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Page 1: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of

Neurodegenerative Disease

P. St George-Hyslop

Centre for Research in Neurodegenerative Diseases,Toronto Western Hospital Research Institute,

University of Toronto,Toronto, Ontario, CANADA

Page 2: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Overview

• Genetics and Biology of Dementias– Alzheimer Disease:

• APP, PS1, PS2, APOE ε4• Other unidentified genes

– Fronto-temporal Dementia (& PSP , CBD)• Tau

– Dementia with Lewy Bodies• APOE ε4

• Current knowledge of known disease causing pathways;

• Application of current knowledge– Prediction of future risks, pharmacogenomics– Design of rational therapeutics

Page 3: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Emerging Concept: neurotoxic intra- or extra-cellular deposition of insoluble proteins (-sheet conformation) is

the cause of many neurodegenerative diseases

DiseaseDisease Protein Protein Enabling eventEnabling event

Alzheimer Disease Alzheimer Disease AAββ ( (ββAPP)APP) ββ- /- /γγ-secretase-secretaseFrontotemporal DementiaFrontotemporal Dementia TauTau ??Creutzfeldt-JacobCreutzfeldt-Jacob PrPSc (PrPc)PrPSc (PrPc) ??Familial EncephalopathyFamilial EncephalopathyNeuroserpinNeuroserpin ??Familial British DementiaFamilial British Dementia ABri (BRI)ABri (BRI) Furin cleavageFurin cleavageParkinson’s DiseaseParkinson’s Disease αα-synuclein-synuclein ??

Page 4: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

What causes Alzheimer Disease?

• Genetic Factors (40% of attributable population risk):– Mutations in genes:

• Amyloid Precursor Protein (APP);• Presenilin 1 (PS1);• Presenilin 2 (PS2);• Apolipoprotein E (APOE ε4);• Other genes on other chromosomes.

• Environmental Factors (± genetic predispositions):– Evidence for specific environmental factors is not robust

• Lower childhood education• Head Injury• Cerebrovascular disease• ?Aluminium

Page 5: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Genetic and “non-genetic” cases are indistinguishable

• Genetic and non-genetic cases have identical:– Clinical features;– Brain pathology; – Brain biochemistry (increased brain levels of

Amyloid β-peptide (Aβ) and tau);– Mortality.

Page 6: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Genetic Determinants of Alzheimer’s Disease

Genetic Determinants of Alzheimer’s Disease

PresenileFamilial ADPresenile

Familial ADSenileSenile

Familial ADFamilial ADSenileSenile

Familial ADFamilial ADSporadicSporadic

ADADSporadicSporadic

ADAD

Presenilin 1Presenilin 1genegene

(chr 14)(chr 14)

age: 25–60 yrsage: 25–60 yrs

Presenilin 1Presenilin 1genegene

(chr 14)(chr 14)

age: 25–60 yrsage: 25–60 yrs

Presenilin 2Presenilin 2genegene

(chr 1)(chr 1)

45–84 yrs45–84 yrs

APPAPPgenegene

(chr 21)(chr 21)

40–65 yrs40–65 yrs

APPAPPgenegene

(chr 21)(chr 21)

40–65 yrs40–65 yrs

APOE APOE 4 allele4 allele(chr 19)(chr 19)

>50 yrs>50 yrs

APOE APOE 4 allele4 allele(chr 19)(chr 19)

>50 yrs>50 yrs

Other genes yet to be identifiedOther genes yet to be identifiedOther genes yet to be identifiedOther genes yet to be identified

Page 7: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

The APP gene encodes a Type 1 membrane protien, a fragment of which accumulates in AD brain

Citron et al. Nature Med. 3: 67-72, 1997

APPCell membrane

Aβ peptide domain

Page 8: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Physiological Endo-proteolytic Processing of APP

-secretase -secretase

Aβ40 >> Aβ42

AICD(?Signalling)

-secretase

Uptake, chaperoning, &degradation of Aβ by neprilysin, IDE, others

APP

Cell membrane

Transcriptional induction

Citron et al. Nature Med. 3: 67-72, 1997Pardossi-Piquard R et al. Neuron 46:541-554, 2005.

Page 9: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Mutations Causing Alzheimer Disease cause mis-processing of APP

-secretase -secretase

AICD(?Signalling)

Extracellular

TM domain

Intracellular

APPmutations

APP

Citron et al. Nature Med. 3: 67-72, 1997

-secretase

Uptake, chaperoning, &degradation of Aβ by neprilysin, IDE, others

Page 10: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

FAD-causing mutations in APP are localized in/around the Aβ peptide domain.

