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 blotTreatment 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

Prion disease

PRNP mutation

PRNP codon 129

PrPres isotype

Histological correlate

Reference

Sporadic CJD (Myoclonic, Heidenhain variants)

None

MM, MV

Type 1

Synaptic and coarse granular PrP staining in cortex.

Parchi et al 1999

Sporadic CJD (Ataxic variant)

None

VV

Type 2A

Plaque-like, focal and perineuronal PrP staining.

Parchi et al 1999

Sporadic CJD (Kuru-plaque variant)

None

MV

Type 2A

Amyloid plaques in the cerebellum.

Parchi et al 1999

Sporadic CJD (Sporadic fatal insomnia)

None

MM

Type 2A

Thalamic atrophy. PrP staining faint and variable.

Parchi et al 1999 Pan et al 2001

Sporadic CJD (Cortical variant)

None

MM

Type 2A (Basic glycans)

Cortical perivacuolar PrP staining.

Parchi et al 1999 Pan et al 2001

Sporadic CJD None VV Type 1 Faint synaptic PrP staining.

Parchi et al 1999

Do different PrPres types replicate with fidelity in vitro?

Soto et al, 2005

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

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

Inoculum Host Host PRNP PrPres (kDa)

None Human FFI(D178, M129) 19

FFI Mouse Tg(MHu2M) 19

FFI Tg(MHu2M) Mouse Tg(MHu2M) 19

None Human fCJD(E200K) 21

fCJD Mouse Tg(MHu2M) 21

fCJD Tg(MHu2M)

Mouse Tg(MHu2M) 21

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 -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 ExampleSulphonated 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

sCJD

CNSPNSOptic nerveRetinaOlfactory epithelium

vCJD

CNSPNSOptic nerveRetina

AppendixLymph nodePeyers’ patchesTonsilSpleenThymus

Wadsworth et al, (2001), Lancet, 358, pp171-80

Head et al, (2004), American Journal of Pathology, 164, pp143-53

Probable pattern of tissue infectivity in vCJD

Onset of symptoms

TIME

CNS Infectivity (perhaps 1,000 times higher than LRS)

LRS Infectivity

INF

EC

TIV

ITY

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 -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 productAlzheimer’s disease APP and A

Creutzfeldt-Jakob disease PrPc and PrPSc

Parkinson’s disease 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