Poster Session PPo5: Basic Mechanisms of Neurodegeneration and Pathology

PPo5-1: Deposition of Multiple Proteins in E200K Genetic Creutzfeldt-Jakob Disease

Gabor G. Kovacs,^1,2 Jérémie Seguin,³ Isabelle Quadrio,³ Romana Höftberger,¹ István Kapás,² Nathalie Streichenberger,³ Anne Gaëlle Biacabe,⁴ David Meyronet,³ Raf Sciot,⁵ Rik Vandenberghe,⁶ Katalin Majtenyi,² Thomas Ströbel,¹ Herbert Budka¹ and Armand Perret-Liaudet³

¹Institute of Neurology; Medical University Vienna; and Austrian Reference Center for Human Prion Diseases; Vienna, Austria; ²Neuropathology and Prion Disease Reference Center; Hungarian Reference Center for Human Prion Diseases; Semmelweis University; Budapest, Hungary; ³Prion Disease Laboratory; Pathology and Biochemistry; Groupement Hospitalier Est; Hospices Civils de Lyon/Claude Bernard University; Lyon, France; ⁴Agence Française de Sécurité Sanitaire des Aliments; Unité ATNC; Lyon, France; ⁵Department of Pathology; University Hospital; Catholic University of Leuven; Leuven, Belgium; ⁶Neurology Department; University Hospital Gasthuisberg; Leuven, Belgium

Key words: E200K mutation, prion protein gene, tau, amyloid-beta, a-synuclein

The E200K mutation is the most frequent prion protein gene (PRNP) mutation detected worldwide that associates with genetic Creutzfeldt-Jakob disease (CJD). We performed a comprehensive neuropathological and biochemical study of brains from 39 individuals carrying the E200K PRNP mutation. Although there was a relatively uniform anatomical pattern of tissue lesioning, the deposition of disease-associated PrP was influenced by the codon 129 constellation, including different or mixed types of PrPres detected by immunoblotting. Some PrP deposition features have not been described in sporadic CJD, like prominent intraneuronal PrP deposition involving also brainstem nuclei. In addition, parenchymal amyloid-β was observed in 53.8% of cases, amyloid angiopathy (A) in 23.07%, phospho-tau immunoreactive neuritic profiles in 92.3%, neurofibrillary degeneration in 38.4%, new types of tau pathology in 33.3%, and Lewy-type α-synuclein pathology in 15.4%. TDP-43 and FUS immunoreactive protein deposits were not observed. Although age-associated and additional neurodegeneration has been described in prion diseases, here we demonstrate intensified and combined neurodegeneration in a genetic prion disease due to a single point mutation, which might become an important model to decipher the molecular interplay between neurodegeneration-associated proteins.

PPo5-2: Temporal Kinetics of Prion Protein Accumulation and Its Effect on Neurotransmitters in the Cerebellum of Guinea Pigs Infected with BSE Prion

Shoichi Sakaguchi,¹ Motohiro Horiuchi,² Yoshio Yamakawa³, Tetsutaro Sata³ and Hidefumi Furuoka¹

¹Department of Pathobiological Science; Obihiro University of Agriculture and Veterinary Medicine; Inada-cho, Obihiro Japan; ²Department of Prion Diseases; Graduate School of Veterinary Medicine; Hokkaido University; Kita-ku, Sapporo Japan; ³Department of Pathology; National Institute of Infectious Diseases; Toyama, Tokyo Japan

Key words: BSE, guinea pig, cerebellum, GABAergic synapse, glutamatergic synapse

Cerebellar lesions in guinea pigs (GPs) inoculated intracerebrally with BSE prion are characterized by severe atrophy of the cerebellar cortex associated with PrPBSE accumulation. In this study, we examined the temporal kinetics of PrPBSE accumulation in the cerebellum of GPs infected with BSE prion and an immunohistochemical fluctuation of GABAergic and glutamatergic synapses using antibody for the vesicular transporter. The GPs inoculated intracerebrally with PrPBSE were sacrificed under anesthesia at 172, 203, 230, 263, 305, 312 and 323 dpi. Immunohistochemical analysis was carried out with anti-prion protein(mAb12F10), GFAP, Calbindin and MAP2 antibodies. For analysis of synapse expression, anti-synaptophysin, GABAergic synapse’s marker (VGAT), glutamatergic synapse’s marker (VGLUT1, VGLUT2) antibodies were used. In the GP control, VGLUT1 positive synapses were seen in the pontine nucleus and cerebellar cortex, while VGLUT2 positive synapses were seen in the pontine, olivary and cerebellar nucleuses and cerebellar cortex. VGAT positive synapses showed similar distribution as VGLUT2 positive synapses except in the pontine nucleus. PrPBSE accumulation was observed in the pontine and olivary nucleuses, and cortex of vestibulocerebellum at on early stage, and then seemed to extend throughout the whole brainstem and cortex of the cerebrocerebellum with time. In PrPBSE accumulated regions, VGLUT1 positive synapses apparently decreased, however, VLUT2 and VGAT positive synapses preserved a normal appearance. In the GPs experimentally infected with BSE, accumulation of PrPBSE were similar to the distribution of VGLUT1 positive glutamatergic synapses, and synapses diminution associated with PrPBSE seemed to reflect a decrease in VGLUT1 positive glutamatergic synapses.

PPo5-3: Tau Protein Inhibits Tubulin Oligomerization Induced by Prion Protein

Krzysztof Nieznanski, Katarzyna M. Osiecka and Hanna Nieznanska

Department of Biochemistry; Nencki Institute of Experimental Biology; Warsaw, Poland

Key words: prion protein, tubulin, tau protein, MAPs

Tubulin is the major building block of microtubules—dynamic cytoskeletal structures involved in numerous cellular functions. In previous studies we have demonstrated that prion protein (PrP) interacts with tubulin and disrupts microtubular cytoskeleton by inducement of tubulin oligomerization. These observations may explain molecular mechanism of toxicity of cytoplasmic PrP in transmissible spongiform encephalopathies (TSEs). Herein, we check whether microtubule associated proteins (MAPs)—molecules regulating stability of microtubules—influence oligomerization of tubulin by PrP. We have found that tubulin preparations depleted of MAPs are more susceptible to oligomerization by PrP than the ones containing traces of these proteins. Tau protein—one of the major members of the MAPs family—significantly reduces the effect of PrP. Furthermore, phosphorylation of tau protein regulates its ability to affect PrP-induced oligomerization of tubulin. We did not observe competition between tau and PrP for binding on tubulin. Hence, we hypothesize that binding of tau protein stabilizes tubulin in the conformation which is less susceptible to oligomerization by PrP. Since elevated phosphorylation of tau leading to loss of its function is observed in tauopathies, our studies my provide possible molecular link between these neurodegenerative disorders and TSEs.

