1. 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

2. 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

3. Emerging Concept: neurotoxic intra- or extra-cellular deposition of insoluble proteins (  -sheet conformation) is the cause of many neurodegenerative diseases DiseaseProtein Enabling event Alzheimer Disease Aβ (βAPP)β- /γ-secretase Frontotemporal DementiaTau? Creutzfeldt-JacobPrPSc (PrPc)? Familial EncephalopathyNeuroserpin? Familial British DementiaABri (BRI)Furin cleavage Parkinson’s Diseaseα-synuclein?

4. 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

5. 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.

6. Genetic Determinants of Alzheimer’s Disease Presenile Familial AD Senile Familial AD Sporadic AD Presenilin 1 gene (chr 14) age: 25–60 yrs Presenilin 1 gene (chr 14) age: 25–60 yrs Presenilin 2 gene (chr 1) 45–84 yrs APP gene (chr 21) 40–65 yrs APP gene (chr 21) 40–65 yrs APOE  4 allele (chr 19) >50 yrs APOE  4 allele (chr 19) >50 yrs Other genes yet to be identified

7. 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 APP Cell membrane Aβ peptide domain

8. 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, 1997 Pardossi-Piquard R et al. Neuron 46:541-554, 2005.

9. Mutations Causing Alzheimer Disease cause mis-processing of APP  -secretase  -secretase AβAβ AICD (?Signalling) Extracellular TM domain Intracellular APP mutations APP    Citron et al. Nature Med. 3: 67-72, 1997  -secretase Uptake, chaperoning, & degradation of Aβ by neprilysin, IDE, others

10. FAD-causing mutations in  APP are localized in/around the Aβ peptide domain. CodonMutationPhenotypeEffect 670/671Lys-Met/ FADβ-secretase cleavage Asn-Leu 692Ala->GlyFAD Fibrillogenesis/toxicity 693Glu->GlnHaemorrhageFibrillogenesis/toxicity Glu->GlyHaemorrhage Fibrillogenesis/toxicity 694Asp->AsnHaemorrhageFibrillogenesis/toxicity 713Ala->ThrFAD 714Thr->IleFADN-truncated Aβ42 715Val->MetFADN-truncated Aβ42 716Ile->Val FADAβ42 717 Val->Ile/Phe FADAβ42 /Gly 723Leu->Pro FADAβ42 Extracellular TM domain Intracellular APP   

11. FAD-causing mutations in  APP are alter the amount or the fibrillogenic potential of Aβ peptide CodonMutationPhenotypeEffect 670/671Lys-Met/ FADβ-secretase cleavage Asn-Leu 692Ala->GlyFAD Fibrillogenesis/toxicity 693Glu->GlnHaemorrhageFibrillogenesis/toxicity Glu->GlyHaemorrhage Fibrillogenesis/toxicity 694Asp->AsnHaemorrhageFibrillogenesis/toxicity 713Ala->ThrFAD 714Thr->IleFADN-truncated Aβ42 715Val->MetFADN-truncated Aβ42 716Ile->Val FADAβ42 717 Val->Ile/Phe FADAβ42 /Gly 723Leu->Pro FADAβ42

12. Mutations Causing Alzheimer Disease cause mis-processing of APP  -secretase  -secretase AA AICD (?Signalling)  -secretase Extracellular TM domain Intracellular APP mutations APP    Citron et al. Nature Med. 3: 67-72, 1997 PS1/PS2 mutations Uptake, chaperoning, & degradation of Aβ by neprilysin, IDE, others

13. 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, 1995 Rogaev et al Nature 376: 775-778, 1995 Citron 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. XDXD X G X GD Contain conserved aspartate residues in transmembrane domains (protease active site).

14. APH-1 Nicastrin PEN-2 Cytoplasm Lumen/ Cell surface Membrane Presenilin Proteins Form a Complex With Nicastrin APH-1 and PEN-2 To Cleave Amyloid Precursor Protein (APP) and generate neurotoxic Aβ peptide. Golgi/ER Presenilin AA  -site ε -site AICD Sherrington, Nature, 1995 Rogaev, Nature, 1995 Katayama, Nature Cell Biol, 1999 Yu, Nature, 2000 Chen, Nature Cell Biol, 2002 Sisodia, Nature Neurosci, 2002 Pardossi-Piquard Neuron, 2005 D Similar presenilin-dependent intramembranous cleavages for: Notch Delta p75 LRP1 SorLA Others... _ Alzheimer Disease

15. Presenilin Mutations Cause Alzheimer Disease by altering γ-secretase cleavage of APP  -secretase  -secretase-42 A  AICD (?Signalling)  -secretase Extracellular TM domain Intracellular APP mutations APP    Citron et al. Nature Med. 3: 67-72, 1997 PS1/PS2 mutations Uptake, chaperoning, & degradation of Aβ by neprilysin, IDE, others

16. Apolipoprotein E and Alzheimer’s Disease APOE has 3 variants:  2,  3,  4;APOE has 3 variants:  2,  3,  4; APOE  2 increased frequency in normal elderly, reduced frequency in AD;APOE  2 increased frequency in normal elderly, reduced frequency in AD; APOE  4 associated with Sporadic/familial AD (dose-dependent relationship with age of onset);APOE  4 associated with Sporadic/familial AD (dose-dependent relationship with age of onset); APOE  4 association not specific to AD, and not all APOE  4 carriers will succumb to disease.APOE  4 association not specific to AD, and not all APOE  4 carriers will succumb to disease. APOE ε4 appears to block removal of Aβ via LRP receptors, causing accumulation of Aβ.APOE ε4 appears to block removal of Aβ via LRP receptors, causing accumulation of Aβ.

