Lecture 5Prion Diseases

David Saffen, Ph.D.Professor/Principal Investigator

Department of Cellular and Genetic MedicineFudan University, Shanghai, China

Email: saffen@fudan.edu.cn

Outline1. Introduction:

Transmissible spongiform encephalopathies (TSEs) aka “prion diseases”

2. Prion diseases in animals:ScrapieBovine spongiform encephalopathy (BSE)Additional TSEs in animals

3. Prion diseases in humans:KuruCreutzfeldt-Jacob Disease (CJD)New variant Creutzfeldt-Jacob Disease (vCJD)Gerstmann-Straussler-Scheinker disease (GSSD)Fatal Familial Insomnia (FFI)

4. Mechanisms of neurodegeneration in prion diseases5. Normal and adaptive functions of prion proteins6. Routes of prion infection and possible opportuities for early

intervention7. Prions and neurodegenerative diseases: common mechanisms

1. Introduction

Transmissible spongiform encephalopathies (TSEs) aka “prions diseases”

• Progressive, fatal neuro-degenerative diseases characterized by ataxia and dementia.

• Caused by “prions”: proteinaceous infectious particles

• The best characterized human prion is “prion protein” (PrP) a normal cellular protein that can undergo conformational changes to produce “self-replicating,” infectious variants

“Sponge-like” appearance of TSE brain;gliosis and loss of neurons are also characteristic of prion diseases

“normal” conformation

infectiousconformation

(Toxic!)

Key TSE and prion investigators

Daniel Carleton Gajdusek (NIH)1923-2008

Nobel Prize 1976Demonstrated that kuru agent

is infectious by transmitting the disease to monkeys

Stanley B Prusiner (UCSF-MS) b1942

Nobel Prize 1997Coined the word “Prion.”

Produced convincing experimentalevidence that prions are by themselves

“self-replicating” and infectious(i.e, independent of DNA or RNA).

Prevalence of human prion diseasesin the United States compared to other

neurodegenerative diseases (2000)

Prusiner SB, 2001

The prion gene (PRNP) and encoded protein (PrP)Human PRNP gene: Chr20p13

Processing, cellular location and putative functions of PrPC

• PrPC is translocated to the endoplasmic reticulum as 254-aa precursor; first and last 23 aa residues are removed, followed by addition of glycosyl-phosphatidylinositol (GPI)-anchor

• Mature protein is localized primarily on the outer surface of plasma membrane; enriched in neurons

• The functions of normal PrPc protein are unknown, although roles in the synapse formation, copper transport, and intracellular signaling have been proposed.

• Double-KO of PrP gene in mice is non-lethal

[ASN-linked]

Conformations and properties of “normal” and

pathogenic

forms of PrP

PrPC PrPSc

“cellular” “scrapie”

a-helix-rich b-sheet-rich

soluble* insoluble*

protease-sensitive

protease-resistant

*in nondenaturing detergents

2. Prion diseases in animals

Scrapie

• A fatal neurodegenerative disease of sheep and goats that was first recognized in Europe 250 years ago.

• Prototypic transmissible spongiform encephalopathy (TSE): causative agent is PrPSc.

• Transmission is thought to occur primarily through contact with placenta and placental fluids from infected ewes.

• Early symptoms include changes in behavior or temperament, scratching or rubbing body against fixed objects, loss of coordination, lip smacking and gait abnormalities.

• Susceptibility is influenced by polymorphisms in PrP gene; most infected sheep have the genotypes, 136VV, 136VA, or 171QQ

Bovine spongiform encephalitis (BSE)“mad cow disease”

• A fatal neurodegenerative disease in cattle characterized by spongiform degeneration of brain and spinal chord

• Transmission is thought to take place primarily by consumption of meat and/or brain-containing feed produced from contaminated cows and sheep

• Transmission to humans who ate meat from infected cattle during a major outbreak in Great Britain in the early 1980’s has been documented

• As of Oct 2009, over 200 human deaths had been linked to the consumption of contaminated beef. Over 4.4 million cows were destroyed in an effort to eradicate BSE in the British Isles.

Additional TSEs in animals

TSE disease Host

Prion

name

Prion

isoform

Chronic wasting

disease (CWD)

elk & deer CWD

prion

MDePrPsc

Feline spongiform

encephalopathy (FSE)

cats FSE

prion

FePrPsc

Transmissible mink

encephalopathy (TME)

mink TME

prion

MkPrPsc

Exotic ungulate

encephalopathy (EUE)

nyala &

greater

kudu

EUE

prion

NyaPrPsc

3. Prion diseases in humans

Kuru• A fatal disease characterized by progressive ataxia &

dementia; brains show spongiform neurodegeneration

• Associated with “mortuary rites” (ritualistic cannibalism) among Fore people of the Eastern Highlands Province of Papua New Guinea

• Kuru Epidemic in middle of the 20th century killed approximately 20% of Fore population: mostly women and children; epidemic ended following the banning of mortuary rites in 1960

• Causative agent is a self-propagating and infectious conformational variant of PrPc.