PPo5-4: Changes In Hsp Gene and Protein Expression in Natural Scrapie Related with Brain Damage

Inmaculada Martín-Burriel,¹ Carmen Serrano,¹ Rosa Bolea,² Jaber Lyahyai,¹ Hicham Filali,² Luis Varona,³ Ane Marcos-Carcavilla,⁴ Cristina Acín,² Jorge H. Calvo,⁵ Magdalena Serrano,⁴ Juan J. Badiola² and Pilar Zaragoza¹

¹Laboratorio de Genética Bioquímica (LAGENBIO); ²Centro de Investigación en Encefalopatías y Enfermedades Transmisibles Emergentes; ³Unidad de Genética Cuantitativa y Mejora Animal; Facultad de Veterinaria; Universidad de Zaragoza; Zaragoza, Spain; ⁴Departamento de Mejora Genética Animal; INIA; Madrid Spain; ⁵Unidad de Tecnología en Producción Animal; CITA; Zaragoza, Spain

Key words: heat shock protein

Heat shock proteins (Hsps) play cytoprotective roles such as apoptosis regulation and inflammatory response control. These proteins also can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits the correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsps in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70 and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the analysed areas. Whereas changes in transcript levels were not observed in the cerebellum and medulla oblongata, a significant decrease of HSP27 and HSP90 was detected in the prefrontal cortex. On the contrary, HSP73 was overexpressed in diencephalons of scrapie animals. Expression changes obtained by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using a stepwise regression. Changes in Hsp gene and protein expression were associated with prion deposition, astrogliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunostaining in Purkinje cells of scrapie sheep. By contrast, controls displayed little or null staining in these cells. The differences observed in gene expression and protein distribution suggest that the analysed heat shock proteins play a role in the natural form of the disease.

PPo5-5: Cerebellar Granule Neurons Loss and PrP^Sc Deposition Pattern in Sporadic Creutzfeldt-Jakob Disease: Variation with M129V PRNP Polymorphism and PrP^Sc Type

Baptiste A. Faucheux,^1,2 Emilie Morain,^1,2 Vanessa Diouron,^1,2 Jean-Philippe Brandel,^2,3 Dominique Salomon,³ Veronique Sazdovitch,^1,2 Nicolas Privat,^1,2 Jean-Louis LaPlance,⁴ Jean-Jacques Hauw and Stephane Haïk^1-3

¹AP-HP; Neuropathology Lab; Salpetriere Hospital; Paris, France; ²ICM-Brain and Spine Institute Research Centre; INSERM UMRS975; CNRS UMR7225; Pierre and Marie Curie University; Paris, France; ³AP-HP; National Reference Unit for Creutzfeldt-Jakob Disease; Salpetriere Hospital; Paris, France; ⁴AP-HP; Molecular Biology Dept; Lariboisiere Hospital; Paris, France

Key words: prion, neurodegeneration, cerebellum, neuron, cell death, Creutzfeldt-Jakob

Neuronal death is a major neuropathologic hallmark in prion diseases, but the mechanisms involved in neurodegeneration are still poorly understood. Although the toxicity of disease-related prion protein (PrP^Sc) has been proposed to play a central role, the association between its deposition and neuronal loss varies with the studied models. We investigated neuronal loss in the granule layer of the cerebellum from cases of the six subtypes of sporadic Creutzfeldt-Jakob disease (sCJD; n = 100) that can be determined according to the M129V PRNP polymorphism and molecular-isotype of PrP^Sc. Numerical density of neurons was estimated with a computer-assisted image analysis system and the accumulation of PrP^Sc deposits was scored. The scores of PrP^Sc deposits of the punctate-type (synaptic type) were correlated with neuron counts-the higher the score, the higher the neuronal loss- in the sCJD-VV2 and other sCJD groups. By contrast, the scores of large PrP^Sc deposits of the focal type were inversely correlated with neuronal counts in the sCJD-VV2 group. Furthermore, scores of large PrP^Sc deposits were not associated with increased neuronal loss in the sCJD-MV2 group. Our results support the hypothesis of putative pathogenic roles for PrP^Sc through neurotoxic mechanisms within the human brain. These findings are in accordance with the view that punctate deposits may point out the anatomical location of neuronal death. The present data also indicate that PrP^Sc aggregation in large deposits may have an effect on neuronal death opposite to that of PrP^Sc punctate deposits.

PPo5-6: Aminoterminally-Truncated Human Prion Protein Fragment hPrP90-231 Forms Intracellular Aggregates and Causes Lysosomal Loss of Impermeability

Stefano Thellung, Alessandro Corsaro, Valentina Villa and Tullio Florio

Department of Oncology; Biology and Genetics Sect; Pharmacology; University of Genova; Genova, Italy

Key words: hPrP90-231, intracellular ggregation, lysosmial permeabilization, cell death

Intracellular accumulation of recombinant hPrP90-231 has been related to cell death in SH-SY5Y neuroblastoma cells. We analysed the cellular alterations induced by hPrP90-231 intracellular accumulation with the aim to study the role of PrPRes intracellular deposits in prion diseases-related neuronal death. Internalisation of hPrP90-231 was determined by confocal live imaging after cell exposure to fluorescein-tagged hPrP90-231. We report that hPrP90-231 forms cytosolic aggregates that increase in size and number along with exposure time; using viable cell probes with selective tropism for intracellular organelles, we observed that hPrP90-231 accumulates in organelles of the endolysosomial system. The possibility that hPrP90-231 intralysosomial accumulation could determine SH-SY5Y cell death was addressed by evaluating proteolysis sensitivity and aggregation state of the internalised prion fragment and lysosomes permeability in hPrP90-231-treated cells. First, we demonstrated that the internalised hPrP90-231 displays a partial resistance to proteolysis and can be recovered almost completely into the detergent-insoluble fraction of total cell proteins. Moreover, total cell lysate resolution through agarose gel electrophoresis, conducted in semi-denaturing conditions, revealed that such hPrP90-231 clusters display features of aggregation. Lysosomal integrity has been evaluated by assessing lysosomes capability to segregate the fluorescent probe Lucifer Yellow (LY). Confocal microscopy analysis showed that, whereas control cells evidenced a punctate pattern of LY fluorescence (index of dye accumulation into healthy lysosomes), hPrP90-231-treated cells showed a more diffuse fluorescent signal, indicating a cytosolic redistribution of LY. In conclusion, these data suggest that exogenous hPrP90-231 accumulates within SH-SY5Y cells in intralysosomial deposits having features of insoluble, protease-resistant aggregates that may trigger a lysosome-mediated apoptosis inducing lysosomes membrane permeabilization.

Acknowledgements

This work was supported by grants from Italian Ministry of University and research (MIUR-PRIN 2008) and Compagnia San Paolo to T.F.

PPo5-7: Dynamin-related Protein 1 via Protein Phosphatase 2A Regulatory Subunit B?2 Promotes Mitochondrial Fission-mediated Neuronal Apoptosis in Prion Knock-down Cell Line

Yu-Mi Roh,¹ Sang-Gyun Kang,² Seung-Bin Cha,¹ Won-Jung Lee,¹ Min-Kyoung Shin,¹ Myoung-Hwan Jung¹ and Han Sang Yoo^1,*

¹Department of Infectious Diseases; College of Veterinary Medicine; BK21 for Veterinary Science and KRF Zoonotic Priority Research Institute; Seoul National University; Seoul, Korea; ²Centre for Prions and Protein Folding Diseases; University of Alberta; Edmonton, Canada

Key words: prion, Ppp2r2b, Drp1, mitochondria, apoptosis

Introduction. Prion diseases are caused by the conversion of normal cellular PrPC into pathogenic isoform (PrPSc), which results in PrPSc accumulation as well as PrPC deficiency in the infected host. The accumulated PrPSc is not the sole factor of prion disease but the reduced PrPC has been reported to contribute to neuronal loss. Focusing on the depletion of PrPC, we investigated a linkage between regulatory Bα2 subunit of protein phosphatase 2A (PP2A/Bα2) mediated-mitochondrial fission and neuronal apoptosis.

Results. The expression of both Ppp2r2b and Drp1 were increased in N2amiRdual cells compared with control cells. In response to serum deprivation, the amount of cytochrome C in cytosol and activity of caspase 3 were increased in N2amiRdual.

