF THE THG-1PIT GENE IN CEREBELLUM GRANULE AND PURKINJE CELLS OF THE MOUSE

 

Bosco A , Canterini S,  De Matteis V, Grillo D, Mangia F  and  Fiorenza MT

 

Department of Psychology, Section of Neuroscience, University "Sapienza" of Rome,  Piazzale Aldo Moro, 5 - 00185 Roma (Italy)

tel. +39-06-49917869; fax +39-06-49917871; E-mail: mariateresa.fiorenza@uniroma1.it

 

Proteins partners interacting with ataxia-causing protein have recently been characterized (Lim et al., 2006), including the protein product of THG-1, namely the human homologue of the murine Thg-1pit, a gene we identified and cloned a few years ago in a screen for genes acting downstream from the LIM-homeodomain transcription factor gene lhx3 (Fiorenza et al., 2001). According to Lim et al. (2006) ataxia causing proteins interaction network, THG-1 interacts with the apoptosisöinducing factor (AIF), the prion protein (PRNP), and sacsin (SACS). These findings pinpoint Thg-1pit as relevant to cerebellum physiopathology. A similar conclusion can also be drawn by our studies on patterns of Thg-1pit expression in the mouse developing and adult cerebellum. In fact, Thg-1pit is highly expressed in cerebellum granule cells starting from very early stages of development/differentiation of these cells (Canterini et al., 2005). In the adult, Thg-1pit becomes also activated in Purkinje cells in an asynchronous manner that follows the maturation of the synaptic contacts these cells establish with granule cells. This feature suggests the involvement of THG-1pit in synaptic function. In agreement with this idea, subcellular localization studies performed in our laboratory by: i) confocal microscopy immunolocalization in cerebellum histological sections, and ii) transfection of granule and neuroblastoma N1E-115 cells with a DNA construct expressing a chimeric eGFP-THG-1pit, have shown that THG-1pit is localized in the cytoplasm, but not the nucleus, at the level of Golgi bodies, neurite extensions and neurite terminals. We are presently identifying THG-1pit protein partners by immunoprecipitation experiments, using Thg-1pit-Myc transfected cell lysates of granule and N1E-115 cells. We expect this approach will give us a novel insight on functional THG-1pit interactions and the cellular pathway(s), in which this factor is involved in cerebellum granule and Purkinje cells.

OC11

 

DEPLETION OF GGA3 STABILIZES BACE AND ENHANCES BETA-SECRETASE ACTIVITY

 

Giuseppina Tesco (1)*, Young Ho Koh (1), Eugene Kang (1), Andrew Cameron (1), Shinjita Das (1), Miguel Sena-Esteves (2), Mikko Hiltunen (1), Shao-Hua Yang (3), Zhenyu Zhong (4), Yong Shen (4), James Simpkins (3) and Rudolph E. Tanzi (1)

 

1 Genetics and Aging Research Unit, Massachusetts General Hospital, Charlestown, MA 02129; 2 Neuroscience Center at Massachusetts General Hospital, Charlestown, MA 02129; 3 Department of Pharmacology & Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107; 4 Sun Health Research Institute, Sun City, AZ, USA.

 

Beta-site APP-cleaving enzyme (BACE) is required for production of the Alzheimer's disease (AD)-associated Abeta protein. BACE levels are elevated in AD brain, and increasing evidence reveals BACE as a stress-related protease that is upregulated following cerebral ischemia. However, the molecular mechanism responsible is unknown. We show that increases in BACE and beta-secretase activity are due to post-translational stabilization following caspase activation. We also found that during cerebral ischemia, levels of GGA3, an adaptor protein involved in BACE trafficking, are reduced, while BACE levels are increased. RNAi silencing of GGA3 also elevated levels of BACE and Abeta. Finally, we found that GGA3 levels are reduced in temporal cortex and cerebellum of AD patients. The decrease in GGA3 levels was more pronounced in the temporal cortex versus cerebellum, which is relatively spared of AD pathology. Importantly, decreased levels of GGA3 were inversely correlated with increased levels of BACE only in the temporal cortex, which is strongly impacted by AD pathology. In contrast, BACE levels were not significantly increased in the cerebellum of AD patients as compared to control subjects. These findings suggest that some subjects have lower levels of GGA3 independently of AD pathology, e.g. in cerebellum. This may then increase risk for AD especially under conditions leading to caspase activation in vulnerable brain regions e.g. due to stroke, which is a risk factor for AD. In summary, we have elucidated a novel GGA3-dependent mechanism regulating BACE levels and beta-secretase activity. Collectively, these findings suggest that events that induce caspase activation e.g stroke, which is a risk factor for AD, may trigger or precipitate AD pathology by lowering levels of GGA3 leading to reduced degradation of BACE, elevated beta-secretase activity, and increased production of Abeta.

P1

 

THE P150 SUBUNIT OF DYNACTIN (DCTN1) GENE IN AMYOTROPHIC LATERAL SCLEROSIS

 

*MŸnch C (1,2), Sperfeld AD (2), Meyer T (3), Ludolph AC (2)

 

(1) Department of Neurology, Jewish Hospital Berlin, Heinz-Galinski-Strasse 1, 13347 Berlin (Germany)

tel. +49-30-49942477; fax +49-30-49942982; email: muench@jkb-online.de

(2) Department of Neurology, University of Ulm, Ulm (Germany)

(3) Department of Neurology, CharitŽ Hospital, Humboldt-University, Berlin (Germany)

 

Retrograde axonal transport of vesicles and organelles involves the multiprotein complex dynactin. Several reports have shown that functional abnormalities of dynactin contribute to the pathogenesis of selective motor neuron degeneration. We performed a mutation screening of the p150 subunit of dynactin (DCTN1) gene in 420 patients with amyotrophic lateral sclerosis (ALS), 200 patients with multiple sclerosis (MS) and 350 unrelated controls. Heterozygous missense mutations of the DCTN1 gene were detected in one apparently sporadic case of ALS (T1249I), one individual with familial ALS (M571T), two patients with familial ALS and two unaffected relatives in the same kindred (R785W). Furthermore, a heterozygous mutation (R1101K) was detected in a patient with ALS and his brother with frontotemporal dementia (FTD). The sequence variants were excluded in 350 non-neurological controls. Given the common features of neurodegeneration in MS, FTD and ALS, we investigated whether sequence variants of the DCTN1 gene may be a contributory factor to neurodegeneration in MS. In MS patients we did not find mutations in the DCTN1 gene. The frequency of a single nucleotide polymorphism (R495Q) was not significantly different between patients and controls. In conclusion, the results support the hypothesis that mutations in the DCTN1 gene may act as susceptibility alleles for neurodegeneration in ALS and FTD but not for MS.

 

P2

 

EXPRESSION OF FMO1, FMO2, FMO3, FMO4 AND FMO5 GENES IN TRANSGENIC MICE MODELS OF ALS

 

Ogliari P.(1), Corato M (1),  Cova E. (1), Cereda C. (1), Bendotti C. (2) and Ceroni M (1)

 

(1) Experimental Neurobiology, Neurological Institute IRCCS C Mondino, Pavia, (Italy)

(2) Laboratory of Molecular Neurobiology, Department of Neuroscience, Istituto di Ricerche Farmacologiche 'Mario Negri', Milano, (Italy)

 

Amyotrophic Lateral Sclerosis (ALS) is an adult-onset, progressive and fatal neurodegenerative disease; his pathogenesis is unknown. Two major hypotheses have been proposed: oxidative stress and excitotoxicity. Flavin-containing monooxigenases (FMO) represent a gene family coding for microsomal enzyme that are involved in the oxidative metabolism of a variety of xenobiotics. This family contains five genes: FMO1, FMO2, FMO3, FMO4, FMO5. Their function is largely studied in liver, kidney and lung but not in nervous system. Recently some experimental data suggest a relation between ALS and FMO genes: 80% reduction of FMOâs mRNA expression in spinal cord and 3â-UTR polymorphisms of FMO1 gene have been observed in sporadic ALS patients. In this work the mRNA levels of FMO1, FMO2, FMO3, FMO4, FMO5 have been investigated in various areas of the nervous system (cerebellum, cerebral hemispheres, brainstem, spinal cord) in mouse by Real-Time PCR. Two different groups have been studied: control (mice C57BL/6J) and SOD1 transgenic mice (mice C57BL/6J with SOD1 G93A mutation). Real-Time PCR with TaqMan probes and Hprt as housekeeping gene has been performed. Analysis of variance has been made with non-parametric tests for the median. This study demonstrated that Fmo1, Fmo2, Fmo4 and Fmo5 genes are expressed in the tissue areas investigated and this expression is sex-dependent. In particular, in G93A males, we observed a lower expression of Fmo2 gene than in WT in all tissue areas; in brainstem, Fmo4 is significantly under expressed in G93A males compared to WT. G93A Female mice expressed significantly greater amounts of Fmo2 and Fmo5 genes in cerebellum and cerebral hemispheres. This work represents the first attempt to analyse Fmo gene expression in nervous system areas and particularly in spinal cord. Although this study show a correlation between the expression of Fmo genes and a transgenic model of ALS but its significance in the onset of ALS disease is unknown.


P3

 

IDENTIFICATION OF GENES THAT REGULATE THE PROCESSING OF THE BETA-AMYLOID PRECURSOR PROTEIN AND ARE CANDIDATES FOR ASSOCIATION WITH LATE-ONSET ALZHEIMERâS DISEASE BY A GENOME-WIDE siRNA SCREEN

 

John Majercak*1, David Stone2, Krista Getty3, Amy Espeseth1, Adam Simon1, Shane Marine3, Erica Stec3, Marc Ferrer3, Steven Bartz2, Adam Gates1, Carrie Wolffe1, Paul Shughrue1, Julja Burchard1, Ken Koblan1, Berta Strulovici3, Daria Hazuda1, Alan Sachs2, Mark Shearman1, Guy Seabrook1,  Jim Ray1

 

1Alzheimer's Research, Merck Research Laboratories, West Point, PA; (2) Molecular Profiling, Rosetta Inpharmatics, Seattle, WA; (3) Automated Biotechnology, Merck Research Laboratories, West Point, PA

 

Mutations causing familial Alzheimerâs disease (AD) shift APP metabolism towards pro-amyloidogenic processing by either elevating total Abeta secretion or changing the ratio of Abeta40/Abeta42 peptides. To identify candidate late onset AD (LOAD) genes that may modulate either the non- or pro-amyloidogenic processing of APP, we used a functional genomics approach to assess 15,200 human genes for their effects on secreted metabolites of APP.  HEK293 cells stably expressing an optimized APP construct were transiently transfected with pools of three siRNA molecules, each targeting a single human transcript and Abeta40, Abeta42, sAPPalpha, and sAPPbeta were measured in the culture medium.  Additional experiments were performed to control for effects on cell viability and non-specific transgene regulation or general secretion. We identified over 800 siRNAs that significantly altered the levels of secreted Abeta. However, many of these siRNAs were false positives due to significant suppression of transgene expression without a direct affect on secretase processing of APP.  In contrast, a subset of siRNAs raised sAPPalpha, lowered beta- site cleavage products without affecting transgene and endogenous APP expression similar to siRNAs targeting the secretases responsible for Abeta production. One of these genes, LRRTM3, maps to a region on chromosome 10 (10q21.3) associated with both elevated plasma Abeta42 and LOAD incidence. LRRTM3 is expressed nearly exclusively in brain regions, including those affected during AD, and siRNAs significantly inhibit beta-secretase mediated amyloidogenic processing of APP in SH-SY5Y neuroblastoma cells and primary neurons. Interestingly, LRRTM3 shares homology with the NOGO receptor (RTN4R), a gene involved in axonal sprouting and a cofactor for APP processing.

P4

 

PRELIMINARY RESULTS OF THE STUDY ON HAPLOTYPE LINKED TO FMR1 GENE IN POLISH ATAXIA PATIENTS AND IN CONTROLS.

 

*Marta Rajkiewicz*, Anna Sulek-Piatkowska, Walentyna Szirkowiec, Wioletta Krysa, Elzbieta Fidzianska, E. Zdzienicka

 

*Institute of Psychiatry and Neurology, Genetic Department,Sobieskiego street 9, Warsaw 02-957, Poland, e-mail: rajkiewi@ipin.edu.pl, tel. +48 22 45 82 567, fax: +48 858 91 69

 

In some triplet repeats expansion diseases, (HD, SCA, DM, FXTAS- Fragile X tremor ataxia syndrome), the linkage disequilibrium between expansion mutation and some closely linked DNA markers is observed. This indicates that such chromosomal background might contain external to the repeat cis-elements that predispose, or drive to trinucleotide expansions.

The aim of our study was an attempt to find the haplotypes specific of instability of CGG repeats in FMR1 gene among our group of patients with ataxia syndrome and in controls.

Among haplotypes found to be in linkage disequilibrium with FMR1 gene we have choosen 5 markers: DXS548, FRAXAC1, (FMR1), ATL1, FRAXAC2 and FMRb and have analyzed them for 41 patients with ataxia symptoms and in 61 controls. In the ataxia patients group we have previously excluded SCA (8 types), HD and DRPLA, and this group was characterized by intermediate size CGG repeats (35 CGG).

The possible markers polymorphisms examined in patients and in controls were: for SNP -ATL1 (A,G), FMRb (A,G), for microsatelite polymorphism: FRAXAC1 (1, 2, 3, 4, 5, *6*), DXS548 (1, 2, 3, 4, 5, 6, 7, *8*), and FRAXAC2 (1, 2+, 2, 3+, 3, 4+, 4, 5+, 5, 6+, 6, 7+, 7, *8+, 8, 9+*). Interestingly we found the microsatellite markers polymorphisms previously not observed in other populations ö marked in bold letters.

The control group was characterized by 4 most frequent haplotypes in the following order: DXS548, FRAXAC1, ATL1, FRAXAC2, FMRb: 7-4-A-7+-G (14,6%), 7-4-G-7-G (9,8%), 7-4-G-7+-G (11%), 7-5-G-9+-G (6,1%).

In the patients group we have indicated 4 most frequent haplotypes: 2-2-G-6-A(13,9%), 7-4-A-7-G (16,7%), 7-4-A-7+-G (13,9%), 7-4-G-7-G (13,9%).

The 2-2-G-6-A and 7-4-A-7-G haplotypes were found only in ataxia patients, and it was not observed in

the control group- the difference being statistically significant.

Interestingly we have also found 2-2-G-6-A haplotype in 2 of 3 patients with FraX syndrome

P5

 

ANALYSIS OF THE MUTATIONS SPECTRUM IN EARLY-ONSET PARKINSON DISEASE PATIENTS. THE USE OF MLPA TECHNIQUE IN DETECTION OF REARRANGEMENTS IN THE PARKINSON GENES.

 

D.Hoffman-Zacharska(1)*, M.Nawara(1), D.Koziorowski(2), J.Slawek (3), A.Friedman(2)

(1)Dept.of Medical Genetics; Institute of Mother and Child, Warsaw;Kasprzaka 17A, 01 211 Warsaw (Poland): e-mail:dhoffman@poczta.wp.pl

(2) Department of Neurology, Faculty of Health Science, Medical University in Warsaw

(3) Dept. of Neurological and Psychiatric Nursing, Medical University in Gdansk

 

Parkinsonâs disease (PD) is one of the most frequent neurodegenerative disorders. Majority of PD cases are sporadic but the monogenic form of PD has been described. Mutations in SNCA, UCHL1 and LRRK2 result in autosomal dominant form of PD. Mutations in PARK2, DJ-1 and PINK1 result in autosomal recessive PD.

Although PD is the disease of aging brain the early onset form of the disease (EO-PD) have been defined (age of onset between 20-40). Mutations in PARK2, DJ-1 and PINK1 contribute to develop this form, but the PARK2 mutations are reported to be the major cause of the EO-PD in familial/isolated cases of J-PD. The frequency of PARK2 mutations is still not known, but it has been established in Europe at 50% in EO-PD families with recessive inheritance and 18% in isolated patient. Over 100 pathogenic variants of PARK2 have been described exon rearrangements (deletions or duplications) and point mutations (missense, nonsense, one/two bp deletion/insertion). Various types of mutations, depending on the ethnic background, are found at variable frequency. In European populations rearrangements and point mutations are found to be equally frequent. Exon rearrangements have been also reported in the DJ-1.

As there is no information about mutations contributing to EO-PD in Polish patients, we have performed such analysis. Following the expectation of the equal number of rearrangements /point mutation in the main gene PARK2 (data for European populations), we decided to start with analysis of gene rearrangements using the multiplex ligation-dependent probe amplification (MLPA).Usage of two available MLPA PD kits allows analysis of PARK2 exons, PACRG exon 1(in intron 1 of PARK2) and some regions of the DJ-1 and PINK1.

The results of the MLPA analysis for 30 EO-PD Polish patients will be presented. The obtained data suggest that rearrangements in PARK2 and other analysed genes are not common type of mutations. It seems that the point mutation analysis as the first step may be the better way to identification of the molecular defect in EO-PD.

P6

 

ASSOCIATION OF FUNCTIONAL MXA PROMOTER POLYMORPHISMS AND THE RISK OF ALZHEIMERâS DISEASE IN CHINESE

 

Tang NLS(1), Ma SL(1), Huang W(1), Lam LCW (2), Chiu HFK(2)

(1) Department of Chemical Pathology, The Chinese University of Hong Kong; Email: nelsontang@cuhk.edu.hk

(2) Department of Psychiatry, The Chinese University of Hong Kong.

Alzheimerâs disease (AD) is the most common form of dementia among the elderly. The cause of AD is still unknown but the involvement of inflammation in its pathogenesis has been supported by several lines of evidence. MxA protein is an interferon-induced protein and it was expressed in senile plaques in AD brain. In this study, we aimed to investigate the association between MxA promoter polymorphisms and the risk of AD in a Chinese population.

 

Two promoter SNPs in the MxA gene were investigated in two sample sets of patients diagnosed with NINCDS-ADRDA criteria of possible and probable AD and 197 age-matched healthy Chinese subjects. The genotypes of the subjects on these SNPs were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Significant associations were detected by chi-square test and haplotype analysis. Functional study on the promoter activities of the MxA SNPs were investigated by a dual luciferase reporter assay.

 

MxA-123 was associated with the risk of AD in clinical AD group (p<0.001) and it might modulate the severity of the disease. -123A/-88T haplotype showed significantly higher MxA promoter activity than the wild-type haplotype, -123C/-88G, in different types of cell line such as SK-N-MC, HeLa and OVCAR-3. Among the cell lines we tested, the increase in MxA promoter activity by -123A/88T haplotype was the highest in neuroblastoma cell line, with 2-fold increase. This might suggest the possible involvement of MxA in the brain. In conclusion, results presented here suggested the functional polymorphisms in MxA gene was associated with the risk of AD.

P7

 

HUNTINGTIN AGGREGATE FORMATION PRECEDES NEURODEGENERATION IN VITRO AND IN VIVO AS DETERMINED BY AGAROSE GEL ELECTROPHORESIS 

 

Andreas Weiss(1)*, Corinna Klein(1), Ben Woodman(2), Kirupa Sathasivam(2), Miriam Bibel1, Etienne RŽgulier(1), Gillian P. Bates(2) and Paolo Paganetti(1)

 

(1)Neuroscience Discovery, Novartis Institutes for Biomedical Research, Basel, Switzerland

(2)Department of Medical and Molecular Genetics, King's College London School of Medicine, London, UK

 

Huntington's disease (HD) is caused by a CAG repeat expansion in the huntingtin (htt) gene producing a mutant protein with a pathogenic polyglutamine insert at the N-terminus. Htt is predicted to adopt an aberrant, aggregation-prone conformation in particular following proteolytic digestion releasing toxic N-terminal fragments. Htt deposits represent a major pathologic hallmark and may become useful biomarkers for disease progression. Therefore, thorough analysis and determination of htt aggregates and breakdown products are essential for testing therapies designed to decrease htt or interfere with its aggregation. Characterization of htt-aggregates in HD models both in vitro and in vivo relies mostly on filter-trap assays or histochemistry: both methods have analytical limitations. DNA-type agarose gel electrophoresis is a simple and robust method to investigate protein inclusions. We applied this technique to study htt-aggregate formation in a neural cell line as well as in primary striatal cells. We then proceeded to investigate the kinetics of htt-aggregate formation in brains and muscles of transgenic mouse models of HD, the R6/2 mice and the Hdh150 mice. In R6/2 mice htt-aggregates were already detected at 2 weeks of age, their amount increased rapidly with time and attained at 14 weeks of age a signal 340-fold above background. Further analysis showed that cytoplasmic, but not nuclear, htt-aggregates grew drastically in relative size during aging. Our method allows for a very simple, rapid and sensitive quantitative and qualitative biochemical analysis of protein complexes in tissue extracts during neurodegeneration.

 

P8

 

ABSENCE OF ASSOCIATION BETWEEN ERYTHROPOIETIN GENE VARIATION AND ALS IN SPORADIC ITALIAN PATIENTS

 

Ghezzi S (1)*, Del Bo R (1), Corti S (1), Santoro D (1), Prelle A (1), Nardini M (1), Siciliano G (2), Mancuso M (2), Murri L (2), Bresolin N (1), and Comi GP (1)

 

(1) IRCCS Foundation Ospedale Maggiore Policlinico Mangiagalli and Regina Elena, Milan; Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, Via F. Sforza, 35 Milano (Italy) tel +39-02-55033843; fax +39-02-50320430; e-mail: serena.ghezzi@fastwebnet.it.

(2) Department of Neuroscience, Neurological Institute, University of Pisa.

 

Increasing evidence points to a pivotal role of erythropoietin gene (EPO) in the pathophysiology of motor neuron disease. In addition to its role in erythropoiesis, EPO exerts neuroprotective effects following the occurrence of ischemic, hypoxic, metabolic neurotoxic or excitotoxix stress in the central nervous system. Recently, it has been reported that EPO delays disease onset in an amyothrophic lateral sclerosis (ALS) model and high EPO levels are present in cerebral fluid from hypoxemic ALS patients.

To investigate the role of EPO as genetic determinant in the susceptibility to sporadic ALS.

222 Italian sporadic ALS patients were consecutively recruited; the 3â hypoxia-responsive element of the EPO gene was amplified and directly sequenced; all patients were also screened for two known EPO gene polymorphisms (C3434T and G3544T). EPO variability was also determined in 204 healthy control subjects matched for age, sex and ethnic background to cases.

No gene mutation within the 3âhypoxia-responsive element was detected; the two EPO polymorphisms were not differently distributed in patients and controls; furthermore, haplotype analysis revealed no association between variants and the risk of ALS.

In conclusion, our data argue against the hypothesis of EPO gene as risk factor for motoneuron dysfunction, at least in Italian patients. Additional studies are required to asses the potential therapeutic effects of EPO in protecting motor neurons against hypoxic stress.

 

P9

 

UNSTABLE (CCTG)n REPEAT TRACT OF LOW RANGE IN A FAMILY WITH CLINICAL SYMPTOMS OF MYOTONIC DYSTROPHY.

 

A. Sulek-Piatkowska (1), A. Lusakowska (2), W. Krysa (1), A. Kostera-Pruszczyk (2), M. Rajkiewicz (1), J. Zaremba (1)

 

(1) Department of Genetics, Institute of Psychiatry and Neurology, Warsaw, Poland

(2) Department of Neurology, Medical Academy, Warsaw, Poland

 

Myotonic dystrophy type 2 (DM2) inherited in autosomal dominant trait is caused by dynamic mutation of CCTGn repeats in intron 1 of the ZNF9 gene on chromosome 3. DM2 is a clinically heterogenous neuromuscular disorder characterized by: muscular dystrophy, myotonia and multisystem involvement. The normal CCTG repeat sequence is composed of complex repeat motif: (TG)n(TCTG)n(CCTG)n of size range from 104 to 176 base pairs. Interruptions of GCTG and TCTG or 1 -2 TCTG in normal motif are observed.

Characteristic feature of the DM2 patients is the loss of interruptions and CCTGn repeats expansion from 75 to 11 000 repeats. Expansions of CCTG produce mRNA with excessive repeats that probably leads to the increase of CUG-binding protein level and abnormal splicing of different genes causing pathological symptoms of DM2.

In a Polish control group composed of 108 healthy individuals the range of repeat motif varied from 122 to 155 bp which corresponds to the results obtained in other populations.

One of our patients suspected of DM was a 7 years old girl who had difficulty in starting of walking when she was 3 years old. She also complained of stiffness of the muscles of lower legs which disappeared after more intense movement. In EMG examination the myotonic discharges were observed. Her father complained of cramps and muscle stiffness but neurological and EMG examinations performed in him were normal.

The molecular test for DM2 revealed  the 197 bp fragment in the affected girl and 189 bp fragment in her father, which  revealed instability and 8 bp expansion at transmission from the father to his daughter. Such a length of the repeat motif was not reported until now and it is unclear wether it is within the range of premutation or mutation of incomplete penetrance. This is also the first report of possible anticipation in DM2.

P10

 

DIFFERENT AGE AT ONSET IN HD FAMILIES WITHOUT TRANSGENERATION (CAG)n EXPANSION.

 

Krysa W., Sulek-Piatkowska A., Zdzienicka E., Hoffman-Zacharska D., Fidziaska E., Rajkiewicz M.,Zaremba J.

 

Department of Genetics, Institute of Psychiatry and Neurology, Warsaw, Poland

 

Huntington disease (HD) is a progressive, late onset, neurodegenerative disorder inherited as an autosomal dominant trait characterised by abnormalities of movement, cognition and emotion with prominent features including  involuntary choreoathetotic movements and dystonia. Cognition impairment and dementia are the features of advanced stages of the disease.

HD is caused by dynamic mutation involving expansion of CAG tract in open reading frame of IT15 gene.

HD like other triplet repeat expansion diseases exhibits the anticipation phenomenon wherein there is the increase in severity and reduction of the age of onset in successive generations. It has been explained by the increase in repeat number when transmitted from affected parents to offspring.

During 11 years of genetic testing for HD in The Institute of Psychiatry and Neurology we have collected over 650 pedigrees affected with HD. Among them we selected 10 parent-child pairs who carry the same mutation with equal CAG repeats number. Interestingly, in every pair they exhibit distinct ages of onset - the difference being up to over 20 years - within single pair parent-child from 25 to 3 years.

Strong negative correlation between CAG repeats number and age of onset in HD is a well known fact. However, as our data show, prediction of the HD first symptoms onset, based on CAG repeats  expansion size, can be misleading even among members of the same family.


P11

 

NOVEL Q23R SOD1 MUTATION ASSOCIATED WITH MUSCLE MITOCHONDRIAL DYSFUNCTION

 

Ronchi D (1)*, Corti S (1), Bordoni A (1), Santoro A (1), Papadimitriou D (1), Lamperti C (1), Lucchini V (1), Magri M (1), Guglieri M (1), Crugnola V (1), Moggio M (1), Bresolin N (1), and Comi GP (1).

 

(1) IRCCS Fondation Ospedale Maggiore Policlinico Mangiagalli and Regina Elena, Milan; Dino Ferrari Centre, Department of Neurological Sciences, University of Milan, Via F. Sforza, 35 Milano (Italy) tel +39-02-5503-3843; fax +39-02-50320430; e-mail: darioronchi@tin.it.

 

To describe the clinical and muscle mitochondrial oxidative defect associated with a novel SOD1 mutation.

 

Mutation in the gene encoding Cu/Zn superoxide dismutase (SOD1) is responsible for 20% of Famililal Amyotrophic Lateral Sclerosis (ALS) patients. Substantial evidence underlines that mitochondrial dysfunction is involved in ALS pathogenesis. In fact, microdeletion of COX subunit 1 has been reported, and recent studies suggest that the toxicity of mutant SOD1 arises from its selective recruitment to motoneuron (MN) mitochondria.

The proband, a 40-year-old man from Bangladesh was investigated for symmetrical limb muscle weakness. The patient s mother died at 35 age for pneumonia, while a maternal uncle presented a referred muscle impairment at the lower limbs. The whole SOD1 coding sequence was amplified by PCR, followed by automated sequence analysis.

The patient reported progressive proximal lower limb muscle weakness by the age of 37, involving after one year also the arms. At age 40 years, neurological examination shows proximal and distal limb muscle weakness with distal atrophy, characterized by lower rather than upper MN impairment. Diffuse fasciculations were present, also in the tongue. EMG showed acute and chronic denervation findings, while brain and spinal MRI was normal. A muscle biopsy showed neurogenic pattern associated with cytochrome c oxidase (COX) deficieny in several muscle fibers, mainly in the atrophic ones. No apoptotic nuclei were found with TUNEL reaction. Direct sequencing revealed a heterozygous mutation CAG to CGG in codon 23 substituting glutamine to arginine in the SOD1 gene (Q23R).

The novel SOD1 Q23R mutation affects a highly conserved aminoacidic position and results in a early-onset ALS phenotype. The mutation is likely to confer to the mutant SOD1 protein a mitochondrial toxicity also in muscle tissue, as demonstrated by partial cytochrome c oxidase dysfunction.

P12

 

SCREENING FOR PEPTIDES THAT INTERACT WITH MUTANT-HUNTINGTIN

 

Carnemolla A* (1), Michelazzi S (1), Del Sal G (2), Persichetti F (1)

 

(1) Neurobiology Sector, International School for Advanced Studies SISSA/ISAS, Area Science Park, S.S. 14 Basovizza - 34012 Trieste; Tel. 040-3756534; Fax 040-3756502 e-mail: calisia@sissa.it

(2)Department of Biochemistry, University of Trieste, Laboratorio Nazionale CIB, Area Science Park, Padriciano 99 - 34012 Trieste.

 

Huntingtonâs disease (HD) is an inherited neurodegenerative disorder characterized by choreiform movements, psychiatric and cognitive decline, and the graded loss of neurons in the striatum. The mutation that causes disease is an expansion of a CAG repeat in the HD gene that extends a segment of glutamine residues in the protein huntingtin (Htt). The abnormal conformation of mutant huntingtin, imposed by the expanded glutamine tract, is likely to induce aberrant interaction with specific cellular targets. Therapies aimed to disrupt such abnormal interactions may therefore be successful in slowing the pathogenic process. 

Here we report the expression of a combinatorial library of constrained 16-residues peptides displayed by the active site loop of E. coli thioredoxin and the use of a yeast two-hybrid system to select those that bind mutant huntingtin, using the N-terminal region of mutant huntingtin (Htt1-550Q60) as bait. We have identified a number of peptides that specifically interact with huntingtin. In silico analysis does not show significant similarity of the isolated peptide aptamers to any known protein, but very interestingly they all share a common positively-charged region. We are currently investigating the ability of the isolated peptides to interfere with the abnormal phenotypes induced by mutant huntingtin in STHdhQ111 cell lines. 


P13

 

THE LOC387715 LOCUS IN ALZHEIMER'S DISEASE

 

Benerini Gatta L (1), Venturelli E (2), Galimberti D (2), Scarpini E (2), and Finazzi D (1,3)*

 

(1) Section of Chemistry, Faculty of Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy.

(2) Department of Neurological Sciences, ãDino Ferrariä Center, University of Milan, Fondazione IRCCS Ospedale Maggiore Policlinico, via F. Sforza 35, 20122 Milan, Italy.

(3) Terzo Laboratorio di Analisi Chimico Cliniche, Spedali Civili di Brescia, P.le Spedali Civili 1, 25123 Brescia, Italy.

 

Age-related macular degeneration (AMD) is the most common cause of acquired visual impairment in the elderly and it is characterized by degeneration of photoreceptors and retinal pigment epithelium. A genome-wide scan showed that the region q26 on chromosome 10 has strong genetic linkage with AMD; further studies pinned down the strongest association with the rs10490924 polymorphism which causes the Ala69Ser substitution in the protein encoded by the putative LOC387715 gene. A nearby region on chromosome 10 has been associated with an increased individual risk of late onset Alzheimer's Disease.  We decided to investigate whether the same locus could be a risk modifier for AD. Firstly, we set up RT-PCR experiments to verify the presence of the specific mRNA in the brain. We could show the presence of a specific transcript in the hippocampus from two different subjects. Other cortical brain areas are under investigation.

Secondly we analyzed the presence and distribution of genomic DNA sequence variations in exon 1 of the putative LOC387715 gene in a population of 131 AD patients and 109 controls. Exon1 covers more than 90% of the entire ORF.

We identified 3 different type of nucleotide substitutions: c+73G>A, c+177T>G and c+270G>A (rs10490924). The c+270G>A (Ala69Ser) polymorphism seems to have a higher incidence in the AD population (25.5% vs 18.8%, allele frequencies), with a borderline statistical significance. We are extendying the analysis to further subjects in order to validate the preliminary results.

P14

 

C.ELEGANS AS A MODEL FOR AXONAL DEGENERATION

 

Di Schiavi E*(1),  Esposito G (1),  Bazzicalupo P (1),  Hilliard MA (2)

 

(1) IGB-ABT, CNR, Via P. Castellino, 111, 80131 Naples, ITALY

Tel. +390816132365;  FAX +390816132350; e-mail: dischiav@igb.cnr.it

(2) The Rockefeller University, 1230 York Avenue, New York

New York 10021-6399 USA

 

The genetic causes and the cellular mechanisms that can trigger axonal degenerative diseases are still largely unknown.  The nematode C. elegans has been widely utilized as a model system to address many neurodevelopmental questions and more recently as a model system for neurodegenerative diseases. To study the effects of the accumulation of specific proteins in neurons (axons and cell bodies), a neuronal defective phenotype that can mimic the one observed in human diseases was obtained by expressing selectively in specific neurons the putative toxic proteins. With this approach cellular models of Polyglutamine-expansion diseases, Parkinson Disease and tauopathy disorders have been obtained in C. elegans. Remarkable findings have come out from these studies including the discovery of the protective role that Resveratrol, a sirtuins activator, has on polyglutamin cytotoxicity in C. elegans as well as on mammalian neurons. However the genes and mechanisms acting intrinsically on the axonal degeneration process have instead not yet been investigated. We would like to use C. elegans neurons as an experimental system to study axonal degeneration and, with a genetic approach, identify the key molecules of this process. We recently developed a modification of the RNA-interference approach that can reduce in chosen C. elegans neurons, that are otherwise resistant to RNAi, the function of genes. It is thus now possible, with this approach, to dissect the role exerted by the gene of interest in specific cells or groups of cells and to study essential genes that could not be analysed because of lethality or sterility. We would like to follow a best candidate approach and look for genes that, once silenced in selected GFP-expressing neurons, will result in an axonal degenerative phenotype. In a second approach we will knock-down many lethal/sterile genes in specific neurons to test their possible role in manteinance of axonal shape and functionality and in neuronal survival.