Papua New Guinea (PNG) Eastern Highlands Province (EHP)

Okapa Subdistrict

Fore tribe women suffering from kuru (1957)

The women are showing upper limb postures adopted to prevent postural tremors. From Philosophical Transactions of the Royal

Society B (Image: 2008 The Royal Society)

Population genetics of Kuru

• Homozygosity at PrP aa residue 129 (129MM or 129VV) is associated with early onset of kuru; by contrast, heterozygosity (129MV) is associated with late-onset or resistance to kuru.

• PrP 129M is the “ancestral” allele; the “derived” allele 129V is thought to have arisen about 500,000 years ago and has attained high frequencies in populations worldwide.

• It has been proposed that kuru-like epidemics have propelled the increase in 129V allele frequencies in these populations. Is this evidence for wide-spread cannibalism in human history?

• Heterozygosity at aa 127 (127GV) was also found to found to strongly correlate with protection against kuru in 129MMindividuals.

• An additional protective polymorphism, E219K, is found at high frequency in the Japanese population, where M129V is present at a very low frequency.

Fore women over the age of 50 who participated in mortuary feasts showed excess heterozygosity and

relatively low homozygosity for M129V

MM MV VV

Expected 8 15 7

Observed 4 23 3

c2 = [Oi-Ei]2/Ei = 8.267;

P = 0.003 (1 df)Fisher’s exact test: P = 0.01

Mead S et al, Science, 2003;Hedrick PW, Science, 2003

N x p2

30 x 0.266

N x 2pq

30 x 0.499

N x q2

30 x 0.233

Expected

(HWE)

8 15 7

p = f(M) = (8+8+15)/60 = 0.5167q = f(V) = (7+7+15)/60 = 0.4833N = 30

Haplotypes containing the 129M and 129V alleles are highly divergent in DNA sequence, suggesting an ancient split; the rapid rise of the 129V allele to high

frequencies in many population around the world may reflect “balancing selection.”

European

Japanese

ForeAfrican

Predicted haplotype geneaologies for four populations:Orange: 129MBlue: 129VPink: 129M, 219K

Note: a “kuru-resistant” PrP allelewas found in every population studied.

* Recent evidence has identified a secondprotective prion variant in the Fore population:G127V. Asante et al, Nature 522, 2015

M129V

11 majorEuropean haplotypes

Mead S et al, Science, 2003;

*

Creutzfeldt-Jacob Disease (CJD)Accounts for approximately 85% of prion disease cases: incidence =1-2/million in general population; Onset: 30-55 years; duration: several months to several years.

Histopathology includes: spongiform neurodegeneration, astrogliosis, and (in about 10% of cases) amyloid plaques containing PrPSc.

Most cases are sporadic and are caused by a spontaneous transition of PrPC to the PrPSc conformation; mutations identified in rare familialforms include: and R178N, V180I, E200K, R208H, V210I, & M232R.

Iatrogenic origins include, the therapeutic use of cadaveric human growth hormone, cornea or dura mater and reuse of surgical instruments or EEG electrodes; most victims of iatrogenic CJD are homozygous 129VV).

Spongiform degeneration Astrogliosis

Prusiner,2001

Variant Creutzfeldt-Jacob Disease (vCJD)

• Caused by eating meat from BSE-positive cows; causative agent is a self-propagating, infectious conformational variant of bovine PrPC (bPrPSc).

• Histopathology: spongiform degeneration; “florid plaques” composed of PrPSc surrounded by vacuoles.

• “Mad cow disease” epidemic in UK has claimed 176 victims to date, most homozygous MM129; M129/V129 heterozygotes seem to be protected.

• Sub-clinical infection in UK estimated at 1/2000! (Based on surveys of surgically removed appendices) Possible public health risk: blood trans-fusions, dentistry etc.

Spongiform degeneration “Florid plaques”

Gerstmann-Straussler-Scheinker (GSS) disease

• Autosomal dominant neurogenerative disorder; usually presents with cerebellar ataxia with pyramidal features, with dementia occurring later than in other prion diseases; onset of disease: 40-60; duration: 3-7 years

• Histopathology: dense core of amyloid surrounded by smaller globules of amyloid

• Caused by mutations in PrPC; identified mutations include: P102L, P105L, P105S, A117V, Y145Stop, N160Stop, F200K, & N217R

PrP amyloid plaques in cerebellum Visualization of plaques after hydrolytic

autoclaving and immunostaining

Fatal Familial Insomnia (FFI)

• Fatal autosomal dominant neurodegenerative disorder; usually presenting with untreatable insomnia, dysautonomia and dementia; selective degeneration of the thalamus; onset typically 40-50 yr; duration of illness: 12 to 16 months.

