SP1-1: Defining the Zoonotic Potential of a TSE Strain
Jean Manson
The Roslin Institute; Roslin, Midlothian, Scotland UK
Key words: zoonosis, prion strain, transgenic model
While the BSE epidemic is now clearly in decline but not yet eliminated, there are still considerable concerns over the potential for human to human transmission of vCJD Epidemiological surveillance of these diseases is clearly of great importance to ensure the future safety of the food chain and to protect the human population from possible transmission of TSEs both from other species and also within the human population. Surveillance in animals has identified “new” or previously unrecognised strains of TSE agents in sheep, cattle and goats and the zoonotic potential of these agents is at present unknown. Additionally Chronic Wasting Disease is currently a major problem in both farmed and wild deer populations in the US. Since we do not yet understand how and why TSE strains mutate or what controls the species barrier to infection we do not know how to predict whether an animal TSE can acquire zoonotic potential. Thus while these agents exist in either farmed or wild populations of animals there is the possibility that these agents can become zoonotic in the future. While epidemiological surveillance is an important tool to identify new diseases or changes in the currently identified diseases, by the time surveillance has picked up such an event it is often too late to stop the initial spread of a disease. It is therefore important to use the current tools available to assess the zoonotic potential of these agents. Transgenic mouse models are numerous and varied and have been used as a major tool in this assessment. Here we discuss the role of these models in understanding the species barrier and the zoonotic potential of the animal TSEs.
SP1-2: The Zoonotic Potential of Animal TSEs: Evidences from Classical Strain Typing in Rodents
Umberto Agrimi, Michele Angelo Di Bari, Gabriele Vaccari and Romolo Nonno
Istituto Superiore di Sanità; Rome, Italy
Key words: zoonosis, prion strain, prion transmission
The data used to infer the zoonotic potential of animal TSE derive from epidemiology, strain typing and risk modelling.
Although currently based on improved animal models, strain typing still refers to the “classical” analysis of the disease characteristics in experimentally-inoculated rodents.
To date, the BSE agent represents the only animal prion with a proven zoonotic potential. Epidemiological evidences showed a relationship between BSE and vCJD epidemics, while strain typing in wild-type mice confirmed that the same agent was responsible for the two diseases. This indicates that, in case of epidemics, epidemiological and strain typing investigations can provide conclusive answers. By contrast, investigating the zoonotic potential of prions in the absence of epidemiological evidences is a more complex task.
Strain typing may reveal similarities between human and animal prions, but their interpretation in terms of risk for humans is not obvious. A major problem resides in understanding to what extent the characteristics observed in a model reflect the inherent properties of a strain rather than its phenotypic expression in that particular host.
In recent years, a huge amount of data has been produced by transmission studies. How to interpret these data with respect to the potential relationship between human and animal prions is crucial.
In our experience, the transmission characteristics of human isolates to voles were different from those of animal isolates, with few notable exceptions. Here, the significance of similarities between human and animal prions in rodent models will be discussed along with possible experimental approaches for their closer investigation.
SP1-4: Evidence from Molecular Strain Typing
Gianluigi Zanusso
Department of Neurological and Visual Sciences; Section of Clinical Neurology; University of Verona; Verona, Italy
Key words: molecular analysis, strain typing, atypical BSE, CJD
In 2001, active surveillance for bovine spongiform encephalopathy (BSE) led to the discovery of atypical BSE phenotypes in aged cattle distinct from classical BSE (C-type). These atypical BSE cases had been classified as low L-type (BASE) or high H-type BSE based on the molecular mass and the degree of glycosylation of of the pathological prion protein (PrPSc). Transmission studies in TgBov mice showed that H-type BSE, C-type BSE and BASE behave as distinct prion strains with different incubation periods, PrPSc molecular patterns and pathological phenotypes. A still unclear issue concerns the potential transmissibility and phenotypes of atypical BSEs in humans. We previously indicated that BASE was similar to a distinct subgroup of sporadic form of Creutzfeldt-Jakob disease (sCJD) MV2, based on molecular similarities and on neuropathological pattern of PrP deposition. To investigate a possible link between BASE and sCJD, Kong et al. and Comoy et al. experimentally inoculated TgHu mice (129MM) and a non-human primate respectively, showing in both models that BASE was more virulent compare to BSE. Further, non-human primate reproduced a clinical phenotype resembling to that of sCJD subtype MM2. Here, we presented a comparative analysis of the biochemical fingerprints of PrPSc between the different sCJD subtypes and animal TSEs and after experimental transmission to animals.