Materials and Methods. The expression level of PP2A/Bα2 gene (Ppp2r2b) and Drp1 were evaluated by quantitative RT-PCR and western blot, respectively, in prion knock-down mouse neuroblastoma cell line (N2amiRdual). Apoptosis was assessed by level of cytosolic cytochrome C and activity of caspase 3. The proportion of apoptotic cells were determined by fluorescence-activated cell sorting of Annexin V-Cy3 bound cells under serum withdrawal condition.

Conclusion. Our new hypothesis is that neuronal loss in prion diseases occurs through depletion of PrPC following increase of PP2A/Bα2, which drives Drp1 to induce mitochondrial fragmentation resulted in neuronal apoptosis by releasing cytochrome C and activating caspase family.

Acknowledgements

This study was supported by BK21, KRF-2006-005-J502901 and Research Institute of Veterinary Science, Seoul National University, Korea.

PPo5-8: The Fate of Mutant PrP in Human Genetic Prion Disease: Linking Autophagy and Aggresome Formation

Lajos László,¹ Gergö Botond,² Herbert Budka² and Gabor G. Kovacs²

Key words: autophagy, aggresome, mutant PrP

In a systematic study of human genetic prion disease with E200K mutation, we observed widespread intraneuronal dot-like PrP immunoreactivity. To evaluate the precise subcellular location and functional implications of this type of immunoreactivity, frontal cortex and medulla oblongata samples from 5 individuals with E200K mutation were investigated by immunohistochemistry for PrP, double immunolabeling for ubiquitin, p62, early endosomal marker rab5, and immunogold electron microscopy for ultrastructural localisation of disease-associated PrP. Intraneuronal PrP immunoreactivity was represented by dots or larger globular structures. These PrP immunoreactive globules were not detectable using antibodies against the N-terminal fragment of PrP. While the small dot-like PrP immunoreactivity was present in early endosomes, some larger globular inclusion-body-like structures showed co-localisation with ubiquitin and occasionally with p62. Ultrastructural examination revealed intense PrP localisation in huge aggresomes and in autophagic vacuoles containing smaller aggresome-like structures (ALIS). Inclusion-body-like intracellular accumulation of PrP is unusual in human prion diseases. The physiological functional coupling of constitutive autophagy to ubiquitin-proteasome system (UPS) provides a complementary housekeeping mechanism for clearance of unwanted proteins, preventing the accumulation of misfolded proteins, mitigating ER stress, and reducing cell death. Our morphological results support the notion that the permanent production of mutant PrP undermines this sophisticated collaborative defence mechanism in human genetic prion disease. Overwhelming of the UPS shifts the balance towards autophagy and ubiquitinated inclusion-body formation. However, formation of aggresomes is considered to be also cytoprotective. This first report of intraneuronal PrP inclusion bodies in a genetic prion disease parallels events in other neurodegenerative diseases.

PPo5-9: Differential Gene Expression in BSE-inoculated Macaques

Ann-Christin Schmädicke,¹ Lisa Gasperini,² Dirk Motzkus,¹ Cristiano Corona,³ Maria Novella Chieppa,³ Chiara Porcario,³ Stefano Gustincich,² Cristina Casalone,³ Gabriela Salinas-Riester,⁴ Lennart Opitz⁴ and Giuseppe Legname²

¹Unit of Infection Models; German Primate Centre; Göttingen, Germany; ²Neurobiology sector; International School for Advanced Studies (SISSA); Trieste, Italy; ³Istituto Zooprofilattico Sperimentale del Piemonte; Liguria e Valle d’Aosta (IZSTO); Turin, Italy; ⁴DNA Microarray Facility; University Medicine; Göttingen, Germany

Key words: prion, BSE, gene expression, non-human primates, genome microarray, neurodegeneration

Prion diseases are a class of fatal neurodegenerative disorders characterized by abnormal protein deposits. Despite many recent advances in prion research, the molecular mechanisms causing neuronal death have not been established yet. We have used a macaque model of BSE-infection to identify dysregulated transcripts in infected vs. uninfected animals. For our analysis we have isolated high quality RNA from the gyrus frontalis region of seven BSE-infected and five non-infected control macaques, respectively. The RNA was labeled and used for whole genome microarrays (Affymetrix). Bioinformatic analysis revealed that not more than 100 transcripts were significantly up or downregulated more than twofold. Beside others, we found upregulation of α1-antichymotrypsin, that has also been described in scrapie-infected mice (Miele et al. PLoS One 2008) and Alzheimer’s disease. We are currently validating the most regulated candidates using quantitative RT-PCR. Our systematic approach will identify genes that can provide novel insights into prion disease or neurodegeneration. The use of non-human primates (Macaca fascicularis) is the model of choice to expand this study to human CJD and vCJD.

PPo5-10: Manipulation of Endogenous Glycosaminoglycans to Affect Uptake and Trafficking of Prions in Genetic Prion Disease

Jeremy M. Welton¹, Laura Ellett¹, Andrew F. Hill^2,4, Steven J. Collins^1,3,4 and Victoria A. Lawson^1,4

¹Department of Pathology; The University of Melbourne; Australia; ²Department of Biochemistry and Molecular Biology; Bio21 Institute; The University of Melbourne; Australia; ³The Australian National Creutzfeldt-Jakob Disease Registry; Australia; ⁴Mental Health Research Institute of Victoria; Australia

Key words: glycosaminoglycans, chlorate, immunofluorescence, genetic prion disease, P101L

Prion diseases are transmissible, neurodegenerative disorders affecting both humans and animals. The principle characteristic of prion diseases is the aggregation and deposition of misfolded form (PrP-D), of the host encoded prion protein (PrP-C). A subset of prion diseases is associated with mutations within the prion protein gene. Glycosaminoglycans are one of the major binding partners identified in PrP-D infection and are reported to facilitate binding and entry of the PrP-D into cells. Modification of glycosaminoglycans has been shown to profoundly alter the outcome of cell based models of prion infection. In this project the influence of a mutation in the prion protein gene on prion disease was investigated.

The RK13 (rabbit kidney epithelial) cell line was used to investigate the effect of mutations in the prion protein gene on glycosaminoglycan mediated prion infection. Following chlorate treatment, to reduce endogenous glycosaminoglycan sulfation, wild-type PrP had a predominantly perinuclear localization. In contrast P101L PrP localization was unaltered. This result suggests that PrP carrying a genetic mutation interacts differently with glycosaminoglycans. The requirement of glycosaminoglycan expression for PrP-D binding and infection of cells expressing the P101L mutation, and the ability to cure P101L prion infected cells was investigated.

Understanding the effect of the cellular environment on genetic prion disease may identify molecular events involved in the development of disease, and targets for prophylactic and therapeutic intervention.

PPo5-11: Inducible Neuronal PrP Knockout Mice Reveal Potential Therapeutic Window for TSE Intervention

Barry M. Bradford,¹ Alison S. Marshall,¹ Deborah A. Brown,¹ Dorothy J. Kisielewiski,¹ Nadia L. Tuzi,² Pedro Piccardo,³ Alan Clarke,⁴ V. Hugh Perry⁵ and Jean C. Manson¹

¹The Roslin Institute and R(D)SVS; The University of Edinburgh; Neuropathogenesis Division; Roslin Biocentre; Roslin, UK; ²University of Edinburgh; Biology Teaching Organisation; Darwin Building; Kings Buildings; Edinburgh, UK; ³Center for Biologics Evaluation and Research; Food and Drug Administration; Rockville, USA; ⁴Cardiff School of Biosciences; Life Sciences Building; Museum Avenue, Cardiff UK; ⁵School of Biological Sciences; University of Southampton; Boldrewood Campus; Southampton, UK

Key words: neurodegeneration, tamoxifen inducible Cre/LoxP, PrP knockout

It is known that expression of the host form of PrP protein (PrP^C) is required for susceptibility to and the establishment of transmissible spongiform encephalopathies (TSEs). We have produced a Cre/LoxP conditional PrP knockout mouse model in which administration of tamoxifen results in the removal of PrP^C expression from neurones only. Using this model we have previously demonstrated that removal of PrP^C expression from neurones prior to infection with the ME7 mouse-adapted Scrapie strain dramatically altered pathology, preventing spongiosis and neuronal loss in specific neuronal populations, and greatly extended disease incubation period.