 

P15

 

CHLORIDE INTRACELLULAR CHANNEL 1 (CLIC1) PLAYS A CENTRAL ROLE IN REGULATING FREE RADICAL GENERATION BY MICROGLIA IN RESPONSE TO BETA-AMYLOID

 

Milton RH (1), Abeti R (1), Duchen MR (1), S.N. Breit (2), and (*)Mazzanti M. (3)

 

(1)Dept. of Physiology, University College London, WC1E 6BT, UK; (2)Centre For Immunology, St Vincent's Hospital and University of NSW, 2010 AUSTRALIA, (3) Dept. Biomolecular Sciences and Biotecnology, Via Celoria 26, University of Milano, I20133, Italy. Tel:0039 02 50314958, Fax 0039 02 50314932, e-mail michele.mazzanti@unimi.it

 

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimerâs disease, for example, is characterized by an accumulation of beta-amyloid protein (AB) in neuritic plaques which are infiltrated by reactive microglia and astrocytes. Microglia, the immune cells of the CNS, are activated by AB and, among many other responses, produce reactive oxygen species (ROS) through activation of the plasmalemmal NADPH oxidase. Generation of ROS by microglia is thought to contribute to the cell death seen in AD. We have previously shown that AB activates a chloride current mediated by the protein CLIC1 (Novarino et al, 2004). We now show that AB-induced microglial ROS production is contingent upon the channel activity of CLIC1. Rates of ROS generation were measured using hydroethidium fluorescence from BV2 and primary microglial cells. Suppression of CLIC1 protein expression using inhibition of the CLIC1 chloride current using IAA-94, replacement of extracellular Cl- with impermeant anions, transfecting microglia cells with specific siRNA, or using an antibody to the channel protein, all significantly reduced the ROS response to AB (p<0.01).

CLIC1 is essentially a soluble cytosolic protein. Imaging the distribution of a CLIC1-GFP fusion protein and immunofluorescence studies showed that AB promoted translocation of CLIC1 from the cytoplasm to the cell membrane. We propose that CLIC1 translocates from the cytoplasm and inserts into the membrane to form a chloride selective channel where it serves to compensate the charge generated by NADPH oxidase function and so facilitate sustained microglial ROS production in response to AB.

P16

 

DROSOPHILA HTRA2 FUNCTIONS DOWNSTREAM OF THE PINK1/PARKIN PATHWAY

 

Begum RN (1), Tain LS (2), Plun-Favreau H (3), Martins LM (4), Downward J (3), Whitworth AJ (2) and Tapon N (1)

 

(1) Apoptosis and Proliferation Control Laboratory, Cancer Research UK, London Institute, 44 Lincoln's Inn Fields, London, United Kingdom

(2) Department of Biomedical Sciences, University of Sheffield, Sheffield S10 2TN, United Kingdom

(3) Signal Transduction Laboratory, Cancer Research UK, London Institute, 44 Lincoln's Inn Fields, London, United Kingdom

(4) Cell Death Regulation Laboratory, MRC Toxicology Unit, Lancaster Road, Leicester LE1 9HN, United Kingdom

 

Parkinson's disease (PD) is one of the commonest forms of neurodenegenerative disorders. It is characterised by loss of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc), and the formation of Lewy bodies (LB), which are intracytoplasmic aggregates of alpha-synuclein. Genes firmly implicated in familial PD thus far are alpha-synuclein, Parkin, DJ-1, LRRK2, PINK1, UCHL1 and ATP13A2. The study of these genes has led to considerable progress in the understanding of PD. Impaired mitochondrial function is a common trait in PD patients, and is likely to play a significant role in pathogenesis of the disease. Drosophila has recently emerged as a leading model to study PD5. Mutations in Drosophila parkin (park) and pink1 cause reduction in lifespan, loss of dopaminergic neurons, degeneration of flight muscles, male infertility and abnormal mitochondrial morphology and function. The pink1 mutant phenotype can be rescued by park overexpression, suggesting for the first time that these proteins could function in the same pathway, likely influencing mitochondrial homeostasis. The mammalian Omi/HtrA2 protein is a mitochondrial protease that has been associated with parkinsonism. Mutation or deletion of mouse HtrA2 result in a parkinsonian phenotype, degeneration of striatal neurons and mitochondrial dysfunction. Furthermore, mutations in HtrA2 have been identified in PD patients. Here, we show that deletion of the Drosophila homolog, dHtrA2, share phenotypic similarities with the pink1 and park mutants. In addition, dHtrA2 overexpression rescues the pink1 and park mutant phenotypes. We propose that dHtrA2 may function in maintaining mitochondrial integrity downstream of the Pink1/Parkin pathway.

 

P17

 

THE FAD-LINKED PRESENILIN-2 MUTATION T122R INCREASES ENDOPLASMIC RETICULUM CALCIUM LEAKAGE IN FAD FIBROBLASTS AND DIFFERENT MODEL CELLS.

 

Brunello L (1), Zampese (1), Ghidoni R (2), Binetti G (2), Pizzo P (1), and *Fasolato C (1)

 

(1) Department of Biomedical Sciences, University of Padua, Padua (Italy) Tel. +39-049-8276065; Fax: +39-049-8276049; E-mail: cristina.fasolato@unipd.it

(2) NeuroBioGen Lab-Memory Clinic, IRCCS S. Giovanni Di Dio-FBF, Brescia (Italy)

 

We have shown that in Familial Alzheimerâs Disease (FAD), the mutations in presenilin-1 (PS1), P117L and M146L, and presenilin-2 (PS2) M239I, N141I and T122R, reduce the endoplasmic reticulum (ER) and Golgi apparatus (GA) Ca2+ level in various model cells. The phenomenon, which is mimicked by over-expression of wild-type presenilins, is also consistent with the reduced cytosolic Ca2+ release, induced by depletion of intracellular stores, that we previously found in human FAD fibroblasts (1-3). Furthermore, our findings are also consistent with the role of Ca2+ leak channels, recently suggested for wild-type presenilins (4). However, in contrast with the "Ca2+ overload" hypothesis for AD (4,5), the above mentioned FAD-linked PS mutations increase rather than decrease ER Ca2+ leakage.  The present study focuses on the type of Ca2+ dysregulation which is induced by PS2-T122R, among the strongest mutants affecting intracellular Ca2+ stores. Ca2+ dynamics at the cytosolic, ER and GA level were studied following transient or stable expression of PS2-T122R together with suitably targeted aequorins in mouse embryonic fibroblasts (MEFs) knockout for both presenilins and in the human neuroblastoma cell line SH-SY5Y. Experiments were designed to exploit the involvement of IP3/ryanodine receptors, Ca2+ ATPase and translocon as possible PS targets which could directly be responsible for the reduced Ca2+ level. Preliminary results indicate that PS2-T122R increases the ER leakage by a mechanism independent of the translocon complex and possibly involving ryanodine receptors.

 

1. Zatti et al. Cell Calcium 39, 539-550, 2006.

2. Zatti et al. Neurobiol. Dis. 15, 269-278, 2004.

3. Giacomello et al. Neurobiol. Dis. 18, 638-648, 2005.

4. Tu et al. Cell 126:981-993, 2006.

5. Smith et a

l. Cell Calcium, 38, 427-437, 2005.

P18

 

ROLE OF PPP4R2 IN APOPTOSIS AND NEURITOGENESIS

 

Bosio Y(1) , Di Cunto F (1)

 

1)Departement of Genetic, Biology and Biochemistry,,University of Turin ,Molecular Biotechnology Center, via Nizza 52 10126 Turin +39-011-6706410; e-MAIL ylenia.bosio@unito.it

 

Phosphatase 4 is a ubiquitous serine /threonine phosphatase that rules many various cellular functions including mitosis, apoptosis , DNA repair. The regulation of these different activities in the cell is due at the interaction of the enzyme with different regulatory subunits that give specificity of function and cellular districts.

One of these is the regulatory subunit 2 of phosphatase 4 (PPP4R2), a protein of 50 kda, which functions are mainly unknown; PPP4R2 has been identified previously  as interactor of  Survival of Motor  Neuron complex (SMN complex).

The latter is defective in spinal muscular atrophy , a disease that affects  the motor neurons of spinal cord ; the main function of this SMN complex is the regulation of different aspects in RNA metabolism  and splicing , an essential feature of all cell types, so is not clear the reason for which the lack of SMN  leads to a so selected disease. Recently is appearing that SMN has fundamental rules in neuronal apoptosis and neuritogenesis.

Therefore is  important to know the molecular partners  of SMN and their neuronal specific features. Since is known that  PPP4R2 cooperates with SMN complex in the right maturation of the splicing machineâs components, we have studied the effects of overexpression of PPP4R2 also in apoptosis and neuritogenesis.

We find that overexpression of PPP4R2 inhibits basal and  induced apoptosis in  neuronal  but not epithelial cell lines , showing that this effect is cell-type specific; this protective effect is enhanced when is transfected the C-terminal portion of the protein , indicating that this could be the domain responsible of the protection.;moreover overexpression of  the N- terminal mutant of PPP4R2  leads to impaired neuritogenesis in PC12 cells treated with NGF.

These data indicate that PPP4R2 is involved in important neuronal functions and will be important to demonstrate if  PPP4R2 could be a functional  partner of SMN also  in these cellular processes

P19

 

A YEAST TWO-HYBRID APPROACH TO STUDY THE ROLE OF DJ-1 IN PARKINSON'S DISEASE

 

S. Zucchelli*, Z. Scotto Lavina, R. Calligaris, S. Vilotti, R. Foti, M. Biagioli,  M. Pinto, C. Casseler, L. De Maso and S. Gustincich.

 

The Harvard-Armenise Foundation Laboratory, Center for Genetic of Regeneration and NeuroDegeneration (GRAND), Sector of Neurobiology, International School for Advanced Studies (S.I.S.S.A.-I.S.A.S.), AREA Science Park, S.S.14 Km163,5, 34012 Basovizza (TS), Italy

Phone 040 3756507, FAX 040 3756502 Email zuc@sissa.it

 

Mutations in the PARK7/DJ-1 gene were found to be associated with autosomal recessive early-onset Parkinsonâs Disease (PD). Genetic data suggest that a lack of functional DJ-1 leads to neurodegeneration. We hypothesize that an important feature of the neurodegenerative process may involve an altered pattern of protein interactions. To dissect the molecular events related to DJ-1 function, we have isolated a group of DJ-1 interactors from a human foetal library. Among those identified in our laboratory, a subset of interactors was already described (Daxx, Abstrakt, SUMO-1, Ubc9), confirming the reliability of our screen.We also found some previously unidentified DJ-1 partners, such as TRAF and TNF Receptor Associated Protein (TTRAP). TTRAP was previously isolated as an interactor of CD40, a member of the TNF receptor family. It also interacts with TNF-Receptor p75, CD30 and TRAF6. TTRAP inhibits the TNF-Receptor p75-mediated transcriptional activation of NF-kB. TTRAP may act both as a transcriptional regulator and as an endonuclease sharing significant structural homology and biochemical activity with APE1/Ref-1, a major player in cellular oxidative stress response. We are now planning to study the role of TTRAP in cellular and animal models of PD.

P20

 

REDOX, LIPIDS AND MOLECULAR EVENTS TRIGGERED BY POLYQ-EXPANDED PROTEINS

 

Bertoni A. (1), Giuliano P. (2), Rotoli D. (3), Ulianich L. (3), Castaldo I. (1), Di Jeso B. (4), Scorziello A. (5), Adornetto A. (5), Santillo M. (6), Cocozza S. (1), Avvedimento V.E. (1)

 

(1) Department of Molecular and Cellular Biology and Pathology, School of Medicine, Federico II University of Naples, Naples Italy; (2) Department of Clinical Immunology National Cancer Institute, G. Pascale Foundation, Naples Italy; (3) Institute of Endocrinology and Experimental Oncology ãG. Salvatoreä, Italian National Research Council, Naples, Italy; (4) Laboratory of General Pathology, Departement of Biological and Environmental Sciences and Technologies, University of Lecce, Lecce Italy; (5) Division of Pharmacology, School of Medicine Federico II University of Naples, Naples Italy; (6) Departement of Neuroscience, Unit of Physiology, School of Medicine Federico II University of Naples, Naples Italy.

 Polyglutamine diseases are caused by a (CAG) trinucleotide repeat expansion that is translated into an abnormally long polyglutamine tract (polyQ). Although the expanded proteins, found so far, are expressed in all tissues, the death induced by these proteins involves selectively only some types of neurons.. The hallmark of these diseases is represented by the accumulation of aggregates containing the polyQ proteins.

 To determine a possible common pathogenetic feature, induced by polyQ-expanded proteins, we have developed a neuronal system that recapitulates the cellular phenotype found in these diseases. Cells expressing synthetic fusion protein containing 43 polyglutamines (HA-43Q-GFP) accumulate nuclear aggregates, inhibit cAMP and NGF dependent trascription and differentiation. The protein containg 17 Q repeats, homologous to the normal allele, failed  to induce aggregates and did not inhibit neuronal differentiation.  By using a conditional inducible system, we modulated the expression of the pathological protein and analyzed the early and late events linked to the its expression and the reversibility of the pathological processes. As soon as 3 h after the onset of the expression of the 43Q expanded protein, there was a burst of ROS produced by the membrane oxidase. Later, ROS decreased, but remained higher than in control cells. By 3 days, the oxidative wave, originated in the membrane, propagated to the mitochondria, which lowered their electrochemical gradient across the membrane. In 1 to 2 weeks, the wave hit the ER and later the nucleus, where there was a global depression of neuronal specific trascription. One selective marker associated to ROS production, induced by the polyQ expanded protein , was a oxidative DNA damage. DNA damage was evident as soon as 1 day of continuous expression of the expanded protein. DNA damage was also found in  fibroblasts cell lines originated from Huntington and SCA-2 patients. In these cells and in neurons expressing 43Q protein,  DNA damage and other signatures of polyQ expanded proteins, were completely inhibited by specific oxidase inhibitors.

  We conclude that a common and primary mechanism, originating in the plasma membrane, induced by polyQ proteins, set off ROS that progressively propagate to the nucleus and other cellular organelles.

P21

 

USE OF STEROIDOGENIC ACUTE REGULATORY PROTEIN (STAR) IN STUDY OF MITOCHONDRIAL ATP-DEPENDENT PROTEASES POTENTIALIALLY INVOLVED IN FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS (ALS) CYTOPATHOLOGY

 

Bahat A* (1), Eimerl S (1), Granot Z (1), Cantoni L (2) and Orly J (1)

 

(1) Department of Biological Chemistry, The Alexander Silberman Institute of Life Scieces, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel, tel. 972-54-427-2948; E-mail:orly@vms.huji.ac.il

(2)  Laboratory of Molecular Pathology, Istituto di Ricerche Farmacologiche Mario Negri, 20157 Milan, Italy, tel. +39 02 39014423; E-mail: cantoni@marionegri.it

 

A percentage of the familial cases of amyotrophic lateral sclerosis (ALS) shows mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Normally, SOD1 is found mainly in the cytosol, but it can be present also within the intermembrane space and in the matrix of mitochondria. A hallmark of ALS cytopathology is the presence of cytosolic and mitochondrial SOD1 inclusions, also found in murine and cellular familial ALS models.  Importantly, studies in neuronal tissues have shown that mutant SOD1 is preferentially associated with the mitochondria, suggesting that accumulation of mutated SOD1 in these organelles may underlie its cell specific toxicity and motoneuron selectivity in ALS.  Our working hypothesis assumes that mitochondrial SOD1 is cleared from the intra-mitochondrial compartments by ATP-dependent proteases and a yet-to-be defined impairment of such a mechanism may take place in motor neuron mitochondria expressing mutant forms of SOD1.  Since very little is known about the protein quality control systems in mammalian mitochondria, we present herein a strategy we have developed to identify the ATP-dependent Lon protease as the predominant matrix enzyme readily degrading an authentic protein substrate, StAR.  Steroidogenic acute regulatory protein (StAR) is indispensable for steroid hormone synthesis and must be rapidly removed following its import into the mitochondrial matrix.  Degradation assays of murine StAR expressed in mutant bacteria strains, as well as cell-free assays of purified StAR and human Lon protease suggest that both bacterial and human Lon efficiently degrade StAR.  The physiological relevance of these observations was further supported by Lon over-expression and siRNA knockdown experiments in green monkey kidney COS cells. We plan to adopt the strategy described above to explore a potential role for mitochondrial proteases in turnover of mutant forms of SOD1 in motor neuron-like cell models of familial ALS.

P22

 

SULFORAPHANE PROTECTS AGAINST 6-HYDROXYDOPAMINE-INDUCED DOPAMINERGIC CELL APOPTOSIS

 

Tarozzi A (1*), Morroni F (1), Merlicco A (1), Angeloni C (2), Hrelia S (2), Cantelli-Forti G (1), Hrelia P (1)

 

(1) Department of Pharmacology, University of Bologna, via Irnerio, 48 - 40126 Bologna (Italy), tel. +39-051-2091795; Fax +39-051-248862; E-mail: andrea.tarozzi@unibo.it

(2) Department of Biochemistry \"G. Moruzzi\", University of Bologna, Bologna (Italy)

 

Parkinson's disease (PD) is a neurodegenerative disorder with a selective loss of dopaminergic neurons in the substantia nigra. Evidence suggests that oxidative stress is one of the major factors responsible for the dysfunction or apoptosis of dopaminergic neurons. Isothiocyanates (ITCs), present in cruciferous vegetables, are known as cancer chemopreventive agents and strong inducers of  phase II detoxification enzymes. Among the various ITCs, sulforaphane (SUL) shows interesting ability to decrease aging-related CNS inflammation in rats. In this study, we investigated the neuroprotective effects of SUL in a neuronal cell model of Parkinson. An experimental approach using a pulse/chase treatment of human dopaminergic SH-SY5Y cells with 6-OHDA, a PD specific neurotoxin to determine neuronal apoptosis, has been applied. Treatment of SH-SY5Y cells with SUL prior to 6-OHDA-treatment showed a significant dose-dependent inhibition of apoptotic events, such as mitochondrial activity loss, activation of caspases, translocation of phosphatidylserine and DNA fragmentation increase. These highlights resulted from a strong increase of glutathione levels and other phase II detoxication and antioxidant enzymes, such as glutathione-S-transferase, glutathione reductase and NADPH-quinone reductase. Our results also demonstrated that treatment of SH-SY5Y cells with SUL after 6-OHDA-treatment showed a significant decrease of apoptotic events. These neuroprotective effects were abolished by PI3K (LY294002) and MEK1 (PD98059) inhibitors. In particular, the treatment of SH-SY5Y with sulforaphane induced an increase of phospho-ERK1/2 and -Akt levels. Taken together, these results show that SUL protects against 6-OHDA toxicity by decreasing the oxidative stress and activating the neuronal survival pathways. In conclusion, our data suggest that SUL and other ITCs could be interesting candidates for development of prevention and/or treatment of PD.

P23

 

GENERATION OF MOUSE MODELS OF PARKINSONIAN SYNDROME BY SPECIFIC ACTIVATION OF AN ENDOGENOUS SUICIDE MECHANISM RESPONSIVE TO METABOLIC AND OXIDATIVE STRESS.

 

R.Parlato(1), C.Rieker(1), G.Kreiner(1), D.Engblom(1), I.Grummt(2), G.SchŸtz(1)

 

(1)Dept. Molecular Biology of the Cell I, German Cancer Research Center,

Im Neuenheimer Feld 581, D-69120 Heidelberg, Germany

Phone:    +49.6221.42 34 37; Fax:      +49.6221.42 34 70; e-mail: r.parlato@dkfz.de

(2) Dept. Molecular Biology of the Cell II, German Cancer Research Center

 

Oxidative stress and mitochondrial dysfunction have been implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson«s disease. We applied an unexplored approach to generate genetic mouse mutants affected by chronic loss of specific neuronal types, taking advantage of  an ãintracellular suicide mechanismä activated in response to metabolic and oxidative stress. This approach is based on the genetic ablation of the transcription factor TIF-IA that blocks the synthesis of ribosomal RNA and leads to p53-mediated apoptosis. Given the increasing evidence of a crucial role of the transcription factor p53 in the pathophysiology of several human neurodegenerative diseases, we used the conditional inactivation of the gene encoding TIF-IA by the Cre-LoxP system to induce selective loss of different types of neurons in the developing as well as in the adult brain. Here we report that disruption of the nucleoli in dopaminergic neurons by transgenic mouse lines expressing the Cre recombinase in dopaminergic neurons, results in the generation of mouse mutants showing most of the Parkinsonâs disease symptomatology, like progressive degeneration of nigrostriatal dopaminergic neurons, depletion of dopamine in the striatum and typical motor dysfunctions. Moreover, we demonstrate that nucleolar-dependent activities play a protective role in survival of dopaminergic neurons in basal conditions and upon neurotoxic oxidative stress by interfering with mitochondrial function. The mutant mice presented here provide novel tools to test treatment strategies to impede or cure pathologies related to degeneration of dopaminergic neurons. In addition, our study indicates that cellular changes associated with nucleolar pertubation may recapitulate some changes associated with neurodegenerative disorders in response to oxidative stress.


P24

 

IN VITRO MECHANISMS OF NEUROINFLAMMATION IN A CO-CULTURE SYSTEM OF ASTROCYTES AND MICROGLIA FROM NEONATAL HAMSTERS

 

Formentin EAM* (1), Servida F (1), De Luigi A (2), Ponti W (1), Poli G (1)

 

(1) Department of Veterinary Pathology, Hygiene and Public Health- Faculty of Veterinary Medicine, University of Milano, Via Celoria 10 - 20133 Milano, Italy. Tel.+39-02-50318091; fax+39-02-50318089; E-mail: elena.formentin@unimi.it

(2) Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milano, Italy

 

It is proven that the inflammatory events mediated by microglial activation partecipate to pathogenesis of neurodegenerative diseases. Among these, Transmissible Spongiform Encephalopathies (TSE) are characterized by the accumulation of prion protein (PrPres) in the central nervous system, associated to spongiosis and reactive astrogliosis. Astroglial activation is accompanied by morphological changes, cell proliferation and release of various soluble mediators of neuroinflammation, such as cytokines, Reactive Oxygen Species (ROS) and Nitric Oxide (NO). Many findings suggest that understanding glial-astrocyte interactions and mechanisms involved in regulation of microglial activation are important steps toward identification of therapeutic targets and development of effective treatment strategies for neurodegenerative conditions. In this work we developed an astrocyte-microglial co-culture system from neonatal hamster brains to study the molecular events related to microglial activation (NO and ROS production) and the pro-inflammatory cytokine expression (IL-1beta, TNF-alfa by real-time RT PCR). The hamster model is commonly used for in vivo studies of TSE pathogenesis but these studies lack of direct evidence of the functional properties exerted by activated microglia. Astrocyte-microglial co-cultures were acutely and chronically exposed to the peptide HuPrP 106-126, endowed of neurotoxic and fibrillogenic properties. Lipopolisaccharide (LPS) and H2O2 were used as positive control of glial activation, while HuPrP 106-126 scrambled as negative control. An increased expression of pro-inflammatory cytokines after 6h of incubation with HuPrP106-126 followed by a decrease after 24h was observed, while NO and ROS production increased till 24h. These results differ from others obtained in murine in vitro models and support the hypothesis that activated astroglia assumes a pro-inflammatory phenotype also in chronic neurodegenerative diseases.


P25

 

PROTECTIVE EFFECTS OF GUANOSINE AGAINST BETA AMYLOID PEPTIDE (25-35) - INDUCED APOPTOSIS IN HUMAN NEURONAL CELLS

 

Merlicco A (1*), Tarozzi A (1), Morroni F (1), Cantelli-Forti G (1), Hrelia P (1)

 

(1) Department of Pharmacology, Alma Mater Studiorum - University of Bologna, via Irnerio 48, 40126 Bologna, Italy, tel. +39-051-2091782; fax +39-051-248862; E-mail: amerlicco@inwind.it

 

Neurodegeneration in Alzheimer's disease (AD) is associated with abnormal accumulation of neurotoxic beta-amyloid (Abeta) protein, which causes apoptosis of neuronal cells. Guanosine (GUA) and other nonadenin-based purines have many neurotrophic and neuroprotective effects, such as promotion of neurite outgrowth, increased release of NGF and protection of astrocytes against apoptosis induced by staurosporine. However, the neuroprotective efficacy of GUA in AD is still not well established. In this study, we investigated the neuroprotective effects of GUA against Abeta protein-induced apoptosis in a human neuronal SH-SY5Y cells. In particular, the apoptosis in term of mitochondrial activity loss and translocation of phosphatidylserine was induced by 3 h treatment of SH-SY5Y cells with 1 microM of Abeta peptide (25-35), a neurotoxic core of Abeta protein. Treatment of SH-SY5Y cells with GUA (12,5-75 microM) in presence of Abeta (25-35) showed a strong dose-dependent inhibitory effects on Abeta (25-35) induced apoptotic events. The maximum inhibition of mitochondrial damage (66%) and translocation of phosphatidylserine (64%) was observed with 75 microM of GUA. Next, to investigate whether neuroprotection of GUA can be ascribed to its ability to increase heat shock proteins (HSP) and proteasome activity levels, we used KNK437 and lactacystin, specific inhibitors of HSP70 and proteasome, respectively. We found that the antiapoptotic effects of GUA were abolished by lactacystin but not KNK437. Interestingly, the treatment of SH-SY5Y with GUA (75 microM) induced a strong increase of proteasome activity. Thus, the neuroprotective effects of GUA against Abeta-induced apoptosis of SH-SY5Y cells is mediated, at least partly, via proteasome activation. However, further studies wil

l be required to elucidate its cellular and molecular mechanisms at neuronal level. In conclusion, these findings suggest a role for GUA as a potential drug in the treatment of AD.

P26

 

INCREASED FORMATION OF GLUTATHIONE MIXED DISULFIDES IN A MOTOR NEURON-LIKE CELL MODEL FOR FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS

 

DâAlessandro G *(1), Tartari S (1), Rizzardini M (1), Babetto E (2), Conforti L (2) and Cantoni L (1)

 

(1)Laboratory of Molecular Pathology, Dept. of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche "Mario Negri" via Eritrea 62, 20157 Milan (Italy)

(2) Babraham Institute, Cambridge (UK)

 

Mutant forms of Cu/Zn superoxide dismutase (SOD1) are associated with familial amyotrophic lateral sclerosis (FALS1), a neurodegenerative disease selectively affecting motor neurons. Aberrant oxidative chemistry is one possible reason for the toxic gain of function attributed to the mutant SOD1 to explain motoneuron degeneration. The formation of mixed disulfides between glutathione and the cysteines of some proteins (Pr-SSG), a process known as protein glutathionylation, appears greater during oxidative/nitrosative stress and can be taken as a measure of its occurrence. Furthermore, protein S-glutathionylation is currently considered a mechanism of redox- and NO-mediated signal transduction.

This study determined the levels of Pr-SSG in a conditional motor neuron-like cell model for FALS1 developed in our laboratory. We generated conditional NSC-34 cell lines expressing either a high level of wtSOD1 or a high/low level of G93ASOD1, a mutant form of human SOD1. Pr-SSG were measured spectrophotometrically in these lines and in the untransfected control line, without/with doxycycline (dox) in order to modulate the expression of human SOD1s. After four weekly passages in culture without dox, G93ASOD1 cells with the full expression of the transfected protein had a significant higher level of Pr-SSG than controls (p<0.001), while the increase in wtSOD1 cells was smaller (p<0.05). We also analyzed the effects of different amounts of G93ASOD1 protein. Pr-SSG increased comparably in cells with a low or a high level of G93ASOD1, suggesting different rates of protein glutathionylation at high levels of mutant protein. On lowering the expression of human SOD1 with dox, Pr-SSG also decreased. These results suggest that changes in Pr-SSG levels might be a novel aspect of motor neuron responses to cytotoxicity due to mutant G93ASOD1.

Financial support was provided by MIUR, FIRB, Protocol RBIN04J58W_000

P27

 

ADAPTIVE RESPONSE TO STABLE EXPRESSION OF HUMAN MUTANT G93A SUPEROXIDE DISMUTASE 1 IN MOTORNEURON-LIKE CELLS INVOLVES ALTERATIONS OF THE GLUTATHIONE POOL

 

Tartari S* (1), DâAlessandro G (1), Babetto E (2), Rizzardini M (1), Conforti L (2) and Cantoni L (1)

 

(1)Laboratory of Molecular Pathology, Dept. of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche "Mario Negri", via Eritrea 62, 20157 Milan (Italy)

(2) Babraham Institute, Cambridge (UK)

 

Amyotrophic lateral sclerosis (ALS) is a disease causing selective degeneration of motor neurons. Mutations in the Cu/Zn superoxide dismutase gene (SOD1) are associated with some cases of familial ALS (FALS1), oxidative stress being one of the mechanisms likely to be involved in the toxicity of this mutant gene. Glutathione is the main low-molecular-weight thiol in mammalian cells, with important antioxidant properties and a regulatory function of the cellular redox system. It also has a neuroprotective role and a low level might be one factor in FALS1 motor neuron pathology.

This study investigated how chronic exposure to mutant G93ASOD1 affects the pool of reduced glutathione (GSH) and glutathione disulfide (GSSG) in a conditional motor neuron-like cell model of FALS1. Cell lines with a high level of wtSOD1 and a high or low level of G93ASOD1 were cultured for four, seven and ten weekly passages. GSH and GSSG were determined spectrophotometrically in the total cell lysates. Parallely, an untransfected control cell line was studied, with constant levels of GSH and GSSG and a GSH/GSSG ratio of 100:1. At the fourth passage, GSH and GSSG were significantly higher than in controls in all the transfected cells; however, the largest increase (about three-fold) in GSH was in the high-G93ASOD1 cells; while GSSG increased more (about 2.5-fold) in the wtSOD1 cells. At the tenth passage, the GSH increases were no longer evident, while GSSG remained high. These changes affected the GSH/GSSG ratios: in G93ASOD1 cells the environment was more reductive at the early passages; while in wtSOD1 cells the only change was at the tenth passage, when the ratio was lower, suggesting a more oxidative environment. In conclusion, G93ASOD1 alters the glutathione pool differently from the wtSOD1 and exposure time influences this adaptive response.

Financial support was provided by MIUR, FIRB, Protocol RBIN04J58W_000

Keywords: amyotrophic lateral sclerosis; glutathione; G93A Cu/Zn superoxide dismutase.

P28

 

IDENTIFICATION OF HTRA2/OMIâS SUBSTRATES IN MITOCHONDRIA

 

Goo HG(1)*, Seong YM (1), Rhim H (2), and Kang S(1)

 

(1) Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, (Korea)

tel.82-2-3290-3949: mail : skang@korea.ac.kr

(2) Department of Biomedical Sciences/ Research Institute of Molecular Genetics The Catholic University of Korea, 137-701, (Korea)

 

 HtrA2/Omi is a mitochondrial protease that is released into the cytosol during apoptosis to antagonize inhibitors of apoptosis (IAPs) and contribute to caspase-independent cell death. Recent studies showed that a mutation of HtA2/Omi gene causes mitochondria dysfunction. And these mitochondria dysfunction cause neurodegeneration and parkinsonâs phenotype. In this study, we used the mouse mutant mnd2 (motor neuron degeneration 2: missense mutation Ser276Cys in the protease domain of HtrA2/Omi) to find out HtrA2âs substrates in mitochondria. We tried to compare the protein expression differences between normal mouse mitochondria and mouse mutant mnd2 mitochondria by two-dimensional gel electrophoresis. Our results showed that several protein spots were more expressed in mouse mutant mnd2 mitochondria. In these proteins, two proteins are being analyzed using molecular and biochemical metho

ds. Based on these results, we will discuss a novel function of HtrA2/Omi in mitochondria.

P29

 

TARGETED DELETION OF THE GENE ENCODING THE HEME-BINDING PROTEIN HEMOPEXIN CAUSES BRAIN IRON MISDISTRIBUTION IN MICE

 

Noemi Morello*(1),  Elisabetta  Tonoli(2), Federica Logrand(1), Sharmila Fagoonee(1), Emilia Turco(1), Lorenzo Silengo(1), Alessandro Vercelli(2), Fiorella Altruda(1) and Emanuela Tolosano(1)

 

(1)Molecular Biotechnology Center, University of Torino, Via Nizza 52, 10126 Torino, Italy, Tel. +39 0116706417, Fax +39 0116706432;

(2)Dept. of Anatomy, Pharmacology and Forensic Medicine, University of Torino, corso M. D'Azeglio 52, 10126 Torino, Italy.

 

Hemopexin is an acute phase plasma glycoprotein with the highest heme binding affinity  (Kd < 10-9 M). Hemopexin is mainly produced in the liver and released into the plasma where it is involved in heme scavenging through receptor-mediated endocytosis. This system provides protection against free heme-mediated oxidative stress, limits access by pathogens to heme, and contributes to iron homeostasis by recycling heme iron.

Hemopexin synthesis is not restricted to the liver parenchyma, but it is also locally expressed in the sciatic nerve, skeletal muscle and human brain. Recently, Hemopexin has been found in human cerebrospinal fluid and a comparative proteomics analysis has demonstrated an increase in Hemopexin in subjects with Alzheimerâs disease.

In the present study, we have analyzed  iron homeostasis in the brain of  Hemopexin-null mice. We have demonstrated that Hemopexin-null two

 months old mice have greater iron deposits in discrete regions of basal ganglia compared to wild-type controls. Prominently affected areas include caudate-putamen and globus pallidus. Counts of iron-positive cells on consecutive sections show that in Hemopexin-null mice, the number of iron-loaded cells is 2-folds greater than that of wild-type animals. Iron-loaded cells have been identified as oligodendrocytes. Moreover, biochemical analysis shows that both L- and H-ferritin expression is decreased in Hemopexin-deficient brain. Finally, we measured lipid peroxidation as an index of oxidative stress. We show that lipid peroxidation is significantly higher in the brain of Hemopexin-null mice compared to wild-type controls.

