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The New Biology of Brain TraumaEvidence for prions causing many neurodegenerative diseases & approaches to creating novel therapeutics

Institute for Neurodegenerative DiseasesUniversity of CaliforniaSan Francisco, CA

Stanley PrusinerFebruary 14, 2013Institute for Neurodegenerative Diseases

Suicide in United States - 2013• 20,000 of the 30,000 deaths from guns in US in 2010 were suicides. Guns are more effective than pills: suicide with guns is 85% and with pills only 2%.

• Suicide is the 3rd leading cause of death among teenagers.• In 2012, 349 active-duty US military personnel committed suicide.

• A VA study for 2010-12 found ~8000 US veterans commit suicide annually – about 22 per day or 1 every hour. About 2/3 of veterans committing suicide are over 50.

• Investigations of frontotemporal dementias (FTDs) and tau prions seem to offer new approaches to the study and possibly the prevention of many suicides.

A Tale of Disruptive Thoughts• 60 y.o. white female from Marin county was

admitted to UCSF neurology service with difficulty speaking, performing complex movements and remembering recent events. Over the next 3 months, her condition deteriorated—she became unable to move her extremities and was mute before dying.

• Her clinical diagnosis of Creutzfeldt-Jakob disease (CJD) was confirmed at autopsy and by transmission of CJD to a monkey at the NIH 1.5 years later.

• CJD was thought to be caused by a slow-acting virus. Eventually, my colleagues and I showed that prions not viruses cause CJD.

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1974 - 1982: Enriching fractions for scrapie infecti vity from rodent brain required 8 years and led to many disco veries

The absence of any evidence for a virusprompted me to introduce the term "prion”in order to distinguish the unprecedented scrapie pathogen from both viruses and viroids.

The reaction of many scientists to the word “prion” and the idea of “infectious proteins” was less then enthusiastic!

Prions

Discovering prions form amyloid suggested plaques and tangles in other neurodegenerative

diseases might also be etiologic….Neurodegenerative diseases, prion

proteins and inheritanceNeurodegenerative Diseases (NDs) Causative Prion

ProteinsPercent Inherited

Mad cow, scrapie, kuru & Creutzfeldt-Jakob disease (CJD)

PrP 10-20

Alzheimer’s disease (AD) Aβ � tau 10-20Parkinson’s disease (PD) α-synuclein 10-20Fronto-temporal dementia (FTD) & post-traumatic FTD called chronic traumatic encephalopathy (CTE)

tau, TDP43, FUS(progranulin)

10-20

Amyotrophic lateral sclerosis (ALS) SOD1, TDP43,FUS 10-20Huntington’s disease (HD) huntingtin 100

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Different proteins become transformed into prions and each causes a distinct set

of neurodegenerative diseases

Alzheimer’sAβ plaque

FTD, CTE, ADtau tangle

Parkinson’sα-synucleinLewy body

Huntington’snuclear

inclusion

Prions explain many facets of the neurodegenerative diseases (NDs)

• Each ND is caused by a different protein that turns into a prion.

• The self-propagation of prions explains the steady progression of NDs.

• Spread of neurodegeneration is due to the movement of prions from one neuron to another.

• Prions explain the many different forms of NDs: infectious, inherited, sporadic and even post-traumatic.

• Late-onset inherited neurodegen-eration requires a second event.

Third Judgment of Paris• In 1874, the impressionists held their first exhibition in Paris on their way to achieving worldwide acclaim.

• French oenophiles chose both a California cabernet and chardonnay over some exalted French wines in 1976. This surprising adoration for California wines forever changed global viticulture.

• In February 2012, a meeting in Paris was convened to mark the remarkable convergence of experimental findings showing that many different neurodegenerative diseases are caused by prions. For the first time, this creates a unified view of these devastating illnesses.

Transmission of Alzheimer’s to monkeys• In 1970s, monkeys inoculated with

brain homogenates prepared from 52 AD patients; 0/33 sporadic AD cases transmitted to monkeys but 2/18 cases of fAD transmitted after ~50 months showing spongiform changes like those in CJD (Goudsmit et al. 1980).