As a further application of this model, neuronal PrP knockout was induced in groups of mice at various time-points post intra-cerebral ME7 infection to assess the impact upon disease pathogenesis. Results indicate that early in the incubation period there are time-points where neuronal PrP^C expression is essential for ‘normal’ disease progression, but at later stages removal of neuronal PrP^C no longer influenced disease incubation period. This critical time-point occurs after known pathologies such as synaptic loss have become established within the central nervous system.

These results assist in informing us of the basic mechanisms in TSE neurodegeneration and also of a ‘time-window’ when potential therapeutic intervention involving the reduction of PrP^C expression may be possible in TSE diseases. The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy.

PPo5-12: Mitochondrial Dysfunction via Differential Modulation of Mitochondrial Fusion/Fission Proteins in the Brains of Scrapie-Infected Mice

Hong-Suc Choi,^1,2 Jae-Min Oh,¹ Hae-Young Sin,¹ Jae-Kwang Jin,¹ Eun-Kyoung Choi,¹ Richard I. Carp³ and Yong-Sun Kim^1,2

¹Ilsong Institute of Life Science and ²Department of Microbiology; College of Medicine; Hallym University; Republic of Korea; New York State Institute for Basic Research in Developmental Disabilities; Staten Island, NY USA

Key words: mitochondrial fusion, fission, scrapie, prion disease

Mitochondrial dysfunction, a common characteristic of neurodegenerative diseases, is one of the prominent features of prion diseases and is induced by oxidative stress in scrapie-infected animal models. In previous studies, we have found mitochondrial dysfunction with elongated mitochondria in the brains of scrapie-infected mice compared to controls. However, the mechanisms underlying mitochondrial dysfunction are still unclear. To examine whether the modulation of mitochondrial dynamics is involved in mitochondrial dysfunction in prion disease, we investigated the expression patterns of mitochondrial dynamic proteins in the brains of control and scrapie-infected mice. Immunoblot analysis revealed that upregulated Mfn1 was found both in whole brain and in dissected brain regions including cerebral cortex and hippocampus of scrapie-infected mice. In addition, the increased expression levels of Fis1 and Mfn2 were found in the striatum, respectively. However, Dlp1 was significantly reduced in the hippocampus of scrapie-infected brain. We also found a very rare distribution of Fis1 and Dlp1 and increased immunogold-labeled Opa1 in the mitochondria of scrapie-infected hippocampus. Finally, we observed abnormal mitochondrial elongation and damaged mitochondria in the hippocampus of scrapie-infected brain. These results suggest that mitochondrial dysfunction caused by abnormal regulation of mitochondrial dynamic proteins may lead to neuropathological changes in prion diseases.

Acknowledgements

This work was supported by a grant (Code #20080401034016) from BioGreen 21 Program, Rural Development Administration and by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs (A085082) Republic of Korea.

PPo5-13: Neuron Dysfunction is Induced by Mutated Prion Protein and Reversed by Sirtuin Activation in C. elegans

Nicolas Bizat,^1,2 Stephane Haïk,¹ Patrice Beaudry,^1,3 Jean-Michel Peyrin,⁴ Jean-Louis Laplanche² and Christian Neri⁵

¹Université Pierre et Marie Curie; ²Université Pierre et Marie Curie; ³Université Paris Denis Diderot; UMR 975; Centre de Recherche de l’Institut du Cerveau et de la Moelle Epinière; G.H. Pitié-Salpêtrière; CNRS UMR 7225; Paris, France; ⁴Université Pierre et Marie Curie; CNRS UMR 7102; Neurobiologie des processus adaptatifs; Paris; ⁵Institut National de la Santé et de la Recherche Médicale (Inserm) Unit 894; Laboratory of Neuronal Cell Biology and Pathology; Paris, France

Introduction. The activity of cellular prion protein (PrP) involves a variety of ligands and signal pathways. The study of PrP molecules carrying pathogenic mutations may provide insight into the mechanism of prion neurotoxicity. Caenorhabditis elegans is a nematode with well defined neurons connexions, showing about 45% genes homology with human, but without PrP homolog.

Results. The expression of octarepeat-expanded PrP in these mechanosensory neurons led to a progressive loss of touch test response, without causing cell death and that was associated to PrP clustering, PrP partial resistance to proteinase K digestion and PrP insolubility in detergent. Loss of function of src-2 reduced PG13-PrP neurotoxicity while loss of function of sir-2.1/SIRT1, showed aggravation. Increased transgenic expression of sir-2.1 in worm reversed PG13-PrP neurotoxicity. Resveratrol, a polyphenol known to act through sirtuins for neuroprotection, reverse mutant neurotoxicity in sir-2.1 dependant manner.

Methods. We previously identified a family with GSS phenotype disease, associated to eight extra octarepeats in PrP protein (PG13-PrP). We expressed this mutated PrP in six mechanosensory neurons of the nematode. A touch test explored the physiological worm motility, and so these neurons dysfunctions.

Conclusion. Fyn contributes to the mediation of PrP mutant neurotoxicity. Sirtuins activation mitigates the neurotoxic effects. The nematode C. elegans is a suitable model to study the molecular mechanisms of cellular dysfunctions induced by mutant prion proteins and to identify modulator molecules that may protect neuron from the detrimental effects of misfolded PrP conformers.

PPo5-14: Alteration of Secretory Protein Trafficking as Possible Pathogenetic Mechanism in Inherited Prion Diseases

Elena Restelli,^1,2 Susanna Mantovani^1,2 and Roberto Chiesa^1,2

¹Dulbecco Telethon Institute and ²Department of Neuroscience; Mario Negri Institute for Pharmacological Research; Milan, Italy

Familial prion diseases are dominantly inherited neurodegenerative disorders linked to mutations in the prion protein (PrP) gene on chromosome 20. PrP mutations are associated with defined clinical and neuropathological phenotypes: Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI) or Gerstmann-Sträussler-Scheinker (GSS) syndrome. There is evidence that the pathogenic mutations favor PrP misfolding, but how this leads to neurological dysfunction, and how different PrP mutations may encode the information to specify distinct disease phenotypes is still unknown. We found phenotypic differences in cultured neurons expressing CJD-, FFI- and GSS-linked mutations, with different mutant PrPs accumulating in different compartments of the secretory pathways and producing specific abnormalities in intracellular organelles. On the basis of these observations, we hypothesized that mutant PrP misfolding in the secretory pathway may interfere with the secretory transport of proteins important for synaptic transmission, and that the phenotypic heterogeneity may be due to different mutants affecting different steps of intracellular trafficking.

To investigate this hypothesis, we set-up the conditions to test the efficiency of the membrane trafficking system by monitoring the intracellular transport of well-established cargo reporters, such as temperature-sensitive vescicular stomatite virus, in immortalized cell lines and primary neurons expressing different mutant PrPs.

Preliminary observations are consistent with the hypothesis that intracellular accumulation of mutant PrP may cause abnormalities of secretory trafficking.