SP1-5: In Vitro Studies for Evaluating Prion Transmission Between Species
JoaquÍn Castilla,1-3 Natalia Fernàndez-Borges,1,2 Francesca Chianini,4 Suzette A. Priola,5 Glenn Telling,6 Enric.Vidal,7 Jorge de Castro,3 Louise Gibbard,4 Hugh W. Reid,4 Kristin McNally,5 Scott Hamilton,4 Tanya Seward,6 MartÍ Pumarola,7 Mark P. Dagleish4 and Rachel Angers6
1CIC bioGUNE; Parque tecnologico de Bizkaia; Bizkaia, Spain; 2IKERBASQUE; Basque Foundation for Science; Bizkaia, Spain; 3Department of Infectology; Scripps, Florida USA; 4Moredum Research Institute; Scotland, UK; 5Rocky Mountain Laboratories; Laboratory of Persistent Viral Diseases; NIAID; NIH; Montana, USA; 6Department of Microbiology; Immunology and Molecular Genetics; University of Kentucky; Lexington, Kentucky USA; 7Centre de Recerca en Sanitat Animal (CReSA); UAB-IRTA; Campus de la Universitat Autonoma de Barcelona; Barcelona, Spain
Key words: in vitro replication, pmca, prion, scrapie, transmissible spongiform encephalopathy (TSE)
One of the characteristics of prions is their ability to infect some species and not others. This phenomenon is known as the transmission barrier. In general, the transmission barrier is expressed by an incomplete attack rate and long incubation times which become shorter after serial inoculation passages. The absence of natural TSE cases and/or failed experimental transmissions has suggested that some species could be resistant for prion diseases. Unfortunately, the molecular basis of the transmission barrier phenomenon is currently unknown and we cannot predict the degree of a species barrier simply by comparing the prion proteins from two species. We have conducted a series of experiments using the Protein Misfolding Cyclic Amplification (PMCA) technique that mimics in vitro some of the fundamental steps involved in prion replication in vivo albeit with accelerated kinetics. We have used this method to efficiently replicate a variety of prion strains from, among others, mice, hamsters, bank voles, deer, cattle, sheep and humans. The in vitro generated PrPres possess key prion features, i.e., they are infectious in vivo and maintain their strain specificity. We are using the PMCA to generate infectious PrPres from species hitherto considered to be resistant to prion disease and assay the role that certain amino acids play in the transmission barriers. The correlation between in vivo data and our in vitro results suggest that PMCA is a valuable tool for studying the strength of the transmission barriers between diverse species and for evaluating the potential risks of the newly generated prion species to humans and animals.
SP2-1: Life Cycle of Cytosolic Prions
Ina Vorberg,1 Carmen Krammer,2 Hermann Schätzl3,4 and Julia Hofmann3
1Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE); Ludwig-Erhard-Allee 2; Bonn, Germany; 2Department of Biochemistry; Molecular Biology and Cell Biology; Northwestern University; Evanston, IL USA; 3Institute of Virology; Technische Universität München; Munich, Germany; 4Departments of Veterinary Science and of Molecular Biology; University of Wyoming; Laramie, Wyoming USA
Key words: Sup35, yeast prion, amyloid, transmission, intracellular inclusions
Prions are infectious, self-perpetuating protein aggregates of mammals and lower eukaryotes. In mammals, transmissible spongiform encephalopathies or prion diseases are caused by misfolded assemblies of the cell membrane anchored prion protein PrP. Several other neurodegenerative diseases are also caused by the misfolding of normally soluble proteins into highly ordered protein aggregates, so called amyloid. Recently it was shown that also disease-associated cytosolic protein aggregates can transfer from cell to cell and thus share some features with mammalian prions. But what exactly characterizes a prion in the mammalian cytosol? We have studied the life cycle of synthetic cytosolic prions derived from the prion domain NM of the yeast prion protein Sup35p. Infections of mouse neuroblastoma cells stably expressing soluble NM were accomplished by addition of exogenous recombinant NM fibrils to the cell culture medium. NM prion induction was independent of endogenous PrP expression, as NM aggregates were also induced in PrP knock-out cells. Induced NM aggregates displayed remarkable mitotic stability and were efficiently propagated over multiple passages. NM aggregates replicated with morphologically and biochemically distinct aggregate phenotypes reminiscent to prion strains. Importantly, NM prions were also naturally transmitted between cells, causing induction of heritable NM protein aggregate states in acceptor cells. Transmission was most efficient upon cell-cell contact of donor and acceptor cells and was sensitive to alterations of the cytoskeleton. In conclusion, cytosolic prions follow a life cycle of induction, inheritance and transmission comparable to the life cycle of membrane-bound scrapie prions in cell culture. Our data also reveal that mammalian cells support the replication of cytosolic prions, suggesting that other intracellular protein aggregates might propagate as prions.