These results demonstrate that Hemopexin plays an important role in iron distribution in brain, thus suggesting its implication in iron-related neurodegenerative diseases.

P30

 

NOGO RECEPTOR ANTAGONIZES P75NTR-DEPENDENT MOTOR NEURON DEATH

 

Luc DUPUIS(1), Mariana PEHAR(2), Patricia CASSINA(3), FrŽdŽrique RENE(1), Raquel CASTELLANOS(3), Caroline ROUAUX(1), Leda DIMOU(4), Martin E. SCHWAB(4), Jean-Philippe LOEFFLER(1) , Luis BARBEITO(2) & Jose-Luis GONZALEZ DE AGUILAR(1)

 

(1) Laboratoire de Signalisations MolŽculaires et NeurodŽgŽnŽrescence, INSERM U-692, Strasbourg, France; (2) Departamento de Neurobiolog’a Celular, Instituto de Investigaciones Biol—gicas Clemente Estable; and (3) Faculdad de Medicina, Universidad de la Repœblica, Montevideo, Uruguay; (4) Brain Research Institute, University of Zurich and Department of Biology, ETH Zurich, Zurich, Switzerland

 

The Nogo-66 receptor (NgR) plays a critical role in restricting axon regeneration in the central nervous system. This inhibitory action is in part mediated by a neuronal receptor complex containing p75NTR, a multi-functional receptor also well known to trigger cell death upon binding to neurotrophins such as NGF. We now show that activation of NgR and its downstream effector Rho-kinase are able to regulate motor neuron survival by modulating NGF/p75NTR-induced cell death. We provide pharmacological and genetic evidence that stimulation of the Nogo-66/NgR/Rho-kinase pathway prevents the death-promoting effect of p75NTR both in vitro and in vivo. These findings demonstrate an as yet unknown function of NgR in maintaining neuronal survival that may be relevant for motor neuron development and degeneration.

P31

 

ROLE OF THE DNA DAMAGE RESPONSE IN HUNTINGTONâS DISEASE

 

Merienne K(1)*, Rau F(1), Coin F(2), Egly J.M.(2) and Trottier Y(1)

 

(1)Department of Molecular Pathology, (2)Department of Transcription, IGBMC, CNRS/INSERM/ULP, 67404 Illkirch

 

In Huntingtonâs disease (HD), several pathogenic conditions such as excitotoxicity and mitochondrial defect are sources of reactive oxygen species, which can alter proteins, lipids and also DNA. Oxidative DNA damage is indeed increased in HD, however, whether and how it contributes to the disease remain to be clarified. DNA damages are removed by the DNA damage response, which comprises 4 major pathways, including Base Excision Repair (BER) and Nucleotide Excision Repair (NER), which can remove oxidative lesions, and Double Strand Break Repair (DSBR) and Mismatch Repair (MMR), which modulate CAG repeats stability. Therefore, the various DNA repair pathways might be involved in HD pathogenesis. Using a neuronal cellular model of HD, we show that expression of genes implicated in the various pathways are deregulated in neurons expressing mutant huntingtin (htt), the protein implicated in HD. We further show that NER is deficient in these cells. We indeed find that the luciferase activity of neuronal cells expressing mutant htt and transfected with an UV-damaged luciferase plasmid is decreased, compared to controls. By immunocytofluorescence, we also show that the UV-induced (6-4)PPs lesion is slowly repaired in our HD neuronal cellular model, compared to control cells. Using the ARP assay, which is an ELISA method allowing detection of abasic sites, we further confirm that oxidative DNA damage progressively accumulates in the striatum of R6/2 and R6/1 mice. By quantitative RT-PCR and western-blotting, we also show that genes implicated in NER are deregulated, including XPA, ERCC1 and polymerase beta. Our results suggest that the DNA damage response might be impaired in HD, which could further promote accumulation of lesions, genetic instability and neurodegeneration.


P32

 

DOES MITOCHONDRIAL INVOLVEMENT AFFECT FIBROBLAST FUNCTIONING IN NEURONAL CEROID LIPOFUSCINOSES?

 

Pezzini F* (1), Marconi S (1), Vattemi G (1), Semebenini D (1), Rizzuto N (1), Santorelli FM (2), Simonati A (1)

 

(1) Department of Neurological and Visual Sciences-Neurology, University of Verona, Policlinico GB Rossi, Piazzale LA Scuro, 1 - 37134 Verona (Italy) tel 0039-0458124285; fax 0039-045585933; E-mail:alessandro.simonati@univr.it

(2) Molecular Medicine - IRCCS Bambin Ges Pediatric Hospital, Rome (Italy)

 

Neuronal Ceroidolipofuscinoses (NCLs) are a group of progressive degenerative disorders of the Central Nervous System associated with endolysosomial storage of autofluorescent ceroid and lipofuscin. The presence of peculiar cytosomes is the pathological hallmark in both neural and non neural cells. Most of the clinical forms have onset in childhood with a fatal outcome by the end of the second decade. Cell death of neuronal and glial cells leads to cerebral and cerebellar atrophy; retinal ganglion cell involvement is also common. NCLs are recessively inherited; advances in genetics have allowed to identify 8 different genes and related products. Three forms (CLN1, CLN2, CLN10) are primary lysosomal disorders, due to the dysfunction of specific lysosomal enzymes; in the others the gene defects affect different membrane proteins of the cellular compartments, leading to secondary lysosomal storage. Pathogenetic mechanisms of cytosome formation and cell death are not fully elucidated yet. In this study, we addressed the issues of storage formation and cellular dysfunction in vitro. The fibroblasts of three children affected with late infantile NCL variants, secondary to genetically identified mutation of the CLN1, CLN5 and CLN6 genes, were used. Storage accumulation was monitored by staining cells at different growth times with Lysotracker and by Electron Microscopy. Slowed mitotic rate of the mutated cells was observed. Along time shift of JC-1 fluorescence was observed of the cells which showed pathological swelling, these findings being consistent with increased numbers of mitochondria with depolarized membrane potential. Cytoplasmic cytochrome C staining as well as activated caspases-dependent apoptotic cell death were unremarkable as compared with control cells. Results from this study suggest that depolarization of the mitochondrial membrane potential occurs as a secondary event in the aged NCL fibroblasts. Whether related mechanisms can lead to impaired energy production, and therefore to cellular dysfunction and death, can be a challenging hypothesis to be further investigated.

P33

 

A NOVEL TRANSFERRIN/TFR2-MEDIATED MITOCHONDRIAL IRON TRANSPORT SYSTEM IS DISRUPTED IN PARKINSON'S DISEASE.

 

Pier Giorgio Mastroberardino(1), Eric Hoffman (1) ,Ranjita Betarbet (3), Hye Mee Na (1), Charleen T. Chu (2) and John Timothy Greenamyre (1).

 

(1) Department of Neurology, Pittsburgh Institute for Neurodegenerative Diseases, (2) Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania 15216, (3)Department of Neurology, Center for Neurodegenerative Disease, Emory University, School of Medicine, Atlanta, Georgia 30322

 

How iron accumulates in substantia nigra in Parkinsonâs disease (PD) is unknown. We report a novel transferrin (Tf)-mediated iron transport pathway in mitochondria of substantia nigra dopamine neurons. Transferrin receptor 2 (TfR2) has a previously unrecognized mitochondrial targeting sequence and in cells expressing exogenous TfR2, extracellular Tf is internalized and transported to mitochondria, where its iron is released and incorporated into complex I of the electron transfer chain. In the rotenone model of PD, both Tf and TfR2 undergo intermolecular cysteine oxidation and, in dopamine neurons, there is accumulation of Tf, much of it in mitochondria. These changes are associated with iron deposition in substantia nigra, similar to what occurs in PD. In the normal human substantia nigra, TfR2 is also found in mitochondria of dopamine neurons, and in PD there is a dramatic increase of oxidized Tf in substantia nigra, as predicted by the rotenone model. Thus, we have discovered a novel mitochondrial iron transport system that goes awry in PD.


P34

 

 

NOVEL THERAPEUTICS FOR HD: A HIGH THROUGHPUT PATHWAY SCREEN APPROACH

 

Heitz F

 

Screening Sciences Unit, SienaBiotech, via Fiorentina,1 - 53100 Siena (Italy)

tel.+39-05-77381353; fax +39-05-77381303

E-mail:fheitz@sienabiotech.it

 

Huntingtonâs disease (HD) is a dominantly inherited neurodegenerative disorder caused by an expansion of multiple polyglutamines (>35) at the N-terminal part of the large protein huntingtin (HTT). Expression of mutant huntingtin (µHTT) induces proteolysis at the 5âend, conferring to the polyQ stretch the capacity to aggregate and ultimately cause cytotoxicity in diverse cell types.

At Siena Biotech, we aim at identifying small molecules which inhibit the aggregation process and therefore should be able to slow, halt or reverse disease progression. To this end, we have designed an assay based on a full-length µHTT cell model, taking advantage of the sequestering of the transcription factor CBP by the polyQ-formed aggregates. After having engineered a 293/T-Rex based cell model inducibly expressing full-length µHTT and stably expressing a CRE-Luc reporter gene, we have developed and validated in a HTS format a µHTT-specific pathway screening assay. Briefly, addition of doxycyclin induces µHTT expression followed by formation of aggregates sequestration of CBP and concomitant reduction of reporter activity. Reference compounds which have been shown to inhibit aggregate formation are reversing the µHTT-induced decrease of reporter activity. We have used this assay to screen a diverse library of small organic molecules which allowed us to identify several hit molecules. These compounds are able to specifically reverse µHTT-induced reduction of reporter activity. Details of assay validation and compound characteristics will be presented.

 

P35

 

PROTEOMIC ANALYSIS OF CEREBROSPINAL FLUID FROM AMYOTROPHIC LATERAL SCLEROSIS  PATIENTS

 

Stefano Olivieri (1), Antonio Conti (1), Sandro Iannaccone (3),  Angela Cattaneo (2), Angela Bachi (2), Barbara Sferrazza (3), Stefano Cappa (4) and Massimo Alessio (1)

 

(1) Proteomics Unit,San Raffaele Scientific Institute, via Olgettina,58 - 20132 Milan (Italy)

tel.+39-02-2643.2884; fax +39-02-2643.4153;

E-mail: olivieri.stefano@hsr.it

(2) Mass Spectrometry Unit,San Raffaele Scientific Institute, Milan, Italy

(3) Neurology Department,San Raffaele Scientific Institute, Milan, Italy

(4) Neurological Unit, San Raffaele Scientific Institute, Milan, Italy

 

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of motor neurons in cortex, brainstem and spinal cord.

Similar to other neurodegenerative disease, as Parkinsonâs and Alzheimerâs disease, ALS seems to be a disorder originating from several pathological mechanisms largely unknown.

Moreover, the diagnosis in early stages of this pathology has several limitations due to the absence of specific markers.

For this reason, in order to individuate specific changes due to the presence and the progression of this disease, we perform a comparative analysis of protein expression using Cerebrospinal Fluid (CSF) collected from ALS patients. This analysis has been realized using a proteomic approach in which two dimensional gel electrophoresis (2DE) allows the separation of a large number of proteins on the basis of the isoelectric point and relative mass. The 2D-gel images were analysed using dedicated software and proteins were identified using mass spectrometry.  By comparing proteins from healthy donor versus ALS patients we found differentially expressed protein isoforms that can be involved in pathologic mechanisms or might represent putative diagnostic markers.  In fact we focus our attention on proteins with different levels of expression that have important role in redox regulation, in development of  neuromuscular junction, in neuron degradation and in retinol metabolism. The change of expression of these proteins during the onset and the progression of ALS could be directly associated to possible pathological mechanisms.


P36

 

DIETARY INDUCTION OF OXIDATIVE STRESS IN THE CNS OF APOE-DEFICIENT MICE

 

McCampbell A(1), Marlatt M(1), Wolffe C(1), Finney E(2), Tanis K(2), Levine D(2), Savage M(1), Mitchell TW(3), Barbacci D(4), Stone DJ(2), Majercak J(1), Seabrook G(1), Ray WJ(1)

 

(1) Alzheimer's Research, Merck Research Laboratories, West Point, PA (USA)

(2) Molecular Profiling, Rosetta Inpharmatics, Seattle, WA (USA)

(3) Laboratory Animal Resources, Merck Research Laboratories, West Point, PA (USA)

(4) Proteomics, Merck Research Laboratories, West Point, PA (USA)

 

Human genetic data indicate that apolipoprotein E is an important regulator of neuronal health and function. The ApoE4 variant confers increased risk of Alzheimer's disease, poor prognosis following stroke and head injury, Parkinson's disease, ALS, and other neurological conditions. Similarly, mice lacking the ApoE allele have age related learning and memory deficits and are more susceptible to various neuronal injuries, including oxidative stress. We sought to induce chronic, low-level oxidative stress in mice lacking ApoE and expressing endogenous levels of the human amyloid precursor protein (YAC-APP/ApoE-KO). These mice were placed on a 1% iron, folate deprived diet. After one month, plasma and brain samples were analyzed for signs of oxidative damage. Transcriptional profiling of pooled hippocampal and cortical regions showed a significant elevation of genes involved in oxygen binding and hemoglobin metabolism. APP processing was not broadly altered by the diet, although male mice had significantly elevated A-beta 42 levels. This is consistent with observations of amyloid induction in response to neuronal stress. Lastly, plasma homocysteine levels were elevated, which is consistent with oxidative stress in the circulatory system. Taken together, these data suggest this might provide a model to look at neuronal responses to environmental-induced oxidative stress.

P37

 

THE AMINO TERMINUS OF MUTANT HUNTINGTIN INTEGRATES INTO AND AFFECTS THE EFFICIENCY OF THE BASAL TRANSCRIPTION MACHINERY IN VITRO

 

Hu H* (1), Hogel M (2), Gomez GT (3), Denovan-Wright EM (4)

 

(1)Department of Pharmacology, Dalhousie University, Halifax, N.S.B3H 1X5 (Canada); tel:1-902-4946232;fax:1-902-494-6294;Email:hhu@dal.ca

(2)(3)(4)Department of Pharmacology, Dalhousie University, Halifax, N.S.B3H 1X5 (Canada)

 

Altered transcription is observed in a number of CAG repeat disorders and it appears that the protein context of the polyglutatmine (pQ) repeat is involved in the region- and gene-specific effects on transcription.   Previously, we determined the spatial and temporal pattern of expression of several genes (DARPP-32, ppENK, PDE10A and CB1) that have reduced transcription in young R6 mice, which expressed the amino terminus of human huntingtin with an expanded Q repeat (N-mHtt). We are studying N-mHtt-induced transcriptional dysregulation in these selected genes. It appears that, in general, the time of initial transcriptional dysregulation and rate of change in the levels of steady-state mRNA is dependent on the length of the CAG repeat or amount of N-mHtt expressed or both.  We observed that in all genes analyzed, transcription is repressed to a fraction of the level observed in wild-type animals over a period of weeks and then remained constant.  This suggested that the rate of change in mature mRNA level reflected a time point when transcriptional regulation changed followed by reestablishment of a lower steady-state level of mRNA. It appeared that levels of mRNA in the striatum were reduced to the levels observed in other tissues suggesting that the increased expression of these genes in the striatum, and not constitutive expression, was preferentially affected by N-mHtt. We have analyzed the activity of promoters for these genes in immortalized rat striatal neuronal cell lines (ST14A) and in derivatives of these lines that express the first 548 amino acids of human huntingtin with 22 Q (N548wt) or 128Q (N548hd).  The CMV, DARPP-32, ppENK and PDE10A promoters have decreased activity in stably and transiently transfected cells expressing a minimum of the exon 1 of human huntingtin plus an extended pQ tract. Moreover, the effect of N-mHtt was localized to the smallest active promoter tested; these regions did not contain potential transcription factor (TF) binding sites for TFs that have been shown previously to physically interact with mutant huntingtin such as CREB, SP1, NFkappaB etc. We saw no evidence of altered protein-DNA interaction via gel shift-type assays or DNase I footprinting of the smallest region that showed N-mHtt sensitivity suggesting that N-mHtt does not act as a direct DNA-binding protein or sequester TF that directly bind  to DNA from core promoters. In vitro transcription demonstrated that, even in a chromatin- and cell-free environment, that purified N-mHtt protein directly affected promoter activity. For the CMV promoter, at least, addition of nuclear proteins isolated from brain, but not kidney, enhanced transcription and the relative difference between activities in the presence of 22Q and 89Q. We have isolated the proteins that bind to active promoters. Western blot analysis demonstrated that the amino terminus of human huntingtin with 22Q or 89Q was part of the complex of bound proteins but that only the N-mHtt with 89Q reduced transcription. We are currently analyzing protein-protein interactions within these complexes. Funded by the Canadian Institutes of Health Research, the Nova Scotia Health Research Foundation (scholarship to HH) and the Izaak Walton Killam Trust (scholarship to GG).

P38

 

AMYLOID BETA-BINDING PROTEIN 17BETA-HYDROXYSTEROID DEHYDROGENASE TYPE 10 AND ALZHEIMER DISEASE

 

Kristofikova Z (1), Ripova D(1), Hovorkova P (1) and Horinek A (2)

 

(1) Alzheimer Disease Center, Prague Psychiatric Center, Ustavni 91, 181 03 Prague 8 - Bohnice (Czech Republic)

tel: 420-266 003 164, fax: 420-266 003 160, email: kristofikova@pcp.lf3.cuni.cz

(2) 3rd Internal Department, 1st Faculty of Medicine, Charles University, Prague2 - Albertov (Czech Republic)

tel: 420-224968155

 

It is suggested that intraneuronal accumulation of amyloid beta peptides and their binding to multifunctional mitochondrial enzyme 17beta-hydroxysteroid dehydrogenase type 10 could be involved in pathogenesis of Alzheimer disease. The complex of peptides and enzyme is localized in cytosole and mediates apoptosis. We suppose that estimations of  concentrations of free enzyme or of its complex with peptides by means of ELISA, Western blot or optical biosenzor e.g. in CSF could be used as a perspective biomarker of Alzheimer disease. In the pilot study, we have evaluated the degree of lateralization of 17beta-hydroxysteroid dehydrogenase type 10 mRNA in hippocampi of demented (Alzheimer disease and multi-infarct dementia) or psychotic patients. Our results indicate that enzyme expression is right/left lateralized in controls and changes due to disorders are more marked in the dominant (i.e. left in the majority of cases)hemisphere.  However, our results also indicate that the  observed changes are not specific for Alzheimer disease.

Supported by MSMT (1M0517) and IGA MHCR (NR/9322-3) projects.           


P39

 

ADENOVIRUS-MEDIATED TAU OVEREXPRESSION OR APOPTOSIS IN CEREBELLAR GRANULE NEURONS INDUCES DECREASE AND DETERGENT INSOLUBILITY OF ALPHA-SYNUCLEIN

 

G. Amadoro¤, A. Gentile¤*, V. Corsetti, MT. Ciotti, P. Calissano

 

C.N.R. Institute of Neurobiology and Molecular Medicine, Rome, Italy

¤These authors equally contributed to the work

* C.N.R. Institute of Neurobiology and Molecular Medicine, Via del Fosso di Fiorano 64-65, 00143 Rome, Italy; tel. number: 06/501703235, fax number 06/501703313; email: a.gentile@inmm.cnr.it

 

Aberrant aggregation to form fibrils and insolubile aggregates of alpha-synuclein, has been implicated in the pathogenic processes of many neurological disorders. To study the possible causes of alpha-synuclein aggregation, we treated cerebellar granule neurons by two distinct experimental approaches causing death via necrosis or apoptosis and analyzed the level of alpha;-synuclein in total, detergent insoluble fraction of cellular extracts and in culture medium at different times of treatment.

We have previously reported that the increasing level of the longest human tau isoform (htau 441) is toxic, evoking a NMDA-dependent and caspase-independent neuronal death, when overexpressed by an ad hoc devised adenovirus-mediated infection (1) and that the same neurons die via apoptosis when extracellular K+ concentration is shifted from 25 to 5.0 mM (2). We found that cell death induces a time-dependent loss of intracellular synuclein in both experimental paradigms. We also found that during apoptosis a reduction of secreted immunoblottable alpha-synuclein and a corresponding accumulation of this protein in a Triton-X-100-insoluble pool occur. Moreover, double immunofluorescence stainings of cellular distribution with antibodies directed against alpha-synuclein and synapsin I, or MAP2, showed a specific relocalization of synuclein from nerve terminals to perikarion during apoptosis, without significant changes of the integrity of neuronal cytoarchitecture.

(1) Amadoro et al., P.N.A.S.103(8):2892-7 (2006); (2) DâMello et al., P.N.A.S. 90(23):10989-93 (1993).

P40

 

STEFIN B WT, E31Y AND G4R VARIANT FORM CHANNELS IN PLANAR LIPID MEMBRANES

 

Viero G(1), Rabzelj S(2), Anderluh G(3), Dalla Serra M(1), and Zerovnik E (2)

 

(1) FBK-CNR Institute of Biophysics, Unit at Trento, Via alla Cascata 56/C18, 38050 Povo (Trento), Italy phone. +39 0461 314159; fax +39 0461 314875; email: viero@itc.it.

(2) Joõef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia tel +386 1 477 3753

fax +386 1 477 3984; email:eva.zerovnik@ijs.si

(3) Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia., Slovenia phone: ++386 1 423 33 88; fax: ++386 1 257 33 90; email: gregor.anderluh@bf.uni-lj.si

 

Cytotoxicity of amyloidogenic proteins may be related to perturbation of membrane potential or formation of transmembrane pores, as formulated in the "channel hypothesis" of Alzheimerâs disease. Stefin B represents a very suitable model protein in studies on amyloid-fibril formation of cystatins. In previous studies (Anderluh et al., 2005) interaction of prefibrillar aggregates with predominantly acidic phospholipids membranes was shown and correlated with cytotoxicity. In this study, the formation of pores and their electrophysiological characteristics were studied with planar lipid bilayers, applying the wild type protein at pH 7. Similarly to other amyloid peptides Stefin B wt is able to open cation-selective pores with multiple conductance levels. Besides the wt protein, we explored E31Y polymorphic form and G4R mutant observed in some patients with EPM1. An increased membrane instability was observed after G4R addition but, except for a different pore insertion kinetic, the electrophysiological parameters were very similar to those of the wt. Interestingly, E31Y showed anion selectivity, a strong voltage dependence gating and only two conductance levels with the lack of the highest conductance state observed for the wt.. In order to better understand which prefibrillar/aggregate species form the pores, we investigated the pore forming ability of G4R monomer, dimer and tetramer. Preliminary experiments show that monomers are not able to open pores.

Stefin B is generally overexpressed in neurodegenerative conditions, such as amyotrophic lateral sclerosis, AD and after epileptic seizures, suggesting that it could play a significant role in neuronal protection. In this view, the channel properties of StefinB oligomers could be related to the presence of hyperexcitability in Stefin B defective hippocampal slices and may open new perspectives on a molecular explanation of EMP1 disease (where stefin B acivity is reduced or absent) and physiological role of cystatins.

P41

 

IDENTIFICATION OF AN APP PARTNER WITH A BIOINFORMATIC APPROACH AND EXPERIMENTAL VALIDATION

 

Federico Tommaso Bianchi (1), Vanessa Schubert (2), Paola Camera (1), Carlos Dotti (2) and Ferdinando Di Cunto (1)

 

(1) Molecular Biotechnology Center, Dept. Genetic, Biology and Biochemistry Universitaâ degli Studi di Torino, Via Nizza, 52 - 10100 Torino (italy) tel. 011-6706410; mail: federico.bianchi@unito.it

(2)Cavalieri Ottolenghi Scientific Institute, Universitaâ degli Studi di Torino, Torino (Italy)

 

Alzheimer disease (AD) is a neurodegenerative disorder characterized by a progressive and irreversible decline of cognitive function. A majority of AD is sporadic, although several genetic linkages have also been identified. Altered proteolytic processing of the Alzheimer amyloid precursor protein (APP) is thought to be a major molecular pathogenetic mechanism underlying Alzheimer disease.

Despite extensive efforts to pinpoint the normal function of APP, its function remains elusive.

Roles in cell adhesion, cell proliferation, neuroprotection and neurite outgrowth have been proposed. It has also been suggested that APP might have a role in signal transduction since APP structurally resembles a receptor and is targeted to the cell surface.

To investigate the potentials interactors protein of APP we have used a novel bioinformatic data mining method developed in our lab, referred as CLOE (Coexpression-based Linking of Orthologous ESTs), that allows the identification of transcripts showing evolutionary conserved co-expression in cDNA microarray datasets.

The best candidate molecular partner that we have identified with this method is the heat shock protein Hsp47, a collagen-binding protein that assists the molecular maturation of procollagen.

In this study we are addressing the hypothesis that hsp47 is a molecular partner of App. To this aim we are conducting expression and functional studies.

We have found that Hsp47 is present in body, dendrites and axons of primary hippocampal neurons and in definite stacks in astrocytes. Moreover, we are investigating the possibility that Hsp47 is a new modulator of App processing in neuronal and/or non-neuronal cells types. 


P42

 

THE JOSEPHIN DOMAIN OF ATAXIN-3 ACCOMMODATES UBIQUITIN IN A HYDROPHOBIC CLEFT

 

Nicastro G*, Masino L, Menon RP,and Pastore A.

 

National Institute for Medical Research

Division of Molecular Structure

The Ridgeway, London NW7 1AA, United Kingdom

Ph ++442088162629; Fax ++442089064477; E-mail:gnicast@nimr.mrc.ac.uk

 

Ataxin-3 is a 42-kDa multi-domain protein consisting of an N-terminal josephin domain and an unstructured C-terminal tail which contains a polymorphous polyglutamine (polyQ) tract and two ubiquitin-interacting motifs. Expansion of the poly(Q) tract beyond ~52 residues causes spinocerebellar ataxia type 3 (SCA3) also known as Machado-Joseph disease. The Josephin domain, whose structure was recently solved (1), is a ubiquitin-specific cysteine protease involved in the ubiquitin/proteasome pathway, although no direct description of its complex with ubiquitin is available yet. A distinctive feature of Josephin is the presence of two non-structurally independent sub-domains which form a groove which has been suggested to accommodate the enzyme substrates, among which ubiquitin, and thus to be a functionally important region of the molecule.

With the aims of elucidating the mode of Josephin binding to ubiquitin and investigating the structural basis of the specificity of this interaction, the structure of a ubiquitin-Josephin complex was obtained from an NMR-based molecular docking approach. Using a combination of molecular dynamics and NMR observables, we have mapped the surface of interaction and the molecular dynamics of the complex. We prove that Josephin binds ubiquitin in an open cleft formed between the main body of the domain and a helical hairpin. In the absence of a substrate, the hairpin behaves like a waving hand (2) whereas binding of ubiquitin to the cleft results in a stiffening of the hairpin.

1) Nicastro G., et al. PNAS (2005) 102, 10493-10498.

2) Nicastro G., et al. J. of Biomol. NMR (2006) 34, 267-277.

P43

 

HYPERPHOSPHORYLATED FILAMENTOUS TAU ACCUMULATES IN RETINAL GANGLION NEURONS OF P301S TAU TRANSGENIC MICE

 

Gasparini L(1)*, Crowther RA (2), Martin K (1), Goedert M (2), and Spillantini MG (1)

 

(1) Cambridge Centre for Brain Repair, University of Cambridge - CB2 2PY Cambridge (UK) tel +44 1223 331146; fax +44 1223 331174; E-mail: lg300@cam.ac.uk

(2) Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.

 

Filamentous deposits (e.g. neurofibrillary tangles, NFT) made of the microtubule-associated protein tau  are a major defining pathological hallmark of several neurodegenerative diseases termed "tauopathies", including Alzheimer's disease and cases of frontotemporal dementia. Mutations in the tau gene have been discovered in cases with familial frontotemporal dementia and parkinsonism linked to chromosome 17 and have led to the development of animal models of tauopathy. The P301S tau transgenic mouse is one such model. The expression of mutated P301S human tau in this transgenic mouse is driven by the Thy-1 promoter which results in occurrence of neuronal tau pathology throughout the nervous system. Here we report that P301S transgenic mice developed tau fibrillary pathology and axonopathy in retinal ganglion neurons (RGC), a population of central neurons physiologically expressing Thy-1. RGC of 5-month old P301S transgenic mice expressed human tau in soma and axons. Transgenic tau was hyperphosphorylated as assessed by immunohistochemistry and western blotting of soluble and insoluble tau using AT8, AT100, AT180 and PHF-1 antibodies. Electron microscopy analysis of sarkosyl-insoluble extracts of P301S retinas demonstrated that tau was aggregated into filaments similar to those found in human NFT. Signs of axonopathy were observed in optic nerves of P301S mice. However, the number of RGC was unchanged in transgenic vs C57/Bl6 control mice. Full retinal examinations was performed  by an ophthalmologist (KM) masked to the genetic status of the animals. Marked optic nerve pallor was observed in 4/5 transgenic mice examined compared to 1/5 controls. No other consistent difference were observed between transgenic and control eyes by ophthalmoscopy.

These findings, together with the accessibility of the retina to direct observation and drug delivery, suggest that RGCs of the P301S transgenic tau mouse are an interesting model to investigate tau pathology.

P44

 

SOLUTION STRUCTURE OF THE LRR DOMAIN OF LANP,  A  POTENTIAL  MEDIATOR OF SPINOCEREBELLAR ATAXIA-1 PATHOGENESIS

 

de Chiara C*, Menon RP, and Pastore A

 

National Institute for Medical Research - Division of Molecular Structure- The Ridgeway NW7 1AA, London (UK)

Ph ++442088162629; FAX ++442089064477; E-mail:cdechia@nimr.mrc.ac.uk

 

The Leucine-rich repeat Acidic Nuclear Protein (LANP/Anp32a), a member of the Anp32 family of evolutionary conserved nuclear phosphoproteins, has been suggested as a potential interactor of ataxin-1, the protein responsible for spinocerebellar ataxia of type-1 (SCA1) (1). SCA1 is an autosomal-dominant neurodegenerative disorder associated with expansion of a polymorphic polyQ tract in the gene product ataxin-1 and characterised by ataxia and progressive motor deterioration.  LANP has also been involved in several important signalling pathways including tumour suppression, RNA shuttling, transcriptional regulation, modulation of apoptosis and cerebellar morphogenesis.

The protein architecture of the Anp32 family is characterized by the presence of a highly conserved N-terminal domain containing Leucine-Rich Repeats (LRR), a motif known to mediate protein-protein interactions, and of a C-terminal low complexity highly acidic region. The LRR of LANP has been reported to account for specific interactions with several cellular partners.

With the aim of understanding further its function, we have determined the structure in solution of LANP LRR domain using nuclear magnetic resonance techniques. The structure reveals a typical right-handed solenoidal fold arranged in a horse-shoe shape with a parallel beta-sheet in the concave site. This knowledge allows us to discuss how this domain could recognize specifically its partners.

1)Matilla and Radrizzani (2005) The Cerebellum 4, 7-18.

P45

 

CORTICAL BRAIN DERIVED NEUROTROPHIC FACTOR (BDNF) UPREGULATION IS MEDIATED BY TYPE-1 CANNABINOID RECEPTOR AFTER STRIATAL EXCITOTOXIC LESIONS

 

Zena De March(1)*, Chiara Zuccato(2)*, Carmela Giampˆ(1), Stefano Patassini(1), Monica Bari(3,4) Valeria Gasperi(4,5),  Mauro Maccarrone(4,5),  Maria L.de Ceballos(5),  Giorgio Bernardi(1, 6), Elena Cattaneo(2), and Francesca R. Fusco(1)

 

1Laboratory of Neuroanatomy, Santa Lucia Foundation IRCCS at the European Center for Brain research, Via del Fosso Fiorano 64, 00143 Rome Italy

2Department of Pharmacological Sciences and Centre for Stem Cell Research, University of Milano, Via Balzaretti 9, Milano 20133, Italy

3Department of Experimental Medicine & Biochemical Sciences, University of Rome ãTor Vergataä, 00133 Rome, Italy

4Department of Biomedical Sciences, University of Teramo, 64100 Teramo, Italy, & European Center for Brain Research (CERC)/IRCCS S. Lucia Foundation, 00143 Rome, Italy

5Neurodegeneration Group, Cajal Institute, CSIC, Doctor Arce, 37, 28002 Madrid, Spain

6Department of Neuroscience, University of Rome ãTor Vergataä, Viale Oxford 81, 00133 Rome, Italy

 

An involvement of one particular neurotrophin, namely, the brain-derived neurotrophic factor (BDNF), has been demonstrated in the pathophysiology Huntingtonâs Disease. Type-1 cannabinoid (CB1) receptor has been postulated to upregulate BDNF gene transcription.

To better understand the relationship between CB1 and BDNF levels in a situation where the striatum is degenerating, we studied, by dual label immunofluorescence, the distribution of CB1 and BDNF in cortical neurons projecting to the striatum in our rat  model of striatal excitotoxicity. We completed our study with quantitative analyses of BDNF protein levels and CB1 binding activity.

We show that, two weeks post lesion, cortical neurons contain more BDNF compared to controls and to earlier time points. Such BDNF upregulation coincides with a higher binding activity and an increased protein expression of CB1. We suggest that after excitotoxic lesions, CB1 might, at least transiently, upregulate BDNF in the attempt to rescue striatal neurons from degeneration.