CodonCodon MutationMutation PhenotypePhenotype EffectEffect670/671 Lys-Met/ FAD β-secretase cleavage

Asn-Leu692 Ala->Gly FAD Fibrillogenesis/toxicity693 Glu->Gln Haemorrhage Fibrillogenesis/toxicity

Glu->Gly Haemorrhage Fibrillogenesis/toxicity694 Asp->Asn Haemorrhage Fibrillogenesis/toxicity713 Ala->Thr FAD714 Thr->Ile FAD N-truncated Aβ42715 Val->Met FAD N-truncated Aβ42 716 Ile->Val FAD Aβ42717 Val->Ile/Phe FAD Aβ42

/Gly723 Leu->Pro FAD Aβ42

Extracellular

TM domain

Intracellular

APP

Page 11: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

FAD-causing mutations in APP are alter the amount or the fibrillogenic potential of Aβ peptide

CodonCodon MutationMutation PhenotypePhenotype EffectEffect670/671 Lys-Met/ FAD β-secretase cleavage

Asn-Leu692 Ala->Gly FAD Fibrillogenesis/toxicity693 Glu->Gln Haemorrhage Fibrillogenesis/toxicity

Glu->Gly Haemorrhage Fibrillogenesis/toxicity694 Asp->Asn Haemorrhage Fibrillogenesis/toxicity713 Ala->Thr FAD714 Thr->Ile FAD N-truncated Aβ42715 Val->Met FAD N-truncated Aβ42 716 Ile->Val FAD Aβ42717 Val->Ile/Phe FAD Aβ42

/Gly723 Leu->Pro FAD Aβ42

Page 12: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Mutations Causing Alzheimer Disease cause mis-processing of APP

-secretase -secretase

A

AICD(?Signalling)

-secretase

Extracellular

TM domain

Intracellular

APPmutations

APP

Citron et al. Nature Med. 3: 67-72, 1997

PS1/PS2mutations

Uptake, chaperoning, &degradation of Aβ by neprilysin, IDE, others

Page 13: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Naturally Occurring Mutations in Presenilins Alter APP Processing

Cytoplasm

Lumen

Membrane

• >100 missense/in-frame splicing mutations in PS1 scattered throughout PS1 molecule;

Sherrington et al. Nature 375: 754-760, 1995Rogaev et al Nature 376: 775-778, 1995Citron et al. Nature Med. 3: 67-72, 1997

• Predicted to encode homologous polytopic transmembrane proteins (PS1 and PS2).

• PS1 and PS2 mutations all alter Aβ production – increase Aβ42.

• > 12 mutations in PS2;

• Mutations in PS1 and PS2 often affect orthologous residues.

XD XGXGD

• Contain conserved aspartate residues in transmembrane domains (protease active site).

Page 14: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

APH-1 NicastrinPEN-2

Cytoplasm

Lumen/Cell surface

Membrane

Presenilin Proteins Form a Complex With Nicastrin APH-1 and PEN-2 To Presenilin Proteins Form a Complex With Nicastrin APH-1 and PEN-2 To Cleave Amyloid Precursor Protein (APP) and generate neurotoxic ACleave Amyloid Precursor Protein (APP) and generate neurotoxic Aββ peptide. peptide.

Golgi/ER

Presenilin

A

-site

ε-site

AICD

Sherrington, Nature, 1995Rogaev, Nature, 1995Katayama, Nature Cell Biol, 1999Yu, Nature, 2000Chen, Nature Cell Biol, 2002Sisodia, Nature Neurosci, 2002Pardossi-Piquard Neuron, 2005

D D

Similar presenilin-dependent intramembranous cleavages for:

•Notch•Delta•p75•LRP1•SorLA•Others...

_AlzheimerDisease

Page 15: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Presenilin Mutations Cause Alzheimer Disease by altering γ-secretase cleavage of APP

-secretase -secretase-42

A

AICD(?Signalling)

-secretase

Extracellular

TM domain

Intracellular

APPmutations

APP

Citron et al. Nature Med. 3: 67-72, 1997

PS1/PS2mutations

Uptake, chaperoning, &degradation of Aβ by neprilysin, IDE, others

Page 16: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Apolipoprotein E and Alzheimer’s Disease

• APOE has 3 variants: APOE has 3 variants: 2, 2, 3, 3, 4;4;

• APOE APOE 2 increased frequency in normal elderly, 2 increased frequency in normal elderly, reduced frequency in AD;reduced frequency in AD;

• APOE APOE 4 associated with Sporadic/familial AD 4 associated with Sporadic/familial AD (dose-dependent relationship with age of onset);(dose-dependent relationship with age of onset);

• APOE APOE 4 association not specific to AD, and not 4 association not specific to AD, and not all APOE all APOE 4 carriers will succumb to disease.4 carriers will succumb to disease.

