Stem Cells and the Study of Neurodegeneration Tracy Young-Pearse, PhD September 12, 2014!
Techniques for studying mechanisms of neurological disease Animal models Human subjects Postmortem analyses, imaging Induced pluripotent stem cells
Differential vulnerability to neurodegenerative diseases Alzheimer s disease Parkinson s disease Spinocerebellar ataxia Huntington s disease Frontotemporal dementia ALS (Lou Gehrig s disease) Narcolepsy
Development of stem cells from patients with Alzheimer s disease 1. 11 Schematic modified from Nishikawa et al, 2008
Overview of induced pluripotent stem cells (ipscs) Pancreatic cells Muscle cells Immune cells Neurons and glia Liver cells
Human ipsc neural differentiation ± mast ± dissociation D1 D5 D7 D17 D24 Media ips/es (DMEM/F12) Neural Induction (N2) Neural Induction (N2/B27) Neural Differentiation (N2/B27) +IGF, +camp +BDNF, +GDNF +IGF, +camp aggregates primi2ve NE defini2ve NE neuronal differen2a2on B ips Cells Day 4 Day 10 Day 16 Day 60
Differentiation of human ips cells to neuronal fates ips Cells Day 17 Day 60 Red = Oct4 = pluripotent cells Green = MAP2 = neuronal cells Blue = TOPRO3 = all cells
Living neurons from our patients with neurodegenerative diseases TBR1/MAP2/TOPRO3 MAP2/GFAP TAU/TOPRO3
Vulnerability to degeneration in Alzheimer s disease differs among brain regions Cerebral cortex and hippocampus Memory Thought Awareness Attention Consciousness Cerebellum Movement Balance Coordination Posture
Aβ and Tau accumulate in the cerebral cortex prior to degeneration Aβ42 aggregates Image courtesy of the Alzheimer s Association Hyperphosphorylated tau tangles
Mutation of APP causes early-onset Alzheimer s disease Genome editing with CRISPR/Cas to generate isogenic lines:
The Amyloid Hypothesis of Alzheimer s disease pathogenesis Early onset/dominantly inherited AD! ( familial )! Missense mutations in the APP or! Presenilin 1 or 2 genes! Increased Aβ42 production throughout life! Late onset AD! ( sporadic )! Failure of Aβ clearance mechanisms! (e.g., inheritance of ApoE4; faulty Aβ degradation,! environmental increases in Aβ production, etc.)! Gradually rising Aβ42 levels in brain! Accumulation and oligomerization of Aβ42! MEMORY LOSS/! Subtle effects of Aβ oligomers on synaptic efficacy! COGNITIVE DEFECTS! Microglial and astrocytic activation and attendant inflammatory responses! Altered kinase/phosphatase activities lead to tau-containing tangles! Widespread neuronal/synaptic dysfunction and selective neuronal loss,! with attendant neurotransmitter deficits! DEMENTIA!
APP mutation does not affect neuronal differentiation 6000 Normalized expression 8000 6000 4000 2000 2000 1500 1000 500 Normalized expression 4000 2000 500 400 300 200 100 Control fad 0 MAP2 PSD-95 SYP Synapsin TuJ1 0 CTIP2 Cux1 FoxG1 GAD1 Nestin Pax6 Satb1 Six3 Sox1 Sox2 Tbr1 VGAT VGLUT1 Muratore et al, HMG, 2014
APP mutation leads to increased Aβ42 and Aβ38 production Aβ 42/40 0.6 0.5 0.4 0.3 0.2 Aβ 42/40 0.5 0.4 0.3 0.2 0.1 0.1 0.0 ctl (YZ1) ctl (YK26) fad 1a fad 1b fad 2a fad 2b 0.0 control fad 1250 300 ** 400 Aβ40/total protein 1000 750 500 250 Aβ42/total protein 200 100 Aβ38/total protein 300 200 100 0 ctl DMSO ctl DAPT fad DMSO fad DAPT 0 ctl DMSO ctl DAPT fad DMSO fad DAPT 0 ctl DMSO ctl DAPT fad DMSO fad DAPT Muratore et al, HMG, 2014
