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
 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
Emerging Concept: neurotoxic intra- or extra-cellular
deposition of insoluble proteins (β-sheet conformation) is
the cause of many neurodegenerative diseases

Disease Protein Enabling event

Alzheimer Disease Aβ (βAPP) β- /γ-secretase

Frontotemporal Dementia Tau ?
Creutzfeldt-Jacob PrPSc (PrPc) ?
Familial Encephalopathy Neuroserpin ?
Familial British Dementia ABri (BRI) Furin cleavage
Parkinson’s Disease α-synuclein ?
 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
 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.
 Genetic Determinants of
Alzheimer’s Disease
Presenile Senile Sporadic
Familial AD Familial AD AD

Presenilin 1 APP Presenilin 2 APOE ε 4 allele

gene gene gene (chr 19)
(chr 14) (chr 21) (chr 1)
>50 yrs
40–65 yrs45–84 yrs
age: 25–60 yrs

Other genes yet to be identified

 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

Aβ peptide
domain

APP
Cell
membrane
 Physiological Endo-proteolytic Processing of APP
Citron et al. Nature Med. 3: 67-72, 1997
Pardossi-Piquard R et al. Neuron 46:541-554, 2005.

Aβ40 >> Aβ42

Uptake, chaperoning, &
β-secretase γ-secretase degradation of Aβ by
neprilysin, IDE, others

AICD
(?Signalling)
β
α
γ

APP
Cell Transcriptional
membrane
α-secretase induction
 Mutations Causing Alzheimer Disease cause
mis-processing of APP
Citron et al. Nature Med. 3: 67-72, 1997

APP
mutations
Aβ
Uptake, chaperoning, &
β-secretase γ-secretase degradation of Aβ by
neprilysin, IDE, others

AICD
(?Signalling)
β
Extracellular α
TM domain γ
Intracellular

APP

α-secretase
 FAD-causing mutations in βAPP are
localized in/around the Aβ peptide domain.
Codon Mutation Phenotype Effect
670/671 Lys-Met/ FAD β-secretase cleavage
Asn-Leu
692 Ala->Gly FAD Fibrillogenesis/toxicity
693 Glu->Gln Haemorrhage Fibrillogenesis/toxicity
Glu->Gly Haemorrhage Fibrillogenesis/toxicity
694 Asp->Asn Haemorrhage Fibrillogenesis/toxicity
713 Ala->Thr FAD
714 Thr->Ile FAD N-truncated Aβ42
β
715 Val->Met FAD N-truncated Aβ42
Extracellular α

716 Ile->Val FAD Aβ42

TM domain γ

10 Val->Ile/Phe FAD Aβ42

Intracellular

APP
/Gly
723 Leu->Pro FAD Aβ42
 FAD-causing mutations in βAPP are alter the
amount or the fibrillogenic potential of Aβ peptide

Codon Mutation Phenotype Effect

670/671 Lys-Met/ FAD β-secretase cleavage
Asn-Leu
692 Ala->Gly FAD Fibrillogenesis/toxicity
693 Glu->Gln Haemorrhage Fibrillogenesis/toxicity
Glu->Gly Haemorrhage Fibrillogenesis/toxicity
694 Asp->Asn Haemorrhage Fibrillogenesis/toxicity
713 Ala->Thr FAD
714 Thr->Ile FAD N-truncated Aβ42
715 Val->Met FAD N-truncated Aβ42
716 Ile->Val FAD Aβ42
10 Val->Ile/Phe FAD Aβ42
/Gly
723 Leu->Pro FAD Aβ42
 Mutations Causing Alzheimer Disease cause
mis-processing of APP
Citron et al. Nature Med. 3: 67-72, 1997

APP
PS1/PS2
mutations
mutations
Aβ
Uptake, chaperoning, &
β-secretase γ-secretase degradation of Aβ by
neprilysin, IDE, others

AICD
(?Signalling)
β
Extracellular α
TM domain γ
Intracellular

APP

α-secretase
Naturally Occurring Mutations in Presenilins Alter APP Processing

• Predicted to encode homologous

polytopic transmembrane
proteins (PS1 and PS2). Cytoplasm

• Contain conserved aspartate Membrane

residues in transmembrane
Lumen
domains (protease active site).
• >100 missense/in-frame splicing XD XGXGD
mutations in PS1 scattered
throughout PS1 molecule;

• > 12 mutations in PS2;

• Mutations in PS1 and PS2 often

affect orthologous residues.