• APOE APOE εε4 appears to 4 appears to block removal of Ablock removal of Aββ via LRP via LRP receptors, causing accumulation of Areceptors, causing accumulation of Aββ..

Page 17: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Mutations Causing Alzheimer Disease cause mis-processing of APP

-secretase -secretase

A

AICD(?Signalling)

↓ Uptake, chaperoning, & degradation of Aβ

-secretase

Extracellular

TM domain

Intracellular

APOE 4PS1/PS2mutations

APPmutations

Aaggregatesinto neurotoxicprotofibrils

A accumulates

APP

X

Citron et al. Nature Med. 3: 67-72, 1997

Page 18: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

What’s the evidence for this linear pathway?

Page 19: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Enhancer and suppressor interactions amongst genes causing Alzheimer Disease

St George-Hyslop et al Science 263:536-537, 1994Pastor, P. et al. Ann Neurol 54, 163-9 (2003)

Gene interactions in human patients with AD:Gene interactions in human patients with AD:– APP717 mutation + APOE 4 allele = earlier onset (enhancerenhancer);

– APP717 mutation + APOE 2 allele = delayed onset (suppressorsuppressor);

– PS1E280A + APOE ε4 = earlier disease (enhancerenhancer)

– PS2N141V + APOE ε4 = earlier disease (enhancerenhancer) .

Gene interactions In animal modelsGene interactions In animal models– APP717 mutation + PS10/0 = no disease (suppressorsuppressor);

– APP717 mutation + PS1mutations = enhanced disease (enhancerenhancer).

Page 20: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Suppressor

APP genotype (AA= APP717)APOE Genotype

AA717717/WT

AA717717/WT

AA717717/WTAA717717/WT

WT/WTε2/ε3 ε3/ε3

ε4/ε3ε4/ε3εε2/2/ε3

Elderly (>65yrs old) asymptomatic carrier of APPV717I

mutation

APPV717I + APOE ε2 carrier eventually developed AD, but at >2 SD beyond mean age-of-onset.

Page 21: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Enhancer and suppressor interactions amongst genes causing Alzheimer Disease

St George-Hyslop et al Science 263:536-537, 1994Pastor, P. et al. Ann Neurol 54, 163-9 (2003)

Gene interactions in human patients with AD:Gene interactions in human patients with AD:– APP717 mutation + APOE 4 allele = earlier onset (enhancerenhancer);

– APP717 mutation + APOE 2 allele = delayed onset (suppressorsuppressor);

– PS1E280A + APOE ε4 = earlier disease (enhancerenhancer)

– PS2N141V + APOE ε4 = earlier disease (enhancerenhancer) .

Gene interactions In animal modelsGene interactions In animal models– APP717 mutation + PS10/0 = no disease (suppressorsuppressor);

– APP717 mutation + PS1mutations = enhanced disease (enhancerenhancer).

Page 22: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Enhancer effect of cross-breeding mutant PS1 and mutant APP mice

APP x PS1 mice - 2 months

PS1 mice - 2 months

APP mice – 2 months

Page 23: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Enhancer and suppressor interactions amongst genes causing Alzheimer Disease

• Confirms that the known AD genes really do act in the same biochemical pathway affecting APP processing.

St George-Hyslop et al Science 263:536-537, 1994Pastor, P. et al. Ann Neurol 54, 163-9 (2003)

Page 24: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

What are the other genes?

Page 25: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

General Paradigms for Gene Discovery

CASE : CONTROL ASSOCIATIONLINKAGE BASED

•Difficult to collect families•Expensive•Relatively few assumptions•Robust directly observable results

•Easy to collect sporadic cases•Cheap, quick•Easy to mess up•Requires assumption that cases and controls are from same founder population..

Page 26: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

What are the other AD genes?Case:Control> 100 candidate genes reported to be associated with AD;

Generally had poor track-record of replication (NB: one or two ‘independent replications’ in the face of many non-replications = non-replication);

Family linkage-based methodConfirmed localization of an AD-gene to broad region of chromosome 10 containing several hundred genes (the specific gene remains to be found);

Confirmed localization of an AD-gene to broad region of chromosome 12 containing several hundred genes (the specific gene remains to be found)

Page 27: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

What is the role for themicrotubule associated protein Tau

and neurofibrillary tangles?

Page 28: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.
Page 29: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Fronto-temporal dementia:Fronto-temporal dementia:molecular geneticsmolecular genetics

• Mutations in Tau gene on chromosome 17q in ~10-40% of FTD cases;

• Mutations disturb binding of tau protein to microtubules, causing accumulation of free unbound tau;

• Free unbound tau aggregates into fibrils and these then coalesce into paired helical filaments as the neurofibrillary tangle;

• The tau fibrils then injure cells (but mechanism is unclear).