• In the 1990s, 18/20 marmosets <10 y.o. were Ab positive by immuno-staining after inoculation with sporadic AD, familial AD or Down’s brain homogenates. 0/11 uninjectedmarmoset controls <10 y.o. were Abpositive (Ridley et al. 2006).

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Evidence that Alzheimer’s prions can be experimentally transmitted to transgenic mice• Jucker, Walker et al (2006) hastened Ab amyloid plaque

formation after ic inoculation of Tg(ΔHuAPP) mice with brain homogenates containing Ab amyloid from AD patients or old Tg(APP23) mice expressing the Swedish mutation.

• Eisele, Jucker et al (2010) accelerated Ab amyloid plaque formation after ip inoculation of brain homogenates.

• Watts, Giles et al (2011) showed ic inoculation of AD patient or old Tg(APP23) mouse brain homogenates quickened the appearance of bioluminescence in bigenic mice expressing mutant HuAPP and luciferase. Morales et al inoculated AD brain into Tgmice expressing wt HuAPP and found Aβ deposits.

• Stöhr et al (2012) showed purified Aβ fibrils from old Tg(ΔAPP) mouse brains and synthetic HuAβ40 or a dimer increased in bioluminescence and Aβ amyloid plaque formation.

Amyloid Plaques and Neurofibrillary Tangles• In Alzheimer’s, the brain is filled with plaques and neurofibrillary tangles (NFTs).

• In FTD, the brain only has tangles.

• In Alzheimer’s and FTD, normal proteins (Aβ and tau) become prions when they undergo alternative folding.

• Prions coopt more and more proteins to adopt altered shapes — this slow process becomes self-propagating.

Bioluminescence Imaging of Astrocyte Activation Using Tg(Gfap-luc) Mice

Tg(Gfap-luc)

0 2 4 6 8 10 12 14 16 18 20 22

Time (weeks post inoculation)

Exp

ress

ion

(Log

2)

Bioluminescence imaging (BLI) used as a surrogate biomarker to detect prions in the brains of rodents

Living Tg(Gfap-luc) mice were inoculated with RML prions and

scanned every 2 weeks after a luciferin

injection. Injectedwith prions

no prions

BLI signal increases

Clinical signs

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Kinetics of GFAP deposition in astrocytes of Tg(Gfap-luc) mice after inoculation with RML prions

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Incubation time (days)

PrP

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Incubation time (days)

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GFAP mRNA (blue)Bioluminescence (red)

and control (black)

BLI of Tg(APP23:Gfap-luc) mice inoculated with purified Aβ prions prepared from older Tg(ΔAPP) mice

10 mon post-inoculation

Preparation of synthetic Aβ aggregatesLyophilized Aβ peptides were dissolved at 500 µg/ml in 25 mM NH4HCO3 buffer, pH 8.5 and immediately diluted 1:1 in NaPO4buffer, pH 7.2; aliquots were incubated at 37 °°°°C for 72 h under constant agitation. Below, Ag stain after limited PK digestion of the aggregates and SDS-PAGE.

Characterization of Aβ aggregates byEM and AFM

BLI of Tg(APP23:Gfap-luc) mice inoculatedwith protease-resistant synthetic Aβ prions

3.5

10mM NaPi

Tg(APP23:Gfap-luc)

Limited digestion with proteinase K (50 µg/ml) was performed at 37°C

for 30 min.