SP2-4: Generating Highly Infectious Synthetic Mouse Prions
Fei Wang,1 Xinhe Wang,1 Liang Zha,1 Chonggang Yuan2 and Jiyan Ma1,2
1Department of Molecular and Cellular Biochemistry; Ohio State University; Columbus, OH USA; 2School of Life Science; East China Normal University; Shanghai, China
Recombinant prion protein (rPrP) purified from Escherichia coli was converted to the infectious conformation in the presence of synthetic phospholipid POPG and total RNA isolated from mouse liver. The newly generated recombinant prion (rPrP-res) recapitulates almost all the biochemical characteristics of a naturally occurring prion. When injected into wild-type mice, the rPrP-res causes prion disease with a short incubation period, indicating high infectivity associated with rPrP-res. The properties of rPrP-res caused disease in mice were characterized, which has all the hallmarks of naturally occurring prion diseases. Propagation of rPrP-res in vitro requires the presence of lipid, suggesting an important role of lipid-rPrP interaction in converting rPrP into the infectious conformation. Our biochemical analyses revealed that the middle region of PrP is essential for lipid-induced PK-resistant conformation. Middle region localized PrP mutations and the 129 polymorphism significantly alter lipid-rPrP interaction, supporting the relevance of rPrP-lipid interaction to the pathogenesis of prion disease.
S1-2: Mitochondria Fusion/Fission in the Balance of Alzheimer Disease
Perry G,1 Wang X,2 Su B,2 Moreira PI,3 Smith MA2 and Zhu X2
1University of Texas at San Antonio; San Antonio, TX USA; 2Case western Reserve University; Cleveland, OH USA; 3University of Coimbra; Coimbra, Portugal
Key words: mitochondria, Alzheimer disease, oxidative stress
Mitochondrial function relies on the dynamic balance of morphology, distribution and transport, alterations of which are increasingly implicated in neurodegenerative diseases. Here, we investigated these mitochondrial dynamics in Alzheimer disease (AD). First, the mitochondrial morphology and distribution in human fibroblast from sporadic AD (sAD) patients was found to have abnormal mitochondrial distribution characterized by elongated mitochondria being accumulated in perinuclear areas in around 20% sAD fibroblasts which was in marked contrast to a normally even distribution throughout the cytoplasm in the majority of normal human fibroblasts (0.95%). Interestingly, dynamin-like protein 1 (DLP1), a regulator of mitochondrial fission and distribution, was greatly decreased in sAD fibroblasts. By manipulating DLP1 levels, we demonstrated that DLP1 reduction causes mitochondrial abnormalities in sAD fibroblasts. We then investigated brain biopsy specimens from normal and AD patients and found disease-related changes in mitochondrial morphology and distribution as well as changes in expression levels and distribution of mitochondrial fission and fusion proteins. To understand the underlying mechanisms of these mitochondrial alterations in AD, we overexpressed or knocked down functional DLP1 and other mitochondrial fission/fusion proteins in rat primary neurons. In situations where functional protein changes, including amyloid beta production and oxidative stress, mimicked that in AD, similar changes in mitochondrial morphology and distribution were found as in AD neurons. These findings suggested that elevated oxidative stress and increased amyloid production are potential pathogenic factors that cause impaired balance of mitochondrial fission/fusion.
Acknowledgements
Supported by the NIH and the Alzheimer’s Association.