APP cleavage products increase over differentiation from immature to mature neuronal fates Aβ40/total RNA 30 25 20 15 10 5 5 4 3 2 control fad Aβ42/total RNA 15 10 5 2.0 1.5 1.0 control fad Aβ38/total RNA 20 15 10 5 2.0 1.5 1.0 control fad * 1 0.5 0.5 0 d9 d17 d24 d40 d60 d80 d100 0.0 d9 d17 d24 d40 d60 d80 d100 0.0 d9 d17 d24 d40 d60 d80 d100 Days of Differentiation Days of Differentiation Days of Differentiation 0.7 0.6 control fad 0.5 Aβ 42/40 0.4 0.3 ** 0.2 0.1 0.0 d9 d17 d24 d40 d60 d80 d100 Days of Differentiation Muratore et al, HMG, 2014
APP mutation leads to increased β-secretase cleavage of APP 4.0 3.5 3.0 1500 800 * APPsα/APPsβ 2.5 2.0 1.5 1.0 ** ** * ** APPα/total RNA 1000 500 APPsβ/total RNA 600 400 200 0.5 0.0 ctl-yz1 ctl-yk26 fad-1a fad-1b fad-2a fad-2b 0 Control fad 0 Control fad Muratore et al, HMG, 2014
APP cleavage products increase over differentiation from immature to mature neuronal fates Muratore et al, HMG, 2014
APP subcellular localization is altered in fad ipsc-derived neurons control fad EEA-1/APP/MAP2/DAPI Muratore et al, HMG, 2014
The Amyloid Hypothesis of Alzheimer s disease pathogenesis Early onset/dominantly inherited AD! ( familial )! Missense mutations in the APP or! Presenilin 1 or 2 genes! Increased Aβ42 production throughout life! Late onset AD! ( sporadic )! Failure of Aβ clearance mechanisms! (e.g., inheritance of ApoE4; faulty Aβ degradation,! environmental increases in Aβ production, etc.)! Gradually rising Aβ42 levels in brain! Accumulation and oligomerization of Aβ42! MEMORY LOSS/! Subtle effects of Aβ oligomers on synaptic efficacy! COGNITIVE DEFECTS! Microglial and astrocytic activation and attendant inflammatory responses! Altered kinase/phosphatase activities lead to tau-containing tangles! Widespread neuronal/synaptic dysfunction and selective neuronal loss,! with attendant neurotransmitter deficits! DEMENTIA!
Higher Tau protein levels are observed in fad-derived neurons d35 d100 Muratore et al, HMG, 2014
Aβ-specific antibodies (3D6 and AW7) bind secreted Aβ Aβ bound to antibody: control fad Little unbound Aβ following antibody treatment: : APPs-α and β do not bind to antibody: Muratore et al, HMG, 2014
Aβ-specific antibodies (3D6 and AW7) rescue the Tau phenotype ELISA Total Tau/MAP2 (%Ctl) 300 200 100 0 * IgG 3D6 IgG 3D6 * ELISA Total Tau/MAP2 (%Ctl) 1000 800 600 400 200 0 * PI AW7 PI AW7 * control fad control fad Western Blot Total Tau/MAP2 (%Ctl) 250 200 150 100 50 0 * IgG 3D6 IgG 3D6 * Western Blot Total Tau/MAP2 (%Ctl) 500 400 300 200 100 0 * PI AW7 PI AW7 control fad control fad Muratore et al, HMG, 2014
The Amyloid Hypothesis of Alzheimer s disease pathogenesis Early onset/dominantly inherited AD! ( familial )! Missense mutations in the APP or! Presenilin 1 or 2 genes! Increased Aβ42 production throughout life! Increased APPs-β and Aβ38! Late onset AD! ( sporadic )! Failure of Aβ clearance mechanisms! (e.g., inheritance of ApoE4; faulty Aβ degradation,! environmental increases in Aβ production, etc.)! Gradually rising Aβ42 levels in brain! Accumulation and oligomerization of Aβ42! MEMORY LOSS/! Subtle effects of Aβ oligomers on synaptic efficacy! COGNITIVE DEFECTS! Microglial and astrocytic activation and attendant inflammatory responses! Altered kinase/phosphatase activities lead to tau-containing tangles! Widespread neuronal/synaptic dysfunction and selective neuronal loss,! with attendant neurotransmitter deficits! DEMENTIA!