• Histopatholgy: neuron dropout and gliosis, especially in thalamus and inferior olivary nucleus of brain stem; relative lack of spongiform neurodegeneration

• Pathogenic mutations: R178N + 129M; R178Stop; sporadic cases with no known PrP mutations have also been reported.

Gliosis in thalamus(GFAP staining)

Summary of known human PrP mutations

and polymorphisms in inherited prion diseases

Lloyd, Mead, Collingwe, Brain, 2013

4. Mechanisms of neurodegenerationin prion diseases

Evidence that prions (protein-only) are the sole infectious agents in TSEs

Modified from Soto and Satani, 2010

The infectious agent is too small to be a virus or bacteria.

Infectivity is not blocked by treatments that destroy nucleic acids.

Injection of highly purified PrPSc induces prion disease in animals.

PrPSc is detected only in individuals with prion disease.

All inherited cases of prion disease are associated with PrP mutations.

Transgenic mice over expressing PrPC develop prion disease.

PrP knockout mice are resistant to prion infection.

PrPSc self-propagates in vitro by inducing the misfolding of PrPC.

PrPSc induces recombinant and purified PrPC to become infectious.

Differences between prion “strains” depend upon differences inprion folding

Mechanisms by which prions stimulate neurodegeneration

• The relatively mild pathologies observed in PrP KO mice suggest that loss of function is not the main mechanism

• Rather, the PrPSc form, by itself, or PrPSc in the form of higher molecular weight fibrils or aggregates are implicated

• The conversion of cellular membrane-anchored GPI-PrPC to the GPI-PrPSc form may also result in neurotoxic signaling that eventually kills the neuron to which it is attached.

Models for PrPSc propagation

Tuite MF and Cox BS, Nature Reviews Cell Biology 4, 878-890, 2003

Multiple neurodegenerative pathways mediate TSEs

Soto and Satani, 2010

Putative signaling pathways for PrPSc-induced neurodegeneration

Soto and Satani, 2010

Model for neurodegeneration in prion diseases

Soto and Satani, 2010UPR = Unfolded Protein Response

5. Normal and adaptive functions of prions

Putative roles for normal PrP(in mammals)

• Neuroprotection

• Anti-oxidation

• Binding of copper and other divalent cations, including Fe2+ (within the octapeptide repeat)

• Myelin maintenance

• Neurogenesis

• Circadian rhythm

• Sensitivity of brain to hypoxia, ischemia, seizures

• Immune system: T cell development, macrophage function, hematopoietic stem cell self-renewal

Yeast prions• The yeast translation termination factor Sup35 forms a prion state designated [PSI+], with reduced activity compared the “normal” [psi-] state.

• Cells that contain [PSI+] have different properties that promote survival in certain environments.

• In addition to Sup35,at least 8 additional yeast proteins form prions, including Rnq1, a protein that stimulates other proteins to convert to their prion forms.

Monitoring the [PSI+] prion by a simple color assay. The presence of the prion allows translation read-through of an aberrant stop codon in the ADE2 gene. The consequence of this is that prion-containing cells are white and cells lacking prions are red. (Gary Jones)

Prion epigenetics:

A. “Life-cycle of yeast amyloid

prion;

B. The effect of stress on prion formation and

loss

Halfmann& Lindquist,

2010

Prion phenotypes can result from either loss or gain of function

Prion strains:“mutation” and selection

Collinge J, 2010

Specific prion conformational states (oftencorrelated withdifferent prion aminoacid sequences) self-propagate, givingrise to prion “strains”with distinct bio-chemical propertiesand host range.

Rather thanmaintaining prionswith a specificconformation, however, hosts maymaintain a spectrumof prion states that can be transmitted to secondary hosts withdiffering efficiencies.

6. Routes of prion infection and possible opportunities for early intervention

Aguzzi, Nuvolone & Zhu,Nat. Rev. Immunol., 2013

Aguzzi, Nuvolone & Zhu, Nat. Rev. Immunol., 2013

Peripheral prion replication and the involvement of follicular dendritic cells (FDC)

Secondarylymphoid

Organs (SLOs)

TNFR: tumor necrosis factor (TNF)receptor

PDGFRb = platet-dirived growth factorreceptor-beta

LTa1b2: lymphotoxin-abhetero-trimer produced by lymphoid tissue inducer (LTi) cells or B cells

MFGE8: milk fat globule epidermalgrowth factor 8

FcyRIIB: low affinity immunoglobulin-g-Fc region receptor II-B

CD21, CD35: dendritic cell antigens

FDC = follicular dendritic cell

Prion-induced neuro-

degeneration and potential therapeutic

targets

Aguzzi, Nuvolone & Zhu,Nat. Rev. Immunol., 2013

Soluble (PrP-Fc)2 dimers act as decoys that prevent

the conversion of endogenous PrPC to PrPSc

2 x PrP (lacking GPI attachment sites)