S2-1: Biological Diversity of TSE Agents: The Strain Typing Puzzle
O. Andreoletti,1 J.C. Espinosa,2 H. Cassard,1 C. Lacroux1 and J.M. Torres2
1UMR INRA-ENVT 1225; Interactions Hôte Agent Pathogène; Ecole Nationale Vétérinaire de Toulouse; Toulouse, France; 2Centro de Investigaciôn en Sanidad Animal INIA; Madrid, Spain
Key words: prion strain, prion transmission, transmission barrier, transgenic model
More than half of a century has gone since Pattison described, on the basis of clinical signs, the existence of diversity in the properties of TSE agents.
From those observations a wide number of investigations were carried out, mainly using conventional mice models to classify and characterize TSE strains. The existence of transmission barriers that are limiting the propagation of TSE agent between species has for long being considered responsible for the difficulties into providing a coherent classification of TSE strains.
The more recent development of PrPSc based methodologies and of PRP transgenic mice made believe that TSE agent strains’ mysteries would be soon decipher. However, rather than bringing clarifications on the essence of TSE strains these tools let us catch sight of the complexity and evolutionary abilities of TSE agents.
S2-2: Neuropathology of Animal Prion Diseases Toxic Effects of PrPd, Relationships with Strain and Clinical Disease
M. Jeffrey, G. McGovern, S. Martin, S. Sisó, L. González
VLA (Lasswade); Edinburgh, Scotland UK
Key words: neuropathology, scrapie, BSE, prion protein, immunohistochemistry
Ruminant prion diseases have a wide range of morphological types of PrPd as shown by immunohistochemistry. Different sheep scrapie strains may be distinguished by different proportions of these morphological types of PrPd. Sheep scrapie sources from across Europe have varying disease phenotypes within a single PRNP genotype suggesting significant naturally occurring scrapie strain variation. Strain characterization of prion isolates is often performed in mice. Different BSE sources transmit to mice with constant disease phenotypes. However, when sheep derived cloned murine strains of scrapie are passed into sheep of different breeds and genotypes and then re-isolated into mice, the original phenotype is often lost and murine disease phenotypes differs according to donor sheep breed and PRNP genotype. Similarly, when different stable isolates of sheep scrapie are passaged across sheep of different genotypes the disease phenotype in the recipient is linked to both donor strain and recipient PRNP genotype.
Electron microscopy shows PrPd accumulations within lysosomes, on cell membranes or within the extracellular space. Membrane PrPd is most commonly found on dendrites and is associated with an altered endocytosis caused by poorly excision of PrPd. PrPd on plasma-lemmas may also be transferred to other cells or released to the extracellular space. PrPd-linked membrane changes are found in all “classical” animal prion diseases but they are absent from some transgenic models where mature amyloid dominates the pathology. Mature amyloid depositions are associated with distinct sub-cellular features suggesting that at least two mechanistically separate disease processes may exist depending on the model system.
S2-3: Variably Protease-sensitive Prionopathy: Update on this Novel Human Prion Disease
Pierluigi Gambetti
National Prion disease Pathology Surveillance Center; Institute of Pathology; Case Western Reserve University; Cleveland, OH USA
Key words: VPSPr, protease-sensitive, ladder-like, prionopathy, tau, novel prion disease
Introduction. Initially we described a novel prion disease affecting subjects valine homozygous at codon 129 (129VV) of the prion protein (PrP) gene and associated with an abnormal PrP (PrPDis) that was largely protease-sensitive. We recently reported that an overall similar disease also affects subjects that are 129 methionine/valine heterozygous (129MV) and methionine homozygous (129MM) although clinical histopathological and PrPDis features are distinguishable in the three 129 genotypes. Specifically, although the three PrPDis show the same electrophoretic pattern, they have different degrees of protease resistance and PrP antibody immunoreactivities. None of the VPSPr cases reported to date carries a mutation in the PrP gene open reading frame.
Results. We will report data from the three VPSPr genotypes related to (1) presence of tau and Afl pathology; (2) CSI features; (3) PMCA data on the propensity of PrPDis to act as template and convert PrPC to a similar PrPDis isoform; (4) further comparative analysis with Gerstmann-Sträussler-Scheinker disease (GSS).