• PS1 and PS2 mutations all alter Sherrington et al. Nature 375: 754-760, 1995
Rogaev et al Nature 376: 775-778, 1995
Aβ production – increase Aβ42. Citron et al. Nature Med. 3: 67-72, 1997
 Presenilin Proteins Form a Complex With Nicastrin APH-1 and PEN-2 To
Cleave Amyloid Precursor Protein (APP) and generate neurotoxic Aβ peptide.

AICD Golgi/ER
ε-site

Cytoplasm

D D
Membrane

Lumen/
Cell surface Presenilin PEN-2 APH-1 Nicastrin

γ-site

Alzheimer A
Disease β_
Similar presenilin-dependent
Sherrington, Nature, 1995 intramembranous cleavages for:
Rogaev, Nature, 1995 •Notch
Katayama, Nature Cell Biol, 1999 •Delta
Yu, Nature, 2000
•p75
Chen, Nature Cell Biol, 2002
Sisodia, Nature Neurosci, 2002 •LRP1
Pardossi-Piquard Neuron, 2005 •SorLA
•Others...
 Presenilin Mutations Cause Alzheimer Disease
by altering γ-secretase cleavage of APP
Citron et al. Nature Med. 3: 67-72, 1997

APP
PS1/PS2
mutations
mutations
Aβ42
Uptake, chaperoning, &
β-secretase γ-secretase-42 degradation of Aβ by
neprilysin, IDE, others

AICD
(?Signalling)
β
Extracellular α
TM domain γ
Intracellular

APP

α-secretase
 Apolipoprotein E and
Alzheimer’s Disease
• APOE has 3 variants: ε 2, ε 3, ε 4;
• APOE ε 2 increased frequency in normal elderly,
reduced frequency in AD;
• APOE ε 4 associated with Sporadic/familial AD
(dose-dependent relationship with age of onset);
• APOE ε 4 association not specific to AD, and not
all APOE ε 4 carriers will succumb to disease.
• APOE ε4 appears to block removal of Aβ via LRP
receptors, causing accumulation of Aβ.
 Mutations Causing Alzheimer Disease cause
mis-processing of APP
Citron et al. Nature Med. 3: 67-72, 1997

APP
PS1/PS2 APOE ε4
mutations
mutations
Aβ

β-secretase γ-secretase
↓ Uptake, chaperoning, &
X degradation of Aβ

AICD
(?Signalling)
β
Extracellular α
TM domain γ
Intracellular

APP

α-secretase
Aβ accumulates Aβ aggregates
into neurotoxic
protofibrils
What’s the evidence for this linear
pathway?
 Enhancer and suppressor interactions amongst
genes causing Alzheimer Disease
Gene interactions in human patients with AD:
– APP717 mutation + APOE ε4 allele = earlier onset (enhancer);
enhancer

– APP717 mutation + APOE ε2 allele = delayed onset (suppressor);

suppressor

– PS1E280A + APOE ε4 = earlier disease (enhancer)

enhancer

– PS2N141V + APOE ε4 = earlier disease (enhancer)

enhancer .

Gene interactions In animal models

– APP717 mutation + PS10/0 = no disease (suppressor);
suppressor

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

enhancer

St George-Hyslop et al Science 263:536-537, 1994

Pastor, P. et al. Ann Neurol 54, 163-9 (2003)
 Suppressor

WT/WT A717/WT APP genotype (A= APP717)

ε2/ε3 ε3/ε3
APOE Genotype

A717/WT A717/WT A717/WT

ε2/ε3
2/ ε4/ε3 ε4/ε3

APP
Elderly
V717I (>65yrs
+ APOEold)
ε2 carrier
asymptomatic
eventually carrier AD,
developed of APP
butV717I
at
>2
mutation
SD beyond mean age-of-onset.
 Enhancer and suppressor interactions amongst
genes causing Alzheimer Disease
Gene interactions in human patients with AD:
– APP717 mutation + APOE ε4 allele = earlier onset (enhancer);
enhancer

– APP717 mutation + APOE ε2 allele = delayed onset (suppressor);

suppressor

– PS1E280A + APOE ε4 = earlier disease (enhancer)

enhancer

– PS2N141V + APOE ε4 = earlier disease (enhancer)

enhancer .