Dimerized Fc domain(lacking FcR and complement

protein binding sites)

Meier P, et al, Cell 113, 49-60, 2003

Expression vector

Meier P, et al, Cell 113, 49-60, 2003

7. Prions and neurodegenerative diseases:common mechanisms

Basic mechanisms of protein aggregation

Brundin P et al, 2010

Potential pathways of uptake and release of

protein aggregates

by cells

Brundin P et al, 2010

Principles for progression of neuropathological changes

Brundin Pet al, 2010 ALS

FTLD

Evidence for prion-like properties of key proteins implicated in neurodegenerative diseases

Prusiner SB, Science 336, 2012

References• Prusiner SB, Shattuck Lecture--Neurodegenerative diseases and prions,

The New England Journal of Medicine 344, 1516-1525, 2001

• Wadsworth DF and Collinge J, Update on human prion disease, Biochimica et Biophysica Acta 1772, 598-609, 2007

• Westergard L et al, The cellular prion protein (PrPC): its physiological function and role in disease, Biochimica et Biophysica Acta 1772, 629-644, 2007

• Mastrianni JA, The genetics of prion disease, Genetics in Medicine 12, 187-194, 2010

• Loyd SE, Mead S, Collinge J, Genetics of prion diseases, Genetics and Development 23, 1-7, 2013

• Mead S et al, Balancing selection at the prion protein gene consistent with prehistoric epidemics, Science 300, 640-643, 2003

• Hendrick PH, A heterozygote advantage, Science 302, 57, 2003

• Soto C and Santani N, The intricate mechanisms of neurodegeneration in prion diseases, Trends in Molecular Medicine, in press, 2010

References• Collinge J, Prion Strain Mutations and Selection, Science 328, 1111, 2010

• Angers, RC et al, Prion strain mutation determined b prion protein conformational compatibility and primary structure, Science 328, 1154-1158, 2010

• Halfmann R & Lindquist S, Epigenetics in the extreme: prions and the inheritance of environmentally acquired traits, Science 330, 629-632, 2010

• Aguzzi A, Nuvolone M and Zhu C, The immunobiology of prion diseases, Nature Reviews Immunology 13, 888-902, 2013

• Kraus A, Groveman BR and Caughey B, Prions and the potential transmissibility of protein misfolding diseases, Annual Review Microbiology 67, 543-564, 2013

• Aguzi A and Rajendran L, The transcellular spread of cytosolic amyloids, prions and prionoids, Neuron 64, 783-790, 2009

• Brudin P et al, Prion-like transmission of protein aggregates in neurodegenerative diseases, Nature Reviews Cell Biology 11, 3001-3007, 2010

References• Kim J-S & Holtzman DM, Prion-like behavior of Amyloid-ß, Science 330,

918=919, 2010

• Eisele V et al, Peripherally applied Ab-containing inoculates induce cerebral b-amyloidosis, Science 330, 980-982, 2010

• Masuda-Suzukake M, et al, Prion-like spreading of pathological a-synuclein in brain, Brain, January 13, 1-11, 2013

• Prusiner SB, A unifying role for prions in neurodegenerative diseases, Science 336, 1511-1513, 2012

• Lahiri DK, Prions: a piece of the puzzle?, Science 336, 1172, 2012

• Kim HJ et al, Mutations in prion-like domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and ALS, Nature 495, 467-473, 2013

• King OD, Gitler AD, Shorter J, The tip of the iceberg: RNA-biding proteins with prion-like domains in neurodegenerative disease, Brain Research 1462, 61-80, 2012

Journal Presentation

• Background articles:

1) Palfreman J, The Virus That Could Cure Alzheimer’s, Parkinson’s,

and More, Nova next, Public Broadcasting Service, March 23, 2016

2) Coghlan A, Universal plaque-busting drug could treat various brain

diseases, Daily News, New Scientist, July 19, 2015

• Research article:

Krishanan R et al, A bacteriophage capsid protein provides a

general amyloid interaction motif (GAIM) that binds and remodels

misfolded protein assemblies, Journal of Molecular Biology 426,

2500-2519, 2014

Internet resources

• World Health Organization http://www.who.int/zoonoses/diseases/prion_diseases/en/

• National Institute of Allergy and Infectious Diseases (NIH) http://www.niaid.nih.gov/topics/prion/Pages/default.aspx

• Centers for Disease Control and Prevention http://www.cdc.gov/ncidod/dvrd/prions/

• National Prion Disease Pathology Surveillance Center

• http://www.cjdsurveillance.com/