PPo5-15: Enhanced Susceptibility to Kainate-induced Seizures in a Transgenic Mouse Model of Inherited Creutzfeldt-Jakob Disease

Ilaria Bertani,^1,2 Mattia Maroso,² Susanna Mantovani,^1,2 Annamaria Vezzani² andd Roberto Chiesa^1,2

¹Dulbecco Telethon Institute and ²Department of Neuroscience; Mario Negri Institute for Pharmacological Research; Milan, Italy

Transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders caused by an abnormally folded form of the cellular prion protein (PrP^C). Creutzfeldt-Jakob disease (CJD) is the most common human TSE, recognized clinically by global cortical dementia, motor abnormalities and myoclonus. Tg(CJD) mice express the mouse homolog of the D178N/V129 PrP mutation associated with a familial form of CJD. These mice develop cognitive, motor and neurophysiological abnormalities, associated with PrP deposition, gliosis, and loss of calretinin (CR)-positive neurons in the hippocampus and cerebral cortex (Neuron 60:598-609, 2008). Several data demonstrated that mice lacking PrP^C (PrPko) have increased susceptibility to kainate (KA)-induced seizure, associated with significant cell death in the hippocampus.

Immunohistochemical analysis of presymptomatic Tg(CJD) mice show activation of microglia and increased expression of IL-1beta, in the absence of astrocytic activation or degeneration of CR-positive interneurons. These changes may play an active role in hyperexcitability of the epileptic tissue (Vezzani et al. Brain Behav Immun 2008; 22:797–83). Indeed, EEG analysis of young presymptomatic Tg(CJD) mice after intrahippocampal injection of KA, showed increased number of seizures, and longer time in ictal activity as compared to PrPko and wild-type mice.

Our results show proinflammatory changes in brain of presymptomatic Tg(CJD) mice that may alter their seizure threshold; this phenomenon could explain the increased frequency of seizures observed in CJD patients.

PPo5-16: Characterization of the Properties and Trafficking of Truncated PrP Mutants Associated with Two Different Prion Diseases

Manuela Pozzoli,² Ilaria Bertani^1,2 and Roberto Chiesa^1,2

¹Dulbecco Telethon Institute and ²Dept. of Neuroscience; Mario Negri Institute for Pharmacological Research; Milan, Italy

Familial prion diseases are neurodegenerative disorders linked to point mutations or insertions in the prion protein (PrP) gene. Stop codon mutations have been reported in few patients with PrP cerebral amyloid angiopathy (PrP-CAA). This disease is characterized by PrP amyloid deposition in blood vessels without gray matter spongiosis. Recently, two patients with new stop codon mutations at position 226 (Y226Stop) and 227 (Q227Stop) have been described (Jansen C, et al. Acta Neuropathol 2010; 119:189–97). They showed PrP amyloidosis with different clinicopathological phenotypes. The patient carrying Y226Stop had typical PrP-CAA, whereas the patient with Q227Stop showed an unusual Gerstmann-Sträussler-Scheinker phenotype with cerebral amyloid plaques and neurofibrillary lesions.

To investigate the cellular and molecular basis of this phenotypic difference, we expressed mouse PrP homologues of the 226 and 227 stop codon mutations in different cell lines.

Mouse PrP cDNAs carrying a stop mutation at codon 225 or 226 (homologous to human codons 226 and 227, respectively) were generated by overlapping PCR, and cloned into the expression vector pCDNA3.1. These constructs were expressed in different cells, including HEK293, HeLa and SH-SY5Y. The biochemical properties, metabolic stability and cellular localization of the mutant PrPs were analyzed.

Preliminary results indicate that the mutated proteins are expressed at lower levels compared to wild-type PrP, probably due to proteasomal degradation. Interestingly, the two mutants showed different patterns of cellular localization, suggesting that their different cellular metabolism may underlie the different disease phenotypes.

PPo5-17: A Mutant Prion Protein Causes Impairment of Voltage-gated Calcium Channels

Assunta Senatore,^1,2 Simona Colleoni,² Claudia Verderio,³ Elena Restelli,^1,2 Susanna Mantovani,^1,2 Steven Condliffe,³ Gianluigi Forloni,² Michela Matteoli,³ Marco Gobbi² and Roberto Chiesa^1,2

¹Dulbecco Telethon Institute; ²Istituto di Ricerche Farmacologiche Mario Negri; Milan, Italy; ³CNR Institute of Neuroscience; University of Milan; Italy

Key words: mutant PrP, synaptic dysfunction, voltage-gated calcium channels

Prion diseases are neurodegenerative disorders of infectious, sporadic or genetic origin, due to conversion of the cellular prion protein (PrP^C) into a conformationally altered isoform. Accumulation of misfolded PrP ultimately causes neurodegeneration but the underlying molecular mechanisms are poorly understood. Tg(PG14) mice, expressing a PrP insertional mutation associated with an inherited prion disease, accumulate in their brains a misfolded and aggregated form of the mutant protein. These mice become ataxic and display synaptic degeneration and massive loss of granule neurons in the cerebellum associated with PrP deposition. To explore the hypothesis that mutant PrP alters synaptic function in the cerebellum as a primary pathogenic event, we analyzed the motor behaviour and the cerebellar function of the mice at different ages.

Analysis showed an early impairment of motor coordination correlating with defective glutamatergic synaptic function in the cerebellum. Compared to controls, cerebellar synaptosomes from Tg(PG14) mice showed impaired glutamate exocytosis associated with reduced calcium influx upon depolarization. These defects were also found in primary cultures of cerebellar granule neurons from newborn Tg(PG14) mice. Electrophysiological analysis of these cells detected a significant impairment of voltage-gated calcium channel (VGCC) function which was directly linked to intracellular accumulation and aggregation of mutant PrP. Studies are in progress to clarify the molecular mechanisms by which PG14 PrP misfolding causes the VGCC defect, and identify potential therapeutic targets.

PPo5-18: Redox-iron and Prion Disease Pathogenesis

Neena Singh, Ajay Singh

Case Western Reserve University, Cleveland, OH USA

Key words: iron, prion

We will present data that suggest a major role of redox-iron in prion disease pathogenesis. Our observations on cell and mouse models indicate that prion protein (PrP^C) functions as an iron uptake and transport protein, and absence of PrP^C in PrP knock-out (PrPKO) mice induces systemic iron deficiency, a phenotype that is rescued by expressing PrP^C on the PrPKO background. Pathogenic and non-pathogenic mutations of PrP alter cellular iron status differentially, suggesting a dominant role of PrP^C in cellular iron metabolism. More importantly, aggregation of PrP^C to the PrP^Sc form induces imbalance of brain iron homeostasis in sporadic Creutzfeldt-jakob-disease (sCJD) affected human and scrapie infected animal brains that show upregulation of iron uptake proteins transferrin (Tf) and transferrin receptor in the presence of increased brain iron. The phenotype of ‘apparent’ iron deficiency increases with disease progression, and is probably due to sequestration of iron in a biologically unavailable form in SDS insoluble and heat stable protein complexes that include PrP^Sc and ferritin. There is a direct correlation between PrP^Sc levels and iron deficiency, suggesting that brain iron imbalance results from the combined effect of loss of normal function of PrP^C in iron uptake and sequestration of iron in PrP^Sc-protein complexes. Since iron is highly redox-active and neurotoxic if mismanaged, these observations have significant implications for prion disease pathogenesis.

Our hypothesis implicating iron in prion disease pathogenesis is supported by another report using a structural biology approach (Hwang et al. 2009), and appeared in Nature Journal Club discussion (Kell, August 2009).