Examining Glial Activation in Alzheimer s Disease Muratore et al, PLOS ONE, in press
APPV717I induces GFAP expression in astrocytes-derived from ipscs neuronal markers astrocyte markers
APPV717I induces GFAP expression in human astrocytes Control neurons fad neurons Add wild type human astrocytes (Sciencell) GFAP protein levels normalized to glutamine synthetase (% control)
Aβ secreted from fad neurons induces GFAP expression in human astrocytes Control neuron CM fad neuron CM ctl ID Aβ ID ctl ID Aβ ID human astrocytes (Sciencell)
The Amyloid Hypothesis of Alzheimer s disease pathogenesis Early onset/dominantly inherited AD! ( familial )! Missense mutations in the APP or! Presenilin 1 or 2 genes! Increased Aβ42 production throughout life! Increased APPs-β and Aβ38! Late onset AD! ( sporadic )! Failure of Aβ clearance mechanisms! (e.g., inheritance of ApoE4; faulty Aβ degradation,! environmental increases in Aβ production, etc.)! Gradually rising Aβ42 levels in brain! Accumulation and oligomerization of Aβ42! MEMORY LOSS/! Subtle effects of Aβ oligomers on synaptic efficacy! COGNITIVE DEFECTS! Microglial and astrocytic activation and attendant inflammatory responses! Altered kinase/phosphatase activities lead to tau-containing tangles! Widespread neuronal/synaptic dysfunction and selective neuronal loss,! with attendant neurotransmitter deficits! DEMENTIA!
Differential vulnerability of neuronal subtypes in Alzheimer s disease Susceptible in AD Cortical/hippocampal forebrain neurons ipscs NPCs Cerebellar and lower motor neurons Relatively spared in AD
Directed differentiation of ipscs to alternate neuronal fates rostral neurons Susceptible in AD ipscs NPCs +RA/Shh caudal neurons Relatively spared in AD
ipscs directed to caudal neuronal fates show altered expression profiles Normalized expression 8000 7000 6000 5000 4000 3000 3000 2000 1000 Normalized expression 600 400 200 ** ** ** ** Normalized expression 3000 2000 1000 150 100 50 * ** RA/Shh: 0 - + - + - + Map2 Tuj1 Tau 0 RA/Shh: - + - + - + - + - + - + Fezf2 Tbr1 CTIP2 Reln Dab1 Six3 RA/Shh: 0 - + - + - + - + - + - + HoxB4 HoxB6 En-1 Glra1 Irx3 MMP7
ipscs directed to a caudal neuronal fate secrete a less toxic ratio of Aβ42/40 Aβ/total RNA RA/Shh: 11 Aβ40 10 Aβ42 Aβ38 9 8 7 6 5 4 3 2 1 0 - + - + control fad Aβ 42/40 normalized to -RA/Shh for each line 1.25 1.00 0.75 0.50 0.25 0.00 RA/Shh: - + - + - + - + control (YZ1) ** * * control (YK26) fad #1 fad #2 total Tau/GAPDH (% Ctl) 200 150 100 50 * P-262 Tau/total Tau (% Ctl) 150 100 50 ** ** 0 control fad control fad 0 control fad control fad +RA/Shh +RA/Shh
Neurons susceptible to degeneration in Alzheimer s secrete more toxic Aβ Susceptible in AD Aβ42/Aβ40 Tau Aβ42/Aβ40 ipscs NPCs Relatively spared in AD Tau
Measuring Aβ and sappα levels at the single cell level sappα 86,000 wells Live/Dead assay (or immunostaining, in situ, etc) Aβ Technology developed in JC Love lab (MIT) to study cytokine secretion
Current/Future directions: Measuring Aβ and sappα levels at the single cell level Liao et al, unpublished
Current/Future directions: Measuring Aβ and sappα levels at the single cell level Liao et al, unpublished
Summary of studies of Alzheimer s disease using ipscs 1. Generation of new stem cell (ips) lines from patients with early-onset, familial Alzheimer s disease (fad) 2. In all fates tested, the fad APP V717I mutation leads to increased total Aβ, Aβ38 and Aβ42 generation 3. Aβ generation increases as stem cells differentiate to neuronal fates 4. Tau protein levels are increased in fad neurons directed to a rostral but not caudal neuronal fate and Aβ-specific antibodies are able to rescue this phenotype 5. Aβ from fad neurons stimulates GFAP expression in astrocytes 6. Directing differentiation to caudal fates versus rostral fates alters APP cleavage 7. Microengraving can be used to determine which cell fates secrete the highest levels of Aβ, and compare drug responsiveness in different cell types
Young-Pearse Lab Christina Muratore, PhD Heather Rice, PhD Meichen Liao, PhD Sarah Sullivan, PhD Priya Srikanth Dana Callahan Taehwan Shin Acknowledgments Collaborators Dennis Selkoe, MD Dominic Walsh, PhD Chris Love, PhD Phil De Jager, MD David Bennett, MD