Prion diseases - Basic Science

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1 Prion diseases - Basic Science Dr. David Westaway Centre for Prions and Protein Folding Diseases, University of Alberta david.westaway@ualberta.ca

2 Spongiform change and amyloid in a scrapie-infected brain.

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4 CWD in North America A B C D

5 The spectrum of prion diseases includes three patterns of manifestation Infectious (kuru, iatrogenic CJD, vcjd experimental disease Familial (autosomal dominant genetic). F- CJD, GSS, FFI Sporadic (sporadic CJD, sporadic fatal insomnia). How?

6 Are diseases like scrapie infectious or genetic? HB James Parry, Univ Oxford: genetic, controlled by the recessive s gene Alan Dickinson, Univ Edinburgh: naturally infectious

7 The search for a genomic nucleic acid (Studies in the slow virus era)

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10 Inactivation studies Ionizing radiation target size indicates an infectious particle of 55 kda. UV irradiation indicates that if there is a double-stranded DNA genome it would have a size of about 40 base-pairs. Scrapie agent was resistant to agents that destroy or modify nucleic acids including psoralen, DNAses, RNAses, divalent cations.

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12 PrPSc and PrPC

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16 Gel of the purified prion protein (S= scrapie, N = normal A protease-resistant protein can be visualized in highly purified (~ x) preparations of scrapie infectivity made using detergent insolubility and sucrose gradient fractionations. This is called PrP27-30, because it has a size of kda. Although this species has implications for molecular nature of prion infections, a slightly different type of experiment is done to use PrP27-30 as a diagnostic (below).

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18 Harbingers of the conformational hypothesis PrP encoding gene exon PrP Sc and PrP C have closely related amino acid sequence (1985). The PrP gene has a single uninterrupted coding exon (1986). Low resolution structural analysis reveal PrP C is alpha-helical (1992). Low resolution structural analysis reveal PrP Sc is enriched in β- sheet (1992). Also, PrP Sc amyloid deposits in hamster scrapie stain with congo red dye (1985). Overlapping Endo Lys C proteolytic fragments of PrP Sc reveal no differences from the predicted sequence of PrP C (1993).

19 PrP Sc is host-encoded Intronless gene DNA PrP C Expression, maturation S-S H1 H2 H3 GPI Cu domain Conversion CHO CHO PrP Sc Proteinase K digestion PrP 27-30

20 PrP C PrP Sc Structure α-helix ~43% ~34% β-sheet ~3% ~43% Solubility + - Protease - + resistance + - Genetic and cell biological experiments are in favour of the prion hypothesis too.

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23 PrP C PrP Sc α-helix ~43% ~34% β-sheet ~3% ~43% Solubility + - Protease - + Resistance + - Conformation is the key!

24 Financial Times: BSE PrP β αb αc CHO CHO GPI (TM region) amino acids, $6 billion : $42 million per residue

25 Some issues in prion replication. Does the PrP C get unfolded? (stable protein) and, if so, how? How does the unfolded protein (or native PrP C ) get templated into a PrP Sc - like shape??? PrP C PrP-U? PrP Sc

26 Heterodimer hypothesis (Prusiner) A large energy barrier prevents spontaneous conversion of PrP C to PrP Sc. PrP C is unfolded by a hypothetical molecular chaperone called protein X. The replication intermediate is a PrP C /PrP Sc heterodimer (60 kda). PrP C PrP-U? PrP Sc

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28 Seeding hypothesis (Caughey) There is only a small energy barrier between PrP C and PrP Sc but spontaneous conversion is prevented by a kinetic barrier: conversion is too slow. Once a pre-formed seed of PrP Sc multimers is made the long lag period is avoided and PrP C to PrP Sc conversion takes place rapidly of the surface of the multimeric PrP Sc. As multimers get bigger they fragment and thus can create multiple new seeds. Replication intermediates are big.

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30 All labs agree conversion takes place on the cellsurface or in an early endosomal compartment -distinction from viruses - (Implies an ectochaperone modulates folding)

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32 Variant prion proteins modulate disease susceptibility Mouse PrP gene mutations Sheep PrP gene mutations Human PrP gene mutations

33 Net genotype for polymorphism affects outcome of infectious, sporadic or familial prion diseases Methionine (M)129 Valine (V)129 38% 51% 11% M M M V V V

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35 Birefringent amyloid plaques in a prion disease (GSS) Congo Red staining of Maltese-cross shaped GSS amyloid plaques. Plaques can also be stained with thioflavin S, or with PrP-directed antibodies

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37 Inheritance of a prion mutation tracks with disease in GSS families

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40 Sporadic prion disease No families. No clusters to indicate infectious spread Disease appears out of nowhere Presumably due to spontaneous misfolding of PrP C. Risk factors are unknown. PrP C PrP-U? PrP Sc

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42 Prion strains Strains are isolates of prions that can be distinguished in the same inbred host (typically mice or hamsters) Do not confuse with strains of mice!