Discussion. We will focus on the nature and classification of VPSPr when compared to classic and atypical prion diseases such CJD and GSS, respectively, as well as to other neurodegenerative diseases such as the tauopathies which have been shown to propagate in receptive animals.
Patients and Methods. We have carried out comparative clinical, histopathological and immunohistochemical examinations as well as PrPDis analyses in 29 cases received from the US and Italy (Drs. F. Tagliavini and P. Parchi). Conformation stability assay (CSI) and protein misfolding cyclic amplification (PMCA) were carried out in several of these cases.
Acknowledgements
Supported by NIA AG-14359, NINDS NS052319, CDC UR8/CCU515004 and Charles S. Britton Fund.
S2-4: Neuropathological Concepts in Typing of Human Non-transmissible Dementias
Gabor G. Kovacs
Institute of Neurology; Medical University of Vienna; Austria
Key words: neuropathology, neurodegenerative disease, amyloid-beta, tau protein, alpha-synuclein
Widespread application of immunohistochemical and biochemical investigations initiated a protein-based classification of neurodegenerative diseases. Extracellular deposits comprise deposits with immunoreactivity for amyloid-beta (Abeta) or prion protein (PrP). Major proteins that deposit intracellularly include tau, alpha-synuclein, TAR DNA Binding Protein 43 (TDP-43), or fused in sarcoma protein (FUS). Pathological tau, alpha-synuclein and TDP-43 may deposit in neuronal processes or cytoplasm, whereas alpha-synuclein and TDP-43 aggregates may be seen in the neuronal nucleus as well. Glial cells also show a variety of inclusions: astrocytic tau pathology is different in several disease entitites, furthermore, in oligodendroglial cells tau, alpha-synuclein, TDP-43 or FUS forms thin cytoplasmic inclusions (coiled-body), distinguishable from more massive deposits in some alpha-synucleinopathies and tauopathies. These morphologies may implicate functional differences or different capabilities to co-aggregate further proteins or to reach body fluids at a detectable level. Furthermore, (1) physiological protein forms may become upregulated or there may be deficit in their catabolism; (2) protein deposition may follow stages or phases; (3) upregulation or deposition may also occur in non-neurodegenerative disorders; (4) protein depositions may entrap physiological or pathologically modified forms of other proteins. Thus, for biomarker research, all conditions that alter the detectability of a protein or a pattern of protein deposition, which themselves are not considered to be enough to represent disease entities, should be documented. Defining clusters of patients based on the patterns of protein deposition and biochemically detectable modifications of proteins (“codes”) may have higher prognostic predictive value and may open new avenues for research on pathogenesis.
S2-5: Genetic, Biochemical and Neuroimaging Markers in Preclinical and Clinical Alzheimer’s Disease—Current State and Future Perspectives
David Prvulovic, Harald Hampel
Department of Psychiatry; Psychosomatic Medicine and Psychotherapy; Goethe-University of Frankfurt; Frankfurt, Germany
Key words: Alzheimer disease, dementia, biomarker, neuroimaging, neurogenetics
Alzheimer’s disease (AD) is the most common cause of dementia and its prevalence is estimated to double every 20 years until 2050. Earliest pathophysiological changes in AD are detectable years and even decades before the clinical manifestation of dementia. These preclinical and presymptomatic stages of AD represent a critical period for early diagnosis as they are likely to be the best time points for the application of potential disease modifying treatments, a large number of which are currently in different stages of development and clinical testing. So far, multiple mono- and multicentre studies have established a typical signature of CSF based core AD-biomarkers, consisting of Abeta, t-tau and p-tau that can differentiate between AD and healthy controls with a sensitivity and specificity of 80–90%. Moreover, structural brain imaging markers derived from high-resolution MRI scans have also shown high diagnostic and prognostic accuracy. State-of-the-art approaches using proteomics, functional neuroimaging and neurogenetics may help to develop new biomarkers that will help to further improve early diagnosis of AD and to monitor therapy effects. Novel biomarkers offer the possibility to substantially improve and speed up drug development if they succeed to quickly assess functionally relevant benefits of AD drug candidates and to label ineffective compounds before they enter expensive clinical phase II/III stages. Here we give an overview of established and emerging biomarker research including CSF and blood based biomarker candidates and current advances in neurogenetics. We also discuss multimodal biomarker applications including functional neuroimaging.