�

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Alzheimer’s disease is a double prion disease initiated by Aβ prions

APP � Aβ peptide � Aβ prion � Aβ amyloid fibril � Aβ plaque

tau protein � tau prion � paired helical � neurofibrillaryfilament tangle

�

Aβ & TauPrions

Aβplaques

Tautangles

Aβ prions stimulate the conversion of tau into prions

✚✚✚✚

✚✚✚✚

Many different causes of brain injury provoke neurofibrillary tangle formation

Diseases with Neurofibrillary Tangles• Frontotemporal dementias (FTD), PSP,

CBD, Pick’s disease• Argyrophilic grain disease • Traumatic brain injury (TBI) & chronic

traumatic encephalopathy (CTE, dementia pugilistica)

• Alzheimer’s disease • SSPE & rabies viral infections • Niemann-Pick Type C disease• Postencephalic Parkinson’s disease (PD) • Guam ALS-PD with dementia • Myotonic dystrophy • Familial prion disease - GSS(F198S) &

fCJD with octarepeat expansions

Tau

FTD and CTE occupy an interface between psychiatry and neurology

• FTDs are neurodegenerative diseases caused by the accumulation of tau prions.

• Changes in personality, behavior, executive function and language are hallmarks of the FTDs.

• Behavioral changes range from lethargy to disinhibition and include apathy, social withdrawal and inappropriateness.

• Drug addiction and alcoholism are frequently seen.

• Violent mood swings from euphoria to depression can precede suicide.

FTDs, concussions and tau prions• Tauopathies include inherited and sporadic frontotemporal dementia (FTD) in older adults as well as post-traumatic FTD often in young males.

• Multiple lines of evidence argue that these disorders are caused by tau prions.

• Besides football, soccer and hockey players, boxers and military personnel suffer from post-traumatic FTD also called dementia pugilistica and chronic traumatic encephalopathy (CTE).

• Over 50 athletes and 20 veterans have been diagnosed pathologically with CTE.

Micrograph by Ann McKee

Micrograph by Ann McKee

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NFTs in 27 y.o. marine with PTSD after repeated exposure to blasts from mortars and IEDs

• 27 y.o. US marine with 2 deployments to Fallujah and Ramadi. He was exposed to mortar and IED blasts <50 meters away. Following his 2nd deployment, he developed progressive cognitive impairment, memory deficits, behavioral and mood changes and alcohol abuse. Dx PTSD. He committed suicide 8 months after honorable discharge.

• At autopsy, the gross brain was normal. Microscopically, there were numerous tau positive NFTs in the frontal cortex (Omalu et al, Neurosurgical Focus, 2011).

Self-propagating tau aggregates in cultured cells and Tg(HuTau) mice

• After fibrillization of recombinant wtfragment (243-375 aa) or mutant HuTau, cultured cells were exposed to the fibrils, which induced de novo tau phosphorylation and amyloid fibril formation. The process was self-propagating and coalesced tau fibrils resembled NFTs.

• Tau aggregates from Tg(HuTau,P310S) mice were injected into Tg(wtHuTau) mice where they induced de novo hyperphosphorylatedtau aggregates after 6 months.

• Tg mice expressing mutant (DK280) Hutaurepeat domain (244-372) under control of the Tet-off system initiated the persistent de novo aggregation of wt mouse tau.

Tg(wtHuTau) mice

Tg(HuTau,∆K280) mice

Mutant human tau prions in Tg brain homogenates detected by BLI

Inoculate homogenate containing wild-type or A152T tau prions

Tg(MAPT*P301S,A152T:Gfap-luc)

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Aged Tg(MAPT*P301S) brain

Inoculum

PBS

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PSP brain

Inoculum

Control human brain

Pick's disease brain

Human tau prions in brains of sporadic PSP and Pick’s cases detected by BLI

Inoculate homogenate containing aggregated tau

Tg(MAPT*P301S:Gfap-luc)

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Incubation Periods for Tau Prions in Tg(MAPT*P301S:Gfap-luc) Mice

Brain inoculumMean incubation period ± s.e.m.

(days)

Number of mice

analyzed

P-value compared to uninoculated mice

None 170 ± 5 11 --

Control human 168 ± 4 7 P > 0.05

Aged Tg(MAPT*P301S) 93 ± 8 6 P < 0.001

PSP 88 ± 14 6 P < 0.001

Pick’s disease 83 ± 10 6 P < 0.001

CTE 91 ± 5 8 P < 0.001

Detection of tau, Aβ and α-synuclein prions using bioluminescence imaging in bigenic mice

• Incubation times measured by BLI for bank vole PrPSc prions are as short as ~21 days.

