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

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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 Slide 2 Overview Genetics and Biology of Dementias Alzheimer Disease: APP, PS1, PS2, APOE 4 Other unidentified genes Fronto-temporal Dementia (& PSP, CBD) Tau Dementia with Lewy Bodies APOE 4 Current knowledge of known disease causing pathways; Application of current knowledge Prediction of future risks, pharmacogenomics Design of rational therapeutics Slide 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 A (APP)- /-secretase Frontotemporal DementiaTau? Creutzfeldt-JacobPrPSc (PrPc)? Familial EncephalopathyNeuroserpin? Familial British DementiaABri (BRI)Furin cleavage Parkinsons Disease-synuclein? Slide 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 4); 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 Slide 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 -peptide (A) and tau); Mortality. Slide 6 Genetic Determinants of Alzheimers Disease Presenile Familial AD Senile Familial AD Sporadic AD Presenilin 1 gene (chr 14) age: 2560 yrs Presenilin 1 gene (chr 14) age: 2560 yrs Presenilin 2 gene (chr 1) 4584 yrs APP gene (chr 21) 4065 yrs APP gene (chr 21) 4065 yrs APOE 4 allele (chr 19) >50 yrs APOE 4 allele (chr 19) >50 yrs Other genes yet to be identified Slide 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 A peptide domain Slide 8 Physiological Endo-proteolytic Processing of APP -secretase -secretase A40 >> A42 AICD (?Signalling) -secretase Uptake, chaperoning, & degradation of A 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. Slide 9 Mutations Causing Alzheimer Disease cause mis-processing of APP -secretase -secretase AA AICD (?Signalling) Extracellular TM domain Intracellular APP mutations APP Citron et al. Nature Med. 3: 67-72, 1997 -secretase Uptake, chaperoning, & degradation of A by neprilysin, IDE, others Slide 10 FAD-causing mutations in APP are localized in/around the A peptide domain. CodonMutationPhenotypeEffect 670/671Lys-Met/ FAD-secretase cleavage Asn-Leu 692Ala->GlyFAD Fibrillogenesis/toxicity 693Glu->GlnHaemorrhageFibrillogenesis/toxicity Glu->GlyHaemorrhage Fibrillogenesis/toxicity 694Asp->AsnHaemorrhageFibrillogenesis/toxicity 713Ala->ThrFAD 714Thr->IleFADN-truncated A42 715Val->MetFADN-truncated A42 716Ile->Val FADA42 717 Val->Ile/Phe FADA42 /Gly 723Leu->Pro FADA42 Extracellular TM domain Intracellular APP Slide 11 FAD-causing mutations in APP are alter the amount or the fibrillogenic potential of A peptide CodonMutationPhenotypeEffect 670/671Lys-Met/ FAD-secretase cleavage Asn-Leu 692Ala->GlyFAD Fibrillogenesis/toxicity 693Glu->GlnHaemorrhageFibrillogenesis/toxicity Glu->GlyHaemorrhage Fibrillogenesis/toxicity 694Asp->AsnHaemorrhageFibrillogenesis/toxicity 713Ala->ThrFAD 714Thr->IleFADN-truncated A42 715Val->MetFADN-truncated A42 716Ile->Val FADA42 717 Val->Ile/Phe FADA42 /Gly 723Leu->Pro FADA42 Slide 12 Mutations Causing Alzheimer Disease cause mis-processing of APP -secretase -secretase AA 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 A by neprilysin, IDE, others Slide 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 A production increase A42. > 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). Slide 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 A peptide. Golgi/ER Presenilin AA -site -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 Slide 15 Presenilin Mutations Cause Alzheimer Disease by altering -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 A by neprilysin, IDE, others Slide 16 Apolipoprotein E and Alzheimers 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 4 appears to block removal of A via LRP receptors, causing accumulation of A.APOE 4 appears to block removal of A via LRP receptors, causing accumulation of A. Slide 17 Mutations Causing Alzheimer Disease cause mis-processing of APP -secretase -secretase AA AICD (?Signalling) Uptake, chaperoning, & degradation of A -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 Slide 18 Whats the evidence for this linear pathway? Slide 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 4 = earlier disease (enhancer) enhancer PS2 N141V + APOE 4 = 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). Slide 20 Suppressor A APP genotype (A= APP 717 ) APOE Genotype A 717 A 717 /WT WT/WT 2/3 3/3 4/3 2/ 2/3 Elderly (>65yrs old) asymptomatic carrier of APP V717I mutation APP V717I + APOE 2 carrier eventually developed AD, but at >2 SD beyond mean age-of-onset. Slide 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 4 = earlier disease (enhancer) enhancer PS2 N141V + APOE 4 = 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). Slide 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 Slide 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) Slide 24 What are the other genes? Slide 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.. Slide 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-gen