Mec echanisms hanisms of Sel electiv ective e Cell ell Vul - - PowerPoint PPT Presentation

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Mec echanisms hanisms of Sel electiv ective e Cell ell Vul ulnerability erability in Hum uman an Prion ion Disease ease Christina Sigurdson Department of Pathology UC San Diego Protein misfolding and aggregation is a common feature

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Mec echanisms hanisms of Sel electiv ective e Cell ell Vul ulnerability erability in Hum uman an Prion ion Disease ease

Christina Sigurdson

Department of Pathology UC San Diego

SLIDE 2

Protein misfolding and aggregation is a common feature of neurodegeneration

Alzheimer Parkinson Amyotrophic lateral sclerosis Huntington Creutzfeldt-Jakob

a-synuclein substantia nigra

Modified from Forman et al, Nature Medicine, 2006

PrPSc plaques cerebellum Ubiquitin inclusions spinal cord Amyloid-b plaques cortex Poly-Q inclusions striatum

SLIDE 3

distinct conformations

Prion conformational subtypes: Distinct disease phenotypes occurring in patients

Subtype 2 Subtype 1

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Human prion disease: prion plaques vary in morphology and cells targeted

Sporadic CJD Plaque deposits Perineuronal deposits vCJD Plaques 1 2 3

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Prion protein aggregates vary by morphology and cells targeted in mice

mCWD 22L Me7 RML

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What mechanism underlies the selective cell vulnerability in prion disease?

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Heparan sulfate proteoglycans and prion pathogenesis

OH OH/SO3 CH2OH/SO3 NH/Ac/SO3/H OH/SO3

O O

COOH

Core protein

HS chain

Diverse glycoproteins on cell surfaces and in

the extracellular matrix

Promotes the internalization of PrPC and

propagation of PrPSc

Prolongs prion disease in scrapie-infected

rodents

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Hypothesis: PrPSc interaction with heparan sulfate proteoglycans is a major determinant underlying prion cell tropism

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Defining the HS molecules associated with the most common human CJD subtypes

1) Purify PrPSc from 3 brain regions

Prion-infected brain purification

* * *

2) Identify the PrPSc bound heparan sulfate by liquid chromatography-mass spectrometry (LC/MS)

Quantify N-SO3, 2-O-SO3, 6-O-SO3 groups and N-acetylated, N-sulfated and N-unsubstituted glucosamine residues

GlcNH2

CAA pos CAA neg 1 2 3

mol (%)

Amyloid β: cerebral amyloid angiopathy (CAA) IHC Congo red HE

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How does the sulfation of HS molecules impact prion replication?

Seed PrPSc Increase in PrPSC Sonication PrPC PrPC produced in RK13 cells

Glycosylated
GPI-anchored
3F4-tag

Amplification

f misfolding

Detection of new PrPSc using 3F4 tag Fragmentation

f aggregates

creates new seeds

Heparan sulfate

Different variably sulfated HS

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Heparin and pentosan polysulfate enhance prion conversion in a dose-dependent manner

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Heparin and PPS lead to a decrease PrPSc levels in persistently prion-infected cells

Demonstrates the paradoxical effect of HS – in cell lysate amplification versus in live cells and in vivo

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50% 75%

Challenge with prions Terminal disease Analysis of brain Biochemical properties Histopathology Biological properties

The impact of sulfation of HS chains on prion disease progression

Wild type mice Ext1+/- : shorter HS chain length

Ongoing……

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PrPSc purified for mass spectrometry analysis to

identify the heparan sulfate bound to PrPSc

Pentosan polysulfate promotes PrPSc formation in vitro

in the PMCA assay

Heparin and pentosan polysulfate decrease PrPSc

levels in prion-infected cells in culture

Conclusions

SLIDE 15

Acknowledgements

UC San Diego Patricia Aguilar-Calvo Tim Kurt Cyrus Bett Patricia Gaffney Jun Liu Peter Kobalka Katrin Soldau Jeff Esko Chrissa Dwyer Case Western Reserve University Witold Surewicz National Institutes of Health, NINDS Creutzfeldt-Jakob Disease Family Foundation