Gene interactions In animal models

– APP717 mutation + PS10/0 = no disease (suppressor);
suppressor

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

enhancer
St George-Hyslop et al Science 263:536-537, 1994
Pastor, P. et al. Ann Neurol 54, 163-9 (2003)
Enhancer effect of cross-breeding
mutant PS1 and mutant APP mice
APP mice – 2 months APP x PS1 mice - 2 months

PS1 mice - 2 months

 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, 1994

Pastor, P. et al. Ann Neurol 54, 163-9 (2003)
What are the other genes?
 General Paradigms for Gene Discovery

LINKAGE BASED CASE : CONTROL ASSOCIATION

•Easy to collect sporadic cases

•Difficult to collect families •Cheap, quick
•Expensive •Easy to mess up
•Relatively few assumptions •Requires assumption that cases and controls
•Robust directly observable results are from same founder population..
 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 method

Confirmed 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)
 What is the role for the
microtubule associated protein Tau
and
neurofibrillary tangles?
 Fronto-temporal dementia:
molecular 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).
Conclusions to Be Drawn From the Discovery of
Pathogenic Mutations in Tau in FTD

• Disturbed tau/microtubule homeostasis,

regardless of cause, is toxic to neurons
 Aβ accumulation initiates a biochemical
cascade leading to neuronal death

Dementia

Cause: Aβ peptide
(eg gene
Neuronal Neuronal
accumulation injury dysfunction
defect)
and death

Altered Tau
metabolism
 How is this knowledge applied for
patients?

• Adjunctive Diagnostics
• Therapeutic Targets
 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
 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.
 Prediction of therapeutic response
• Theoretically reasonable;
• Remains to be validated.

Gene 1 Gene 2 Environment factor 1

Step 1 Step 2 Step 3 Step 4 AD

Rx 1 Rx 2
 Using Aβ accumulation pathways as a
target for therapies

Dementia

Cause: Aβ peptide
(eg gene
Neuronal Neuronal
accumulation injury dysfunction
defect)
and death

Altered Tau
metabolism
 Exploiting Knowledge Gained to Create New
Diagnostics and Therapeutics
•Anti-Aβ antibodies to remove Aβ;
•Block enzymes;
•Block aggregation. Dementia

X
Cause: Aβ peptide
(eg gene
Neuronal Neuronal
accumulation injury dysfunction
defect)
and death

Altered Tau
metabolism
Janus et al Nature. 408: 979-982, 2000,
McLaurin et al, Nature submitted, 2004
 How can the amyloid cascade be blocked?

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

Pharma: Pharma: Vaccine:

toxic
Aβ

β-secretase γ-secretase Uptake, chaperone, or

degradation (by neprilysin).

AICD
(?Signalling)
β Pharma
α
γ

APP X
Cell
membrane

Aβ accumulates Aβ aggregates
into neurotoxic
protofibrils
 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.
 Acknowledgements
S. Arawaka
F. Chen A. Bruni,
L. Farrer, F. Checler
P. Fraser JF Foncin,
YJ. Gu Canadian Institutes of Health Research G. Marcon,
H. Hasegawa Howard Hughes Medical Institute M. Mortilla,
M. Ikeda Alzheimer Society of Ontario, A. Orlacchio,
T. Katayama Canadian Genetic Diseases Network E. Paitel
T. Kawarai S. Piacentini,
G. Levesque L. Pinessi,
M. Nishimura I. Rainero,
A. Petit S. Sorbi,
E. Rogaeva R. Tupler,
N. Sanjo G. Vaula
P. St George-Hyslop
D. Westaway
 CONTACT INFORMATION
• Analysis of familial cases:
P. St George-Hyslop, University of Toronto
tel: 416-978-7460
p.hyslop@utoronto.ca

• Animal models (transgenic mice etc):

David Westaway
David.westaway@utoronto.ca

• Reagents (clones, cell lines, antibodies, etc)

P. St George-Hyslop, University of Toronto
p.hyslop@utoronto.ca