Page 30: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Conclusions to Be Drawn From the Discovery of Pathogenic Mutations in Tau in FTD

• Disturbed tau/microtubule homeostasis, Disturbed tau/microtubule homeostasis, regardless of cause, is toxic to neuronsregardless of cause, is toxic to neurons

Page 31: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

A accumulation initiates a biochemical cascade leading to neuronal death

Cause:Cause:(eg genedefect)

AA peptide peptideaccumulationaccumulation

NeuronalNeuronalinjuryinjury

Altered TauAltered Taumetabolismmetabolism

Neuronal Neuronal dysfunctiondysfunctionand deathand death

DementiaDementia

Page 32: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

How is this knowledge applied for patients?

• Adjunctive Diagnostics

• Therapeutic Targets

Page 33: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Prediction of future risk for AD?

• Testing and genetic counselling feasible for: – Highly penetrant forms, with – Clear patterns of inheritance, and – Relatively predictable age-of-onset:

• PS1• APP• Tau

• Testing and genetic counselling not presently feasible/useful for: – Incompletely penetrant forms with variable age-of-onset:

• PS2• APOE• Putative genes on chromosomes 10, 12 etc

– NB: Advent of future therapies may make even fuzzy-risk data from such genes useful

Page 34: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Can Genetics Predict Conversion From MCI To AD?

• Intuitive expectation: – Carrier of AD risk allele with MCI would be more likely to convert to

AD.

• Actual data available only for ApoE

• ApoE ε4 predictive:– Petersen et al, JAMA 274: 538,1995 – Bartrez-Faz et al, JAGS 49: 485, 2001

• ApoE ε4 not predictive:– Marquis et al, Arch. Neurol. 59: 601, 2002– Tierney et al, Neurol. 46: 149, 1996.

Page 35: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Prediction of therapeutic response

• Theoretically reasonable; • Remains to be validated.

ADADStep 1 Step 4Step 3Step 2

Gene 1 Environment factor 1Gene 2

Rx 1 Rx 2

Page 36: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Using A accumulation pathways as a target for therapies

Cause:Cause:(eg genedefect)

AA peptide peptideaccumulationaccumulation

NeuronalNeuronalinjuryinjury

Altered TauAltered Taumetabolismmetabolism

Neuronal Neuronal dysfunctiondysfunctionand deathand death

DementiaDementia

Page 37: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Exploiting Knowledge Gained to Create New Diagnostics and Therapeutics

Cause:Cause:(eg genedefect)

NeuronalNeuronalinjuryinjury

Altered TauAltered Taumetabolismmetabolism

Neuronal Neuronal dysfunctiondysfunctionand deathand death

DementiaDementia

AA peptide peptideaccumulationaccumulationX

•Anti-A antibodies to remove A•Block enzymes;•Block aggregation.

Janus et al Nature. 408: 979-982, 2000,McLaurin et al, Nature submitted, 2004

Page 38: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

How can the amyloid cascade be blocked?

-secretase -secretase

A

AICD(?Signalling)

Pharma:Pharma:

Aaggregatesinto neurotoxicprotofibrils

A accumulates

Citron et al. Nature Med. 3: 67-72, 1997

APP

Cell membrane

Uptake, chaperone, ordegradation (by neprilysin).

Vaccine:toxic

Pharma

X

Page 39: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Conclusions:

• All known genes causing AD modulate APP and Aβ processing;

• Neurodegeneration from mutations in tau prove that tau accumulation is also a toxic event (regardless of whether caused by mutation in tau or due to Aβ accumulation)

• Knowledge of pathway will provide targets for disease-modifying therapies.

Page 40: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

Acknowledgements

Canadian Institutes of Health ResearchHoward Hughes Medical InstituteAlzheimer Society of Ontario,Canadian Genetic Diseases Network

S. ArawakaF. ChenL. Farrer,P. FraserYJ. GuH. HasegawaM. IkedaT. KatayamaT. KawaraiG. Levesque M. NishimuraA. PetitE. RogaevaN. SanjoP. St George-HyslopD. Westaway

A. Bruni, F. CheclerJF Foncin, G. Marcon, M. Mortilla, A. Orlacchio,E. PaitelS. Piacentini, L. Pinessi,I. Rainero, S. Sorbi, R. Tupler, G. Vaula

Page 41: Insights into Basic and Clinical Neurobiology Derived from the Analysis of Genetic causes of Neurodegenerative Disease P. St George-Hyslop Centre for Research.

CONTACT INFORMATION

• Analysis of familial cases:P. St George-Hyslop, University of Torontotel: [email protected]

• Animal models (transgenic mice etc):David [email protected]

• Reagents (clones, cell lines, antibodies, etc)P. St George-Hyslop, University of [email protected]