• For purified and synthetic Aβprions causing Alzheimer’s, they are ~150 days.

• For tau prions causing inherited, sporadic and post-traumatic FTD, incubation times are ~85 days.

• For α-synuclein prions causing Parkinson’s, they are ~150 days.

Disease Prion Incubation times

Scrapie, CJD, mad cow, CWD

PrPSc ~21 d

Alzheimer’s A β ~150 d

FTD, PSP, Pick’s, CTE

tau ~85 d

Parkinson’s, LBD, MSA

synuclein ~150 d

Incubation time is the interval from inoculation to a sustained increase in bioluminescence

Evidence for α-synuclein prions causing Parkinson’s disease (PD)

• When α-synuclein refolds into a β-sheet-rich conformation, it becomes a prion. Synuclein prions assemble into amyloid fibrils, some of which form the exterior surface of Lewy bodies that are the hallmark of PD. Mutations in the α-synuclein gene cause familial PD.

• Toward finding better therapeutics, grafts of fetal substantia nigra(SNc) cells were injected into the striatum of patients with advanced PD. A decade after transplantation, Lewy bodies were found in the grafted fetal cells (Kordower et al, Li et al 2008).

• Human α-synuclein fibrils endocytosed into cultured mouse hippocampal neurons recruit mouse α-synuclein to aggregate (Volpicelli-Daley et al 2011).

• Tg(SCNA,A53T) mice expressing mutant α-synuclein were inoculated with aged Tg(SCNA,A53T) brain homogenates and showed shortened survival (Mougenot et al. 2011; Luk et al. 2012) and bigenic mice exhibited early changes bioluminescence (Watts et al. in prep).

Mutant human α-synuclein prions in Tgbrain homogenates detected by BLI

Inoculate homogenate containing aggregated

α-synuclein

Tg(SNCA*A53T+/-:Gfap-luc)

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NoneSpon. ill Tg(SNCA*A53T+/+)

Inoculum

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Neuropathological Changes in Inocu-lated Tg(SNCA*A53T+/-:Gfap-luc) Mice

Inoculum

Phosphorylatedα-synuclein GFAP Iba1

None

Spon. ill Tg(SNCA*A53T+/+)

162 days postinoculation

Roadmap for discovering effective therapeutics for halting neurodegeneration

Drug discovery is a complex process

Humans

• Need many thousands of compounds in developing one useful drug.• Potential drugs are optimized through many rounds of chemical synthesis.• Need real efficacy in animal models since most drugs fail in humans.

Assays Hits Leads Animals

AMT lead compounds for treatment of Creutzfeldt-Jakob disease

IND81

IND24 IND22

(Kawasaki et al 2007)

PrPSc Drug Pipeline—Diverse ChemicalsLibrary HTS SPC EC50 PK EfficacyLibrary HTS SPC EC50 PK Efficacy

ChemBridge-1SPECS

ChemBridge-2(CNS focused)

Screened:24,000 CB-1

28,000 SPECS

Found 3,100 hits

SPC on 2,033

Compounds

Found 14 scaffolds

EC50 >700 compounds

44 compounds with improved profile in PK

queue

8 testedBrain exposure:

0.5–1 µM (4 AMTs)2.5 µM (1 pyAMT082)3 µM (1 benzothiazole)

4–8 µM (2 Amide-oxazole)

3 compounds2 scaffolds

Compound B

SMDC-256082

SMDC-256163

SAR-by-catalog:Over 300 compounds

Identified 7 potent scaffolds

SAR-by-synthesis:

81 of 84 compounds received

66 tested: 11 cmpds <100nM29 cmpds <500nM

From 6 scaffolds:AMT

Amide Benzoxazole/benzothiazole

PiperazineStilbeneThiazole

1-day mouse PK

3-day mouse PK

Dividing ScN2a Cells

IND-024

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RML-infected FVB-GFAP mice treated with IND024 at 210 mg/kg/day

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Time from inoculation (days)