P46

 

ULTRASTRUCTURAL STUDY OF MOTOR NEURON MITOCHONDRIA IN A MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS AFTER PHARMACOLOGICAL TREATMENTS

 

Gioria M (1), Vitellaro-Zuccarello L (1), De Biasi S* (1), Fontana F (1), Bendotti C (2)

 

(1) Dip. Scienze Biomolecolari e Biotecnologie, Universitˆ di Milano, via Celoria 26, 20133 Milano (Italy) tel. +39-02-50314885; fax +39-02-50314881; E-mail: silvia.debiasi@unimi.it

(2)  Dip. Neuroscienze, Istituto di Ricerche Farmacologiche ãMario Negriä, Milano

 

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons. The discovery that some familial ALS cases involve mutations in the gene coding for Cu,Zn superoxide dismutase (SOD1) led to the development of transgenic (Tg) mice to investigate the etiology of the disease. Several lines of evidence indicate mitochondria as a major target of mutant SOD1 toxicity, since mitochondrial alterations affect motor neurons already at presymptomatic stages.

To contribute to the understanding of the mechanisms leading to motor neuron death in ALS, we investigated the effects of minocycline (a tetracycline derivative that prolongs life span in Tg mice models of ALS) and riluzole (an inhibitor of glutamate release that increases survival of ALS patients) on the ultrastructure of mitochondria in spinal motor neurons of Tg mice bearing the SOD1G93A mutation. The six mice groups examined included Tg mice and non-Tg mice treated with vehicle, with riluzole or with minocycline (n =3 per group).

Ultrastructurally  all Tg mice showed massive vacuolization in spinal motor neurons and neuropil and comparable mitochondrial alterations consisting of swelling, dilatation of intermembrane space and rupture of the outer membrane. A stereological analysis, carried out by the point counting method in motor neuron somata, showed: a) no significant differences among experimental groups in the relative volumes of the mitochondrial compartment, although the volumes were slightly increased in vehicle-treated Tg mice and were furtherly increased in riluzole- and minocycline-treated Tg mice; b) a 20% reduction of small mitochondria and a 12-15% increase of medium-size mitochondria in motor neurons of Tg mice compared to controls, whereas large to giant mitochondria (absent in controls) attained 5-8%; c) pharmacological treatment did not significantly modify the mitochondrial size distribution in Tg mice. Collectively the results indicate that pharmacological treatments do not attenuate the structural mitochondrial alteration in motor neurons of Tg mice.

Supported by Telethon (Italy) GGP06063.

P47

 

MITOCHONDRIAL ACT AS SPATIAL AND TEMPORAL DECODERS OF SYNAPTIC CA2+ SIGNALS

 

Young KW, and Nicotera P

 

MRC Toxicology Unit, Hodgkin Building, University of Leicester, Lancaster Road, Leicester, LE1 9HN.

tel +44 116 2525174, fax +44 116 2525616, e-mail kwy1@le.ac.uk

 

Neuronal Ca2+ signals are fundamental to brain function, controlling a vast array of processes from neurotransmitter release to gene expression. Ca2+ signals require strict regulation as excessive Ca2+ increases, which occur during glutamatergic excitotoxicity, causes neuronal death. Under such conditions mitochondria appear to be major target organelles for excitotoxic Ca2+ signals. However, whether mitochondrial Ca2+ uptake also occurs during physiological Ca2+ signalling in neurons has not been fully addressed. In this current study, we have used high resolution confocal imaging of the Ca2+ probe ratioPericam, targeted to the inner mitochondrial matrix (2mtRP), to examine the spatial and temporal sensitivity of neuronal mitochondria to synaptically evoked Ca2+ signals.

Treatment of primary cultures of hippocampal neurons with the GABAA-receptor inhibitor, picrotoxin, produced repetitive Ca2+ transients which resulted from release of glutamate at synaptic contacts. These synaptically-evoked Ca2+ transients produced rapid, repetitive, fluxes in Ca2+ levels in the inner mitochondrial matrix. Mitochondrial Ca2+ fluxes did not require Ca2+ release from the endoplasmic reticulum, or the presence of an inter-connected mitochondrial network. Ca2+ efflux from the mitochondria appeared to be under the control of the mitochondrial Na+/Ca2+ exchanger. The spatial distribution of mitochondrial Ca2+ uptake was regulated by the extent of synaptic recruitment, with dendritic mitochondria displaying the greater sensitivity to synaptic activation. Mitochondrial Ca2+ fluxes could also be observed in the soma of hippocampal neurons. In these cases all mitochondria appeared equally responsive, regardless of distance from the plasma membrane (the source of the cytosolic Ca2+ signal).

Mitochondria are targets for both physiological and pathophysiological signals. In neurons, synaptic connectivity is reduced in treatments which limit mitochondrial distribution. This current study suggests that mitochondrial Ca2+ levels are constantly altering in response to surrounding synaptic activity. This is likely to regulate neuronal ATP production and provide a mechanism for buffering local changes in cytosolic Ca2+.

P48

 

GLUTAMATE-MEDIATED CELL DEATH IN PRIMARY HIPPOCAMPAL NEURONS DOES NOT INVOLVE MITOCHONDRIAL RELEASE OF AIF

 

Pi–—n LGP*, Young KW and Nicotera P

 

MRC Toxicology Unit, Hodgkin Building, University of Leicester, Leicester, LE1 9HN,UK tel. + 44 1162525571; fax. +44 116 2525616; e-mail: lp54@le.ac.uk

 

Excitotoxicity involves excessive stimulation of glutamate (Glu) receptors and is an important mechanism in neurodegenerative disorders and ischemic stroke. Mitochondria are key targets for death signals in neurons. Release of cytochrome C (cytC) from mitochondria results in the induction of caspase-dependent apoptotic pathways. Mitochondria have also been shown to release factors which triggers caspase-independent cell death like apoptosis-inducing factor (AIF). Once released from mitochondria, AIF translocates to the nucleus and initiates cell death via DNA fragmentation.

This study examines whether, in primary hippocampal neurons, excitotoxic applications of Glu causes significant nuclear translocation of AIF. We observed that the deregulation of Ca2+ homeostasis induced by glutamate treatment (30 micromolar) became essentially irreversible if the Glu stimulation exceeded 20 min. Glu treatment caused a dose-dependent increase in neuronal death mainly via necrosis. This occurred via activation of the NMDA-receptor subtype as no significant cell death was observed in cells pretreated with MK801. The effect of Glu appeared maximal within the 6h of the recovery period. Addition of MK801 during Glu wash-off reduced the amount of necrotic cell death, suggesting that residual or newly released Glu could contribute to the cell death process. To determine whether release of apoptosis-related proteins from the mitochondria was involved in this response, hippocampal neurons were stained for AIF, Hsp70, cytC. Although Glu caused mitochondria fragmentation no major nuclear translocation of AIF was detectable after the treatments employed. In contrast, partial cytC release was observed and could occur in neurons in which AIF was maintained within the mitochondria.

Our data suggests that in hippocampal neurons glutamate-mediated excitotoxicity stimulates a mainly necrotic form of cell death. This process has a rapid onset, and does not appear to involve nuclear translocation of AIF.

P49

 

DREAM HAS A NEUROPROTECTIVE ACTION BY REGULATING CALCIUM PERMEABILITY.

 

Sof’a Domingo, Rosa Gomez-Villafuertes, Malgosia Palczewska, Britt Mellstršm and Jose R. Naranjo.

 

Department of Molecular and Cellular Biology, National Centre of Biotechnology (CNB-CSIC), C/ Darwin 3 - 28049 Madrid (Spain) tel. +34-91-5854913;Fax  +34-91-5854506; E-mail: sdomingo@cnb.uam.es

 

Excitotoxic neuronal death has been associated to several neurodegenerative processes including Alzheimerâs disease, amyotrophic lateral sclerosis and epilepsy as well as to ischemic and traumatic brain injury. DREAM/calsenilin/KChIP3 is a calcium binding protein that plays different roles depending on its intracellular localization. In the nucleus DREAM (Downstream Regulatory Element Antagonist Modulator) functions as a calcium-dependent transcriptional repressor. DREAM regulates the expression of genes related to calcium homeostasis (NCX3) and neuronal viability (c-fos, hrk). We investigated excitotoxic death in cortical primary cultures from transgenic mice (E14) overexpressing a DREAM mutant insensitive to Ca2+ (EFmDREAM). Neuronal excitotoxicity was studied in transgenic and wild type cultures by stimulation of ionotropic glutamate receptors, particularly kainate receptors. Depolarization with high K+ concentrations was also used as a stimulus. In order to assay neuronal death excluding excitotoxic pathways, cultures were incubated with staurosporine. In all the experiments performed, DREAM transgenic neurons showed an increase in neuronal survival suggesting that overexpression of EFmDREAM might be involved in neuroprotection. We have analyzed the molecular mechanisms associated to the neuroprotective effect of mutant DREAM and we found that the expression of different voltage-dependent calcium channel subtypes was reduced in transgenic neurons. Our data suggest that DREAM has a neuroprotective effect through the regulation of intracellular Ca2+ levels.


P50

 

MOTOR NEURON BASAL PROPERTIES AND RESPONSES TO AMPA RECEPTOR-MEDIATED EXCITOTOXICITY IN DIFFERENT PRIMARY CULTURES FROM MOUSE ANTERIOR HORN SPINAL CORDS

 

M. De Paola(1)*, V. Diana(1), P. Bigini(1) and T. Mennini(1)

 

1Department of Molecular Biochemistry and Pharmacology, "Mario Negri" Institute for Pharmacological Research,.

* Laboratory of Receptor Pharmacology, "Mario Negri" Institute for Pharmacological Research, Via Eritrea 62, 20157 Milan, Italy, Tel: +390239014401,  Fax: +39023546277, E-mail address: depaola@marionegri.it

 

Primary motor neuron cultures represent the most valid in vitro model to study the early mechanisms involved in different motor neuron pathologies. Amyotrophic lateral sclerosis (ALS) is a devastating disease characterized by the selective and progressive degeneration of motor neurons. Many events involved in the complex ALS aetiopathogenesis have been study by using primary cultures. Unfortunately, the difficulties in obtaining useful and reproducible culture conditions make it difficult the direct comparison between results reported by different studies.

Here we directly compared the basal morphological properties and the responses to AMPA receptor (AMPAR)-mediated excitotoxicity of mouse spinal cord motor neurons under different culture conditions. Motor neurons co-cultured with a confluent glial layer had significant improvements in axonal length and in soma perimeter and surface, compared both to mixed anterior horn cultures and to purified cultures, suggesting that the presence of more ãmatureä glial cells was determinant to obtain healthier motor neurons.

By immuno-cytochemical and immuno-fluorescent assays we found that both in mixed anterior horn cultures and in co-cultures, lower AMPA or kainate concentrations, but not the higher, lead to the activation of classical apoptotic markers such as the nuclear fragmentation and the activation of the caspase cascade.  The different degenerative pathways induced by AMPAR agonist concentrations suggest that the experimental conditions used for in vitro studies are a key factor that should be deeply considered in order to obtain more valid and reproducible results.


P51

 

ASTROCYTES DIFFERENTIATED FROM ADULT NEURAL STEM CELLS OF WOBBLER MOUSE HAVE REDUCED GLUTAMATE UPTAKE AND DO NOT SUPPORT MOTOR NEURON SURVIVAL

 

Diana V*(1), Fumagalli E(1), Mennini T(1)

 

*Departement of Biochemistry and Molecular Pharmacology, Laboratory of Receptor Pharmacology,Institute of Pharmacological Research Mario Negri, via Eritrea 62, Milan (Italy) Tel. +39-02-39014401, Fax. +39-02-3546277, e-mail diana@marionegri.it

(1)Laboratory of Receptor Pharmacology, Institute of Pharmacological Research Mario Negri, Milan (Italy)

 

Neural stem cells (NSC) are an heterogeneous population of self-renewing, multipotent, immature progenitor cells,  able to differentiate both in glial and neuronal cells. Amyotrophic Lateral Sclerosis (ALS) is a multifactorial, progressive,  neurodegenerative disease.. Altered homeostasis of glutamate  is one of the contributing factors: reduced glial uptake of this amino acid causes an increase of glutamate concentration in extracellular fluid, leading to excitotoxicity and neuronal  death. In this work we used cultured astrocytes derived from adult NSC obtained from control or mutant wobbler mice, a murine model of ALS. NSC isolated as neurosphere from sub ventricular zone (SVZ) of adult mice were dissociated and plated at two different passage (P10 and P20). Differentiated astrocytes were obtained by adding FBS to proliferating medium.   We found a significant decrease of glutamate uptake in cultures obtained from wobbler NSC, probably linked to a different expression or activity of glutamate transporters. By immunocytochemistry  we confirmed a different expression of GLT1 between the two groups. Reduced axonal length and increased mortality of motor neuron (obtained from spinal cord of healthy mice embryos) was observed after  plating on  a wobbler astrocyte layer, compared to motor neurons plated on control astrocytes. Our results suggest that astrocytes from affected mice have primary, intrinsic metabolic alterations that may play a causative role in motor neuron loss rather than being a consequence of the environmental changes (i.e. spinal cord motor neuron degeneration).

P52

 

RESVERATROL PROTECTS AGAINST OXYGEN AND GLUCOSE DEPRIVATION RAT HIPPOCAMPAL ORGANOTYPIC CULTURES AND ACTIVATES AKT AND INACTIVATES GSK-3BETA.

 

Zamin LL (1), Gerhardt D (*1), Horn AP (1), Frozza RL (1), Sim‹o F (1), and Salbego C (1)

 

(1)  Departement of Biochemistry, Federal University of Rio Grande do Sul, Rua Ramiro Barcelos, 2600 ö 90035-003  Porto Alegre (Brazil) tel. +55-51-3308-5569; fax +55-51-3308-5535; E-mail: danieli83@yahoo.com.br

 

The reduction in the supply of glucose and oxygen to the brain that occurs in cerebral ischemia leads to a complex cascade of cellular events that result in neuronal death. Here, we investigated the neuroprotective effect of resveratrol, found in grapes and red wine, in an in vitro model of ischemia. We used organotypic hippocampal slice cultures, treated with resveratrol, and exposed to oxygen-glucose deprivation (OGD). Cellular death was quantified by measuring uptake of propidium iodide (PI), a marker of dead cells. In OGD exposed cultures, treated only with vehicle, about 70% of the CA1 area of hippocampus was labeled with PI, indicating a great percentage of cellular death. When cultures were treated with resveratrol, 10, 25 and 50 uM, this cellular death was reduced to 36, 34 and 28% respectively. To elucidate a possible mechanism by which resveratrol exerts its neuroprotective effect we used LY294002 (5uM) and PD98059 (20uM). The resveratrol (50 uM) neuroprotection was prevented by LY294002 but was not by PD98059. Immunoblotting assay reveled that resveratrol 50 uM induced the phosphorylation/activation of Akt and ERK ¸ and the phosphorylation/inactivation of glycogen synthase kinase-3beta. Taken together, the increase in phosphorylation of Akt and GSK-3beta induced by resveratrol after OGD and the effect of the inhibition of PI3-K by LY294002 leading to a decrease in the neuroprotection mediated by resveratrol, suggest that the PI3-K/Akt pathway together with GSK-3beta are involved in the mechanism by which resveratrol protects organotypic hippocampal slice cultures.

P53

 

EFFECTS OF THE ADENOSINE A2A RECEPTOR ANTAGONIST SCH58621 ON CYCLOOXYGENASE-2 EXPRESSION, GLIAL ACTIVATION AND BDNF AVAILABILITY IN A RAT MODEL OF STRIATAL NEURODEGENERATION

 

Minghetti L* (1), Greco A (1), Potenza RL (2), Pezzola A (2), Blum D (3), Bantubungi K (4), and Popoli P(2)

 

(1) Department of Cell Biology and Neuroscience and (2) Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanitˆ, Viale Regina Elena 299, 00161 Roma (Italia) tel+39-06-49903153; fax +39-06-4957821; E-mail: luisa.minghetti@iss.it

(3) INSERM U815, and (4) INSERM U816, Jean-Pierre Aubert Research Centre, UniversitŽ Lille2, Lille, France.

 

A2A receptors (A2ARs) belong to a family of at least four types of G-protein coupled receptors [A1, A2A, A2B, A3] that mediate the multiple functions of the purine nucleoside adenosine. In the central nervous system, A2A receptors are mainly localized in the striatum, although lower levels of expression are detected in the cortex and in the hippocampus. Besides their role in modulating dopamine-dependent activities in both physiological and pathological conditions, A2ARs are implicated in neuronal cell death associated with excitotoxicity. Given their preferential striatal localization, A2ARs are regarded as suitable targets for the development of neuroprotective strategies for treating disorders that are characterized by basal ganglia dysfunctions, such as Parkinsonâs disease and Huntingtonâs disease (HD). Although A2AR blockade, either by administration of selective antagonists or genetic ablation, is neuroprotective in several experimental settings, the mechanisms elicited by A2AR blockade are only partially known. In the present study, we analyzed the influence of the selective A2AR antagonist SCH 58261 in a rat model of striatal excitoxicity, obtained by unilateral intrastriatal injection of quinolinic acid (QA). We found that SCH 58261 differently affected the expression of cyclooxygenase-2 (COX-2) induced by QA in cortex and striatum. The antagonist enhanced COX-2 expression in cortical neurons while prevented it in striatal microglia-like cells. Similarly, SCH 58261 differently regulated astrogliosis and microglial activation in the two brain regions. In addition, the A2AR antagonist prevented the QA-induced increase in striatal brain derived neurotrophic factor (BDNF) levels. Since COX-2 activity has been linked to excitotoxic processes, and since BDNF depletion has been observed in mouse models as well as in HD patients, we suggest that the final outcome of A2AR blockade is likely to depend on the balance among its various and region-specific effects.

P54

 

RNA APTAMERS TARGETTING GLUTAMATE ION CHANNELS AS ANTI-EXCITOTOXIC DURG CANDIDATES

 

Li Niu

 

Chemistry Department, and Center for Neuroscience Research, State University of New York (SUNY) at Albany, New York (USA). tel. 518-591-8819; fax 518-442-3462; E-mail: lniu@albany.edu

 

Excitotoxicity is a leading pathogenic mechanism ascribed to a number of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Excitotoxicity is induced largely by the excessive activation of AMPA-type glutamate ion channels. Using inhibitors to block the AMPA receptor-mediated excitotoxicity is a long-pursued therapeutic strategy. However, the majority of existing inhibitors of AMPA channels are small organic molecules and are poorly water soluble. NBQX, for instance, is a classical competitive inhibitor for the AMPA/kainate glutamate receptors but failed clinically mainly due to its poor water solubility. Furthermore, inhibitors are routinely assayed with the desensitized receptor form, because conventional kinetic techniques have insufficient time resolutions to study an AMPA receptor, which, upon binding glutamate, opens its channel in the microsecond time domain an

d desensitizes in the millisecond time region. These problems have hampered the development of anti-excitotoxic compounds as effective drugs.

Using systematic evolution of ligands by exponential enrichment (SELEX), we identified a class of aptamers or RNA inhibitors, from a RNA library containing ~10^15 sequences, against GluR2Qflip, a key AMPA receptor subunit that controls the calcium permeability and mediates excitotoxicity. An aptamer is a single-stranded nucleic acid that inhibits a proteinâs function. It does so by folding into a specific three-dimensional structure that dictates high-affinity binding to the target. Furthermore, using a laser-pulse photolysis technique, we screen these aptamers against GluR2Qflip with a microsecond time resolution, sufficient to measure the inhibitory effect of an aptamer with a functional AMPA channel. One of our aptamers was found to have Kd of 4 nM, and this affinity rivals any existing inhibitors, including NBQX. Unlike NBQX, the aptamer is water soluble, and maintains the same potency even at clinically relevant acidic pH. The aptamer represents a unique, water-soluble lead compound with nanomolar affinity for future design of better AMPA receptor inhibitors/drugs for potential therapy of various neurodegenerative disorders. The methods we have developed during this study should be also applicable in general to selection of high affinity inhibitors targeting membrane proteins.


P55

 

THE ROLE OF METALS IN MISFOLDING AND AGGREGATION PROCESSES X-RAY SPETTROSCOPY AND NUMERICAL SIMULATIONS

 

Morante S.

 

Department of Physics, University of Rome "Tor Vergata", Via della Ricerca Scientifica, 1 - 00133 Roma (Italy)

tel. +39-0672594554; fax +39-0672594554; E-mail:morante@roma2.infn.it

 

Metals are essential elements for many of the fundamental activities of cells, with storing, metabolism and trafficking mediated by many proteins via well tuned mechanisms, because of the high toxicity of free ions.

Well identified peptides or proteins undergo a mis-folding process during the development of amyloidosis like beta-peptide in the case of AD and the prion proten in the case of the prion diseases. In both cases an important, but not yet fully elucidated, role is played by transition metals (mainly Cu(II) and Zn(II)). There exist in the literature conflicting statements about the role of Cu(II) and Zn(II) ions in providing protection against or act as promoters of plaques formation.

       In this talk I will present and discuss X-ray absorption spectroscopy (XAS) experiments on PrP [1] and beta-peptide [2] complexed with either Cu or Zn, which allow to identify the geometrical and atomic structure of the relevant metal binding site. I will also discuss the results we obtained in a large scale ab initio simulation of the PrP-metal system, devised to clarify the quantum mechanical basis for the identified metal coordination mode.

For what concerns the analysis of XAS data visible differences among spectra of Zn and Cu samples have been identified, that can be suggestive of a different structural role of the two ions.

On the more theoretical side, by using first principle ab initio molecular dynamics simulations of the Car-Parrinello type, we have thoroughly investigated the Cu coordination mode of the binding sites located in the PrP octarepeat region [3,4]. Simulations have been able to put in evidence a dipeptide entangled arrangement of two HGGG domains with exchange of amide nitrogen bonds between the two Cu centers emerges, which may be indicative of Cu favouring aggregation.

 

1.    S.Morante, C.Poltrich, R.Gonz‡lez-Iglesias, C.Meneghini, W.Meyer-Klaucke, G.Menestrina, M.A.Pajares, M.Gasset (2004) J. Biol. Chem. 279: 11753.

2.    F.Stellato, G.Menestrina, M.Dalla Serra, C.Potrich, R.Tomazzolli, W.Meyer-Klaucke, S.Morante (2006) Eur. Biophys J. 35(4): 340.

3.    S.Furlan, G.La Penna, F.Guerrieri, S.Morante, G.C.Rossi (2006) to be published on Journal of Biological Inorganic Chemistry

4.    S.Furlan, G.La Penna, F.Guerrieri, S.Morante, G.C.Rossi (2006) to be published on Journal of Biological Inorganic Chemistry.

P56

 

THE ROLE OF METAL IONS IN THE FIBRILLOGENESIS OF BETA-AMYLOID

 

*D. Drago (1), M. Bettella (2), L. Cendron (3), G. Tognon (4), P. Zatta (1)

 

(1)CNR-Institute for Biomedical Technologies, Padova Unit Metalloproteins, Department of Biology, University of Padova, Italy

Department of (2)Pharmacy and (3)Chemistry, University of Padova, Italy

 

Metal ions are widely recognized as a key factor for conformational changes and aggregation of Alzheimer's disease amyloid (Abeta). The purpose of this study is to compare the effects of Abeta and Abeta-metal complexes (Al, Zn, Cu, Fe) in human SHSY5Y neuroblastoma cells in terms of cell viability (MTT assay), membrane structure properties (fluorescence anisotropy), cellular and mitochondrial morphology (TEM, confocal microscopy).

No significant toxic effects are observed in neuroblastoma cells after 24h treatment with Abeta and Abeta-metals (Zn, Cu, Fe), except for a significant reduction of cellular viability after treatment with Abeta-Al complex. The effects of Abeta-Al as well as other Abeta-metal complexes are also evaluated in terms of fluorescence anisotropy in order to consider the possible alteration in the membrane structure properties. In this connection, treatment with Abeta-Al is able to increase specifically the membrane fluidity with respect to other Abeta-metal complexes.

Moreover, the toxic role of Abeta-Al is also correlated with a significant alteration in cellular morphology shown by electron microscopy (TEM).

Mitochondrial functionality could be compromised after treatment with Abeta-Al complex as reported in confocal micrographs of neuroblatoma cells stained with Mitotracker Red.

Importantly, the different toxicity between Abeta-Al and Abet or other Abeta-metal complexes seems to be related with the strong difference in the structure aggregates as well shown by electron microscopy (TEM) and by size exclusion chromatography (SEC).

Based on these findings, involvement of Al in Abetaaggregation and consequently increasing neuroblastoma toxicity is clearly demonstrated.

P57

 

MODEL FOR STEFIN B AMYLOID-FIBRILLATION FROM THE KINETICS & SIZE OF THE TOXIC OLIGOMERS.

 

Eva Zerovnik (1), Slavko Ceru (1), Katja Skerget (1), Andrej Vilfan (2), Sasa Jenko Kokalj (1), Sabina Rabzelj (1), Gregor Anderluh (3), Vito Turk (1), Dusan Turk (1) and Rosemary A Staniforth (4)

 

(1) Department of Biochemistry, Molecular and Structural Biology, Joõef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.tel.+386-1-4773753; fax +386-1-4773984; E-mail:eva.zerovnik@ijs.si

(2) Department of Condensed Matter Physics, Joõef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.

(3) Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia.

(4) Department of Molecular Biology and Biotechnology, Krebs Institute, Western Bank, University of Sheffield, S10 2TN Sheffield, U.K.

 

The process of amyloid-fibril formation is believed to be a generic property of proteins. However, proteins differ in the propensities to form amyloid-fibrils and probably each follows a characteristic mechanism in between the two limits of downhill fibrillation and step-wise polymerization/fibrillation with extensive lag phase. In our studies thus far, stefin B and some of its mutants were shown to exhibit a prominent lag phase (Úerovnik et al., 2002, Rabzelj et al., 2005). Here, we present a model of the amyloid fibrillation reaction by stefin B, which was obtained from temperature and protein concentration dependencies of the kinetics.

The model is consistent with structural and morphological data (Jenko-Kokalj et al. 2007, JMB). In this work we concentrated on energetic barriers of kinetically detectable steps.  A high energy of activation for the nucleation phase indicates that nearly complete unfolding (as needed for domain-swapping) occurs prior to fibril initiation. This is followed by an elongation phase, which is limited by a process with an energy of activation in range of proline isomerization.

Molecular characteristics of toxic oligomers are believed to be shared among various amyloidogenic proteins, therefore, the toxicity of prefibrillar states of a non-pathological protein human stefin B (cystatin B) was examined. By testing cell viability and caspase activity, it was shown that the lag phase species obtained at pH 5 and pH 3 (prefibrillar aggregates) were toxic to neuroblastoma cells. Of equal toxicity were the higher-order oligomers obtained at pH 7 by the size exlusion chromatography  (predominantly 12- to 16-mers as compared to standards). In distinction, monomers, dimers and tetramers were not toxic. The toxic higher-order oligomers were the ones which inserted best into the lipid monolayers (similarly to what was shown before for the lower pH prefibrillar aggregates by Anderluh et al., 2005). Diameters of the globular particles making the toxic aggregates and their hydrodynamic radii were determined by AFM and DLS, respectively.

P58

 

PrP82-146 AMYLOID: THE ROLE OF OLIGOMERS IN TRIGGERING NEURONAL DEGENERATION

 

Manzoni C(*), Colombo L (1), Gobbi M (1), Beeg M (1), Tagliavini F (2), Forloni G (1), Salmona M (1)

 

*. Mario Negri Institute for Pharmacological Research, via Eritrea 62, 20157 Milano, Italy, tel. 02 39014-445, fax 02 3546277, E-mail: manzoni@marionegri.it

1. Mario Negri Institute for Pharmacological Research, Milano, Italy,

2. Fondazione I.R.C.C.S. Istituto Neurologico Carlo Besta, Milano, Italy

 

Brain amyloidosis are central nervous system diseases involving neuronal degeneration  as a consequence of one or more endogenous proteins misfolding and taking on the typical beta-sheet rich, protease resistant amyloid conformation. These misfolded proteins are prone to self aggregation, triggering the polymerisation of insoluble amyloid fibrils. The prion pathologies are amyloidosis involving the cellular prion protein. In familial Gerstmann Straussler Scheinker prion disease the amyloid plaques are composed mainly of a prion protein fragment spanning residues 81/82-145/146. The corresponding PrP82-146 human prion peptide has been synthesized and can be considered an in vitro tool for the production of prion amyloid. The current study found that with PrP82-146 the deposition of mature amyloid fibrils was secondary to the production of soluble prefibrillar species showing the features of oligomers. These oligomeric precursors for the PrP82-146 mature amyloid have been characterized by electron microscopy and with a specific anti-oligomer antibody recognition test. In vitro PrP82-146 oligomerization profiles have been described after "freezing" oligomeric solutions by a new photo cross-linking tool (PICUP reaction). In cellular models PrP82-146 oligomerization is essential for peptide toxicity and to alter intracellular calcium fluxes. In conclusion, as previously established for other amyloidosis such as Alzheimerâs disease, the production of prefibrillar oligomers seems essential in triggering amyloid-related neurodegeneration in the case of prion pathologies too.

P59

 

EARLY ALTERATIONS OF GLUTAMATE EXOCYTOSIS IN THE CEREBELLUM OF TRANSGENIC MICE EXPRESSING A PrP INSERTIONAL MUTATION

 

Colleoni S* (1), Senatore A (2,3), Restelli E (2,3), Chiesa R (2,3), and Gobbi M (1)

 

(1)Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche "Mario Negri", Via Eritrea, 62 - 20157 Milano (Italy)

tel.+39-02-39014570; fax +39-02-3546277;E-mail: colleoni@marionegri.it

(2)Department of Neuroscience, Istituto di Ricerche Farmacologiche "Mario Negri", Milano (Italy)

(3)Dulbecco Telethon Istitute

 

Tg(PG14) mice express a mutant prion protein (PrP) containing 14 octapeptide repeats, the human homologue of which is associated with an inherited prion dementia. Mutant PrP accumulates in a synaptic-like pattern throughout the brain and displays biochemical properties reminiscent of PrPSc, the pathogenic isoform of PrP. Tg(PG14) mice develop a progressive neurological disorder characterized pathologically by gliosis and loss of cerebellar granule cells, and clinically by ataxia. Here we tested the hypothesis that mutant PrP expression induces early synaptic dysfunction, which precedes neurodegeneration and clinical symptoms. We evaluated the functional status of glutamatergic and GABAergic nerve endings in the brain areas of Tg(PG14) mice at different stages of neurological illness. As a control, we used age-matched Tg(WT) mice that express wild-type PrP and remain healthy.

Glutamatergic synaptosomes from Tg(PG14) cerebellum, but not from cortex, showed  impaired depolarization-induced release of [3H]D-aspartate (a non-metabolizable glutamate analogue)in presymptomatic 30 day-old mice. By the time mice had advanced disease (at >260 days of age) the K+-induced release was completely impaired. Importantly, there was no impairment of [3H]D-aspartate uptake in synaptosomes from Tg(PG14) cerebellum, excluding non-specific synaptosomal damage as a cause of these findings. No significant differences were found in [3H]GABA uptake or exocytosis between cerebellar synaptosomes of Tg(WT) and Tg(PG14) mice.

These data indicate that PG14 PrP expression is associated with early functional alterations of presynaptic glutamatergic nerve endings in the cerebellum, which degenerate in the later stages of disease. Studies are in progress to clarify the specific mechanism(s) by which PG14 PrP leads to defective depolarization-induced glutamate exocytosis.

P60

 

PRESYMPTOMATIC ALTERATIONS OF CALCINEURIN-DEPENDENT SYNAPSIN-I DEPHOSPHORYLATION IN THE CEREBELLUM OF TRANSGENIC MICE EXPRESSING A MUTANT PRION PROTEIN

 

Senatore A* (1,2), Colleoni S (3), Garofoli A (1,2), Restelli E (1,2), Forloni G (2), Gobbi M (3), and Chiesa R (1,2)

 

(1) Dulbecco Telethon Istitute and (2) Department of Neuroscience, (3) Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea, 62 - 20157 Milan (Italy) tel. +39-02-39014428; fax +39-023546277; e-mail: senatore@marionegri.it

 

Inherited prion diseases are neurodegenerative disorders linked to mutations in the prion protein (PrP) gene on chromosome 20. These mutations favor conformational conversion of the PrP into a beta-sheet-rich isoform that is aggregated and partially protease-resistant. Transgenic mice expressing a mouse PrP homologue of a nine-octapeptide insertion (PG14) associated with a human inherited prion disease, accumulate a form of the mutant protein in their brains that is aggregated and weakly protease-resistant. As this form accumulates, Tg(PG14) mice develop a fatal neurological disorder characterized by dramatic cerebellar atrophy due to loss of synaptic endings in the molecular layer and apoptosis of granule neurons. Deletion of the proapoptotic gene Bax efficiently rescues cerebellar granules, but does not prevent PG14-associated synaptic degeneration and development of neurological illness. Further, the activity of the phosphatase calcineurin, which is involved in regulating synaptic vesicle mobilization via the dephosphorylation of synapsin I, is strikingly reduced in the cerebellum of Tg(PG14) mice. These findings suggest that PG14 PrP deposition may affect normal synaptic function.

To explore this possibility, we carried out biochemical and functional analyses in purified cerebellar synaptosomes from Tg(PG14) mice at different stages of the disease. We found a relative enrichment of PrP in synaptosomes where the mutant protein was highly aggregated. Consistent with the observed reduction in calcineurin activity, depolarization-induced synapsin I dephosphorylation was selectively reduced already in the presymptomatic stage. These results are consistent with the hypothesis that mutant PrP affects the mechanisms governing exocytotic neurotransmitter release, and that this represents an early event in the pathogenesis (see abstract by Colleoni et al).

