James Alibhai

Insights into Mechanisms of Chronic Neurodegeneration.

Chronic neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and prion diseases are characterised by the accumulation of abnormal conformers of a host encoded protein in the central nervous system. The process leading to neurodegeneration is still poorly defined and thus development of early intervention strategies is challenging. Unique amongst these diseases are Transmissible Spongiform Encephalopathies (TSEs) or prion diseases, which have the ability to transmit between individuals. The infectious nature of these diseases has permitted in vivo and in vitro modelling of the time course of the disease process in a highly reproducible manner, thus early events can be defined. Recent evidence has demonstrated that the cell-to-cell spread of protein aggregates by a “prion-like mechanism” is common among the protein misfolding diseases. Thus, the TSE models may provide insights into disease mechanisms and testable hypotheses for disease intervention, applicable to a number of these chronic neurodegenerative diseases.

Prion Protein-Specific Antibodies that Detect Multiple TSE Agents with High Sensitivity

This paper describes the generation, characterisation and potential applications of a panel of novel anti-prion protein monoclonal antibodies (mAbs). The mAbs were generated by immunising PRNP null mice, using a variety of regimes, with a truncated form of recombinant ovine prion protein spanning residues 94–233. Epitopes of specific antibodies were mapped using solid-phase Pepscan analysis and clustered to four distinct regions within the PrP molecule. We have demonstrated the utility of these antibodies by use of Western blotting and immunohistochemistry in tissues from a range of different species affected by transmissible spongiform encephalopathy (TSE). In comparative tests against extensively-used and widely-published, commercially available antibodies, similar or improved results can be obtained using these new mAbs, specifically in terms of sensitivity of detection. Since many of these antibodies recognise native PrPC, they could also be applied to a broad range of immunoassays such as flow cytometry, DELFIA analysis or immunoprecipitation. We are using these reagents to increase our understanding of TSE pathogenesis and for use in potential diagnostic screening assays.

The Mechanisms of Prion Disease

Prion diseases are enigmatic, neurodegenerative disorders affecting several mammalian species. The family of diseases include scrapie of sheep, bovine spongiform encephalopathy (BSE), Creutzfeldt-Jakob disease (CJD) of humans and chronic wasting disease (CWD) in deer and elk. Prion diseases are believed to be caused by the misfolding of an endogenous host protein and the abnormal, protease-resistant isoform of this protein is suggested to represent the infectious entity. Through many transmission experiments, we know much about the course of prion diseases and the ensuing pathology in the central nervous system. However, few of the causative mechanisms have been deciphered at a level that allows prediction of the disease characteristics of novel prion strains or that allows rational design of chemo-therapeutics. This review article outlines the state of our knowledge of prion disease mechanisms from the concept of protein misfolding, through the factors that may encode different strains of disease, mechanisms of neuronal loss and the ways that disease are transmitted.

Human stem cell-derived astrocytes replicate human prions in a PRNP genotype-dependent manner

Prions are infectious agents that cause neurodegenerative diseases such as Creutzfeldt–Jakob disease (CJD). The absence of a human cell culture model that replicates human prions has hampered prion disease research for decades. In this paper, we show that astrocytes derived from human induced pluripotent stem cells (iPSCs) support the replication of prions from brain samples of CJD patients. For experimental exposure of astrocytes to variant CJD (vCJD), the kinetics of prion replication occur in a prion protein codon 129 genotype–dependent manner, reflecting the genotype-dependent susceptibility to clinical vCJD found in patients. Furthermore, iPSC-derived astrocytes can replicate prions associated with the major sporadic CJD strains found in human patients. Lastly, we demonstrate the subpassage of prions from infected to naive astrocyte cultures, indicating the generation of prion infectivity in vitro. Our study addresses a long-standing gap in the repertoire of human prion disease research, providing a new in vitro system for accelerated mechanistic studies and drug discovery.

Gene Targeted Transgenic Mouse Models in Prion Research

The production of transgenic mice expressing different forms of the prion protein (PrP) or devoid of PrP has enabled researchers to study the role of PrP in the infectious process of a prion disease and its normal function in the healthy individual. A wide range of transgenic models have been produced ranging from PrP null mice, normal expression levels to overexpression models, models expressing different species of the Prnp gene and different mutations and polymorphisms within the gene. Using this range of transgenic models has allowed us to define the influence of PrP expression on disease susceptibility and transmission, assess zoonotic potential, define strains of human prion diseases, elucidate the function of PrP, and start to unravel the mechanisms involved in chronic neurodegeneration. This chapter focuses mainly on the use of the gene targeted transgenic models and summarizes the ways in which they have allowed us to study the role of PrP in prion disease and the insights they have provided into the mechanisms of neurodegenerative diseases.

Disease-associated protein seeding suggests a dissociation between misfolded protein accumulation and neurodegeneration in prion disease

ABSTRACT Chronic neurodegenerative diseases, such as prion diseases or Alzheimers disease, are associated with progressive accumulation of host proteins which misfold and aggregate. Neurodegeneration is restricted to specific neuronal populations which show clear accumulation of misfolded proteins, whilst neighbouring neurons remain unaffected. Such data raise interesting questions about the vulnerability of specific neuronal populations to neurodegeneration and much research has concentrated only on the mechanisms of neurodegeneration in afflicted neuronal populations. An alternative, undervalued and almost completely unstudied question however is how and why neuronal populations are resilient to neurodegeneration. One potential answer is unaffected regions do not accumulate misfolded proteins, thus mechanisms of neurodegeneration do not become activated. In this perspectives, we discuss novel data from our laboratories which demonstrate that misfolded proteins do accumulate in regions of the brain which do not show evidence of neurodegeneration and further evidence that microglial responses may define the severity of neurodegeneration.