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IND024 treatment of Tg mice overexpressing MoPrPor chimeric Hu/MoPrP inoculated with RML prions

Tg(Hu/MoPrP)1014 miceTg(MoPrP)4053 mice

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IND024 treatment of Tg mice overexpressing chimeric Hu/MoPrP or HuPrP inoculated with CJD prions

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Tg(Hu/MoPrP)1014 mice Tg(HuPrP)2669 mice

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IND024 efficacy in Tg(Elk) mice inoculated with chronic wasting disease (CWD) elk prions

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Mice were inoculated with RML prions & treated with the drug IND024. While the incubation time doubled, a new strain of prions emerged, designated RML[D].

RML[VV]

RML[VD]RML[V]

RML BrainHomogenates

RML[D]

RML[DV]

RML[DD]

Green bars signify times when the drug IND024 was given orally

N2a CAD5RML[D] RML RML[D]

µM IND024: 0 2.5 5 10 20 0 2.5 5 10 200 0.5 1 1.5 2

RML[DD]

0 2.5 5 10 20

N2a CAD5RML RML[DV] RML RML[DV]

µM IND024:0 2.5 5 10 20 0 2.5 5 10 20 0 2.5 5 10 20 0 2.5 5 10 20

Infection of N2a and CAD5 cells with RML prions from mice treated with the 2-AMT IND024

Alternative mechanisms to explain drug-induced protein inheritance

Strain Selection• Numerous strains of prions

exist in a single inoculum but one or a few predominate.

• Selection pressure such as survival of the host may select for a particular property such as shorter incubation times.

• The drug IND024 selected for PrPSc prions with extended incubation times and resistance to the drug.

Conformational Mutagenesis• Relatively few stains are

present in most inocula.• Drug selection may create

new prion strains by increasing the conformational diversity of PrPSc.

• The drug IND024 changed the conformation of PrPScresulting in extended incubation times and resistance to the drug in cultured cells.

Green = Amide; Red = Piperazine; Yellow = Benzoxazole; Purple = Stilbene; Blue = Other; Brown = AMT; Orange = Indoleglyoxylamide; Black = Cmpd B Analog;

Rainbow = IND24; new compounds being tested for efficacy in Tg mice

Increased potency for many new anti-PrPScprion compounds with EC50 values of <100 nM

0.001

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Tau High Content Screening of SH-SY5Y Human Neuroblastoma Cells for Tau Reduction

DMSO Hit (51% reduction) No tau mAb

• SH-SY5Y human neuroblastoma cells treated with 10 µM test compound for 3 days

• 39,894 compounds screened to date (1/18/13): 625 hits ( ≥ 40 % reduction of tau, < 50 % reduction # of cells)

• 1.5 % hit rate IND4904

Early Diagnosis and Evaluation of Drug Efficacy in Humans

• In the early stages of the FTDs, the symptoms are primarily psychiatric. Often such symptoms are difficult to quantify.

• To address this, it is necessary to be able to measure the levels of tau in living patients. PET imaging offers a non-invasive technology.

• Tau PET reporters will be used for diagnosis and measuring the efficacy of tau-lowering drugs in patients.

Some impressive advances in neurodegenerative diseases (NDs)

• Molecular genetic and neuropathological studies have identified about a dozen different proteins that feature in the NDs.

• Growing body of evidence argues that these etiological proteins become transformed into prions.

• Prions cause late onset NDs including Alzheimer’s and Parkinson’s as the prions accumulate.

• There is not a single drug that halts or even slows one ND of the CNS.

Chorus of Naysayers – how can prions cause AD and PD? It’s impossible!

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Developing imaging and therapeutics for neurodegenerative diseases

• The new Sandler Neurosciences building provides space for expanding brain imaging and novel CNS therapeutics research.

• We are creating the largest group of chemical biologists in the world, who are focused on drugs for the early diagnosis, prevention and treatment of neurodegeneration.

• The discovery of conformational mutagenesis argues that only cocktails of anti-prion drugs will halt or even slow neurodegeneration.