P61

 

BEHAVIOR OF SKIN FIBROBLASTS IN HUNTINGTONâS DISEASE (HD): POSSIBLE ROLE OF TISSUE TRANSGLUTAMINASE 

 

Melone MAB (1),  Barone MV (2),  Petillo O (3), Calarco A (3),  Torpedine A (3),  Margarucci S (3), and Peluso G (3)

 

(1) Department of Neurological Sciences, Second University of Naples, First Division of Neurology, School of Medicine, Policlinico Federico II,Isola 8, Ed. 10, Via Sergio Pansini, 5 - 80131 Napoli tel.  +39-081-5666809/6810;fax +390-81-5666805; E-mail: marina.melone@unina2.it

(2) Department of Paediatrics, University "Federico II", Naples (Italy)

(3)Institute of Protein Biochemistry-CNR, Naples (Italy)

 

Identification of huntingtin(htt)-interacting proteins suggests that htt might function as a scaffold involved in orchestrating sets of proteins for signaling processes and intracellular transport. The abnormal interactions of mutant-htt with these signaling molecules could affect cell viability, chemotaxis, wound healing and increase caspase activity. We generated a cellular model by using dermal fibroblasts both of  HD patients cultured in different conditions. In particular, we examined the cell growth and the effects of all-trans retinoic acid (RA) and calcium ionofore (CI) both on cell tTGase expression and activation and on aggregate and apoptosis induction. Therefore, by using Boyden chamber in presence of 10% FBS, or 1 microM fMLP, as chemoattractants, we studied cell locomotion of HD fibroblasts. RA treatment of HD fibroblats  stimulates the synthesis of TGase protein, and the activity of TGase, as shown by transglutaminase activity in lysates of RA-treated (80 ± 10 nmol of putrescine incorporated/mg of casein/mg of protein) and untreated cells (20 ± 6 nmol putrescine incorporated/mg of casein/mg of protein). Western blot analysis of cell extracts using anti-huntingtin antibody showed that virtually all of the monomeric huntingtin in RA-exposed HD cells was absent and replaced by larger immunologically cross-reactive species. Furthermore, control fibroblasts exposed to RA did not show the alteration in the size of huntingtin, demonstrating that the effect is dependent upon polyglutamine repeats. Caspase transcript was not detected in control fibroblasts while low levels of caspase 6, 3, were detected in HD fibroblasts. Besides,  we found that HD cells lose the wound healing and exhibit a strong decrease of their cell growth and migration ability when compared to healthy cells.These findings suggest that abnormal interactions of mutant-htt with these signaling molecules could affect cell viability, chemotaxis, wound healing and increase caspase activity.

P62

 

ATOMIC FORCE MICROSCOPY ANALISYS OF ATAXIN-3 PREFIBRILLAR AGGREGATES AND AMYLOID FIBRILS

 

Riva M (1), Baserga A (2), Bottani CE (2), Tortora P (1), Regonesi ME (1)*

 

(1) Department of Biotechnology and Bioscience, University of Milano-Bicocca, Piazza della Scienza, 2 -20126 Milan (Italy) tel. +39-02-64483437; fax +39-02-64483450; E-mail: mariaelena.regonesi@unimib.it

(2)NEMAS-Center for NanoEngineered MAterials and Surfaces, Department of Nuclear Engineering, Politecnico di Milano, Via Ponzio 34/3 20133 Milan (Italy)

 

Polyglutamine (poly-Q) diseases are hereditary neurodegenerative disorders caused by the expansion of a CAG repeat in genes coding for a set of unrelated proteins. CAG repeats code for poly-Q stretches, which are pathogenic when they become expanded, i.e., exceed a characteristic threshold, generally 40 residues. Expanded repeats are thought to cause structural changes in the affected proteins, leading to aberrant interactions with resultant formation of extra- and intranuclear aggregates. Here, we report investigations on ataxin-3 (AT-3), a protein whose expanded variants are responsible for spinocerebellar ataxia type 3. AT-3 consists of a folded Josephin domain (1-182 aminoacid) followed by two ubiquitin-interacting motifs and a C-terminal poly-Q tract. We investigated different variants, i.e., the murine wild-type carrying six consecutive glutamines (AT-3Q6), the human wild type of twenty four glutamines (AT-3Q24), two truncated forms, one at the residue 291 (AT-3/291) and another at the residue 182 (AT-3/182) and an expanded form carrying thirty six glutamines (AT-3Q36). We submitted these proteins to a steady temperature increase, from 37¡C to 85¡C, to accelerate amyloid fiber formation. By atomic force microscopy (AFM) we observed that, in keeping with a previous report [Ellisdon et al. (2006) J. Biol. Chem. 281, 16888-16896], AT-3 forms protofibrils but not mature amyloids, for which a longer poly-Q stretch is required. These observations highlight the intrinsic amyloidogenic potential of the protein, irrespective of the poly-Q stretch. Under the same experimental conditions, the truncated variants also generated protofibrils, as supported by thioflavine T fluorimetry measurements. These results suggest that the Josephin domain is the key player in the aggregation, while the poly-Q tract in its pathological expansions is required for triggering the subsequent growth of mature fibrils from the starting short protofibrils.


P63

 

ANALYSIS OF THE MOLECULAR PATHOGENESIS OF UPR IN CMT 1B MICE

 

D'Antonio M (1), Musner N* (1), Pennuto M (1), Tinelli E (1), Quattrini A (3), Feltri ML (1) and Wrabetz L (1)

 

(1) Myelin Biology Unit, San Raffaele Scientific Institute-DIBIT, 20132 Milano

(2) Department of Neurology, San Raffaele Scientific Institute, 20132 Milano, Italy

 

Myelin Protein Zero (MPZ,P0) is the most abundant glycoprotein of peripheral nerve. Mutations in P0 cause several human inherited neuropathies with defects in myelin. Diverse mutations cause different phenotypes, suggesting gain of function mechanisms. Deletion (S63del) or conversion of serine 63 to cysteine (S63C) results in Charcot Marie Tooth 1B disease or Dejerine-Sottas syndrome, respectively. We showed that if expressed in mouse together with wild type P0, either mutant P0 produces a demyelinating neuropathy that mimics the corresponding human disease. S63del never reaches the myelin sheath and is retained in the endoplasmic reticulum (ER)-Golgi complex. Accumulation of S63del in the ER triggers an unfolded protein response (UPR) in a dose-dependent fashion, indicating a toxic gain of function. Ablation of the UPR mediator CHOP restores the motor deficit in S63del mice suggesting that the UPR is pathogenetic. However, the mechanisms by which this is achieved remain to be clarified. Considering that CHOP is a transcription factor, gene profiling of S63del and S63del/CHOP -/- mice followed by GeneSpring and L2L analysis was performed. In this study we show that the strong up-regulation of Derlin protein family in S63del and S63del/CHOP-/- could be potentially involved in the clearance of mutant P0. This suggests that the endogenous activation of ER associated degradation (ERAD), at least in part mediated by Derlins, could be beneficial for the recovery of ER stress in both S63del and S63del/CHOP-/- mice. Furthermore we show that prolonging translational inhibition could be beneficial specifically in S63del/CHOP-/- mice. In fact mRNA and protein levels of GADD34, a re-activator of protein synthesis after ER stress, are specifically up-regulated in S63del and restored to wt levels in S63del/CHOP-/- mice. Therefore we propose that the ablation of CHOP, impeding GADD34 up-regulation, prolongs the translational block, which could prove beneficial for Schwann cells.

P64

 

DISSECTING THE ROLE OF PHOSPHORYLATION IN MODULATING ALPHA-SYNUCLEIN FIBRILLISATION

 

Paleologou KE(1)*, Fredenburg RA (3), Schmid A(1),  Moniatte M (2), Chiappe D (2), Pignat V (1), Lansbury, Jr. PT(3), and Lashuel HA (1,2)

 

(1)Laboratory of Molecular Neurobiology and Neuroproteomics, Brain and Mind Institute, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland, Tel: +41 21 69 39652; Fax:. +41 21 693 96 65, E-mail: ekaterini.paleologou@epfl.ch

(2)The EPFL proteomic core facility, School of Life Sciences, Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.

(3)Harvard Center for Neurodegeneration  and Repair, Center for Neurologic Diseases, Brigham and Womenâs Hospital and Department of Neurology, Harvard Medical School, 65 Landsdowne St., Cambridge, MA 02139.

 

Alpha-synuclein is the major component of Lewy bodies (LBs), which in turn constitute the main neuropathological feature of Parkinsonâs disease (PD). The finding that alpha-synuclein isolated from LBs is phosphorylated at Serine 129 of the protein highly suggests that the phosphorylation of alpha-synuclein is of pathogenic nature. Previous studies aiming at elucidating the role of alpha-synuclein phosphorylation in the pathogenesis of PD relied on comparing the physical and toxic properties of the wild type (WT) protein to mutants designed to mimic the phosphorylated (S129E) and nonphosphorylated (S129A) forms of alpha-synuclein. In the present study, we investigated the biophysical properties together with the structural and functional significance of phosphorylation at Serine 129 and Serine 87 using purified mono- and multiphosphorylated forms of alpha-synuclein prepared by in vitro phosphorylation of the WT and phosphorylation-mimicking mutants of alpha-synuclein with casein kinase I (CK1). The fibrillization and membrane binding properties of the purified phosphorylated forms of WT and phosphorylation-mimicking mutants were compared to that of the corresponding unphosphorylated form in vitro. A comparison of our findings to previously reported data and their implications for the mechanism of pathogenesis in PD are also discussed.

P65

 

'IN VITRO' AGGREGATION OF THE ANDROGEN RECEPTOR WITH AN EXPANDED POLYGLUTAMINE TRACT

 

Palazzolo I.(1, 4), Bolzoni E.(1), Gliozzi A(2), Relini A.(2), Beeg M.(3), Salmona  M.(3), Poletti A.(1)

 

 (1) Inst Endocrinology, Centre of Excellence on Neurodegenerative Diseases, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. (2) Department of Physics, University of Genoa, Genoa, Italy Inter-University Research Centre on the Molecular Basis of Neurodegenerative Diseases, (3) Institute of Pharmacological Research Mario Negri, Milan, Italy. (4) NIND, NIH, Bethesda, MD, USA

 

Spinal and Bulbar Muscular Atrophy (SBMA), or Kennedy disease, is a neurodegenerative disease linked to a polyglutamine expansion  (PolyGln) in the androgen receptor protein (AR). This mutation induces the protein to aggregate and to form intracellular inclusions which may be toxic to the motor neurons. The AR inclusions, are similar to those found in other neuronal disorders, linked to mutated proteins, suggesting that common mechanisms of toxicity are present.

It has already been shown that the PolyGln tract acquires a beta-strand conformation. The expanded PolyGln sequence (>40 repeats) compromises the global folding of the protein; the expanded tract may be exposed on the surface of the protein and may be involved in an abnormal number of intramolecular interactions, starting the aggregation process. This first modified protein structure may then generate further intermolecular interactions stabilizing the inclusions. In fact, the process seems to be reversible in the initial  phases, when the aggregates are still soluble; then, the misfolded PolyGln proteins polymerize in a mature amyloid-like fibers which generate insoluble inclusions. In this work, we investigated in vitro the conformation of the ARs in solution. We generated different chimeras of GST-tagged AR (GST-ARQn) from which we released the ARQn by Thrombin cleavege. Then, we analyzed their structure using the Circular Dichrism. The Far-UV CD spectra of ARQ0 and ARQ22 are similar to each other. The differences in the ARQ48 spectra compared to the others are based on the decrease of alpha-helix content and concomitant increase of the beta-sheet amount. Therefore the expanded PolyGln AR (ARQ48) presented a decreased fraction of protein folded as alpha helices and an increased portion showing an antiparallel beta-sheet when correlated to the normal protein (ARQ22) which presents most of the chain involved in alpha-helices structures. By analyzing the structure of an artificial chimera without PolyGln tract (ARQ0), we demonstrate that the changes in the secondary structure are due to the abnormal PolyGln expansion. We then studied the effect of the mutation in the formation of amyloid like fibrils using the Atomic Force Microscopy. In these analysis we observed that the expanded PolyGln AR, but not the wild type protein, generate fibrils which increased their size in a time-dependent manner. Moreover, ARQ22 and ARQ0 mostly appear as glomerular structures or short, thin non mature protofibrils. Of interest is the fact that only ARQ48 has the capability to form mature fibrils during a prolonged incubation at 37¡C for several days. With this experiment, we demonstrated in an in vitro system that the PolyGln abnormal expansion in the AR leads to protein misfolding and aggregation, and we pointed out a system to validate further studies on anti-aggregant molecules. Grants Telethon - Italy (#GGP06063),  MIUR-FIRB (#RBAU01NXFP); MIUR-Cofin (2005057598_002),  University of Milan-FIRST,  FONDAZIONE CARIPLO.


P66

 

ANALYSIS OF THE UBIQUITIN/PROTEASOME SYSTEM IN HUNTINGTON'S DISEASE USING GFP-BASED REPORTER SUBSTRATES

 

Maynard C.J., Dantuma, N.P.

 

Department of Cell and Molecular Biology, The Medical Nobel Institute, Karolinska Institutet, S-17177, Stockholm, Sweden

 

Impaired function of the ubiquitin/proteasome system (UPS) has been postulated both as a cause and a consequence of Huntingtonâs disease (HD) pathogenesis.  Insufficient UPS degradation of mutant huntingtin (Htt) fragments may initiate the accumulation of this toxic fragment.  Alternatively, overwhelming the UPS machinery with misfolded Htt fragments or sequestration of proteasomes and other crucial UPS components into the nuclear inclusions may hinder the degradation of other proteasome substrates, causing a general impairment of UPS-dependent proteolysis.  We use complementary in vivo and in vitro model systems to monitor the functionality of the UPS in parallel with HD pathogenesis

We have generated a panel of different yellow-fluorescent protein (YFP) reporters of UPS function for in vitro analysis.  By co-transfecting cell lines expressing various YFP-UPS substrate reporters with constructs encoding mutant Htt, we are able to monitor the efficiency of degradation of different classes of UPS substrate during the progression from soluble Htt, to nuclear inclusions and cell death. With this assay, we found that mutant Htt differentially affects degradation of different classes of UPS substrate.

Using our recently developed transgenic mouse model for monitoring UPS function, crossed with transgenic mouse models of HD, we are able to monitor UPS functionality in parallel with the progression of HD pathology directly in mouse brain.  In the R6/1 and R6/2 mouse models of HD we observed no global impairment of UPS proteolysis early or late in HD pathogenesis. Our results suggest that although acute overexpression of mutant Htt in cell lines impairs degradation of some classes of UPS substrate, there is no global or chronic impairment of UPS functionality in the brain of these mouse models of HD.

This work was supported by the Wallenberg foundation, the HighQ Foundation, Nordic Centre of Excellence, and the Swedish Research Council.

P67

 

PPAR gamma-INDEPENDENT INHIBITION OF PI3K/AKT BY 15-DEOXY-delta12,14-PROSTAGLANDIN J2 INCREASES P27KIP PROTEIN LEVELS IN IMMORTALIZED LYMPHOCYTES FROM ALZHEIMERâS DISEASE PATIENTS.

 

*òrsula Mu–oz, Fernando BartolomŽ and çngeles Mart’n-Requero

 

Department of cellular and Molecular Pathophysiology, Centro de Investigaciones Biol—gicas. CSIC. Ramiro de Maeztu 9 28040,Madrid, Spain

 

Cyclin-dependent kinase inhibitor p27kip1 (p27), a critical determinant for cell cycle progression, is an important regulation target of mitogenic signals. It is believed that failure of cell cycle control might play a role in the pathogenesis of Alzheimerâs disease (AD). We previously reported that immortalized lymphocytes from AD patients, show an enhanced proliferative activity, associated with down-regulation of this CDK inhibitor. Treatment of AD cells with the PPARgamma ligand, 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) prevented the serum-mediated stimulation of cell proliferation and increased p27 protein levels.

The purpose of this investigation was to elucidate the mechanism of 15d-PGJ2-induced p27 accumulation. It was observed that 15d-PGJ2 increased the stability of the p27 protein in AD cells without affecting the p27 half-life of p27 in control cells. Treatment of AD cells with 15d-PGJ2 had no effect on cellular proteasome activity nor resulted in accumulation of ubiquitin-tagged proteins. 15d-PGJ2 decreased the enhanced phosphorylation of p27 at Thr 187 observed in AD cells. 15d-PGJ2 decreased the cyclinE/CDK2 kinase activity in AD cells by a mechanism dependent on PI3K/Akt activation. This asseveration finds support in the following observation, first, inhibitin of PI3k/Akt by Ly294002 had similar effects that 15 d-PGJ2 on p27 phosphorylation and cell proliferation, and second 15d-PGJ2 inhibits Akt activation in AD lymphoblasts. 

These effects of 15d-PGJ2 seem to be independent of PPAR; activation since the analog 9,10-dihydro-15dPG2 that retain PPARgamma agonist activity is not able to inhibit Akt and p27 phosphorylation in AD cells. These results also indicate that the alfa,beta-unsaturated carbonyl group in the cyclopentenone ring of 15d-PGJ2 is a requisite for the effects of 15-dPGJ2 in AD cells.

P68

 

PROTEASOMAL INHIBITION AND APOPTOSIS REGULATORY CHANGES IN HUMAN ISOLATED LYMPHOCYTES: THE SYNERGISTIC ROLE OF DOPAMINE.

 

Bazzini E (1), Samuele A (1), Granelli M (1), Levandis G (1), Armentero MT (1), Nappi G (2) and Blandini F (1).

 

(1)Laboratory of Functional Neurochemistry, Neurological Institute C. Mondino, Pavia, Italy

(2)Department of Neurology and Otorhinolaryngology, University of RomeãLa Sapienzaä, Rome, Italy

 

Abnormal deposition of protein aggregates and increased susceptibility to apoptotic cell death may result from defects in the activity of the ubiquitin-proteasome system (UPS); neurotoxicity related to UPS defects seems to require dopamine to be fully expressed. The aim of this study was to investigate the pro-apoptotic effects caused by proteasomal activity inhibition, as well as the synergistic effect of dopaminergic stimulation in human lymphocytes isolated from healthy volunteers. Cells were incubated 20 hours at 37¡C, with: 1) lactacystin, 2) increasing concentrations of dopamine or 3) mixture of dopamine and lactacystin. Activities of proteasome 20S and pro-apoptotic caspases-3 and -9 and levels of anti-apoptotic Bcl-2 were measured with fluorimetric or immunochemical assays, while a "DNA diffusion" assay was used to determine the apoptosis. Incubation of lymphocytes with lactacyst in, which caused reduction of proteasomal activity, was associated with activation of caspases. A clear, dose-dependent reduction of proteasomal activity was also seen in the presence of increasing doses of dopamine, which was accompanied by a slight dose-dependent increase of caspases activities and Bcl-2 levels. Both effects on proteasome and caspase activities were enhanced when cells were simultaneously exposed to lactacystin and elevated concentrations of dopamine. Apoptosis was detected in all treated samples, but not in controls, without significant differences among the treatment groups; however, the association of dopamine and lactacystin induced a clear reduction in the number of cells being analysed, pointing to marked cytotoxicity.

Our data confirm the potentiation of cytotoxicity related to proteasome inhibition, in the presence of dopaminergic stimulation.

P69

 

NEURODEGENERATION AND AXONAL DEVELOPMENT: THE DUAL ROLE OF THE MITOCHONDRIAL PARAPLEGIN-AFG3L2 COMPLEX

 

Maltecca F (1*), Cassina L (1),  Magnoni R (1), Cox G.A. (2), GuŽnet J.L. (3), Previtali S.C. (4), Quattrini A. (4) and Casari G. (1,5)

 

(1) Human Molecular Genetics Unit, San Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan (Italy) tel.+39-02.2643.4951; fax: +39-02.2643.4767; E-mail:maltecca.francesca@hsr.it

(2) The Jackson Laboratory, Bar Harbor, ME, USA 

(3) Institut Pasteur, Paris, France

(4) Department of Neurology, San Raffaele Scientific Institute, Milan, Italy

(5) Vita-Salute San Raffaele University, Milan, Italy

 

Axonal degeneration of the longest motor and sensory axons is the main pathological feature of hereditary spastic paraplegia (HSP). Mutations of paraplegin, a nuclear-encoded mitochondrial metalloprotease, cause a recessive form of HSP. We showed that paraplegin co-assembles with a highly homologous protein, AFG3L2, to form a functional complex (the m-AAA protease) in the inner mitochondrial membrane. Lack of this complex in HSP primary fibroblasts causes a reduced complex I activity and an increased sensitivity to oxidative stress. The Spg7-/- mouse model shows a relatively mild and slowly progressive phenotype. We are characterizing two different mutant mouse models defective in Afg3l2. In spite of the close functional association of AFG3L2 with paraplegin, either Afg3l2 mutation leads to an extremely severe neurological syndrome. Actually, both Afg3l2 mutants show a dramatic neuromuscular phenotype beginning at P7 with hindlimbs paraparesis which progresses until complete tetraparesis and death, generally at P16-18. Contrasting to the paraplegin-deficient model, in Afg3l2 mutants we observe impressive widespread reduction of fiber density in the spinal cord, associated with a decreased axon diameter of the remaining fibers. These alterations have been found in motor and sensory areas, with no evidence of active axonal degeneration and demyelination. The number of motor and sensory neurons is conserved, suggesting an impairment of axonal development. Mitochondrial morphology abnormalities are also detected and enzymatic activities of the respiratory chain complexes are strikingly impaired in Afg3l2 models, denoting the organellar origin of the defect. Again, differently from the paraplegin-deficient model, these abnormalities are predominantly present in cell bodies and proximal regions and not only in synaptic terminals and distal region of axons.

Recently, we have shown that an active homo-oligomer complex containing AFG3L2 is present in mitochondria of paraplegin-deficient mice. The greatly divergent phenotypes of Afg3l2-deficient mice compared to the paraplegin mutant substantially agree with the molecular evidences that assign to AFG3L2 protein a more structural and versatile role within m-AAA complexes crucial for mitochondrial metabolism and axonal development.


P70

 

INVOLVEMENT OF THE UBIQUITIN-PROTEASOME PATHWAY AND AUTOPHAGY IN THE CLEARENCE OF MUTANT ANDROGEN RECEPTOR IN THE SPINAL AND BULBAR MUSCULAR ATROPHY

 

Rusmini P*., Simonini F., Bolzoni, E., Poletti A.

 

Institute of Endocrinology, Center of Excellence on Neurodegenerative Diseases of  the University of Milan (Italy) and InterUniversity Center on Neurodegenerative  Diseases (Universities of Florence, Rome and Milan, Italy),Via Balzaretti 9, 20133 Milan. angelo.poletti@unimi.it

 

Spinal and bulbar muscular atrophy is a motorneuronal disorder caused by an expansion of the CAG repeat localized in the first exon of the androgen receptor (AR), coding for an expanded polyglutamine (polyQ) tract. The AR in its inactive state is confined in the cytoplasm, while the binding with its ligand Testosterone (T) induces nuclear translocation.

Aggregates are a typical hallmark of SBMA; they contain the mutant protein, are ubiquitylated and sequestered proteasome subunits, suggesting that the degradative machinery may be involved in their formation.

We have studied the role of the two main proteolytic pathways used by cells to degrade cellular proteins: the Ubiquitin Proteasome Pathway (UPP), and the autophagy.

We demonstrated that the UPP is impaired when the ARpolyQ is in the soluble and unbounded conformation. Surprisingly, T treatment,which induced ARpolyQ aggregation and restored normal UPP activity possibly by confining the mutant AR into the inclusion. Thus we have studied if these inclusions might be a cellular attempt to degrade the mutant proteins through the alternative autophagic pathway.

Western blot analysis showed that the treatment with an autophagy inhibitor, 3-Methyl-adenine (3-MA), led to ARpolyQ accumulation, both in the inactive and active conformations. Moreover, filter retardation assay showed that T treatment led to the formation of a large amount of insoluble species, while 3-MA co-treatment increase the ARpolyQ accumulation in the insoluble forms. These data strongly suggest that autophagy is involved in the degradation of ARpolyQ, since its inhibition led to an accumulation of the mutant AR.

Therefore, both UPP and autophagy contribute to the degradation of ARpolyQ. The overload of the UPP activity may activate the autophagy to compensate the UPP impairment, in the attempt to remove the mutant and misfolded protein. Understanding the balance and the link between these two degradative systems might provide important information to design novel therapeutic approach for SBMA.

 

Grants Telethon - Italy (GGP06063), MIUR-FIRB (# RBAU01NXFP); MIUR-Cofin (2005057598_002), University of Milan-FIRST, FONDAZIONE CARIPLO.


P71

 

ER STRESS (PERK/eIF2ALPHA) STIMULATES THE CONSTITUTIVE AUTOPHAGE, WHICH  DEGRADES ABNORMAL PROTEIN AGGREGATES INCLUDING POLYGLUTAMINE AGGREGATES

 

Momoi T (1), Kouroku Y (1), and Fujita E (1)

 

1Divisions of Development and Differentiation for Human Disease, National Institute of Neuroscience, 4-1-1 Ogawahigashi-machi, Kodaira, Tokyo 187-8502, Japan

 

Expanded polyglutamine 72 repeat (polyQ72) aggregates impair the retrotranslocon of the ER and induce endoplasmic reticulum (ER) stress-mediated cell death with caspase-12 activation and autophagy formation. Here we examined this relationship and showed that the ER stress stimulates the constitutive autophagy formation and ER stress-mediated constitutive autophagy plays as a cellular defense system by degrading polyQ aggregates. Rapamycin, a stimulator of autophagy, inhibited the polyQ72-induced cell death with caspase-12 activation. PolyQ72, but not polyQ11, stimulated Atg5-Atg12-Atg16 complex-dependent LC3 conversion from LC3-I to -II, which plays a key role in constitutive autophagy. The eIF2alpha A/A mutation, a knock-in to replace a phosphorylatable Ser51 with Ala51, and dominant-negative PERK inhibited polyQ72-induced LC3 conversion. PolyQ72 as well as ER stress stimulators upregulated Atg12 mRNA and proteins via eIF2alpha phosphorylation. Furthermore, Atg5 deficiency as well as the eIF2alpha A/A mutation increased the number of cells showing polyQ72 aggregates and polyQ72-induced caspase-12 activation. Thus, when retrotranslocon/ERAD system is impaired by polyQ aggregates, polyQ-induced ER stress (PERK/eIF2alpha phosphorylation) upregulates Atg12 mRNA, constitutively stimulating the LC3 conversion, and stimulates the autophaosome formation, which degrades the abnormal protein aggregates. Here, we propose two ER-associated degradation systems (ERAD), ubiquitin/proteasome ERAD(I) and constitutive autophagy/lysosome ERAD(II). Mutant aggregates including polyQ aggregates are degraded by the constitutive autophagy/lysosome ERAD(II), as an alternative to ERAD(I).

P72

 

THE TRANSCRIPTIONAL REPRESSOR DREAM REGULATES APP PROCESSING IN THE BRAIN

 

Gomez-Villafuertes R* (1), Domingo S (1), Savignac M (1), Galea P (2), Pruss R (2), Mellstršm B (1), and Naranjo JR (1)

 

(1) Department of Molecular and Cellular Biology, National Centre of Biotechnology (CNB-CSIC), C/ Darwin, 3 - 28049 Madrid (Spain) tel.+34-91-5854913; fax +34-91-5854506; E-mail: mrgomez@cnb.uam.es

(2) TROPHOS S.A., Parc Scientifique de Luminy, Luminy Biotech Entreprise-case 931, 13288 Marseille cedex 9  (France).

 

Presenilin/gamma-secretase complex is a membrane-associated aspartyl protease that is involved, among other things, in the transmembrane processing of the amyloid precursor protein (APP). The cleavage of APP generates amyloid-beta peptides, whose deposition in the brain is a characteristic of Alzheimerâs disease (AD). DREAM (also named calsenilin and KChIP3) is a calcium binding protein that interacts with the C-termini of both presenilin 1 (PS1) and presenilin 2 (PS2). Moreover, the multifunctional DREAM acts as a transcriptional repressor in the nucleus and binds to A-type voltage-gated potassium channels in the plasma membrane. Previous studies reported that overexpression of DREAM enhances the activity of presenilins, suggesting that DREAM may be one of the regulatory factors for the gamma-secretase complex, although the mechanism involved is unknown. Here we show that DREAM is able to regulate APP and the gamma-secretase complex at the transcriptional level. DREAM expression was not modified in the cortex of transgenic mice overexpressing amyloid precursor protein mutants (APPsw and APPswInd mutations), and PS1, PS2 and APP mRNA levels were normal in the cortex and hippocampus of DREAM+/- mice. However, transgenic mice expressing a DREAM mutant protein (EFLx-mutant-DREAM), which is insensitive to Ca2+ and is not regulated by protein-protein interaction, showed an increase in presenilins, nicastrin and APP brain expression. Additionally, EFLxmDREAM transgenic mice cortex showed altered levels of amyloid-beta40 peptide.


P73

 

ALTERATION OF NUCLEAR ROS PROTECTION, IN MODELS OF FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS (fALS).

 

Sau D.*(1), De Biasi S.(4), Vitellaro L. (4), Crippa V.(1,) Bolzoni E. (1), Onesto E. (1), Simonini F. (1), Riso P (2), Bendotti C. (3), Poletti A. (1).

 

(1)Inst. of Endocrinology, Centre of Excellence on Neurodegenerative Diseases, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. Tel/Fax 02-5031.8215/04 angelo.poletti@unimi.it

Inter-University Research Centre on the molecular basis of neurodegenerative diseases

(2)Department of Food Science and Microbiology (DiSTAM), Human Nutrition Unit, University of Milan;  (3)Institute of Pharmacological Research Mario Negri, Via Eritrea 62- 20157 Milan, Italy.

(4)Department of Biomolecular Sciences and Biotechnology, University of Milan,ViaCeloria 26- 20133 Milan, Italy.

 

Amyotrophic lateral sclerosis is a progressive neurodegenerative disorder characterized by the selective loss of  both upper and lower motor neurons. About 10% of cases are inherited and, of these, 20% are linked to mutations of the gene coding for Cu/Zn Superoxide Dismutase (SOD1), one of the major intracellular antioxidant enzyme. In a transgenic mice model for fALS, expressing a mutant SOD1 (G93A_SOD1), we found that mutant SOD1 was mainly present in the cytoplasm of motor neurons, while, in control animals, detectable amounts of hwtSOD1 were also observed in the nuclei of motor neurons. Transfecting different SOD1 constructs (coding for both wt and mutant protein) in immortalized motor neurons (NSC34), we have confirmed this peculiar distribution of the mutant SOD1, and observed the formation of cytoplasmic and nuclear inclusions. Ubiquitinated cytoplasmic aggregates are a typical feature of ALS; recently, nuclear aggregates have been also described; we have, therefore, analyzed the activity of Ubiquitin-Proteasome-Pathway (UPP), responsible for misfolded protein clearance, in the two subcellular compartments.  The data have shown that only the cytoplasmic UPP was compromised. The effect on genomic DNA integrity, of G93A_SOD1 exclusion from nuclei was analyzed using a COMET assay (Single Cell Gel Electrophoresis); after induction of oxidative stress with H2O2, a higher nuclear DNA damage was found in cells expressing mutant SOD1 if compared to cells expressing wtSOD1. The data indicated that G93A-SOD1 toxicity might arise fro m an initial misfolding (gain-of-function) generating nuclear deprivation of the active enzyme (loss-of-function in the nuclei), and consequently DNA damage due to ROS, a process that may be involved in ALS pathogenetic process.

 

Grants Telethon - Italy (#GGP06063), MIUR-FIRB (#RBAU01NXFP); MIUR-Cofin (2005057598_002), University of Milan-FIRST, FONDAZIONE CARIPLO.


P74

 

ATAXIN-2 LOCALIZES AT THE ENDOPLASMIC RETICULUM AND CO-SEDIMENTS WITH POLYSOMES

 

van de Loo S, Nowock J, Hilker R and Auburger G

 

Clinic for Neurolgy of the JWG-University Frankfurt/Main, Schleusenweg 2-16, 60528 Frankfurt/Main tel.+49-69-6301-7428; Email: auburger@em.uni-frankfurt.de

 

Objective: To understand the physiological functions of ataxin-2, we have analyzed its subcellular distribution by immunocytochemistry and subcellular fractionation.

 

Background: Spinocerebellar ataxia type 2 (SCA2) is a neurodegenerative disorder with pronounced atrophy of cerebellar Purkinje, ponto-olivary, nigral and motoneurons, caused by the unstable expansion of a polyglutamine domain in the disease protein ataxin-2.

 

Methods: Immunocytochemical analyses were performed on COS-7 cells and on primary cultures of murine hippocampal neurons. Endoplasmic reticulum and polysomal fractions were generated using subcellular fractionantion and ultracentrifugation experiments of mouse brain homogenates.

 

Results: Ataxin-2 immunostaining was exclusively observed in the cytoplasm, particularly at a prominent structure adjacent to the nucleus, and in punctae towards the cell periphery. The pathogenic form of ataxin-2 with an expanded polyQ domain showed the same distribution pattern. Double-labelling and confocal microscopy identified the juxtanuclear structure as endoplasmic reticulum (ER). Further, ataxin-2 was found to colocalize in part with endosomes. Ultracentrifugation experiments found ataxin-2 to co-sediment with the polysomal fraction.

 

Conclusions: These results are in agreement with recent findings that ataxin-2 is recruited to stress granules which represent transient structures of stalled mRNA synthesis under environmental stress. For the ataxin-2 homologue of drosophila, an association with polyribosomes has also been shown. These data in conjunction with the protein architecture of ataxin-2 suggest that ataxin-2 is involved in mRNA processing and/or regulation of translation.

P75

 

THE EXPRESSION OF NEUROSECRETION IS GOVERNED BY REST

 

D'Alessandro R (1), Klajn A * (1), Stucchi L (1), Podini P (2), Marzella R (3), Malosio ML (2) and Meldolesi J (1) (2)

 

(1)  Vita-Salute San Raffaele University, Center of Excellence in Cell Development, DIBIT, via Olgettina 58, 20132 Milan (Italy); tel. +39.02.26434825; fax +39.02.26434813; e-mail: klajn.andrijana@hsr.it

(2) Scientific Institute San Raffaele, Milan (Italy)

(3) Department of Genetics and Microbiology, University of Bari, Bari (Italy)  

 

A specific function acquired by neurons/neurosecretory cells during differentiation is the ability to synthesize and store neurotransmitters/hormones in dense core granules and synaptic like vesicles, and to discharge them by regulated exocytosis. Previously, we have identified two defective rat PC12 clones that although maintaining many of the properties typical of this neurosecretory cell line, are completely incompetent for neurosecretion. We demonstrated that the defect of the PC12 clones is primarily, if not exclusively, due to hyper-expression of the Repressor Element 1-Silencing Transcription factor, REST, and that reacquisition of some neurosecretion characteristics depends on the induced miss-function of REST. Fusion between the defective cells and human lymphocytes, yielded hybrids in which neurosecretion was partially rescued. BHC80, a member of the co-repressor BRAF-HDAC complex of REST was considered as a strong candidate gene responsible for this. BHC80 gene is mapped on chromosome 11, present in all hybrid clones where the re-expression of the neurosecretion took place. The increase of BHC80, induced by the co-expression of the rat (PC12) and human (lymphocytes) unbalances the equilibrium of the complex, which alters inhibitory function of REST (Iwase et al., 2004). The results were confirmed also by transient transfection of BHC80 cDNA in the defective clones. The role of REST in the maintenance of neurosecretion in differentiated cells has been investigated also by stable transfection of REST in wt PC12 and its dominante negative constructs in the detective clones, studied as such or after application of REST siRNA or blockade of histone deacetylase, a major enzyme recruited by REST. Also these results showed that changes of REST are sufficient to ind

uce the rise and fall of the neurosecretory properties of the cells. These effects appear to be due various mechanisms affecting differentially the expression of the vesicle/exocytosis gene products.  