Validating human stem cell derived neural cultures as a flexible model system in which to investigate neurodegenerative mechanisms

Until now, the 3-dimensional structure of infectious mammalian prions and how this differs from non-infectious amyloid fibrils remained unknown. Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity. One of the major challenges has been the production of highly homogeneous material of demonstrable high specific infectivity to allow direct correlation of particle structure with infectivity. We have recently developed novel methods to obtain exceptionally pure preparations of prions from prion-infected murine brain and have shown that pathogenic PrP in these high-titer preparations is assembled into rod-like assemblies (Wenborn et al. 2015. Sci. Rep. 10062). Our preparations contain very high titres of infectious prions which faithfully transmit prion strain-specific phenotypes when inoculated into mice making them eminently suitable for detailed structural analysis. We are now undertaking structural characterization of prion assemblies and comparing these to the structure of non-infectious PrP fibrils generated from recombinant PrP

Challenging the central hypothesis that misfolded prion protein accumulation, spread and distribution predicts regions of neurodegeneration

Until now, the 3-dimensional structure of infectious mammalian prions and how this differs from non-infectious amyloid fibrils remained unknown. Mammalian prions are hypothesized to be fibrillar or amyloid forms of prion protein (PrP), but structures observed to date have not been definitively correlated with infectivity. One of the major challenges has been the production of highly homogeneous material of demonstrable high specific infectivity to allow direct correlation of particle structure with infectivity. We have recently developed novel methods to obtain exceptionally pure preparations of prions from prion-infected murine brain and have shown that pathogenic PrP in these high-titer preparations is assembled into rod-like assemblies (Wenborn et al. 2015. Sci. Rep. 10062). Our preparations contain very high titres of infectious prions which faithfully transmit prion strain-specific phenotypes when inoculated into mice making them eminently suitable for detailed structural analysis. We are now undertaking structural characterization of prion assemblies and comparing these to the structure of non-infectious PrP fibrils generated from recombinant PrP

Creutzfeldt-Jakob disease prion propagation in human iPS cells-derived astrocytes

The mechanism by which a random-coil polypeptide folds into the native structure may be critical for the regulation of misfolding diseases. However, how the folding pathway is related to the misfolding pathway remains unclear. Our recent study demonstrated that the native folding pathway of prion protein (PrP) involves at least 4 independent species, including native state (N), unfolded state (U), and 2 types of partially folded states (A and I) (Honda et al., Structure, 2015). Interestingly, one of the partially folded states (A state) readily formed a misfolded aggregate whose rates were strongly correlated with the initial population of the A state (Honda et al., J Biol Chem, 2014). This observation indicated that the formation of the A state may be the initial step in the misfolding pathway. We characterized the structure of the A state using circular dichroism, hydrogen/deuterium exchange coupled with NMR, etc. and found that the Strand 1Helix 1-Strand 2 segment was completely unfolded in the A state, whereas the Helix 2Helix 3 segment retained a native-like helical structure. Our studies revealed how the native structure is altered during the early stage of misfolding and how the misfolding pathway is related to the native folding pathway of PrP. O-02: HET-2s, an engineered, 4-rung b-solenoid protein as a model for the structure of PrP Holger Wille, Andrew Fang, Brian Tancowny, Xiongyao Wang, Lyudmyla Dorosh, and Maria Stepanova

Evaluating the species barriers of TSEs with transgenic mouse models

Dietary exposure to bovine spongiform encephalopathy (BSE) contaminated bovine tissues is considered as the origin of variant Creutzfeldt Jakob (vCJD) disease in human. To date, BSE agent is the only recognized zoonotic prion. Despite the variety of Transmissible Spongiform Encephalopathy (TSE) agents that have been circulating for centuries in farmed ruminants there is no apparent epidemiological link between exposure to ruminant products and the occurrence of other form of TSE in human like sporadic Creutzfeldt Jakob Disease (sCJD). However, the zoonotic potential of the diversity of circulating TSE agents has never been systematically assessed. The major issue in experimental assessment of TSEs zoonotic potential lies in the modeling of the ’species barrier’, the biological phenomenon that limits TSE agents’ propagation from a species to another. In the last decade, mice genetically engineered to express normal forms of the human prion protein has proved essential in studying human prions pathogenesis and modeling the capacity of TSEs to cross the human species barrier. To assess the zoonotic potential of prions circulating in farmed ruminants, we study their transmission ability in transgenic mice expressing human PrP (HuPrP-Tg). Two lines of mice expressing different forms of the human PrP (129Met or 129Val) are used to determine the role of the Met129Val dimorphism in susceptibility/resistance to the different agents. These transmission experiments confirm the ability of BSE prions to propagate in 129MHuPrP-Tg mice and demonstrate that Met129 homozygotes may be susceptible to BSE in sheep or goat to a greater degree than the BSE agent in cattle and that these agents can convey molecular properties and neuropathological indistinguishable from vCJD. However homozygous 129V mice are resistant to all tested BSE derived prions independently of the originating species suggesting a higher transmission barrier for 129V-PrP variant. Transmission data also revealed that several scrapie prions propagate in HuPrP-Tg mice with efficiency comparable to that of cattle BSE. While the efficiency of transmission at primary passage was low, subsequent passages resulted in a highly virulent prion disease in both Met129 and Val129 mice. Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion. These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions.