Overview Nature of the infectious particle in TSE TSE strains Role of PrPC in disease Potential therapeutic targets Implications for other neurodegenerative diseases

Prion Diseases: Transmissible Spongiform Encephalopathies Fatal neurodegenerative diseases in man and mammals Transmissible under natural and experimental conditions Lengthy incubation period with no conventional host response Characteristic neuropathology with spongiform change in grey matter Associated with conversion of PrPC to PrPSc

Prion diseases of humans and animals Scrapie in sheep and goats Transmissible mink encephalopathy Chronic wasting disease in deer & elk Bovine spongiform encephalopathy Feline spongiform encephalopathy Kuru Creutzfeldt-Jakob disease Gerstmann-Straussler-Scheinker disease Fatal familial insomnia Variant Creutzfeldt-Jakob disease

Protein-only version of the prion hypothesis “Prions are transmissible particles that are devoid of nucleic acid and seem to be composed entirely of a modified protein (PrPSc).” “The normal, cellular PrP (PrPC) is converted into PrPSc through a post-translational process during which it acquires a high beta-sheet content.” Prusiner SB, Proc Natl Acad Sci USA 1998;95:13363-83

Role of PrPC in TSE PrPC is required for disease propagation and neuropathology PrPC with GPI anchor to cell membrane transduces or potentiates the neurotoxicity of TSE infection Tg PrP null mice do not propagate TSE infectivity Tg mice expressing only anchorless PrPC can propagate TSE infectivity, but with greatly reduced neuropathology and clinical effects

Infectious particle in prion diseases Nonfibrillar particles between 300-600 kDa (mass equivalent to ~14-28 PrP molecules) Other molecular constituents? Cofactors for infectivity – sulphated GAG or nucleic acids?

PrPres Isotype by Western blot Treatment with proteinase K results in N-terminal truncation of PrPres Distinct isotypes of PrPres characterize different forms of CJD Isotypes differ in extent of truncation and degree of glycosylation site occupancy

Multiple conformations of PrPSc? “In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrPSc.” (Prusiner) Is there evidence for heritable structural diversity in different prion diseases?

PRNP codon 129 genotype frequencies MM MV VV Normal 37% 51% 12% population Sporadic CJD 71% 15% 14% vCJD 100% - -

Idiopathic human prion diseases

Do different PrPres types replicate with fidelity in vitro? When human PrPC is converted to PrPres in a PMCA reaction the product has both the conformation and the glycosylation ratio of the in-put PrPres Soto et al, 2005

Cellular co-factors & conversion: mammalian RNA Mammalian brain extracts contain RNA that stimulate the conversion of PrPC to PrPSc in a modified PMCA reaction (Deleault et al, Nature 2003;425:717-720)

Conservation of PrPres isotype following transmission to mice Telling et al 1996

Conservation of targeting following transmission to mice FFI transmitted to Tg(MHu2M)Prnp0/0 mice Thalamic pathology fCJDE200K transmitted to Tg(MHu2M)Prnp0/0 mice Cortical pathology Telling et al 1996

Aspects of PrPSc structure that might encipher strain properties Extent of structural re-arrangement (conversion to b-sheet) at the N-terminus. Presence of methionine or valine at codon 129 Presence or absence of bound divalent cations (Cu2+) Extent of of asparagine-linked glycosylation site occupancy Composition and complexity of attached glycans

Pathogenic mechanism If we accept the centrality of of the conversion of PrPC to PrPSc in the pathogenic process, then there are in principle three possible alternatives: The loss of an essential function of PrPC The acquisition of a toxic function by PrPSc Production of toxic intermediate or by-product

Neurodegenerative mechanism Hope 2000

Problems with anti-TSE therapy Which compound(s) to use? What route of delivery to use? Is peripheral treatment required? How long to treat?

Approaches to treatment of TSE Prevention of PrPC conversion Dissolution of PrPSc aggregates Enhanced PrPSc clearance Neuronal rescue?

Strategies to prevent PrPC conversion Inhibition of expression by RNA interference Binding to site(s) for physiological ligands, resulting in PrPC clustering and internalisation from cell surface

Compounds with in vivo anti-TSE activity Class/compound Example Sulphonated dyes Congo red Sulphated glycans pentosan polysulphate Cyclic tetrapyrroles porphyrins Polyene antibiotics amphotericin B Quinolenes quinacrine Metal chelators penicillamine Tetracyclines doxycyline

Detection of PrPSc in the peripheral tissues in CJD vCJD CNS PNS Optic nerve Retina Appendix Lymph node Peyers’ patches Tonsil Spleen Thymus sCJD CNS PNS Optic nerve Retina Olfactory epithelium Wadsworth et al, (2001), Lancet, 358, pp171-80 Head et al, (2004), American Journal of Pathology, 164, pp143-53 In certain prion diseases PrPSc accumulation has been shown to occur not on in the CNS, but also peripheral tissue Understanding the peripheral pathogenesis of the different forms of CJD is important for the differential diagnosis of these diseases and provides us with information for assessing the potential risk of iatrogenic spread vCJD is known to differ from other human prion diseases in the greater extent to which PrPSc can be detected in the peripheral tissues In contrast, PrPSc is largely confined to the Central Nervous System in sporadic CJD These two patterns have been thought to be due to the different routes of infection in variant and sCJD. In the case of vCJD the infection works its way in from the periphery, whereas in sCJD, the disease occurs within the central nervous system and migrates out by centrifugal spread.

Neurodegenerative disease and aberrant protein deposition Classical neuropathology identifies abnormal histological structures which are diagnostic for particular conditions. Nuclear and cytoplasmic inclusion bodies and extracellular amyloid deposits Proteinaceous, fibrillar, and rich in b-pleated sheet secondary structure “Fatal attractions” between abnormally folded forms of specific normal cellular proteins resulting in specific neurodegenerative diseases A common feature of Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis and prion diseases

Neurodegenerative diseases associated with abnormal protein conformations (toxic gain of function) Disease Gene product Alzheimer’s disease APP and Ab Creutzfeldt-Jakob disease PrPc and PrPSc Parkinson’s disease a synuclein Huntingdon’s disease Huntingtin Machado-Joseph disease Ataxin 3 (SCA 3)

Neuronal vulnerability to “toxic gain of function” Neurones are post-mitotic cells which cannot be replaced (liable to damage by increasing DNA mutations?) Unique metabolic demands - some neurones have to maintain an axon over 1m in length Functional plasticity Environment subject to control by many other structures, including astrocytes and the blood-brain barrier

Review Nature of the infectious particle in TSE TSE strains Role of PrPC in disease Potential therapeutic targets Implications for other neurodegenerative diseases