P76

 

INVESTIGATION OF SERCA, PAX6 AND SP1 GENE EXPRESSION IN LENS CELLS DERIVED FROM A CATARACTOUS MYOTONIC DYSTROPHY PATIENT.

 

Lonigro R.(*), Bregant E. (1), Spelat R. (2), Passon N. (1), Pertile G. (3), Lanzetta P. (4), Damante G. (1)

 

(*) Department of Biomedical Sciences and Technologies, University of Udine. P.le Massimiliano Kolbe N¡2, 33100 Udine. Tel. 0432 494370; Fax 0432 494379; E-mail rlonigro@makek.dstb.uniud.it

(1) Department of Biomedical Sciences and Technologies, University of Udine.

(2) Department of Experimental and Clinical Pathology and Medicine, University of Udine.

(3) U.O. of Ophthalmology, "Sacro Cuore" Hospital, Negrar (Verona).

(4) Department of Ophthalmology, University of Udine.

 

Steinert's Myotonic Dystrophy (DM1) is an autosomal dominant inherited disorder due to the expansion of CTG repeat at the 3' untranslated region of the DMPK gene on human chromosome 19. DM1 is a multisistemic neuromuscular disease and symptoms are not limited to skeletal muscle, in fact DM patients can develop pre-senile cataract. Although cataract is a characteristic feature of DM1, little is known of the underlying mechanisms. Previous studies implied calcium homeostasis and SERCA (sarco/endoplasmic calcium ATP-ase pumps) proteins expression and function in cataract development. Furthermore, over-expressed Pax-6 transcription factor, a key regulator of eye development, causes cataract  in lens of transgenic mice. In this study we analyzed, by RT-PCR, SERCA genes and PAX6 gene expression level in primary coltures of epithelial lens cells derived from a DM1 cataract, seven different age-related cataracts and in two human lens cell lines (CE11560 and CD5A) as control. The results obtained demonstrate: a) The epithelial lens cells, as well as CE11560 and CD5A cell lines, express SERCA2 (SERCA2b and SERCA2a isoforms) and SERCA3 genes, but not SERCA1 gene. b) In almost all of the aged and DM1 cataract derived cells, the SERCA mRNAs are less expressed compared to the expression of control cell lines. Particularly, SERCA2a isoform is almost undetectable in DM1 cells. c) On the contrary, PAX6 gene expression is up-regulated, compared to the control cells, especially in the DM1 cells (6 fold over CE11560, 3 fold over CD5A expression level). Since SERCA2 gene is ubiquitously expressed and Sp1 transcription factor as been demonstrated to trans-activate SERCA2 gene promoter in cardiomyocytes of neonatal rats, we investigated Sp1 gene expression level in the previous described experimental system. Sp1 expression level is reduced in age-related cataracts with respect to the control cells and this data well correlate with SERCA2 gene expression level. Interestingly, the lens cells derived from the DM1 patient does not exhibit a significant reduction of Sp1 gene expression. These results would suggest the existence of different mechanism/s responsible of SERCA2 gene expression down regulation in cataract, some of which can be peculiar of DM1 lens cells.

P77

 

PERTURBATION IN NUCLEIC ACID METABOLISM AND NEURODEGERATION

 

Smith, CL (1), Bolton, A (1), Abdolmaleky, H (1)

 

(1) MBRL, Boston University, 44 Cummington Street,

Boston, Mass 02215 tel. 1 - 617 571 3068: fax. 1-617 236 0232

 

Our research focuses on understanding the cause(s) of schizophrenia that would account for the multiple, and seemingly disparate, environmental and genetic factors linked to this and similar diseases.  Our comparative DNA studies on affected monozygotic twins uncovered a linked between schizophrenia and somatic genomic instability. Other research uncovered a heretofore-unknown link between schizophrenia and the 120 interspersed fragile sites spread throughout the genome. Fragile site regions are sensitive to perturbations in nucleic acid metabolism, composed of simple or complex repeating sequences, and are especially prone to instability, retroviral insertions and epigenetic changes. Our studies on dopamine metabolism demonstrated the importance of epigenetic (DNA methylation) gene regulation for dopamine metabolism in the brain of both controls and individuals with schizophrenia and bipolar disease.  The epigenetic studies examined gene expression as a function of genotype and epigenetic promoter methylation status in post-mortem brain samples. Epigenetic changes to DNA are an important cellular response to the environment that records exposure history.  Our hypothesis that genetic and/or environmental factors that perturb nucleic acid metabolism leads to neurodegeneration provides a simple explanation for results by us and others that converging on the sulfur, methionine, and folate pathways  


P78

 

PROHIBITIN INTERACTS WITH RNF2 AND REGULATES DIRECTLY AND INDIRECTLY E2F1 TRANSCRIPTIONAL ACTIVITY.

 

Choi D*(1), Lee SJ(1), and Kang S(1)

 

(1)Graduate School of Life Sciences and Biotechnology, Korea University,5-ka, Anam-dong, Sungbuk-ku Seoul 136-701 (Korea) tel.+82-2-3290-3949; E-mail:skang@korea.ac.kr

 

Recently, it was reported that prohibitin was abnormally expressed in the substantia nigra and frontal cortex in Parkinson`s disease. In this study, we show that the prohibitin protein interacts with RNF2, the Hip2-interacting protein, and that RNF2 regulates the prohibitin-mediated E2F1 transcriptional activity through the p16-CDK4/cyclin D Rb pathway. We found by co-immunoprecipitation experiments that prohibitin interacts with RNF2. Interestingly, the expressed amounts of RNF2 and prohibitin were affected interdependently at a post-transcriptional level. Furthermore, knock-down of either RNF2 or prohibitin by the RNAi technique increased the amount of p16, whereas overexpression of either RNF2 or prohibitin did not affect the expression of the p16 protein. Cell proliferation also was regulated by the prohibitin-RNF2 interaction. Based on these results, we will discuss a novel pathway of E2F1 activity regulation and a role of prohibitin in the pathogenesis of Parkinson`s disease.

P79

 

CALCIUM HOMEOSTASIS AND MITOCHONDRIAL DYSFUNCTION IN STRIATAL NEURONS OF HUNTINGTONâS DISEASE

 

Lim D* (1,2), Fedrizzi L (2,3), Tartari M (4), Zuccato C (4), Cattaneo E (4), Brini M  (2,3), Carafoli E (1,2)

 

(1) Venetian Institute of Molecular Medicine, Via Orus, 2 - 35129 Padua (Italy) tel.+39-049-7923242; fax +39-049-7923250; E-mail:dlim@bio.unipd.it

(2) Department of Biochemistry; University of Padova, Padova (Italy)

(3) Department of Experimental Veterinary Science, University of Padova, Padova (Italy)

(4) Department of Pharmacological Sciences, University of Milan, Milan (Italy)

 

Huntingtonâs disease (HD) is a genetic neurological disorder caused by the expansion of the polyQ repeats at the amino-terminus of huntingtin. Disturbances in the regulation of cellular Ca2+ homeostasis and of mitochondrial Ca2+ handling have been described and claimed to cause the apoptotic death of striatal spiny neurons in the disease. The homeostasis of Ca2+ and the mitochondrial metabolism, thus, have been studied in STHdhQ111 cells, precursors of striatal neurons from a mouse model of HD. Using targeted photoprotein aequorin we analysed cytoplasmic and mitochondrial Ca2+ responses to two G alpha/q-coupled receptors agonists, ATP and bradykinin (BK). Ca2+ transients induced by ATP were strongly decreased, but those induced by BK were increased. This was due to altered transcription of the purinergic and BK receptors as revealed by real-time PCR analysis. The transcription of the components of the phosphatidyl-inositol (PI) cycle myo-inositol monophosphotase 1 and inositol polyphosphatase, of the InsP3-producing phospholipase C beta, and of the InsP3R1 was altered, delaying the production of InsP3 and decreasing the liberation of Ca2+ in STHdhQ111 cells. The transcription of the components of the PI cycle and of the InsP3-mediated signaling was altered in a similar way in the striatum of the parent KI-HdhQ111 mouse HD model. The mitochondrial membrane potential and the ability of mitochondria to handle Ca2+ was compromised in STHdhQ111 cells, but only when they were stressed by the inhibition of  complex II of the respiratory chain. The inhibition made mitochondria peculiarly vulnerable to Ca2+, as it impaired their ability to handle large Ca2+ loads. The transcription of enzymes glutathione peroxidase 1, catalase and superoxide dismutase 1 that degrade reactive oxygen species (ROS), which also stress and damage mitochondria, increased in the striatal cells, lowering ROS concentration.

P80

 

INHIBITED  NUCLEAR EXPORT IN MOST KNOWN POLYGLUTAMINE DISEASE PROTEINS: COMMONALITY OF DISEASE MECHANISM?

 

 Xia, J (1),Taylor, J (1),  Atwal RS (1) and Truant R (1).

 

(1) McMaster University, Department of Biochemistry and Biomedical Sciences, Hamilton, Ontario, CANADA L8N3Z5. truantr@mcmaster.ca, www.RayTruantlab.ca.

 

Several of the known polyglutamine expansion disease proteins have now been determined to have the ability to import and export from the nucleus as part of their normal biological function. These include the ataxin-1 protein in spinocerebellar ataxia type 1, SCA1, the ataxin-7 protein in SCA7, the ataxin-3 protein in Machado-Joseph disease, and the Huntingtonâs disease protein, huntingtin.  Others have defined nuclear import and export signals in Atrophin-1 protein in DRPLA and the androgen receptor (AR) in SBMA. In each case the nuclear import of the protein is not affected, but export is inhibited, resulting in nuclear accumulation of mutant proteins.  For ataxin-7 and huntingtin, we see that increased nuclear levels of mutant protein correlate better with increased toxicity than the presence or absence of polyglutamine-mediated aggregates.  This suggest that one common mechanism of polyglutamine expansion in a nuclear shuttling protein may be to cause an increase in nuclear levels of the polyglutamine disease protein.  However, this does not explain the exact mechanism of toxicity within the nucleus, or the specific toxicity between these diseases.

       Here, we provide evidence that ataxin-7 and huntingtin proteins are signaled to the nucleus.  Huntingtin nuclear entry is highly regulated by four independent signal sequences spaced throughout the entire 350KDa protein that involve ER retention, nuclear localization, nuclear export, and nuclear export via mRNA.  We have now precisely defined all of these signals in huntingtin, and demonstrate that huntingtin nuclear export may be mRNA dependent, through a known pathway used by other mRNA export proteins.  Together with evidence of ataxin-1 RNA interaction in vivo, this suggests that for SCA1 and Huntingtonâs disease, some level of disease specificity may be at the level of mRNA affected by the mutant proteins.  This work may place SCA1 and HD into a larger family of known genetic neurologic diseases in with specific mRNA export is affected. Together with unique cell signaling pathways, these two mechanisms may define the precise disease specificity in these polyglutamine expansion diseases.


P81

 

REGULATION OF SPG4 EXPRESSION:  TRANSCRIPTIONAL AND TRANSLATIONAL MECHANISMS FOR THE SYNTHESIS OF TWO SPASTIN ISOFORMS

 

Giuseppe Mancuso 1, Elena Riano 1, Elena I. Rugarli 1,2

 

1 Division of Biochemistry and Genetics, Istituto Neurologico Besta via Temolo Libero 4 20126 Milan, Italy; Tel. 02/23942621; Fax. 02/23942619; E-mail: manq.hsp@libero.it; 2 DNTB, Universitˆ Milano-Bicocca, Milan, Italy

 

Hereditary spastic paraplegia (HSP) is a heterogeneous genetic disease characterized by selective axonal degeneration of the corticospinal axons. The gene SPG4, encoding spastin, is mutated in about half of dominant cases of HSP. The pathogenetic mechanism of axonal degeneration in patients with SPG4 mutations still awaits elucidation. Previous studies from our and other laboratories have shown that spastin is involved in microtubule severing. Furthermore, we found that the SPG4 locus synthesizes two spastin isoforms, of 68 and 60 kDa respectively, through usage of two different translational start sites present in exon 1. Both isoforms are imported into the nucleus, but the 68 kDa isoform contains two overlapping nuclear export signals that efficiently drive export to the cytoplasm. Albeit both the long and short spastin isoforms are able to act as microtubule severing proteins, the nuclear role of spastin is still unclear. To further understand how the two spastin isoforms are generated, we studied the regulatory region of the SPG4 gene in different cell lines. We found that the 5âUTR has an important role for transcriptional activity both in HeLa and SH-SY-5Y cell lines. Moreover, both the 5âUTR and a region of the SPG4 coding sequence located between the first and second methionine, alone and in combination, display significant promoter activity in HeLa cells, but not in SH-SY-5Y. These data suggest that a cryptic promoter in the first exon of the SPG4 gene may drive the production, in non neuronal cells, of a shorter mRNA able to translate only the 60 kDa spastin isoform. Consistently we found a shorter spastin transcript, specific for the short isoform in HeLa cells. These data suggest that the production of the two spastin isoforms is regulated also transcriptionally, providing a mechanism to control the relative levels of the two isoforms in different tissues.

P82

 

DISTINCT SUBREGIONS AFFECTING TRANSCRIPT STABILITY ARE PRESENT IN THE HUMAN CDK5R1 3'UTR

 

Moncini S* (1),Venturin M (1), Bevilacqua A (2), Ratti A (3,4), Nicolin A (2), and Riva P (1)

 

(1)Department of Biology and Genetics, Medical Faculty, University of Milan ö via Viotti 3/5 20133 Milano, (ITALY) phone +39-02-50315862, FAX +39-02-50315864, e-mail:paola.riva@unimi.it

(2)Department of Pharmacology, Chemotherapy and Medical Toxicology, University of Milan, Milano (ITALY)

(3)Department of Neurological Sciences, University of Milan

(4)Department of Neuroscience, `Dino Ferrari' Centre, University of Milan-IRCCS Istituto Auxologico Italiano, Cusano Milanino (MI)(ITALY)

 

Human CDK5R1 encodes for p35, a neurone-specific activator of CDK5, which is involved in neuronal migration and differentiation during CNS development. The active CDK5-p35 complex is involved in phosphorylation of neurofilaments and its aberrant hyperactivity has been shown to be implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimerâs and Parkinsonâs diseases. CDK5R1 has also been proposed as a candidate gene for mental retardation. The remarkable size of CDK5R1 3âUTR suggests a role of this region in the control of CDK5R1 expression by post-transcriptional regulatory elements modulating mRNA stability or translation efficiency. Bioinformatics showed a high conservation in mammals and predicted several AU-Rich Elements (AREs). CDK5R1 3âUTR was cloned at the 3â end of the Renilla luciferase reporter gene to perform Dual Luciferase assays: the construct showed a decreased luciferase activity in six transfected cell lines. The quantitative analysis of luciferase mRNA suggests that CDK5R1 3âUTR affects mRNA stability. We identified five 3âUTR subregions reducing the luciferase activity in some instance with a cell line-dependent way. We also identified, by deletion analysis, a type I ARE displaying a stabilizing effect in two neuroblastoma cell lines. Our findings evince the presence of both destabilizing and stabilizing regulatory elements in CDK5R1 3âUTR. We are now attempting to identify, by REMSA and immunoprecipitation assays, stabilizing neuronal proteins binding the type I ARE, with the final aim of verifying the functionality of this element. The fine tuning of CDK5R1 expression by 3âUTR may play a role in CNS development and functioning. The finding of altered 3âUTR regulatory elements might help to clarify the molecular basis of some neurological diseases and, in perspective, the validated regulatory elements may represent new pharmacological targets, for enhancement of the treatment of neurodegenerative diseases.

P83

 

EXPRESSION OF TGFbeta1 IN MUSCLE OF A MOUSE MODEL OF FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS

 

Onesto E. (1), Galbiati M. (1), Mariotti R. (2), Bentivoglio M. (2), and Poletti A. (1)

 

(1) Institute of Endocrinology, Centre of Excellence on Neurodegenerative Diseases, University of Milan, Via Balzaretti 9, 20133 Milan, Italy.

tel 02-5031.8215; fax 02-5031.8204; E-mail: angelo.poletti@unimi.it

Inter-University Research Centre on the Molecular Basis of Neurodegenerative diseases (Universities of Florence, Rome and Milan, Italy)

(2) Department of Morphological and Biomedical Sciences (University of Verona, Italy)

 

Amyotrophic lateral sclerosis (ALS) is an adult-onset disease that causes degeneration of motor neurons; a familial form (fALS) is characterized by a dominant mutation in superoxide dismutase (SOD) 1 gene. The loss of motor neurons causes atrophy of denervated skeletal muscles. Recent data have raised the issue of a high risk for ALS among Italian male soccer players, hypothetically due to vigorous physical activity, microtrauma, or use of illegal anabolic performance-enduring substances. It has been shown that not only motor neurons, but also other non-neuronal cell types may contribute to the disease onset and progression, and signalling at the neuromuscular junction could also play a role; indeed, muscle denervation is one of the earliest events detectable in animal models of fALS. TGFbeta family proteins are involved in muscle development and maintenance and in neuron survival; here we focused on a diffusible factor produced by the muscle, transforming growth factor (TGF) beta 1. Infact it has been shown that serum TFGbeta1 level is high in patients with long progression rate of ALS.

We have evaluated the modulation of TGFbeta1 expression, using real time PCR analysis, in gastrocnemius muscle of wild type (wt) or mutant SOD1(G93A) mice sampled at different times of disease progression. We also investigated whether the synthetic anabolic steroid nandrolone, a common doping agent, is able to modify these levels. As control, we also tested the expression of MyoG and Atrogin, two markers known to be modulated both in muscular atrophy and in ALS. We observed that the expression of these genes was up-regulated in mutant mice both at disease onset and at advanced clinical stage. Nandrolone administration did not modify the expression of these two muscular markers. The expression of TGFbeta1 in wt mice remained unchanged at different ages. In SOD1(G93A) mice, we observed a marked increase in TGFbeta1 expression at disease onset and the levels were further increased with disease progression, doubling those measured at onset time. Nandrolone treatment did not induce any variation in TGFbeta1 mRNA levels in wt mice, while it significantly increased TGFbeta1 expression of in SOD1(G93A) mice disease onset. It remains to be determined whether the modulation of TGFbeta1 is due to the presence of mutant SOD1 in muscular cells or is a consequence of muscular denervation.

 

Grants: Telethon - Italy (#GGP06063), MIUR-FIRB (#RBAU01NXFP), MIUR-Cofin (2005057598), FONDAZIONE CARIPLO, University of Milan-FIRST.

P84

 

ALLELE SPECIFIC SILENCING OF THE MUTATED HUNTINGTON ALLELE IN PATIENT DERIVED FIBROBLASTS

 

van Bilsen PHJ (1), Jaspers L* (1), Kaemmerer WF (2)

 

 (1) Medtronic Bakken Research Center B.V, Endepolsdomein 5-6229GW Maastricht (The Netherlands) tel. +31-43-3566779; fax. +31-43-3566519; E-mail: leonie.jaspers@medtronic.com

(2) Medtronic Inc., Materials and Biosciences Center, Minneapolis (USA)

 

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by the expression of mutant huntingtin protein (htt).  Suppression of htt expression using RNA interference might be an effective therapy.  However, if reduction of wildtype protein is not well-tolerated in the brain, it may be necessary to suppress just the product of the mutant allele.  We present an siRNA that selectively reduces the endogenous mRNA for a heterozygous HD donorâs pathogenic allele by approximately 80% by specifically targeting a single nucleotide polymorphism (SNP) located several thousand bases downstream from the disease-causing mutation.  We further present a method using just a heterozygous patientâs own mRNA to determine which SNP variants correspond to the mutant allele.

P85

 

REDUCTION IN BDNF mRNAS AND PROTEIN LEVEL IN HUMAN HUNTINGTON'S DISEASE CORTEX

 

Chiara Zuccato*, Manuela Marullo, Paola Conforti, Marzia Tartari and Elena Cattaneo

 

Department of Pharmacological Sciences and Center for Stem cell Research, University of Milan, via Balzaretti 9, 20133, Milano; tel +30 02 5031 9673; fax +30 02 5031 8284; email: chiara.zuccato@unimi.it.

 

Dysfunction of striatal neurons is a cardinal feature of Huntington's disease (HD). Survival and differentiation of these neurons depend upon binding of cortically-derived Brian-Derived Neurotrophic Factor (BDNF) with high-affinity (trkB) and low-affinity (p75NTR) neurotrophin receptors produced within striatal neurons. While several mouse models of HD show a decrease in cortical BDNF gene transcription and a reduction in BDNF trophic support to striatal neurons, studies on a limited number of human postmortem HD cortices have produced conflicting data. These studies indicate both the presence of a defect in cortical BDNF gene transcription and a defect in the transport of this neurotrophin from the cortex to striatum and, consequently reduced BDNF support to striatal neurons. Here we provide new evidence indicating a statistically significant reduction in BDNF mRNA and protein level in cerebral cortex from 20 HD subjects versus 17 controls and independently from disease stage or pathological CAG repeats. These data further support the notion of an impaired BDNF production in human HD cortex as a consequence of an expanded CAG into the HD gene. Analyses of the most well characterized BDNF mRNA isoforms reveal for the first time that downregulation of transcription from BDNF promoter exon II and IV occurs in human HD cortex from early symptomatic stage further suggesting that loss of BDNF may contribute to disease progression.

 

Support: Huntington's Disease Society of America Coalition for the Cure, HighQ Foundation, Telethon, Fondazione Cariplo

 

P86

 

ROLE OF ANDROGENS ON ABC-HALF TRANSPORTERS IN X-LINKED ADRENOLEUKODYSTROPHY

 

Carissimi R(1), Blasevich M (1), Cappa M (2), Uziel G (3), Mattavelli* S(1), Petroni A (1)

 

(1)Depart. of Pharmacological Sciences, University of Milan, via Balzaretti 9, 20133 Milan, (Italy) tel.+39-02-50318307; fax +39-02-50318284;E-mail:anna.petroni@unimi.it

(2)Depart. of Pediatrics Medicine, Childrenâs Hospital Bambino Ges, IRCCS, Rome, (Italy)

(3) Child Neurology Dep., Istituto Neurologico C. Besta, Milan, (Italy)

 

Adrenoleukodystrophy (ALD) is a rare, inherited metabolic disorder. This disease is associated with progressive demyelination. Due to defective peroxisomal beta-oxidation, Very long chain fatty acids (VLCFA), the biochemical markers of the disease, accumulate in plasma, fibroblasts nervous system, adrenal cortex and different tissues.

The mutated gene (ABCD1, Xq28), encodes for the peroxisomal ABC half-transporter ALDP. It has been shown that gene ABCD2, which presents the closest relation to ABCD1, and the other related genes ABCD3 and ABCD4 can compensate the impaired function of ALDP and that different agonists are able to induce their expression, partially compensating the pathological abnormalities of the disease such as enhancing VLCFA beta-oxidation.

We have evaluated the effect of the androgen dihydrotestosterone (DHT) and 5 alpha-androstan-3 alpha, 17beta-diol (3 alpha-diol), as a promising therapeutic approach, on the expression of the ABC half-transporters encoded by ABCD2 and ABCD3 genes, in fibroblasts drawn from controls and from two affected brothers. The two patients presented the same mutation in exon 9 but had different clinical manifestations, one patient being asymptomatic and the second one severely affected.

When the cells were stimulated with the testosterone metabolites only the patient with the severe form showed a significant increase in ABCD2 mRNA levels, whereas ABCD3 expression remained unchanged in both cases.

P87

 

ENDOGENOUS LIGANDS FOR TREM2 RECEPTOR

 

Stefano L. (1), Meldolesi J. (1)

 

(1) Vita-Salute San Raffaele University, DIBIT, via Olgettina 58, 20132 Milan, Italy. Tel.: +39 022 643 4825; Fax: +39 022 643 4813 ; e-mail meldolesi.jacopo@hsr.it; stefano.luisa@hsr.it.

 

Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as Nasu-Hakola disease, is a recessively inherited disease characterized by early onset dementia associated with bone cysts. Palonevaâs group has established the molecular background of PLOSL by identifying mutations in DAP12 and TREM2 genes.

The PLOSL gene DAP12 encodes a transmembrane adaptor protein which seems to play a role in triggering and amplifying inflammatory responses by interaction with different receptors expressed in several cell types.

The second PLOSL gene, TREM2 (or Triggering Receptor Expressed on Myeloid cells-2) encodes one of the DAP12-associated receptors belonging to the innate immune receptor TREM family.

It was suggested that the receptor binding of an unknown TREM2 ligand induces the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain of DAP12, starting an intracellular signaling cascade which surprisingly leads not to the stimulation, but to the repression of the inflammation process.

In order to identify the TREM2 receptor, pull down experiments were performed on cell surface-biotinylated  N2A cells employing, as a probe, a recombinant protein consisting of the extracellular domain of TREM-2 receptor conjugated to the cristallyzable fraction of human IgG.

Pull down products were subject to western blot analysis and silver staining, resulting in the identification of different chaperonins as possible ligands for TREM2 receptor.

P88

 

TARGETING SIRTUIN 2 MICROTUBULE DEACETYLASE FOR DEVELOPING PARKINSONâS DISEASE THERAPY

 

Tiago Fleming Outeiro (1), Eirene Kontopoulos (2), Steve Altman (1), Irina Kufareva (3), Katherine E. Strathearn (4), Allison M. Amore (1), Catherine B. Volk (4) Michele M. Maxwell (1), Anne B. Young (1), Jean-Christophe Rochet (4), Pamela J. McLean (1), Ruben Abagyan (3), Mel B. Feany (2), Bradley T. Hyman (1), and Aleksey Kazantsev (1), *

 

(1)  Harvard Medical School and  MassGeneral Institute for Neurodegenerative Disease,  Massachusetts General Hospital,  Bldg 114-3300, 16th St., Charlestown, MA 02129-44-04, USA,  Email: akazantsev@partners.org,  Phone: (617) 726-1274,  Fax:     (617) 724-1480; (2) Harvard Medical School and Brigham and Womenâs Hospital, (3) Scripps Research Institute,(4) Purdue University

 

Aggregated mutant misfolded proteins, a hallmark of Parkinsonâs, Huntingtonâs, Alzheimerâs and other neurodegenerative diseases, are known to be associated with microtubules and cytoskeleton components. Phenotypic screens for small molecules affecting protein aggregation yielded hit-compound, rescuing aSyn mediated cytotoxicty and proteasome deficiency in cellular models of neurodegeneration. Biochemical profiling identified compound selective inhibitory activity against microtubule deacetylase sirtuin 2 (SIRT2), a member of the histon deacetylase (HDAC) family, class III. SIRT2 is a ubiquitously expressed cytoplasmic protein, responsible for deacetylation of alpha-tubulin, a key component of microtubules.

In follow-up studies we identified a lead-series of SIRT2 inhibitors, which were subjected for potency optimization. Next, we tested selected entities in alpha-synuclein cell-based model of Parkinsonâs disease (PD). Novel SIRT2 inhibitors reduced alpha-synuclein cytotoxicity in a dose-dependent manner. The rescuing effect was correlated with the coalescence of small multiple alpha-synuclein (aSyn) aggregates in the cell into a few larger inclusions. Pharmacological rescue of aSyn cytotoxicity was confirmed genetically by selective siRNA knock-out of SIRT2 expression. Further, we observed neuroprotective effects of two lead-inhibitors for primary dopaminergic neurons, expressing mutant (A53T) aSyn. And lastly, the same lead-inhibitors of SIRT2 rescued dopamine neurons from aSyn cytoxicity in an in vivo fly model of PD. Our results demonstrated neuroprotective effects of targeting SIRT2 activity, possibly by modulating microtubule stability and aggregation. Based on identified series of SIRT2 inhibitors, we plan to develop potent and select brain-penetrable inhibitors with optimal ADMET (Adsorption, Distribution, Metabolism, Excretion, Toxicology) properties, and test efficacies of lead-candidates in mouse PD models. Identified efficacious small molecules will be treated as therapeutic leads and subjected for further drug development and inclusion in phase I clinical trials.

P89

 

CELL AUTONOMOUS DEGENERATION AND SYSTEMIC GLIAL TOXICITY IN DROSOPHILA MODELS OF DENTATORUBROPALLIDOLUYSIAN ATROPHY (DRPLA)

 

Charroux B(1)¤, Montrasio S(2), Peyre E (1) Napoletano F(2) and Fanto M (2)*¤

 

(1) IBDML, Campus de Luminy Case 907, F-13288 Marseille Cedex 9, France. E-mail:charroux@ibdml.univ-mrs.fr

(2) Dulbecco Telethon Institute, DIBIT-San Raffaele Scientific Institute, Via Olgettina 58, I-20132 Milan, Italy. Tel. +39.02.2643.4846; Fax +39.02.2643.4855; E-mail:m.fanto@hsr.it

¤ These authors contributed equally and are co-corresponding authors.

 

Dentatorubropallidoluysian Atrophy (DRPLA) is a neurodegenerative disease caused by the expansion of a polyglutamine tract in the Atrophin-1 gene. As for the other diseases of the polyglutamine family the precise mechanisms through which neurodegeneration and the neurological manifestations arise, are not clear. To address these issues we have generated 3 different Drosophila models for DRPLA by expressing wt and mutated forms of: full length human Atrophin-1; a truncated version of human Atrophin-1; Drosophila Atrophin.

Interestingly whereas the full length Atrophin-1 is kept at low levels and brings about limited changes in the fly the other two forms recapitulated most phenotypical manifestations found in other Drosophila models for polyglutamine pathologies.

We have analysed cellular degeneration in the photoreceptor neurones of the Drosophla retina in which our genetic and ultrast

ructural analysis suggests a critical role for autophagy, whereas there is little evidence that apoptotic cell death is induced.

At the systemic level human Atrophins and one form of Drosophila Atrophin with expanded polyglutamines display a glial specific toxicity which leads to a sharp decrease in organism viability. Despite glial intracellular autophagic modifications, similar to those found in photoreceptor neurones, a different yet unidentified mechanism appears to be responsible for the effect on viability.

P90

 

IDENTIFICATION OF ENDOGENOUSLY NITRATED TYROSINE IN CYTOSKELETAL PROTEINS

 

Nonnis S*(1),Taverna F(1),Ronchi C(1),Grassi E(1),Cappelletti G(2), Negri (1),Tedeschi G(1).

 

(1)D.I.P.A.V.- Section of Biochemistry, University of Milano, Via Celoria 10, 20100 Milano, Italy. Tel. +39-0250318127; fax +39-0250318123.

(2)Department of Biology, University of Milano, Via Celoria 26, 20133 Milano, Italy.

 

Nitric oxide (NO) is a signalling molecule involved in numerous physiological and pathophysiological events. Some actions of NO are mediated directly by protein modifications, including the nitration of tyrosine and tryptophan residues. Although the accumulation of nitrated proteins correlates well with many disease states and it is considered a marker of oxidative stress under pathological conditions, substantial evidence has accrued that protein tyrosine nitration is a post-translational modification playing a role in physiological processes, including signal transduction. We have recently reported that tyrosine nitration of proteins is implicated in the signalling pathway triggered by NO during nerve growth factor (NGF)-induced neuronal differentiation. The study was carried out in PC12 cells that have been widely used to investigate neuronal cell fate, including survival, proliferation, differentiation and apoptosis. During this process  we described that the cytoskeleton becomes the main cellular fraction containing nitrotyrosinated proteins and we identified alpha-tubulin, peripherin and TAU as the major targets for nitration after 5 days of NGF-induced differentiation (1).

In the present study a parallel study is carried out on rat brain to determine if cytoskeletal nitration is restricted to cellular models or it is also present  in vivo in physiological conditions. The results clearly show that some cytoskeletal proteins contain nitro-tyrosines in vivo suggesting novel functional roles for protein nitration in physiological processes.

Acknowledgments: this work was supported by grants from University of Milano (FIRST 2005-2006)

 

[1] Cappelletti G, Ma

ggioni MG, Tedeschi G, et al. (2003) Protein tyrosine nitration is triggered by nerve growth factor during neuronal differentiation of PC12 cells. Exp. Cell. Res. 288:9-20

[2] Tedeschi, G., Cappelletti, G., Negri, A., Pagliato, L., Maggioni, M.G., Maci, R. and Ronchi, S. (2005) Proteomics 5, 2422-2432.

[3] .Cappelletti G, Tedeschi G, Maggioni MG, et al. (2004) The nitration of tau protein in neurone-like PC12 cells. FEBS Lett. 562:35-39

[4] Tedeschi G., Cappelletti G., Nonnis S., Taverna F., Negri A., Ronchi C., Ronchi S. (2007) Tyrosine nitration is a novel post-translational modification occurring on  the neural intermediate filament protein peripherin. Neuroch. Res. In press.

P91

 

A CELL CULTURE MODEL TO INVESTIGATE THE ROLE OF MICROGLIA IN AMYOTROPHIC LATERAL SCLEROSIS (ALS).

 

Padovano V, Massari S., and Pietrini G

 

Department of Pharmacology, School of Medicine, University of Mila, Via vanvitelli, 32 - 20129 Milano (Italy)

 

Despite the selectivity of motoneuron damage in amyotrophic lateral sclerosis (ALS), increasing evidence indicates a participation of microglial cells to motoneuron degeneration in human and murine ALS. Microglia are the resident immune&#8722;competent cells of the CNS; notious stimulus may elicit microglia activation and release of toxic factors that accelerate neuronal degeneration and death. However, it is unknown how microglia communicates damage to motoneurons.

To unravel the contribution of microglial cells to the pathogenesis of ALS, we are testing the hypothesis of altered pathways of secretion induced by ALS linked mutant superoxide dismutase (mtSOD1) expression in microglia. To this purpose we have recently established cell culture models consisting in human microglial N9 cell lines stably transfected with wild type or G93ASOD1.

Our data indicate that the expression of mutant SOD1 increases the release of potentially toxic molecules including mtSOD1, whereas the expression of even higher amount of wt SOD1 does not change the pattern of secretion in N9 microglial cells. Moreover, we have also data suggesting that mt SOD1 is released via unconventional pathways not involving the endoplasmic reticulum-Golgi complex route. We are now performing experiments aimed at identifying the secretory pathway of mutant SOD1, which is a crucial step in order to develop appropriate therapeutic strategies to prevent microglial toxicity.


P92

 

ATAXIN2 INTERACTS WITH ENDOPHILIN A

 

 Nonis FD*(1),  Tanaka S(2), Auburger G(1) and  Nowock J (1)

 

(1)Section Molecular Neurogenetics, Dept. of Neurology, J. W. Goethe University Medical School, Frankfurt/M, Germany; tel +49 69 6301 6330; fax +49 69 6301 7142;

E-mail: davidnonis@yahoo.com.ar

(2)Research and Development Department of Invitrogen Corporation,Yokohama Kanazawa High-Tech Center, Yokohama, Japan

 

Spinocerebellar ataxia type 2 (SCA2) is an autosomal-dominant neurodegenerative disorder which is caused by the expansion of a coding CAG triplet repeat domain beyond a critical threshold (~34 CAGs) in the ataxin-2 gene. The same type of  mutation is shared by an increasing number of otherwise dissimilar genes, and the respective disorders are viewed collectively as Îpolyglutamine diseasesâ. The expanded polyglutamine domains of proteins cause  neuronal dysfunction which progressively leads to a selective loss of neurons in  brain areas through mechanisms that remain unveiled. To elucidate its cellular function, we have used full-length ataxin-2 as bait in a yeast two-hybrid screen of human adult brain cDNA to find interactors. We found endophilin A1 and A3, two brain-specific isoforms of the endophilin A subfamily involved in synaptic vesicle endocytosis and other non-endocytic functions. Co-immunoprecipitation verified this association in mouse brain. In vitro binding experiments narrowed the binding interfaces to two proline-rich domains on ataxin-2, interacting with the SH3 domain of endophilin A1/A3.


P93

 

LINKING NEURONAL DEGENERATION TO MICROTUBULE DYNAMICS BY THE PARKINSONISM TOXIN MPP+-MEDIATED MICROTUBULE DESTABILIZATION

 

Cartelli D (1), Ronchi C (2), Maggioni MG (1), Rodighieri S (3), Giavini E (1), and Cappelletti G *(1)

 

(1)   Department of Biology, University of Milan, Via Celoria 26, 20133 Milano, Italy;

(2) Di.Pa.V., Section of Biochemistry-University of Milan, Via Celoria 10, 20100 Milano, Italy;

(3) CIMAINA, Via Celoria 16, 20133 Milano, Italy

* tel. +39-02-50314752; fax +39-02-50314802; E-mail: graziella.cappelletti@unimi.it

 

Recent data indicate that microtubules differently interact with mutated proteins in PD including alpha synuclein (Alim et al. 2004), parkin (Yang et al., 2005), LRRK2 (Biskup et al., 2006), and that the dysfunction of microtubule is involved in the mechanism of action of model drugs in PD, MPP+ (Cappelletti et al., 1999, 2001) and rotenone (Ren et al., 2005). However, the role of tubulin in pathogenetic events remains elusive.

We reported earlier that MPP+, the toxic metabolite of MPTP, binds specifically to tubulin and affects microtubule dynamics by acting as a destabilising factor in vitro (Cappelletti et al., 2005). Our current work is focused on the study of the dynamic behaviour of the microtubular cytoskeleton in NGF-differentiated PC12 cells exposed to MPP+. By analyzing post-translational modifications occurring on tubulin and correlating with stability of microtubules we have shown that MPP+ specifically affects the arrangement of microtubules in cell causing the overall loss of dynamic microtubules in the distal region of the neurite. By FRAP (fluorescence recovery after photobleaching) experiments of YFP-tubulin in live PC12 cells we examined tubulin dynamics and found that MPP+ induces a significant reduction of tubulin mobility at the neuronal tip and along the neurite. Finally, in the attempt to link microtubule dysfunction to loss of microtubule-dependent functions, we investigated organelle transport along neuritis and found that MPP+ elicits a significant impairment.

Since dynamics is crucial in microtubule biological functions, we hypothesise that the altered dynamic behaviour of microtubules caused by MPP+ could profoundly affect the functionality of neurones and, consequently, represent a novel pathogenetic pathway triggering neuronal cell death in PD.

P94

 

BETA-AMYLOID PEPTIDE TOXICITY IN ORGANOTYPIC HIPPOCAMPAL SLICE CULTURE INVOLVES AKT/PKB, GSK-3BETA, AND PTEN

 

Nassif  M. (1), Horn A.P. (*,1) , Hoppe J. (1), Gerhardt D. (1), Frozza R.L. (1), Zamin L.L. (1), Sim‹o F. (1) and Salbego C. (1)

 

(1)  Departement of Biochemistry, Federal University of Rio Grande do Sul, Rua Ramiro Barcelos, 2600 ö 90035-003  Porto Alegre (Brazil) tel. +55-51-3308-5569; fax +55-51-3308-5535; E-mail: anapaulahorn@yahoo.com.br

 

Alzheimerâs disease is an irreversible neurodegenerative disorder associated with progressive cognitive and memory loss. Genetic and molecular evidences support a role of beta-amyloid peptide (Abeta) in the pathogenesis of the disease. In the present study we investigated the toxicity induced by exposuring organotypic hippocampal slice cultures to Abeta25-35 (25 uM) for 1, 3, 6, 12, 24 and 48 hours. In addition, we investigated the involvement of the PI3-K pathway proteins Akt/PKB, GSK-3beta, and PTEN in this toxicity. Cellular death was quantified by propidium iodide uptake and proteins were analyzed by immunoblotting. Our results showed a significant cellular death after a 48 hours Abeta peptide exposure. The 6 hours-treatment with Abeta peptide resulted in an increase in the phosphorylation state of Akt and GSK-3beta proteins. Twelve hours after exposure the phosphorylation decreased dramatically. However, after 24 hours, GSK-3beta phosphorylation presented a new increase, while the phosphorylation of Akt protein remains low. The immunocontent of PTEN protein presented an increase after 24 hours and it was maintained in 48 hours. These results suggest an involvement of Akt dephosphorylation/inactivation in the toxicity induced by the Abeta25-35 peptide in organotypic slice hippocampal culture, probably induced by an increase in PTEN immunocontent, since this phosphatase is the main negative regulator of PI3-K/Akt pathway. Taken together, our results provide more information about the molecular mechanisms involved on Abeta peptide toxicity.


P95

 

PERIPHERAL INFLAMMATION AND NEUROPROTECTION: SYSTEMIC PRETREATMENT WITH COMPLETE FREUND'S ADJUVANT REDUCES 6-HYDROXYDOPAMINE TOXICITY IN A RODENT MODEL OF PARKINSON'S DISEASE

 

Armentero MT (1),Levandis G (1), Nappi G (1,2, Bazzini E (1), and Blandini F (1)

 

(1)Laboratory of Functional Neurochemistry, Neurological Institute ãC. Mondinoä, Via Mondino, 2 - 27100 Pavia (Italy) tel.+39-0382-380333/365; fax +39-0382-380286; E-mail: marie.armentero@mondino.it

(2) Department of Neurology and Otorhinolaryngology, University of Rome ãLa Sapienzaä, Roma (Italy)

 

Complete Freund's adjuvant (CFA), a pro-inflammatory agent, was inoculated, subcutaneously, to SpragueöDawley rats prior to the intrastriatal injection of 6-hydroxydopamine (6-OHDA). Animals were sacrificed 7 and 28 days following 6-OHDA injection; neuronal damage, glial activation and cytokine levels, within the nigrostriatal system, were then investigated. Nigrostriatal degeneration induced by 6-OHDA was accompanied by early microglial and astroglial activation, which preceded the onset of dopaminergic cell loss, in the SNc, without significant changes in cytokine levels. CFA pretreatment markedly reduced the SNc neuronal loss and associated microglial activation, as well as the rotational response to apomorphine. These changes were associated with moderate, transient increases in the nigrostriatal levels of glial-cell-derived neurotrophic factor (GDNF) and pro-inflammatory cytokines, including interleukin (IL)-1&#945;, IL-1; and IL-6. Our results show that prior delivery of a peripheral, pro-inflammatory stimulus induces neuroprotection, in a rodent model of Parkinson's disease, possibly through the modulation of cytokine production at the nigrostriatal level.


P96

 

ATAXIN-2 INTERACTS WITH "SIMILAR TO GOLGIN-LIKE"

 

Eich F* (1), Nowock J (1), Auburger GA (1)

 

(1) Section Molecular Neurogenetics, Dept. of Neurology, Klinikum der Johann Wolfgang Goethe-UniversitŠt, Theodor-Stern-Kai 7, 60322 Frankfurt am Main (Germany) tel. +49-69-63017416; fax +39-69-63017142; E-mail: F.Eich@med.uni-frankfurt.de

 

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant hereditary neurodegeneration. In the disease protein Ataxin-2, the expansion of a polyglutamine domain to a length beyond 32 glutamines leads to clinical symptoms. We attempted to obtain clues on the physiological function of ataxin-2 through the identification of its protein interaction partners in a yeast-two-hybrid screen. "Similar to golgin-like" was identified as an candidate interactor of Ataxin-2. "Similar to golgin-like" is a novel protein and not characterised so far. It contains a region homolgous to the coiled coil domain of Golgin-67, a protein of the golgin family. The golgins are a heterogenous group of proteins which are mostly located on the cytoplasmic side of the Golgi apparatus. Some of these proteins bind Rab GTPases via their coiled coil domains, while others are dynamically involved in exchange between membrane surface and cytoplasm. The golgins are necessary as structural support for Golgi cisternae and tether events in membrane fusion. Ataxin-2 has been previously reported to localize at the Golgi apparatus, but an understanding of its role in this compartment depends on future functional testing.

P97

 

THE PARKINSON'S DISEASE ASSOCIATED GPR37 RECEPTOR INTERACTS WITH DAT TO MODULATE DOPAMINE UPTAKE AND BEHAVIOURAL RESPONSES TO DOPAMINERGIC DRUGS

 

C. Di Pietro*, E. Golini, S. Mandillo, R. Matteoni, D. Marazziti, G. P. Tocchini-Valentini

 

Institute of Cell Biology, CNR, Via E. Ramarini, 32 ö 00016 Monterotondo Scalo, (Italy) tel.+39 06 90091251; fax +39 06 90091260; E-mail: chiara_dipietro@ibc.cnr.it

 

GPR37 is an orphan G-protein coupled receptor that has been shown to be a substrate of parkin and its insoluble aggregates have been found accumulated in brain samples of Parkinson's disease patients. To investigate the receptor's functions, we generated homozygous Gpr37 null mutant mice.

The Gpr37-/- mice show: reduction of body weight, reduction in striatal dopamine (DA) content, specific locomotor and motor coordination deficits in the open field and rotarod tests, increased sensitivity to amphetamine treatment and resistance to the nigro-striatal degeneration induced by MPTP treatment.

The pre-synaptic dopamine transporter (DAT) plays a crucial role in controlling the concentration of DA at nigro-striatal synapses and several proteins have been shown to modulate DAT function and affect the post- and pre-synaptic signalling pathways that are mediated by dopamine D1 and D2 receptors.

Our experiments with transfected cells showed that GPR37 colocalises with DAT and co-immunoprecipitation experiments indicated that the DAT protein is precipitated only in the presence of the GPR37 protein. In parallel in vitro assays showed that the DAT activity is enhanced in Gpr37-/- mice and ongoing experiments are evaluating the modulation of DAT cell surface expression. Ex vivo fractionation studies revealed that GPR37 and DAT are both enriched in mouse striatum synaptic membranes.

Behaviour analysis experiments showed that Gpr37-/- mice exhibit an increase of amphetamine-induced and a decrease of cocaine-induced locomotor activity, as well as reduced catalepsy induced by dopamine receptor antagonists. Additionally, the startle response to acoustic stimuli is reduced in Gpr37-/- mice of both sexes, while no differences between genotypes were observed in the prepulse inhibition of startle.

Our results reveal a novel role for putative peptidergic G-protein coupled receptors, such as GPR37, in modulating DAT expression, function and the nigro-striatal dopaminergic signalling.

P98

 

BRAIN LIPID METABOLISM ALTERATIONS IN ATAXIN-2 KNOCK-OUT MICE

 

Lastres-Becker I (1)*, Brodesser S (2), Sandhoff K (2), Azizov M (1), Auburger G (1)

 

(1) Section of Molecular Neurogenetics, Dept. of Neurology, Building 26, 5th floor, J.W. Goethe-UniversitŠt Medical School, Theodor Stern Kai 7, 60590 Frankfurt am Main, (Germany) tel. +49-69-6301-7416;  fax +49-69-6301-7142; e-mail: Lastres-Becker@med.uni-frankfurt.de

 (2) LIMES, Membrane Biology & Lipid Biochemistry Unit, Lipid Biochemistry Lab, University of Bonn, KekulŽ-Institut fŸr Organische Chemie und Biochemie, Bonn, (Germany)

 

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant disorder characterized by progressive degeneration of cerebellar Purkinje cells and other selected neurons, as well as a profound loss of myelin lipids particularly in spinocerebellar tracts. SCA2 is one of the neurodegenerative diseases that are caused by a CAG/polyglutamine expansion. Ataxin-2, the product of the SCA2 gene, is a 140 kDa cytoplasmic protein that is found broadly in brain and other tissue types. Three different lines of ataxin-2 knock-out (KO) mice were generated, in order to understand the physiological role of ataxin-2 protein in mammalian. Gross morphology of the brain at 3 months of age showed no alterations between WT and KO mice. Brain lipids were analysed in cerebellum and neocortex (as control tissue). Selectively in the cerebellum, the levels of sphingomyelin were reduced and the levels of ceramide, GD1a and GM1, increased. No changes were observed in the cortex. These brain lipid alterations in the SCA2 KO mouse are in the sphingomyelin cycle and the ganglioside biosynthesis. Ataxin-2 could be involved in the activation of the SMases in the mouse cerebellum. In order to verify this hypothesis, the transcript levels of A-SMase and N-SMase and of several lipid related proteins were analysed in the cerebellum. No changes for both sphingomyelinases, but an increase was observed for brain lipid binding protein (BLBP), which is supposed to be involved in fatty acid uptake, transport, and targeting, and a decrease was observed for PPAR-delta, which regulates genes implicated in neurotoxicity. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors involved in the transcriptional regulation of key metabolic pathways such as lipid metabolism, adipogenesis, and insulin sensitivity. In conclusion, the absence of ataxin-2 leads to specific lipid metabolism alterations in brain and indicates a functional role of ataxin-2 for lipid homeostasis.

P99

 

BLOCKADE OF METABOTROPIC GLUTAMATE RECEPTORS COUNTERACTS L-DOPA INDUCED DYSKINESIA IN A RODENT MODEL OF PARKINSON'S DISEASE. 

 

Levandis G* (1), Bazzini E (1), Armentero MT (1), Nappi G (2), Blandini F (1)

 

(1)Laboratory of Functional Neurochemistry, Neurological Institute C. Mondino, Pavia, Italy; tel.+39-0382-380333;

E-mail:giovanna.levandis@mondino.it

(2)Department of Neurology and Otorhinolaryngology, University of RomeãLa Sapienzaä, Rome, Italy

 

It has been shown recently that glutamatergic overactivity may be involved in L-DOPA-induced motor complication (dyskinesias) in Parkinsonâs disease (PD). The present study examines the influence of a selective non-competitive mGluR5 antagonist, 2-methyl-6-phenylethynyl-pyridine (MPEP) on the chronic L-DOPA-induced dyskinesias (LIDs) in rats with extensive lesion of nigral dopamine neurons. Rats received two stereotaxical injections of 6-hydroxydopamine (6-OHDA) into the right ascending dopaminergic medial forebrain bundle (MFB) and one week later they were tested for apomorphine-induced rotations. Contralateral turns were counted for 45 minutes and only those rats that showed a rotational response were used. Four weeks after the apomorphine test, animals were randomly divided in three subgroups of treatment: 1) daily systemic injection of vehicle (saline, i.p.); 2) daily administration of L-DOPA (6 mg/kg in saline, i.p.) plus Benserazide (15 mg/kg in saline, i.p); 3) daily administration of MPEP (1.5 mg/kg in water, i.p) followed, 30 minutes later, by injection of L-DOPA (6 mg/kg) plus Benserazide (15 mg/kg). L-DOPA-induced abnormal involuntary movements (AIMs) were recorded three times a week for 21 days. Rats were killed 3 days after the last L-DOPA injection and sections cut throughout the striatum were processed for FosB/DeltaFosB immunostaining (marker of neural activation). Tyrosine hydroxylase immunostaining was used to assay the nigrostriatal damage. Our results show that there is a strong positive correlation linking the AIMs score to the number of FosB/DeltaFosB immunoreactive cells in the striatum. MPEP reduced dramatically the AIMs and caused a significant decreases of FosB/DeltaFosB expression. The present data suggest that mGluR5 receptors may be directly involved in the neural mechanism underlying AIMs; pharmacological antagonism of mGluR5 may be therefore proposed as a novel approach to prevent LIDs in PD.

P100

 

INVESTIGATION ON THE ROLE PLAYED BY ALPHA-SYNUCLEIN IN THE EXOCYTIC CYCLE.

 

Bellani S (1), Sousa VL (2), Meldolesi J (1)(2) and Chieregatti E (2)

 

(1) Dept. of Neuroscience, DIBIT, Vita-Salute San Raffaele University, via Olgettina, 58 - 20132 Milan (Italy) tel. +39-02-26434825; fax +39-02-26434813; E-mail:bellani.serena@hsr.it  (2) Scientific Institute of San Raffaele, Milan (Italy)

 

Alpha-synuclein is a presynaptic protein supposed to modulate the traffick of neurosecretory vesicles. Evidences support the role of alpha-synuclein in neurodegeneration: its mutated forms A30P and A53T are related to juvenile parkinsonism and alpha-synuclein is the major component of Lewy bodies. Here we investigated the role played by alpha-synuclein and its mutated forms on the organization of actin cytoskeleton and on the docking of dense core granules. We found that alpha-synuclein modulates the dynamics of actin both in vitro and in vivo. Using an in vitro fluorimetric assay, we observed that A30P accelerates the rate of actin polymerization in a calcium-dependent way and that its effect is dose-dependent. Using the pore-forming toxin streptolysin-O, we delivered recombinant A30P into living neuroendocrine cells (N2a). The delivery of the mutant A30P causes aggregation of actin, with a change in cell morphology. This result is consistent with the experiments performed in an epithelial cell line (MDCK) stably transfected with a Lac-switch derived inducible system of vectors to control the expression of alpha-synuclein. We found that A30P interferes with the re-establishment of the actin network after latrunculin-induced depolymerization, causing chaotic polymerization. By an assay that reconstitutes docking/fusion processes with cell fractions, we also observed that alpha-synuclein, and in particular the A30P mutant, inhibits the calcium-dependent docking of dense core granules to the plasma membrane. In the future, we will focus on the possible activity of alpha-synuclein on other aspects of vesicles trafficking, such as dense core granules exocytic fusion. We are also investigating for alpha-synuclein putative binding partner(s) on dense core granules and/or on the plasma membrane.

P101

 

NEUROINFLAMMATION AND NEURONAL NETWORKS ACTIVATION INVOLVED IN LEARNING AND MEMORY

 

Rosi S (1), Milliken HL (1)

 

Departments of Physical Therapy and Rehabilitation Science and Neurological Surgery

Division of Brain and Spinal Cord Injury Center

University of California San Francisco

1001 potrero Av San Francisco General Hospital, Bld#1, Room 101. San Francisco, CA, 94110

 

Neuroinflammation is associated with a variety of neurological diseases, such as Alzheimer disease (AD), and is reliably detected by the presence of activated microglia. In early AD, high numbers of activated microglia are observed, particularly in brain regions involved in memory. The ability of neurons to alter their transcriptional programs in response to synaptic input, also known as synaptic plasticity, is of fundamental importance to the mechanisms underling learning and memory. Previously, we showed that neuroinflammation, induced by chronic lipopolysaccaride-infusion in young rats, led to a significant increase in the number of neurons expressing the behaviorally-induced immediate early gene Arc in hippocampal regions that showed activated microglia. Arc is transcribed in neurons that are part of stable neural networks activated during spatial exploratory behaviors. Given the role of Arc regulation in synaptic plasticity and memory, we hypothesized that neuroinflammation alters synaptic activity associated with spatial learning and memory.  To test this hypothesis, we used a novel and sensitive technique, cellular compartmental analysis of temporal activity by fluorescence in situ hybridization (catFISH). With this technique we could to assess the activity history of neurons in the hippocampus and entorhinal cortex following different behavioral paradigm. Our data suggest that the CA1 area of the hippocampus is not able to compensate for the neuroinflammation-induced alteration of activity in the DG and CA3 areas. We are currently identifying the temporal dynamics of Arc transcription and Arc protein translation in the different layers of the entorhinal cortex and as it relates to the extent of microglial activation. Understanding how the presence of activated microglia affects synaptic plasticity is of critical importance for the development of strategies to prevent cognitive dysfunctions associated with initial stages of AD.

P102

 

MONITORING SIMULTANEOUS SUBCELLULAR DYSFUNCTIONS IN HD PATHOGENESIS USING THE LIVE CELL IMAGING

 

Abu-Baker A* (1), Laganire J (1), and Rouleau G (1)

 

(1)Center for the Study of Brain Diseases, CHUM Research Center - Notre Dame Hospital. J.A. de Sve Pavillion, Room Y-3612, 1560 Sherbrooke Street East. Montreal, QC. H2L 4M1, CANADA.

 

Huntingtonâs disease (HD) is an autosomal progressive neurodegenerative disorder. HD is caused by expansion of a CAG repeat coding for polyglutamine (polyQ) in the N terminus of huntingtin (htt) protein. The pathogenic mechanisms induced by polyQ-htt are still not clearly understood. A striking array of cellular and molecular mechanisms has been proposed to contribute to HD pathogenesis. Elucidating pathogenic mechanisms underlying HD can be challenging because the relevant changes, and the primary signals can be very early, small, fast and difficult to be detected by standard biochemical or molecular biological assays. One problem that investigators face is distinguishing primary from secondary events in HD pathogenesis. It will be important to discern which alterations truly underlie the disease process and which ones are epiphenomena or compensatory changes. Furthermore, it will be important to dissect the causal links between relevant alterations. It is therefore important to study the molecular mechanisms of HD as they naturally occur in living cells.

In this study, we used the live cell imaging approach to shed the light, in real-time, into the subcellular dysfuntions as they occur in our cellular HD model. We used our established cellular HD model in which Hela cells were transiently transfected with wild-type htt Q25 or mutant htt Q78 fused to cyan fluorescence protein. Using a combination of  in vivo automated microscopy, special multi-color subcellular organelle vectors, and openlab image analysis software, we were able to resolve the dynamics of specific selected two HD subcellular events as they occur: mitochondrial and transport dysfunctions. DsRed-mitochondria (red), EGFP-tubulin (green) were co-transfected with htt constructs, and time-lapse movies were recorded for different samples as early as 5 hours post-transfection. Time-lapse experiments tracked changes (morphology and dynamics) in mitochondria and microtubules simultaneously in real time as early as mutant htt is expressed over a period of time. Whether inclusion bodies or diffuse mutant htt protein directly impair the subcellular functions in HD is still controversial issue. Using our tools, we were able to determine whether a specific subcellular dysfunction is triggered by the inclusion bodies formation or by the diffuse mutant htt expression. Furthermore, we were able to elucidate the relationship between intermediate changes in the subcellular organelles and the long-term fate of a cell in real time over a period of time in our HD model, as the microscopic robotic stage allowed parallel tracking of multiple fields, both within a single well and in multiple wells. For example, mitochondrial  dysfunction was gradually occurring as the cell death happened in HD cell model.

Identifying the pathways that are altered in response to the mutant htt protein is crucial for understanding the cellular processes impacted by the disease as well as for the rational development of effective pharmacological interventions.

P103

 

PENTYLENTETRAZOLE KINDLING INDUCED HIPPOCAMPAL DAMAGE IN RATS IS MEDIATED BY CELL CYCLE ACTIVATION

 

Popova MS, Stepanichev MU, Pavlova TV and Gulyaeva NV

 

Department of Functional Biochemistry of the Nervous System, Institute of Higher Nervous Activity and Neurophysiology Russian Academy of Sciences, 5a Butlerov str., Moscow 117485, Russia.

 

Short periods of repeated seizure activities in the adult human brain are accompanied by neuronal damage and cell loss. The mechanisms of seizure-induced neuronal death are not completely understood. Reactivation of cell cycle events in mature neurons was hypothesized to be one of the mechanisms resulting in the cell death in quite a few of neurodegenerative diseases, as well as in epilepsy. Pentylenetetrazol (PTZ) kindling in rats is one of the models reproducing short repeated seizure in the animals. It has been shown that PTZ kindling is accompanied by neuronal loss in the hippocampal fields. The aim of the present study was to investigate the mechanisms of hippocampal damage after PTZ kindling in rats. Male Wistar rats were used for the study. The animals were injected with PTZ at a subconvulsive dose of 40 mg/kg i.p. three times per week. Damaged neurons with altered morphology and signs of degeneration were observed in all cell areas of the hippocampus of PTZ-kindled rats. A moderate neuronal loss in the CA1 region, hippocampal hilus and dentate gyrus was demonstrated. No signs of apoptosis (TUNEL staining, active caspase-3 and chromatin condensation) in the damaged hippocampal neurons were found in kindled rats. However, damaged neurons were positive for cyclin B1 and cdc2/cdk1 immunostaining, both being the markers of G2 cell cycle phase. These data are in accordance with Nagy and Esiri (1998), who demonstrated the presence of cyclin B1 in the hippocampi of patients with temporal lobe epilepsy. Our  data show the relevance of PTZ kindling model for studying  mechanisms of neuronal death in epilepsy and suggest that cell damage and death during PTZ kindling may be mediated by the activation of cell cycle machinery.

Supported by RBRF grant and RAS grant "Fundamental Medicine"

P104

 

CHARACTERIZATION OF ATAXIN-3 PROTEOLYTIC FRAGMENTS IN TRANSFECTED CELLS

 

Pastori Valentina*1, Somaschini Alessio1, Tedeschi Gabriella2, Taverna Francesca2, Nonnis Simona2 Fusi Paola1 Pozzi Chiara1

 

1)Dipartimento di Biotecnologie e Bioscienze Universitˆ di Milano-Bicocca, P.za della Scienza 2, 20126, Milano, Italy, Tel. 0264483409, fax 0264483565, e-mail: paola.fusi@unimib.it.

2)Dipartimento di Patologia Animale, Igiene e Sanitˆ Pubblica, via Celoria 10 20133, Milano, Italy

 

Ataxin-3 (AT-3) is the protein responsible for spinocerebellar ataxia type 3 (Sca3), also known as Machado Joseph disease. The most widely accepted pathogenic model predicts that a proteolytic event generates a C-terminal fragment of the protein, containing the polyQ stretch, endowed with a toxic function leading to the formation of intracellular inclusions. In order to study AT-3 intracellular proteolysis, different forms of this protein (both pathological and non pathological) were overexpressed in transiently transfected COS7 cells. Normal murine AT-3 (AT-3Q6), pathological human AT-3 (AT-3Q72) and two truncated forms (AT-3&#916;291-end, lacking the poly-Q, and AT-3&#916;262-end, lacking both the poly-Q and a putative NLS) were studied. All cDNAs were cloned in pcDNA3.1/myc-His, in frame with an HA N-terminal epitope and a c myc C-terminal epitope. Subcellular localization was assessed in COS7 trasnfected cells, through confocal microscopy and western blotting. Results showed that both AT-3Q6 and AT-3Q72 localize in the cytoplasm, as well as in the nucleus, while the truncated forms were found mainly in the cytosol. This suggests that neither the NLS nor the poly-Q stretch are essential to nuclear import, although they might be increase import rate. Moreover sub-cellular fractionation showed that ataxin-3 is extensively proteolyzed, leading to the appearance of a number of fragments with molecular masses ranging from 40 to 6 kDa. Edman degradation and mass spectrometry analysis of fragments showed that AT-3Q6, At-3Q72 and AT-3&#916;291-end are cleaved inside the Josephin domain. Moreover, AT-3Q6 was found to be also proteolyzed at different caspase cleavage sites located in the unstructured C-terminal domain. AT-3Q72 was found to be much more resistant to proteolysis; our data suggest that this might be due to the presence of the expanded poly-Q stretch, which alters the protein structure to the point of hampering proteolytic cleavage.

P105

 

HUNTINGTIN-DEFICIENT ZEBRAFISH EXHIBIT DEFECTS IN IRON UTILISATION AND DEVELOPMENT

 

Lumsden AL*(1), Henshall TL(1), Dayan S(1), Lardelli MT(1), Richards RI(1).

 

(1) School of Molecular and Biomedical Science, ARC Special Centre for the Molecular Genetics of Development, The University of Adelaide.

(*) Presenting author.

Address: Molecular Life Sciences Building, The University of Adelaide. SA 5005, AUSTRALIA.

Ph: +61-8-83037557, Fax: +61-8-83037534, email: amanda.l.lumsden@adelaide.edu.au

 

   Huntingtonâs disease is one of nine neurodegenerative disorders caused by expansion of CAG repeats encoding polyglutamine in their respective, otherwise apparently unrelated proteins. Despite these proteins having widespread and overlapping expression patterns in the brain, a specific and unique subset of neurons exhibits particular vulnerability in each disease. It has been hypothesized that perturbation of normal protein function contributes to the specificity of neuronal vulnerability, however the normal biological functions of most of these proteins including the HD gene product, Huntingtin (Htt), are unclear.

   To explore the roles of Htt, we have used antisense morpholino oligonucleotides to observe the effects of Htt deficiency in early zebrafish development. Knockdown of Htt expression resulted in a variety of developmental defects. Most notably, Htt-deficient zebrafish had hypochromic blood due to decreased haemoglobin production, despite the presence of iron within blood cells. Furthermore, transferrin receptor 1 transcripts were increased, suggesting cellular iron starvation. Provision of iron to the cytoplasm in a bio-available form restored haemoglobin production in Htt-deficient embryos. Since erythroid cells acquire iron via receptor-mediated endocytosis of transferrin, these results suggest a role for Htt in the release of iron from the endocytic compartments.

   Iron is required for oxidative energy production, and defects in iron homeostasis and energy metabolism are features of HD pathogenesis that are most pronounced in the major region of neurodegeneration. It is therefore plausible that perturbation of Httâs normal role in the iron pathway (by polyglutamine tract expansion) contributes to HD pathology, and particularly to its neuronal specificity. 


P106

 

DREAM: FROM CALCIUM HOMEOSTASIS TO GENE TRANSCRIPTION, A MULTIFUNCTIONAL PROTEIN

 

Fedrizzi L (1,2)*, Lim D (1), Naranjo JR (3), Brini M (1,2), and Carafoli E (1)

 

(1) Department of Biochemistry, University of Padova, V.le G. Colombo, 3 - 35121 Padova (Italy), tel. +39-049-8276135; fax. +39-049-8276125; E-mail: laura.fedrizzi@unipd.it

(2) Department of Experimental Veterinary Science, University of Padova, Padova (Italy)

(3) Departamento de Biolog’a Molecular y Celular, Centro Nacional de Biotecnolog’a, CSIC, Madrid (Spain)

 

DREAM, an EF hand protein that belongs to the Neuronal Calcium Sensor proteins family, acts as transcriptional repressor of target genes. In addition to this role, DREAM has been identified as Ca2+-dependent interacting protein of Presenilin (and named Calsenilin) and as modulator of Kv potassium channels (and named KChIP3).

We investigated the influence of DREAM/Calsenilin/KChIP3 on Ca2+ homeostasis and on the expression of proteins involved in Ca2+ signalling. We generated stable clones of neuronal cells overexpressing DREAM wt or EFmDREAM, a mutant unable to bind Ca2+ and thus acting as dominant negative. First, we measured Ca2+ concentrations in the different cell compartments using the recombinant Ca2+-sensitive photoprotein aequorin. We observed a reduction in the ER Ca2+ content and a decrease in the Ca2+ influx through the plasmamembrane channels in both DREAM clones. Several works reported a correlation between ER Ca2+ homeostasis dysfunction and neurodegenerative diseases (e.g. Alzheimer disease), and, in particular, recently it was demonstrated that Presenilin could act as passive Ca2+ leak channel in the ER membrane. To investigate a possible functional role of DREAM in modulating the channel function of Presenilin we analyzed the effects of DREAM and Presenilin co-expression on ER Ca2+ content. Our data shown a dramatic loss of the ability to accumulate Ca2+, indicating that the two proteins have a cumulative role. Second, we investigated by qRT-PCR and Western blotting the levels of Calreticulin, Calnexin and BiP, well known as ER Ca2+ buffering proteins but also as indicator and key component of the ER stress response. We found that Calreticulin and Bip protein levels were reduced of about 30% in the DREAM expressing clones. Parallel analysis on mRNA from cerebella of EFmDREAM transgenic mice had revealed a decrease in the transcriptional levels of these chaperones, suggesting that DREAM could act at different levels to control ER Ca2+ homeostasis.