PPo5-19: Generation and Characterization of Transgenic Mice Expressing PrP Mutations Linked to Inherited Creutzfeldt-jakob Disease and Fatal Familial Insomnia

Susanna Mantovani,^1,2 Ilaria Bertani,^1,2 Gianluigi Forloni² and Roberto Chiesa^1,2

¹Dulbecco Telethon Institute; ²Department of Neuroscience; Mario Negri Institute for Pharmacological Research; Milan, Italy

Key words: prion, fatal familial insomnia, mouse model

Fatal familial insomnia (FFI) and a subtype of familial Creutzfeldt-Jakob disease (CJD178) are linked to the D178N mutation in the gene encoding PrP. The disease phenotype is determined by the M/V polymorphism at codon 129 of the mutant allele: D178N/M129 segregates with FFI, while D178N/V129 with CJD. We have generated transgenic (Tg) mice expressing the mouse PrP homologue of the CJD178 mutation (D177N/V128) containing the epitope tag for monoclonal antibody 3F4. These mice synthesize a misfolded PrP and develop pathological features of CJD178, including motor dysfunction, memory impairment, and EEG abnormalities (Dossena, et al. Neuron 2008; 60:598–609).

Our objective is to generate new Tg lines expressing D177N/V128 and D177N/M128 PrP without the 3F4 tag.

cDNAs encoding the mutant PrPs were cloned into a transgenic vector containing the mouse PrP promoter, and Tg mice were generated by random transgenesis. We identified nine Tg(CJD) mice carrying the D177N/V128 (16%) and six Tg(FFI) carrying the D177N/M128 transgene (7.8%). One Tg(CJD) and one Tg(FFI) founder, expressing high transgene levels (4X) died with neurological symptoms at 165 and 109 days of age. Four Tg(CJD) and two Tg(FFI) founders did not transmit the transgene. We obtained five Tg(CJD) lines with expression levels ranging from 0.4 to 2.3X, and four Tg(FFI) with expression from 0.7 to 1.1X. Both mutant PrPs showed abnormal biochemical properties, including detergent insolubility and mild proteinase-K resistance. Behavioral, neuropathological and electrophysiological analyses are in progress to verify if the Tg(CJD) and Tg(FFI) mice recapitulate the phenotypes of the human diseases.

PPo5-20: Immunohistochemical Characterization of BASE Plaques by Confocal Microscopy

Elena Vallino Costassa, Chiara Porcario, Debora Corbellini, Maria N. Chieppa, Alice Z. Perazzini, Tiziana Avanzato, Ferdinando Pulitano, Maria D. Pintore, Barbara Iulini, Cristina Casalone and Cristiano Corona

CEA; Istituto Zooprofilattico del Piemonte; Liguria e Valle d’Aosta; Turin, Italy

Key words: BASE, plaque, characterization

Atypical low-type BSE (L-type) appears to be phenotypically different from classical BSE (C-BSE) because of distinct immunohistochemical and immunobiochemical properties of PrP^Sc. Regarding immunohistochemical features, the four Italian L-type BSE cases detected to date are the only in which a careful description of PrP^Sc deposits throughout the brain has been reported. Unique feature presented by these cases was an unusual PrP^Sc deposition pattern appearing as amyloid plaques, mainly localized at the forebrain level. Because of this distinguishing hallmark, Italian L-type BSE has also been named Bovine Amyloidotic Spongiform Encephalopathy (BASE).

Aim of the present study was to characterize BASE plaques by investigating possible constituents co-localizing with PrP^Sc and by evaluating the relationship between brain glia and plaques morphology. At first BASE plaques have been immunohistochemically characterized by a panel of different antibodies (Beta amyloid, Tau, APOE) and immunostaining was revealed by confocal microscopy. Secondly, a quali-quantitative analysis was performed to investigate the presence and, in case, the activation of the plaque-surrounding astrocytes and microglia. Interesting results were obtained particularly regarding morphological aspects of BASE plaques and about the detection of a prominent activation of the glial cells neighbouring PrP^Sc plaque deposits.

PPo5-21: Proteomic Analysis of Synaptosomes from Patients with Sporadic Creutzfeldt-Jakob Disease

Martin Nowak,^1,* Joanna Gawinecka,^1,* Julie Carimalo,¹ Walter Schutz-Schaeffer,² Abdul R. Asif³ and Inga Zerr¹

¹National Reference Center for TSE Surveillance; ²Department of Neuropathology; ³Department of Clinical Chemistry; Medical Center Georg-August University; Göttingen, Germany

*These authors contributed equally to this work.

Key words: sporadic Creutzfeldt-Jakob disease, proteomics, synaptosome

Introduction. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common form of transmissible spongiform encephalopathy affecting humans. Disease phenotype is mainly influenced by the methionine/valine (M/V) polymorphism at codon 129 in the PRNP gene and by the presence of two major types of protease-resistant form of PrP leading to 2 different profiles in western blot (types 1 and 2). MM1 and VV2 represent the most frequent sCJD subtypes. Recent data strongly suggest that synaptic degeneration is the first step of prion neuropathogenesis and it is responsible for the onset of the clinical signs. However, the precise mechanism underlying this process is not completely understood. In this study, we aimed to provide an overview of the synaptic alterations occurring in the brain from MM1 and VV2 sCJD patients.

Results. Our preliminary data demonstrate that 31 and 13 spots displayed a significantly different regulation in MM1-sCJD and VV2-sCJD patients compared to controls. In MM1 subtype, 19 protein spots were found to be downregulated and 12 upregulated. Moreover, 8 and 5 protein spots showed VV2-specific down- and upregulation, respectively.

Patients and Methods. Synaptosome fractions were isolated from the brain of 5 MM1, 5 VV2 patients and 5 age-matched healthy controls then subjected to 2-D Fluorescence Difference Gel Electrophoresis. A protein spot was recognized as differentially regulated in sCJD, when its densitometric analyses showed at least 2-fold change in abundance. Currently, gel plugs containing proteins of interest are analyzed using mass spectrometry.

Conclusion. We think that our study will give clues for a better understanding of the mechanisms underlying synaptic degeneration.

PPo5-22: MicroRNA-146a is Induced in the Brains of Prion Infected Mice and Functions as a Modulator of Microglial Activation

Stephanie A. Booth, Reuben Saba,^1,2 Rhiannon L.C.H. Huzarewich,¹ Sarah Medina¹ and Kathy Surynicz¹

¹Molecular PathoBiology; Public Health Agency of Canada; ²Department of Medical Microbiology and Infectious Diseases; University of Manitoba; Winnipeg, MB Canada

Key words: microRNA, microglia, miR-146a

Microglia, the resident immune effector cells of the brain, adopt an ‘activated’ state during prion disease. This is characterized phenotypically by shortened and extensively branched processes, and by the concomitant induction of a variety of cell-surface and cytoplasmic molecules. Microglia can have a beneficial effect by secreting neurotrophic factors and perhaps by phagocytosis of prion deposits, or on the other hand activation can be neurotoxic. The stimuli and signaling pathways that lead to these specific modulations of function are as yet unknown. MicroRNAs (miRNAs), small non-coding RNA molecules, play an important role in the regulation of immune processes. We established overexpression of miRNA-146a in prion-infected mouse brain tissues concurrent with the onset of prion deposition and the appearance of activated microglia. Expression profiling of central nervous system-derived cultures showed miR-146a is preferentially expressed in cells of microglial lineage. Prominent upregulation of miR-146a was evident in the microglial line EOC 13:31 following stimulation; this reached maximum level after 24–48 hours coinciding with the return to basal levels of stimulation-induced cytokines. Functional genomic studies using miR-146a mimics and antimers revealed significant transcriptional perturbations, including downstream mediators of the pro-inflammatory transcription factor Nuclear Factor-KappaB (NF-κB), and the JAK-STAT signaling pathway. MiR-146a expression was ultimately able to regulate the protein levels of a number of cytokines. Microarray analysis also predicts a role for miR-146a regulation of morphological changes in the microglial response to stimulation. MiR-146a appears to be an important regulator of the immune response and microglial ‘activation’ state during prion-induced neurodegeneration.

PPo5-23: CREB-induced microRNAs and Implications on Neurodegeneration in Prion Disease

Anna Majer,^1,2 Kathy J. Surynicz,¹ Sarah J. Medina,¹ Kathy Frost¹ and Stephanie A. Booth^1,2

¹Molecular PathoBiology Unit; Public Health Agency of Canada; Winnipeg, MB; ²Department of Medical Microbiology; University of Manitoba; Winnipeg, MB

Key words: CREB, microRNA, hippocampus, neurodegeneration, prion

The mechanisms by which neuronal connections are severed, leading to degeneration remains unclear in prion diseases. A novel class of gene regulators, the microRNAs, may have important roles to fine-tune expression of key genes involved in neuronal damage and death. Previously we determined extensive mRNA and microRNA transcription profiles in hippocampal neurons identifying numerous temporal changes over the course of the disease. Immediate early genes were among the first genes upregulated during the preclinical period whereas immune response genes predominated at later time points. Bioinformatic analysis of these genes suggested the engagement of neuronal plasticity- and survival-related pathways during early stages of neurodegeneration. One of the key regulatory molecules to be chronically deregulated was the transcription factor, cAMP response element binding protein (CREB). Either chronic activation, or inhibition, of CREB can lead to damage and loss of neurons. Induction of intermediate early gene expression suggests an increase in excitability of hippocampal neurotoxicity leading to excitotoxicity and inflammation.

The aim of this study is to identify CREB-induced genes and miRNAs that are dysregulated in prion disease and investigate their function on cellular mechanisms of prion-induced neurodegeneration. Microarray and real-time PCR analysis revealed numerous deregulated mRNAs that can be induced by activated CREB. Similarly, a number of miRNAs whose deregulation was associated with disease were found to be induced by CREB following its overexpression in vitro. Gain- and loss-of-function phenotypes are used to further identify genes and processes regulated by some of these miRNAs in terms of neuronal viability.

PPo5-24: Growth of Prion Plaques and Spongiform Aggregates on Lipid Membranes

Philip J. Robinson and Teresa J.T. Pinheiro

Department of Biological Sciences; University of Warwick; Coventry, UK

The prion protein (PrP) is GPI-anchored to the cell membrane where prion conversion occurs. Therefore it is thought that the membrane microenvironment surrounding PrP will influence the process of prion conversion. Our recent study investigates how membrane lipid composition drives PrP into flat plaque-like aggregates or larger spongiform assemblies. The morphology and mechanism of growth of prion aggregates on membranes is shown to be influenced by the lipid composition of the membrane. Atomic force microscopy is used to image in situ the aggregation of prions on supported lipid bilayers composed of mixtures of the zwitterionic lipid, POPC, and the anionic lipid, POPS, both of which occur in neuronal membranes. PrP on membranes containing POPS acquires β-sheet structure, whereas on POPC membranes it retains its α-helical structure. The aggregates formed on POPC membranes have uniform dimensions and do not disrupt the lipid bilayer. The presence of POPS results in larger prion aggregates with a distinctive sponge-like morphology that disrupt to lipid membrane. The data provides new insight on prion aggregates on membranes, which are representative of the complex array of prion deposits found in diseased brain.

PPo5-25: Subcellular Localization of Peptidylarginine Deiminase 2 and Citrullinated Proteins in Brains of Scrapie-infected Mice: Nuclear Localization of PAD2 and Membrane Fraction-Enriched Citrullinated Proteins

Byungki Jang,^1,2 Eun-Young Kim,¹ Hae-Young Shin,¹ Jin-Kyu Choi,¹ Nguyen Du Phuong Thao,¹ Byung-Hoon Jeong,¹ Akihito Ishigami,^3,4 Naoki Maruyama,⁴ Richard I. Carp,⁵ Yong-Sun Kim^1,2 and Eun-Kyoung Choi¹

¹Ilsong Institute of Life Science; Hallym University, Anyang, Republic of Korea; ²Department of Microbiology; College of Medicine; Hallym University; Chuncheon, Republic of Korea; ³Department of Biochemistry; Faculty of Pharmaceutical Sciences; Toho University; Chiba, Japan; ⁴Aging Regulation; Tokyo Metropolitan Institute of Gerontology; Itabashi-ku, Tokyo Japan; ⁵New York State Institute for Basic Research in Developmental Disabilities; Staten Island, New York USA

Key words: PAD2, citrullination, scrapie, electron microscopy, subcellular fraction

Although peptidylarginine deiminases (PADs) and citrullinated proteins contribute to the pathogenesis of neurodegenerative diseases, detailed subcellular localization of PAD2 and citrullinated proteins are poorly mapped in brain under normal and abnormal status. To explore this issue, we performed subcellular fractionation and electron microscopic analysis using brains of normal and scrapie-infected mice. PAD2 was abundantly present in cytosol and weakly in microsomal and mitochondrial fractions, and expression in these fractions was higher in brains from scrapie-infected mice. Despite the relatively low PAD2, citrullinated proteins were present at high levels in scrapie-infected microsomal and mitochondrial fractions. Surprisingly, increased expression of PAD2 and accumulated citrullinated proteins in nuclear fractions were found in scrapie-infected brains. Moreover, electron microscopic analysis revealed that increased PAD2 and citrullinated proteins in scrapie-infected brains were widely distributed in most cellular compartments including mitochondria, endoplasmic reticulum, glial filaments, nucleus and Golgi apparatus in astrocytes and neurons of hippocampal region. Taken together, we report for the first time the nuclear localization of PAD2 and detailed subcellular localization of PAD2 and of citrullinated proteins in scrapie-infected brains. Our findings suggest that different subcellular compartmentalization of PAD2 and citrullinated proteins may have profoundly different physiological roles in normal and neurodegenerative conditions.

Acknowledgements

This study was supported by a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A085082) and a grant (Code #20080401034016) from BioGreen 21 Program, Rural Development Administration, Republic of Korea.

PPo5-26: A Typical Neuropathological sCJD Phenotype with Abundant Cerebral Kuru-type Plaques Sparing the Cerebellar Cortex

Ellen Gelpi,¹ Josep M^a Soler Insa,² Elena Martínez- Saez,³ Jordi Yagüe,⁴ Carlos Nos,⁷ Raquel Sanchez-Valle^4,5 and Isidro Ferrer⁶

¹Neurological Tissue Bank; University of Barcelona; ²Neurology Department; Hospital St. Joan de Deu, Manresa; ³Vall d’Hebron Research Institute and Pathology Department; ⁴CJD-Unit; ⁵Alzheimer disease and other cognitive disorders Unit; Department of Neurology; Hospital Clínic; ⁶Institut de Neuropatologia; Hospital Universitari de Bellvitge; ⁷General Subdirectorate of Surveillance and Response to Emergencies in Public Health; Department of Public Health in Catalonia; Barcelona, Spain

We describe an atypical neuropatholgical phenotype of a sporadic Creutzfeldt-Jakob disease (CJD) case.