17. Mutations Causing Alzheimer Disease cause mis-processing of APP  -secretase  -secretase AA AICD (?Signalling) ↓ Uptake, chaperoning, & degradation of Aβ  -secretase Extracellular TM domain Intracellular APOE  4 PS1/PS2 mutations APP mutations A  aggregates into neurotoxic protofibrils A  accumulates APP X    Citron et al. Nature Med. 3: 67-72, 1997

18. What’s the evidence for this linear pathway?

19. Enhancer and suppressor interactions amongst genes causing Alzheimer Disease St George-Hyslop et al Science 263:536-537, 1994 Pastor, P. et al. Ann Neurol 54, 163-9 (2003) Gene interactions in human patients with AD: Gene interactions in human patients with AD: enhancer –APP 717 mutation + APOE  4 allele = earlier onset (enhancer); suppressor –APP 717 mutation + APOE  2 allele = delayed onset (suppressor); enhancer –PS1 E280A + APOE ε4 = earlier disease (enhancer) enhancer –PS2 N141V + APOE ε4 = earlier disease (enhancer). Gene interactions In animal models suppressor –APP 717 mutation + PS1 0/0 = no disease (suppressor); enhancer –APP 717 mutation + PS1mutations = enhanced disease (enhancer).

20. Suppressor A APP genotype (A= APP 717 ) APOE Genotype A 717 A 717 /WT WT/WT ε2/ε3 ε3/ε3 ε4/ε3 ε2/ ε2/ε3 Elderly (>65yrs old) asymptomatic carrier of APP V717I mutation APP V717I + APOE ε2 carrier eventually developed AD, but at >2 SD beyond mean age-of-onset.

21. Enhancer and suppressor interactions amongst genes causing Alzheimer Disease St George-Hyslop et al Science 263:536-537, 1994 Pastor, P. et al. Ann Neurol 54, 163-9 (2003) Gene interactions in human patients with AD: enhancer –APP 717 mutation + APOE  4 allele = earlier onset (enhancer); suppressor –APP 717 mutation + APOE  2 allele = delayed onset (suppressor); enhancer –PS1 E280A + APOE ε4 = earlier disease (enhancer) enhancer –PS2 N141V + APOE ε4 = earlier disease (enhancer). Gene interactions In animal models suppressor –APP 717 mutation + PS1 0/0 = no disease (suppressor); enhancer –APP 717 mutation + PS1mutations = enhanced disease (enhancer).

22. 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

23. 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)

24. What are the other genes?

25. General Paradigms for Gene Discovery CASE : CONTROL ASSOCIATION LINKAGE 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..

26. 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)

27. What is the role for the microtubule associated protein Tau and neurofibrillary tangles?

29. 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).

30. Conclusions to Be Drawn From the Discovery of Pathogenic Mutations in Tau in FTD Disturbed tau/microtubule homeostasis, regardless of cause, is toxic to neuronsDisturbed tau/microtubule homeostasis, regardless of cause, is toxic to neurons

31. A  accumulation initiates a biochemical cascade leading to neuronal death Cause: (eg gene defect) A  peptide accumulation Neuronalinjury Altered Tau metabolism Neuronaldysfunction and death Dementia

32. How is this knowledge applied for patients? Adjunctive Diagnostics Therapeutic Targets

33. 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

34. 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.

35. Prediction of therapeutic response Theoretically reasonable; Remains to be validated. AD Step 1 Step 4 Step 3 Step 2 Gene 1Environment factor 1Gene 2 Rx 1Rx 2

36. Using A  accumulation pathways as a target for therapies Cause: (eg gene defect) A  peptide accumulation Neuronalinjury Altered Tau metabolism Neuronaldysfunction and death Dementia

37. Exploiting Knowledge Gained to Create New Diagnostics and Therapeutics Cause: (eg gene defect) Neuronalinjury Altered Tau metabolism Neuronaldysfunction and death Dementia A  peptide accumulation X Anti-A  antibodies to remove A  Block enzymes; Block aggregation. Janus et al Nature. 408: 979-982, 2000, McLaurin et al, Nature submitted, 2004

38. How can the amyloid cascade be blocked?  -secretase  -secretase AA AICD (?Signalling) Pharma: A  aggregates into neurotoxic protofibrils A  accumulates Citron et al. Nature Med. 3: 67-72, 1997 APP    Cell membrane Uptake, chaperone, or degradation (by neprilysin). Vaccine: toxic Pharma X

39. 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.

40. Acknowledgements Canadian Institutes of Health Research Howard Hughes Medical Institute Alzheimer Society of Ontario, Canadian Genetic Diseases Network S. Arawaka F. Chen L. Farrer, P. Fraser YJ. Gu H. Hasegawa M. Ikeda T. Katayama T. Kawarai G. Levesque M. Nishimura A. Petit E. Rogaeva N. Sanjo P. St George-Hyslop D. Westaway A. Bruni, F. Checler JF Foncin, G. Marcon, M. Mortilla, A. Orlacchio, E. Paitel S. Piacentini, L. Pinessi, I. Rainero, S. Sorbi, R. Tupler, G. Vaula

41. 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