P107

 

ANTI-INFLAMMATORY ACTIVITY OF ESTROGEN IN ACUTE AND CHRONIC BRAIN INFLAMMATION

 

Pozzi S, Benedusi V, Vegeto E, Maggi A

 

Center of Excellence on Neurodegenerative Diseases, Department of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. Fax: 0039.02.50318284

e-mail: adriana.maggi@unimi.it

 

Activation of microglia cells is the hallmark of  acute and chronic neurodegenerative disorders, such as ischemia, Multiple Sclerosis (MS) and Alzheimer Disease (AD), characterized by inflammatory events. Recent studies reported that 17b-estradiol (E2) acts as a neuroprotective agent in brain by both targeting neurons and inhibiting the brain inflammatory reactions. In our lab we recently developed an experimental model of brain inflammation, in which LPS (lipopolysaccharide), an inflammatory agent, is injected in the cerebral ventricles, resulting in a transient and localised acute neuroinflammatory reaction. We used this system to investigate the E2 anti-inflammatory activity in the central nervous system and demonstrated that E2 is a potent inhibitor of microglia reactivity in several regions of the brain, including cortex, hippocampus and noncortical areas and that hormone administration results in a significant reduction of the expression of inflammatory markers, such as TNF-a, MCP-1 and MIP-2. Our data thus show that estrogen is able to quench acute brain inflammation in vivo, in agreement with data published by other groups on neuroinflammatory processes such as ischemia or experimental allergic encephalomyelitis (EAE). On the other hand, we used the APP23 transgenic mice, expressing the human amyloid precursor protein (APP) with a mutation reported in familial AD, in order to understand the role of E2 in chronic neurodegenerative diseases. In this animal model of AD microglia displays the characteristic activated morphology and immunoreactive phenotype induced by the chronic deposition of the b-amyloid peptide (Ab). We observed that ovariectomy increases microglia activation at Ab deposits whereas chronic replacement with E2 in ovarectomized APP23 mice reduced neuroinflammation. Thus, chronic neuroinflammatory events, associated with neurodegeneration, are also targeted by hormone action. Future studies using synthetic estrogenic compounds will be discussed.

P108

 

CHARACTERISATION OF SCA3 MUTANT MICE WITH A DOMINANT TOXIC EFFECT

 

Jeannette HŸbener (1), F. Vauti (2), H.-H. Arnold (2), C. Funke (1), M. Bonin (1,3), Th. Schmidt (1), P. Teismann (4), K. Grundmann (1) and Olaf Riess(*)(1)

 

(1)Department of Medical Genetics, University of TŸbingen, 72076 TŸbingen(Germany), te. +49-7071-29-76458, fax +49-7071-29-5171, olaf.riess@med.uni-tuebingen.de

(2)Department of Cell- and Molecular Biology, Technical University of Braunschweig, 38106 Braunschweig (Germany) (3)Microarray Facility, 72076 TŸbingen (Germany)

(4)School of Medical Sciences, University of Aberdeen, AB24 3FX Aberdeen, UK

 

Spinocerebellar Ataxia Type 3 (SCA3), is an autosomal dominant neurodegenerative disorder caused by polyglutamin-expanded ataxin-3, whose function is still unknown. In order to further evaluate the function of ataxin-3 in vivo, we analysed ataxin-3 mutant mice generated by a gene trap (GT) approach. The plasmid GT-vector betaGeo2 was integrated into the intron 8 of the ataxin-3 gene The polyglutamin repeat in exon 10 is missing in these ataxin-3 mutant mice. Homozygous mutant mice (Atx3gt/gt) survive embryogenesis and do not show obvious developmental defects.

Immunohistochemistry analysis revealed a high number of ataxin-3 positive cytoplasmatic inclusion bodies in all brain regions at the age of 3 months. Neurodegeneration of neurons in the cerebellum could be detected by different immunohistochemistal stainings.

Metabolic screens at the age of 3 months and one year, respectively, implicate an impact in the dopaminergic metabolism but exclude disturbances of the serotonin metabolism. Other screens of blood parameters, immunology, hearing and eyesight did not reveal discrepancies between the different genotypes.

Over the whole lifespan of 12 months we screened for neurological symptoms by means of the modified SHIRPA test and by footprint analysis. We further looked for motor performance and coordination on the rotarod and the beam walking test. From birth until the age of seven months we were not able to detect any behavioural differences between heterozygous and homozygous mutant mice in motor coordination and motor learning on the rotarod and beam walking test compared to their wild type littermates. Heterozygous and homozygous mutant mice developed a neurological phenotype at the age of one year. At first we could detect a dramatic reduction of the body weight followed by neurological symptoms e.g. tremor, clasping, stereotypes and ataxia. Two weeks after onset of symptoms both heterozygous and homozygous mutant mice died.

These results demonstrate that the whole ataxin-3, a widely expressed protein, does not seem to be essential for development of the ataxin-3 mutant mice. In contrast they develop a phenotype in adulthood and a premature death consistent with a neurodegenerative disease. We therefore believe that the first 8 exons of the ataxin-3 protein, containing the Josephin-domain, have a dominant toxic effect in the neuronal cells.

P109

 

PROTECTIVE DYSLIPIDEMIA IN AMYOTROPHIC LATERAL SCLEROSIS

 

Anissa FERGANI (1,2), Luc DUPUIS (1,2), Hugues OUDART (3), Jose-Luis GONZALEZ DE AGUILAR (1,2), Bastien FRICKER (1,2), FrŽdŽrique RENE (1,2), Jean-Franois HOCQUETTE (4), Dominique BONNEFONT-ROUSSELOT(5), Randa BITTAR (5), Lucette LACOMBLEZ (6), Vincent MEININGER (6) & Jean-Philippe LOEFFLER (1,2)

 

(1) Inserm, U692, Laboratoire de Signalisations MolŽculaires et NeurodŽgŽnŽrescence, Strasbourg, F-67085 France

(2) UniversitŽ Louis Pasteur, FacultŽ de MŽdecine, UMRS692, Strasbourg, F-67085 France; (3) Centre d'Ecologie et Physiologie EnergŽtiques, UPR9010 CNRS, 23 rue Becquerel, 67087 Strasbourg Cedex, France; (4) Equipe Croissance et Metabolismes du Muscle, Unite de Recherches sur les Herbivores, INRA, Centre de Clermont-Ferrand/Theix, 63122 St Genes-Champanelle, France; (5) Laboratoire des Lipides, Groupe Hospitalier PitiŽ-Salptrire (AP-HP), 47-83 Boulevard de lâH™pital, 75651 Paris Cedex 13, France; (6) FŽdŽration des Maladies du Systme Nerveux, Centre rŽfŽrent maladie rare SLA, H™pital de la PitiŽ-Salptrire, 47-83, Boulevard de l'H™pital 75651 Paris, France

 

Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease causing motor neuron degeneration, muscle atrophy, paralysis and death. Defective energy homeostasis has also been associated with ALS. Many patients and related animal models display unexpectedly increased energy expenditure, and a highly energetic diet can, at least in mice, increase the lifespan of the animals and promote motor neuron survival. Here we show that a large subset (~50%) of patients is hyperlipidemic and that bearing an abnormally elevated LDL/HDL ratio significantly correlates with increased survival. In an animal model of ALS, lipid metabolism was deeply altered due to an increase in peripheral clearance of triglycerides-rich lipoproteins, probably caused by skeletal muscle hypermetabolism which lead to decreased post prandial lipidemia. This decreased post prandial lipidemia was reverted by the protective high fat regimen. Altogether, our results show that increased lipid availability is protective for both ALS patients and animal model.

P110

 

INTEGRATION OF NEURAL PROGENITORS INTO THE CIRCUITS OF THE NEONATAL BRAIN

 

Muzzi P(1)*, Kilb W(2), Kodirov S(2), Bossi M(3), Mirabelli M(1), Gilardini A(3), Amedeo MR(1), Tredici G(3), Luhmann H(2), and Vercelli A(1,4)

 

(1) DAFML, University of Turin, corso Massimo DâAzeglio 52, 10126 Torino (Italy); tel.+390116707736; fax +390116705931; e-mail: patrizia.muzzi@unito.it

(2) Institute of Physiology, University of Mainz, D

(3) Department of Neurosciences and Biomedical Technologies, Bicocca University of Milan, Monza, I

(4) National Institute of Neuroscience, Torino, I

 

We transplanted embryonic neural progenitors into the brain of neonatal and adult mice, and showed that, after neonatal transplantation, they survive, migrate and integrate into the host neural circuits.

Cells from the ganglionic eminence (GE) and the dorsal telencephalon (DT) of mice of different embryonic ages (E12.5, E14.5, E17.5) expressing green fluorescent protein (EGFP) were injected into the right lateral ventricle of newborn, P7 and adult mice. A few days to more than one year from transplantation, the host brains were reconstructed for EGFP-positive cells, and some sections immunoreacted against CR, CB or PV, MAP2, GFAP or MBP, or embedded for Electron Microscopy. In a separate set of experiments, EGFP-positive projection neurons were retrogradely labeled with fluororuby from the thalamus. In another set of experiments, coronal brain slices were prepared from 3-wk-old mice which had received EGFP+-cells at P1 and whole-cell voltage- and current-clamp recordings were obtained from EGFP+ neurons.

EGFP+ cells could be observed in all animals which received the transplant around birth, either isolated or in groups. They often produced cell masses, in the ventricles, in the corpus callosum or on the cortical surface. Their phenotype was strictly dependent from where progenitors were dissected: GE-derived cells were suggestive of interneurons and oligodendrocytes. DT cells gave a larger variability of phenotypes, some of which suggestive of pyramidal neurons. We found spiny cells in the basal ganglia, in the septal nuclei and in the hippocampus. EGFP+ axons were abundant in the thalamus. Fluororuby retrogradely labeled cortical EGFP-positive neurons from the thalamus. Electrophysiology on brain slices showed that EGFP+ cells were integrated into the synaptic network of the host brain. EM analysis of EGFP-immunoreacted cells showed synaptic contact on DAB-filled profiles.

P111

 

A NEW TRANSGENIC MOUSE MODEL OF MACHADO-JOSEPH DISEASE PRESENTING MOTOR IMPAIRMENT AND BRAIN PATHOLOGY

 

Silva-Fernandes A (1), Costa MC (1), Costa C (2), Maciel P (1)

 

(1) Institute of Health Sciences, School of Health Sciences, University of Minho, Braga, (Portugal) tel.+351-253-604835; fax +351-253-604831; E-mail: anabelasf@ecsaude.uminho.pt

(2) Neurology Department, Hospital Fernando Fonseca, Amadora, Portugal

 

Machado-Joseph disease (MJD) is a late-onset autosomal dominant neurodegenerative disorder characterized mainly by a progressive ataxia, caused by an expansion of the CAG repeat in the ATXN3 gene. MJD is characterized by a selective neuronal death affecting several regions of the cerebellum and brainstem. Other hallmark of MJD is the presence of intracellular aggregates in neurons of these areas. We have generated a transgenic mouse model expressing the mutant ataxin-3 under the control of a general expression promoter (pCMV), and obtained two lineages: lineage A carrying 94 CAGs and two transgene copies, and lineage B presenting 83 CAGs and approximately ten transgene copies. These lineages express the mutant ataxin-3 in peripheral tissues and in the central nervous system. As in MJD patients, the expanded CAG tract display intergenerational and somatic instability. To study the behavioural phenotype we have performed the Rotarod test and the SHIRPA protocol. Hemizygous and homozygous transgenic mice of lineage A presented motor coordination impairment at 16 weeks of age, given by the significant decrease in the latency to fall in the rod. Additionally, transgenic mice from lineage A presented a reduced locomotor activity beginning at 24 weeks of age. Until 72 weeks of age, no motor impairment was found in the lineage B, suggesting that the repeat length may be more relevant than gene dosage for this disease manifestation. Moreover, brains of transgenic mice from lineage A revealed cell loss and atrophy and neurons carrying cytoplasmatic aggregates positive for ataxin-3 in several MJD affected areas, namely the lateral dentate nucleus. In conclusion, this transgenic mouse model exhibits important features of MJD including the presence of a motor phenotype, somatic and intergenerational instability of the CAG repeat, along with pathological features suggesting that it could be useful in the study of MJD pathogenesis and for therapy development.

P112

 

THE ROLE OF SPASTIN IN THE AXONAL CYTOSKELETON

 

Elena Riano1, Monica Martignoni 1, Elena I. Rugarli1.2

 

1- Division of Biochemistry and Genetics, Istituto Neurologico Besta, Milano, Italy; 2- DNTB, Universitˆ Milano-Bicocca, Milano, Italy

 

Hereditary spastic paraplegia (HSP) is a heterogeneous genetic disease characterized by selective axonal degeneration of the corticospinal axons. The gene SPG4, encoding spastin, is mutated in about half of dominant cases of HSP. The pathogenetic mechanism of axonal degeneration in patients with SPG4 mutations still awaits elucidation. Previous studies from our and other laboratories have shown that spastin is involved in microtubule severing. Microtubules are essential for axonal growth and maintenance, and a fine regulation of their dynamics may be especially crucial in the long processes of cortical motoneurons. The structure and dynamics of the highly organized cytoskeletal arrays in axons are essential to orchestrate the intracellular transport events that organize the neuronal cytoplasm, and for directing the endocytotic and exocytotic traffic. One attractive hypotesis is that degeneration of corticospinal axons may be due to the loss of a regulatory function of spastin in the axonal cytoskeleton.

To begin to dissect the role of spastin in neurite outgrowth, we performed genetic manipulations in NSC34 cells, a murine immortalized cell line. Downregulation of spastin with short interfering RNA duplexes markedly decreased spastin expression levels and surprisingly associated with an increase in the number of cells showing neurites longer than 50 micron and in a increase in the average length of the processes. The same phenotype is observed using a vector encoding a short hairpin RNA specific for spastin and GFP as a reporter. In contrast, spastin overexpression significantly decreased the amount of acetylated tubulin, and suppressed neurite outgrowth. To obtain this phenotype an intact AAA domain is required. These results suggest that microtubule severing may regulate neuronal length in vivo and that tight regulation of spastin levels is required for development and maintenance of axonal connections. To exert its function, spastin is recruited to specific cellular subcompartments, indicating a tight regulation of its action in vivo.

P113

 

RELATIONSHIP BETWEEN AMYLOIDOGENESIS AND MEMBRANE IONIC PERMEABILITY

 

Schininˆ ME (1), Maras B (1), Principe S (2) Cardone F (2), Pesci D (3), Jodice C (*)(3)(4) and Mazzanti M (5)

 

(1) Dip. di Scienze Biochimiche, Universitˆ \"La Sapienza\", Roma (Italy).

(2) Dip. di Biologia Cellulare e Neuroscienze, Istituto Superiore di Sanitˆ, Roma (Italy).

(3) Dip. Biologia, Universitˆ ãTor Vergataä, Roma (Italy). Via della Ricerca Scientifica ö 00133 Roma (Italy). tel.+39-06.72594321; fax +39-06.2023500; e-mail: carla.jodice@uniroma2.it

(4) CIMN (Universities of Florence, Rome ãTor Vergataä and Milan, Italy)

(5) Dip. di Scienze Biomolecolari e Biotecnologie, Universitˆ di Milano (Italy)

 

A main feature shared by several neurodegenerative disorders is the deposition of protein aggregates (amyloid) generated in a disease-specific manner from structurally unrelated proteins. Alzheimer's disease is characterized by a progressive deposition of the beta-amyloid (AB), proteolytically derived from a membrane precursor protein. Intraneuronal protein aggregates are characteristic of "polyglutamine diseases", such as Spinocerebellar Ataxia type 1, neurodegenerative disorders caused by an expanded polyglutamine (polyQ) tract in the disease protein. Transmissible spongiform encephalopathies are invariably associated with the accumulation of a pathological form of the prion protein (PrPsc) that is the main component of highly infectious purified fractions. Several studies showed that amyloidogenic peptide stimulation promoted atypical inflammatory events mediated by astrocytes and microglial activation and contribute to neurodegeneration. Strings of oligomers (protofibrils), and not mature fibrils, could be the key toxic species because of their tendency to interact with cell membranes. Recently, we started a collaborative funded project (MIUR, PRIN2005, prot. 2005054591) with the aim to compare the role of different amyloidogenic polypeptides, i.e. AB, PrPsc and three different polyQs of ataxin-1, in the neurodegenerative processes. Particularly, the main aim was to prove the occurring of changes at the plasma membrane concerning the ability of amyloids to alter ionic permeability. Using the Tip-Dip procedure on an artificial bilayer system, challenged with AB, PrP and polyQs, we were able to demonstrate the ability of all the three amyloid proteins to colonize the selected lipid bilayers. Particularly, data on the effect of disaggregating agent (dimethylsulfoxide) on the prion protein interaction suggest that membrane permeabilization occurs via oligomeric aggregates. The selectivity of the ion gates formed by amyloid peptides is under investigation.

P114

 

EXPRESSION OF TBP WITH 64 CAGS CAUSES CEREBELLAR PHENOTYPE IN TRANSGENIC MICE

 

Nguyen HP (1) , Then F (2), Osmand A (3), Wolburg H (4), Ott T (1), Golub Y (1), Bauer C (1), Teismann P (5), Hennek T (1), Krainc D (2), Riess O (1), Bauer P (1)

 

(1) Department of Medical Genetics, University of Tuebingen, Tuebingen, Germany, e-mail:hoa.nguyen@med.uni-tuebingen.de

(2) Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, USA

(3) Department of Medicine, University of Tennessee, Knoxville, USA

(4) Department of Pathology, University of Tuebingen, Tuebingen, Germany

(5) Institute of Medical Sciences, University of Aberdeen, Aberdeen, Scotland

 

SCA17 is a progressive neurodegenerative disease leading to cerebellar ataxia and dementia.

Several accessorial symptoms such as Parkinsonism, dystonia, and psychiatric disturbances commonly aggravate the disease course. Genetically, a CAG/CAA expansion in the TATA binding protein (TBP) is expanded in SCA17 patients, leading to an expanded polyglutamine chain in this ubiquitously expressed transcription factor.

We have generated transgenic mice which express a 64 CAG/CAA repeats containing human TBP (Q64TBP) gene under the control of the truncated human prion protein promoter (PrP).

Transgene protein expression throughout different brain regions (cortex, basal ganglia, cerebellum, and brain stem) was clearly demonstrable. Onset of motor dysfunction (Accellerod) started by 6 months and progressed with age. By electron microscopy and immunohistochemical methods we were able to detect neurodegeneration and aggregation selectively in the cerebellum. We will present detailed morphological and phenotypical data for this rodent model of SCA17, which enables us to further study the pathogenesis of this progressive neurodegenerative disease.

P115

 

HDAC6 INHIBITION COMPENSATES FOR THE TRANSPORT DEFICIT IN HUNTINGTON'S DISEASE BY INCREASING TUBULIN ACETYLATION

 

Juliette D. Godin1,2,* Jim P. Dompierre1,2, BŽnŽdicte C. Charrin1,2, Fabrice P. Cordelires1,2,3, Stephen J. King4, Sandrine Humbert1,2 and FrŽdŽric Saudou1,2,3

 

(1)Institut Curie, Orsay, F-91405 France. tel. +33 169867128, fax +33 169863017, juliette.godin@curie.u-psud.fr

(2)CNRS UMR 146, Orsay, F-91405 France.

(3)Plate-forme Imagerie Cellulaire et Tissulaire, Institut Curie, CNRS UMR 146, Orsay, F-91405 France.

(4)Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA.

 

A defect in microtubule-based transport contributes to the neuronal toxicity observed in Huntingtonâs disease (HD). Histone deacteylase (HDAC) inhibitors show neuroprotective effects in this devastating neurodegenerative disorder. We report here that HDAC inhibitors, including trichostatin A (TSA), increase vesicular transport of brain-derived neurotrophic factor (BDNF) by inhibiting HDAC6, thereby increasing acetylation of lysine 40 of alpha-tubulin. Microtubule (MT) acetylation in vitro and in cells causes the recruitment of the molecular motors dynein and kinesin-1 to MTs. In neurons, acetylation of lysine 40 of alpha-tubulin increases the flux of vesicles and the subsequent release of BDNF. We show that tubulin acetylation is reduced in HD brains and that TSA compensates for the transport and release defect phenotypes that are observed in disease. Our findings reveal that HDAC6 inhibition and acetylation at lysine 40 of alpha-tubulin may be therapeutic targets of interest in disorders such as HD in which intracellular transport is altered.

P116

 

NEUROTROPHINS AS MODULATORS OF CHOLINERGIC MARKERS AND NEUROTROPHIN RECEPTORS EXPRESSION IN RAT BASAL FOREBRAIN CHOLINERGIC NEURONS

 

Formaggio E., Dalfini A.*, Chiamulera C., Fumagalli G.

 

University of Verona, Section of Pharmacology, Dept. of Medicine and Public Health, P.le Scuro, 10, 37134 Verona (Italy) tel. +39 045 802 7608, fax +39 045 58 1111, E-mail: adalfin@libero.it

 

Alzheimer disease (AD) is characterized by a progressive loss of cognitive functions due to specific neurodegeneration and death of basal forebrain cholinergic neurons (BFCN). BFCNs innervate cortical and hippocampal areas and retrogradely transport NGF, which sustains their survival and differentiation. An impairment in NGF production and/or transport is retained to be one of the factors involved in neurodegeneration in AD. NGF retrograde transport is mediated by neurotrophin receptors (NTRs) so an imbalance in their expression could drive neurodegeneration.

The scope of our study was to test whether exogenous neurotrophins alter NTRs and cholinergic markers expression on BFCN.

To address this question we set up in vitro cultured BFCNs. We found that our cultures expressed  NTRs (p75-NTR, TrkA, TrkB, TrkC) and cholinergic markers (ChAT and CHT) mRNA and proteins. By using immunofluorescence, we observed complete p75-NTR, and partial TrkA, neuronal colocalization with cholinergic markers.

We found that NGF (100ng/ml) induced a significant and BFCN-specific increase of the number of ChAT and p75-NTR positive neurons. BDNF (100ng/ml) and KCl (25mM) induced a significant increase of the number of p75-NTR positive neurons due to higher expression levels in GABAergic neurons.

NGF and KCl also induced significant BFCN-specific increase of p75-NTR protein expression and fluorescence signal spreading along neuronal processes due to neurite outgrowth or to signal spreading in existing neurites.

In conclusion, we developed and validated an in vitro model of cholinergic neurons. Specifically, both NTs and KCl induced  p75 NTR expression in BFCN, suggesting that this NTR may undergo to significant changes after trophic and activity-dependent stimulations.

P117

 

SELECTIVE DOPAMINERGIC ALPHA-SYNUCLEIN PATHOLOGICAL CHANGES FOLLOWING GLUCOSE DEPRIVATION 'IN VITRO': IMPLICATIONS FOR PARKINSON'S DISEASE

 

Bellucci A (1), Tognazzi N (1), Collo G (1), Missale C (1) and Spano PF (1)

 

Division of Pharmacology, Dept. Biomedical Sciences and Biotechnologies, University of Brescia, Italy

 

Progressive degeneration and intraneuronal Lewy bodies (LB) made of filamentous alpha-synuclein in the dopaminergic cells of the substantia nigra (SN) are key pathological features of Parkinson's disease (PD). Since recent positron emission tomography (PET) studies (Sansone et al., J. Neurol. Neurosurg. Psychiatry 77: 425-426)  showed that the rate of glucose metabolism in the brain of PD patients is correlated to dopamine (DA) levels and that low glucose metabolism can be considered an antemortem diagnostic method to distinguish LB dementia from Alzheimerâs disease (Higuchi et al., Exp. Neurol. 162: 247-256) we studied the effect of glucose deprivation (GD) in dopaminergic-differentiated SH-SY5Y cells and primary mesecephalic neurons to investigate the existence of a correlation between decreased glucose metabolism, alpha-synuclein misfolding and selective dopaminergic degeneration. Fur

thermore, we evaluated the effect of DA and D2R/D3R agonists treatment on GD-treated cells. We found that DAT and alpha-synuclein interact in dopaminergic-differentiated cells, and that GD induced a reduction in cell viability and DAT levels, increased alpha-synuclein total content while decresed its membrane-bound fraction. At the same time, after GD insult, we observed the occurrence of alpha-synuclein filamentous aggregation and the formation of alpha-synuclein-/DAT-positive inclusions. DA treatment exacerbated the effects of GD, while DA-uptake blockade and dopaminergic D2 receptors (D2R)-agonist treatment exerted neuroprotective effects, by recovering cell viability and counteracting  DAT decrease and alpha-synuclein increase. Conversely, D2R antagonists exacerbated the effects of GD and DA treatment by further increasing the number of alpha-synuclein inclusions and decreasing cell

viability in GD/DA-treated dopaminergic cells. These data indicate that glucose hypometabolism may be involved in PD pathogenesis by influencing DAT/alpha-synuclein trafficking.

P118

 

EFFECT OF PHYSICAL EXERCISE AND THE STEROID NANDROLONE ON MOTONEURONS COMMITTED TO NEURODEGENERATION

 

Kassa RM *(1), Mariotti R (1), Cupidi C (2), and Bentivoglio M (1)

 

(1) Department of Morphological and Biomedical Sciences, University of Verona Strada Le Grazie 8-37134 Verona (Italy)tel.+39-045-8027164; fax +39-045-8027163; E-mail: roman@anatomy.univr.it

(2) Experimental Medicine, University of Palermo, Palermo (Italy)

 

Adult-onset and selective degeneration of both upper and lower motoneurons characterizes amyotrophic lateral sclerosis (ALS). No exogenous risk factor/s has been unequivocally identified so far. Occurrence of ALS in high-profile athletes, including recent reports of increased incidence among Italian soccer players, have added to the suspicion of an association between sports and ALS due to several potential risk factors. In this scenario, the impact of performance-enhancing drugs should also be taken into consideration. We investigated the potential effect of strenuous exercise and the anabolic steroid nandrolone (19-nortestosterone), both separately and in combination, on and around lumbar motoneurons of SOD1(G93A) transgenic (Tg) mice which provide a murine model of familial ALS, and their wild-type (Wt) littermates. Mice from both genotypes were assigned to four paradigms: a) intense exercise on a treadmill; b) chronic treatment with nandrolone; c) combined treadmill running and nandrolone treatment; d) no running and no treatment. All Tg mice were sacrificed at disease onset ascertained with motor tests. Glial reactivity and motoneuron loss were examined with immunohistochemistry. The results indicated in SOD1(G93A) mice: i) marked microglial activation in the lumbar ventral horn of running-onlyâ ones; ii) increased astrocytic activation in the ventral horn of nandrolone-onlyâ treated ones; iii) significant reduction in the number of lumbar motoneurons of nandrolone-onlyâ treated ones; iv) no changes in the number of motoneurons of Îrunning-onlyâ and combined running and nandroloneâ treated mice compared to sedentary, untreated ones. Altogether these data suggest that chronic nandrolone treatment may exert a detrimental effect on SOD1-mutant lumbar motoneurons which may be reverted by combining with strenuous exercise. The findings also suggest that in SOD1-mutant mice under these circumstances activated astrocytes may have a neurotoxic effect. (Supported by COFIN 2005)

P119

 

IN SEARCH OF PERIPHERAL BIOMARKERs OF HUNTINGTON DISEASE

 

Tarditi A1*, Mariotti C2, Bachoud-LŽvi A-C3, Varani K4, Abbracchio MP5, Borea PA4, Tabrizi S6,Rosser A7, Maccarrone M8, Peschanski M9, DiDonato S2, Cattaneo E1

 

1Laboratory of Stem Cell Biology and Pharmacology of Neurodegenerative Disease, Department of Pharmacological Sciences and Centre for Stem Cell Research, University of Milano, via Balzaretti 9, 20133 Milan- Italy, tel +39 0250318333; fax +39 0250318284; email: alessia.tarditi1@unimi.it; 2Division of Biochemistry and Genetics and of Neuroepidemiology, National Neurological Institute Carlo Besta, Milan; 3INSERM U841, team 1 \" Neuropsychologie Interventionnelle\", Creteil 94010, France; 4Department of Clinical and Experimental Medicine, Pharmacology Unit, University of Ferrara; 5Department of Pharmacological Sciences, University of Milan;;  6Institute of Neurology, University College London , UK; 7School of  Biosciences, University of Cardiff, UK; 8Department of Biomedical Sciences, University of Teramo; 9INSERM/UEVE UMR 861, I-STEM,Genopole Campus 1, France.

 

In HD and in other polyQ linked diseases there is the urgent need of biomarkers which could allow to track the disease progression and to monitor the effect of pharmacological compounds. In fact the genetic mutation, a CAG expansion on the huntingtin gene (The Huntington's Disease Collaborative Research Group 1993), can be considered as \"trait marker\" of disease since its presence indicates that the disease will manifest without provided information on symptoms onset and disease progression. Recent discoveries of molecular pathways and molecules that are specifically affected in HD brain cells and mouse  models and that are present also at peripheral level, has led to the  prediction that such molecules/pathways might be similarly affected in peripheral cells from HD patients, thus representing peripheral biomarker of disease. In particular the identification of easily detectable disease-related changes in symptomatic and presymptomatic subjects is essential on one side to track the clinical state of patients and on the other to develop and monitor treatments aimed to delay disease onset and progression. Based on the evidence that the cortico-striatal synapse is importantly affected in HD (see Progress in Neurobiology, whole issue, Jan-Feb 2007) and that many effectors of neuronal activity  which are altered in HD are also present at peripheral level, we have conducted three hypothesis-driven studies on a large cohort of HD and control subjects. In particular we will report our data on i) the A2A receptor binding activity (Bmax), ii) the peripheral endocannabinoids system, and in particular the fatty acid amide hydrolase (FAAH) level and iii) Brain Derived Neurotrophic Factor (BDNF) levels.

P120

 

PROGRESSIVE REDUCTION OF CHOLESTEROL BIOSYNTHETIC PATHWAY IN THE R6/2 MOUSE MODEL OF HUNTINGTON'S DISEASE

 

M. Valenza1*, V. Leoni2, A. Tarditi1, C. Mariotti2, I. Bjorkhem3, S. DiDonato2 and E. Cattaneo1

 

1 Department of Pharmacological Sciences and Centre for Stem Cell Research, University of Milano, via Balzaretti 9, 20133, Milano-Italy; tel +39 0250318353; fax +39 02 50318284; email marta.valenza@unimi.it

2 Carlo Besta Neurological Institute, Milan, Italy

3 Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska University Hospital, Huddinge, Sweden

 

Huntington's disease (HD) is an adult onset neurodegenerative disorder caused by a CAG expansion in the HD gene and characterized by the clinical triad of movement disorder, dementia and psychiatric disturbance.

We have recently reported a significant reduction in mRNA levels of genes critical for the cholesterol biosynthetic pathway in brains from HD mice and patients, pointing to a biological dysfunction of this pathway. In agreement with those findings, here we show that the brain content of the cholesterol precursors lathosterol and lanosterol and the activity of 3-Hydroxy-3-methylglutaryl CoA reductase are progressively decreased over time in the brain of R6/2 transgenic mice with respect to control mice. We also confirmed our previous findings (Valenza et al., J. Neurosci. 2005) demonstrating that total cholesterol content as measured by an enzymatic method is significantly reduced with an outcome that reflects closely what provided in terms of lathosterol and lanosterol level measured by isotopic dilution Mass Spectrometry (MS). However, isotopic dilution MS reveals no changes in steady-state levels of brain cholesterol between mutant and control mice. Therefore the two methods produce different results, with the enzymatic method based on cholesterol oxidase providing information about total sterols but failing to detect absolute values of total cholesterol as instead one can reveal with MS.

Despite the steady-state levels of total cholesterol are similar in both genotypes, maybe due to long half-life of cholesterol in the brain, these results demonstrate that multiple steps of the cholesterol biosynthetic cascade are affected in the brain of R6/2 mice within twelfth week of survival.  This defect is an early event that progresses with disease evolution and generates lower levels of neo-synthesized cholesterol and its intermediates, which may affect aspects of the disease.

 

 

 

P121

 

REGIONAL PATTERNS OF CEREBRAL GLUCOSE METABOLISM IN SPINOCEREBELLAR ATAXIA TYPE 2, 3 AND 6: A VOXEL-BASED FDG-POSITRON EMISSION TOMOGRAPHY ANALYSIS

 

Wang PS (1,2,3), Liu RS (4), Yang BH (4), Soong BW (1,2)

(1) The Neurological Institute, Taipei Veterans General Hospital, Taiwan, Email: bwsoong@vghtpe.gov.tw

(2) Department of Neurology, National Yang-Ming University School of Medicine, Taipei, Taiwan.

(3) The Neurological Institute, Taipei Municipal Gan-Dau Hospital, Taiwan

(4) Department of Nuclear Medicine, National PET/Cyclotron Center, Taipei Veterans General Hospital, Taiwan

 

The purpose of this study was to investigate the regional patterns of cerebral metabolic deficits by voxel-based FDG-PET analysis in patients with distinct spinocerebellar ataxia (SCA) genotypes, including SCA type 2 (SCA2), SCA3, and SCA6. Nine patients with SCA2, 12 with SCA3, seven with SCA6, and 23 healthy control subjects were recruited. The clinical severity of the patients' cerebellar ataxia was evaluated according to the International Cooperative Ataxia Rating Scale. The brain glucose metabolism was evaluated with 2-[fluorine 18]-fluoro-2-deoxy-d-glucose (FDG) positron emission tomography. Group data were analyzed and compared by voxel-based analysis. In SCA2, FDG utilization was significantly reduced in the cerebellum, pons, parahippocampal gyrus and frontal cortex. In SCA3, FDG metabolism in the cerebellum, parahippocampal gyrus of the limbic system, and lentiform nucleus was decreased. In SCA6, FDG metabolism was diminished in the cerebellum and the frontal and prefrontal cortices. On group comparisons, while all SCAs have impaired cerebellar functions, the cerebellar FDG metabolism was most severely compromised in SCA2. Instead, the FDG metabolism in the lentiform nucleus and medulla was characteristically worst in SCA3. There was no brainstem involvement in SCA6.