A 64-year-old man presented, 5 months before death, rapidly progressive behavioural disturbances, memory complaints, disorientation, and language alterations. No cerebellar signs were present. MRI showed diffuse atrophy and hyperintensities in parietal, occipital, temporal, and frontal cortices and left caudate nucleus in T2 and FLAIR. No periodic sharp wave complexes were observed in the EEG. A repeated 14-3-3 assay was positive after a first negative result performed 3 weeks before.

Neuropathology showed classical CJD changes affecting all cerebral lobes and basal ganglia with small cortical foci of large confluent vacuoles. Cerebellar cortex was relatively well preserved. The most striking feature was the presence of abundant Kuru-type plaques in the cerebral cortex and white matter, and only isolated in cerebellar white matter sparing cerebellar cortex. Immunohistochemistry showed, in addition to unicentric plaques, diffuse synaptic and patchy perivacuolar, as well as plaque-like and periaxonal PrPres deposits. Western blot studies demonstrated PrPres type 2 in frontal cortex and very weak signal in cerebellum. No PRNP mutations were detected, and codon 129 showed methionine homozygosity.

This case can not be classified according to the current sCJD classification (Parchi et al. 2009) as it shares histopathological features of both, the mixed MM1/2 and MV2K + 2C subtypes. In contrast, the patient presented clinically as classical MM1 sCJD. This case highlights the complexity of the phenotypic and isotype correlations. It would be of great interest to collect atypical cases to elucidate further mechanisms modulating phenotypic manifestations.

PPo5-27: Both Macro- and Microautophagy Contribute to Widespread Neuronal Degeneration in Hamsters Infected with the Echigo-1 Strain of Creutzfeldt-Jakob Disease and Mice Infected with the Fujisaki Strain of Gesrtmann-Sträussler-Scheinker Disease

P. Liberski,^1,3 Beata Sikorska^1,3 and Paul Brown²

¹Department of Molecular Pathology and Neuropathology; ³Department of Oncology; ²CEA/DSV/iMETI/SEPIA; Fontenay-aux-Roses, France

Key words: autophagy, fujisaki strain, GSS, echigo-1 strain

While the etiology of prion diseases or transmissible spongiform encephalopathies is widely regarded as understood, its pathogenesis is a real “black box” as practically nothing is known on a subject of neuronal degeneration in TSEs. Both apoptosis and autophagy have been implicated in a process of neurodegeneration, but no data to delineate details of those processes have ever been published. To this end, we performed electron microscopic studies on a well characterized strain of CJD, the Echigo-1, and targeted the development of macro- and microautophagy in this model. Autophagic vacuoles were observed in several cellular compartments. In neuronal cell bodies, large autophagic vacuoles were seen. These were indistinguishable from those described by us in experimental GSS and scrapie and chronic wasting disease and in mule deer. The cytoplasm of some neurons was punctuated by electron-lucent cytoplasm “holes” containing “floating” small autophagic vacuoles.

The major target of autophagy was dystrophic neuritis i.e., enlarged neuritic processes—mostly dendrites but also axonal terminals and preterminals. They contain numerous double- or multiple-membrane bound autophagosomes or autophagolysosomes and large multivesicular bodies. Multivesicular bodies were also observed within autophagic vacuoles and large multivesicular bodies were seen within synaptic terminals. Some dystrophic neuritis were filled almost completely with multivesicular bodies.

PPo5-28: Gene Expression Changes in the Medulla Oblongata of Naturally Infected Scrapie Sheep

Hicham Filali,¹ Inmaculada Martín-Burriel,² Frank L. Harders,³ Luis Varona,⁴ Jaber Lyahyai,² Martí Pumarola,⁵ Juan J. Badiola,¹ Alex Bossers³ and Rosa Bolea¹

¹Centro de Investigación en Encefalopatías y Enfermedades Transmisibles Emergentes; ²Laboratorio de Genética Bioquímica (LAGENBIO); ³Central Institute for Animal Disease Control (CIDC-Lelystad); The Netherlands; ⁴Unidad de Genética Cuantitativa y Mejora Animal; Facultad de Veterinaria; Universidad de Zaragoza; Zaragoza, Spain; ⁵PRIOCAT Laboratory; Centre de Recerca en Sanitat Animal (CReSA); UAB-IRTA; Campus de la Universitat Autònoma de Barcelona; Bellaterra, Barcelona, Spain

Key words: natural scrapie, gene expression, medulla oblongata

The precise molecular and cellular mechanisms that underlie the pathogenesis of scrapie and other prion diseases are unknown. The identification of genes which show differential expression during prion infection could help us to identify novel risk genes as well as to find the abnormal intra- or intercellular pathways that are responsible for the pathogenesis of prion diseases. In addition, it may allow the identification of new biomarkers other than the prion protein. The objectives of the present study were to identify genes that are differentially expressed in the medulla oblongata (MO) of the sheep brain between scrapie-symptomatic and non-affected animals.

The gene expression patterns from these two groups are compared using the custom CVI high-density sheep oligo microarray.

Over 350 probes displayed significant expression changes greater than 2-fold. From these probes we identified 148 genes, many of which encode proteins that according to gene ontology classification are involved in immune response, ion transport, cell adhesion, and transcription. We also confirmed regulation of many earlier published regulated genes in experimental scrapie animal models. Finally, we investigated the relationship between gene expression profiles and the appearance of the main scrapie related lesions: prion deposition, gliosis and spongiosis. In this context, the potential impacts of linked gene expression changes in MO during scrapie development are discussed.

PPo5-29: Neuropathological and Biochemical Characterization of Unusual Cases of Creutzfeldt-Jakob Disease in Young, Prnp 129 mm Subjects

Fabio Moda,¹ Giorgio Giaccone,¹ Giuseppe Di Fede,¹ Alessandro Terruzzi,² Silvia Suardi¹ and Fabrizio Tagliavini¹

¹Fondazione IRCCS Istituto Neurologico Carlo Besta; Milano, Italy; ²Policlinico S. Marco; Bergamo, Italy

Key words: sporadic Creutzfeldt-Jakob disease, immunohistochemistry, prion disease, prion protein, PRNP, PrP types

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common prion disease of humans with the greatest incidence between 60 and 70 years of age. Different polymorphisms (Methionine/Valine) at codon 129 of PRNP gene and PrP^Sc type are associated with different disease phenotypes. Approximately 95% of the sporadic 129MM CJD patients have PrP^Sc type 1 (Parchi classification). Young patients with sCJD are extremely rare, and most of them are 129VV with type 1 PrP^Sc deposition in the brain. We observed two young patients with CJD (age at death 25 and 34 years) who had not the characteristics of variant CJD neither an history of risk factors for iatrogenic transmission. Both were MM at codon 129, without mutation of the PRNP gene. The clinical course was rather long (26 and 28 months), dominated by behavioral disturbances in the early stage and remarkable for the absence of hallmarks of CJD at CSF analysis, MRI and EEG. These two atypical CJD patients were subjected to biochemical and immunohistochemical analysis. Preliminary results evidenced: (i) presence of type 2A PrP^Sc with the immunohistochemical counterpart of diffuse, finely granular, synaptic pattern of staining, without any focal perivacuolar PrP deposition; (ii) sparing of basal ganglia and cerebellum; (iii) absence of reactivity with a monoclonal antibody (12B2) specific for type 1 PrP^Sc. These findings may contribute to clarify the relationship between type of PrP^Sc, patterns and entity of PrP deposition, distribution and severity of the neuropathological changes and their role in the pathogenesis of prion diseases.