43 Distinguishing prion strains (1) No nucleic acid to genotype Time-lapse to disease Pattern of neuropathology

44 Distinguishing prion strains (2) Sizes of protease-resistant PrP fragments on a blot Pattern of PrP glycosylation on a blot Concentration of denaturant needed to render the protein protease-sensitive

45 Strains are encrypted by different PrP Sc conformations Stable but distinct incubation time to disease onset, neuropathology, sizes of protease-resistant PrP fragments

46 Prion vs. viral infections Facts Often (but not always!!!) have spongiform pathology post mortem. Sometimes have CNS amyloid, but there other CNS amyloidoses Will typically have protease-resistant PrP. No genome.

47 Prion vs. viral infections in practice If agent is DNAse-resistant does it automatically mean it is a prion infection? If agent is RNAse-resistant does it automatically mean it is a prion infection? If agent is protease-resistant does it automatically mean it is a prion infection? No genome, therefore no PCR assays. Absence of a nucleic acid is not a useful criterion for day-to-day diagnosis to distinguish from viral infections. But most types of prion infections will have protease-resistant PrP seen on a gel (NB: need to use an antibody too for this western blot assay).

48 Prion Neuroinvasion Systemic route of PrP Sc to the CNS, centripetal spread

49 Prion Neuroinvasion (contd) CNS to retina or muscle, centrifugal Synapse jumping by PrP Sc may distinguish prion disease from nontransmissible amyloidoses Cleavage adjacent to or within the GPI anchor followed by receptormediated endocytosis? GPI anchor excision & insertion into membrane of the next cell Endocytosis and vesicle escape mediated by N-terminal PrP sequences

50 Neuronal targeting Different prion diseases affect different sub-cellular targets Spongy change Synaptic deposition of PrP Sc Extracellular plaques made of PrP Sc Lysosomal perturbations? Different shapes of PrP Sc have different trafficking and perturb different receptor mediated events?

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52 Part II: Prion protein physiology PrP C function Doppel, the dark shadow Shadoo, shadow of PrP

53 A: Neurophysiology of PrP C 17 PrP binding proteins identified to date. Signal transduction Neuronal apoptosis Binding to transition metals and GAGs Mainly on the cell-surface. PrP k/o (Prnp 0/0 ) mice have two phenotypes. (G.Millhauser)

54 Number of PrP copper sites at neutral ph Substrate n Methodology Reference PrP Equil. Dialysis (Brown et al, 1997) PrP CD (Viles et al, 2000) SHa ESI-MS (Whittal et al, 2000) PrP DEPC (Qin et al, 2002) PrP ESI-MS (Kramer et al, 2001) PrP ,5 DEPC (Qin et al, 2002) PrP EPR (Cereghetti et al, 2001) PrP C A B C Insert or, = F-CJD CHO CHO GPI

55 The PrP-interactor proteome Time-controlled transcardiac perfusion cross-linking: pull-down with α-prp Fab HuM-D18 Schmitt-Ulms et al, Nature Biotechnology 6: (2004)

56 Regular PrP k/o mice Look pretty normal Have normal CNS development, very mild white matter changes in old age Have disputed circadian rhythm deficits Have disputed electrophysiological deficits in the hippocampus Have accentuated response to insults

57 Stroke models and hypoxia: PrP C, and PrP k/o mice Infarct size, tetrazolium blue staining PrP staining near ischemic lesions

58 Irregular PrP k/o mice Prnp 0/0 line (wt) Zrch I NPU Ngsk Rcm 0 ZrchII Riken Ataxia No No No Yes Yes Yes Yes Neo Dpl Actually activation of the next gene! - and blocked by reintroduction of PrP C

59 NMR structures of PrP and Doppel (Huaping Mo, Jane Dyson)

60 Doppel protein made on developing sperm Wild type Knock-out

61 Three members in the PrP supergene family, 2 expressed in the CNS PrP C (Human Chr 20) Octarepeat region (TM region) αa αb CHO CHO αc GPI Brain + other tissues Doppel (Human Chr 20) αa αb/b αc CHO CHO GPI Testis, PNS, adult cerebellum Shadoo (Human Chr 10) Basic repeat region (TM region) CHO GPI Areas of the Brain, retina

62 Sho Mouse Shadoo GPI a) c) e10 e11 e12 e13 e14 e15 e16 e17 20 Sho Empty vector b) 28 Wild-type Sho PI-PLC (U) * d) α-rsho α-sho(86-100) P1 P2 Adult PNGaseF Sho Sho Sho α-prp PrP

63 Interlocking expression of PrP C and Shadoo a b c d e Patrick Horne f g h i j k α-shadoo peptide 04SH-1 α-shadoo peptide 06SH-3 α-recomb. Shadoo 06rSH-1 α-moprp 7A12 α-haprp 3F4 α-calbindin

64 PrP C and shadoo trigger an ancient neuroprotective pathway neuroprotective PrP Octarepeats αa CHO GPI αb CHO αc CHO GPI Shadoo Random coil Need an unstructured domain: a site for protein/ protein interactions? Need a polyvalent binding site: scavenging or sensing? Action against different types of toxic stimuli