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Schizophrenia Research Forum Live Webinar - June 28, 2017 - Rusty Gage
- 3. LV-SynP-HTG Rabies-EnvAΔG-mCherry D0 D7 D12 NTP Co-twin SZ 0 2 4 6 8Presynaptic/Postsynaptic Rabies Virus Tracing * * ** * * NTP: Neurotypical Co-twin: Non-affected siblings SZ: Schizophrenia-affected siblings
- 4. Lineage-specific differentiation Advantages of Lineage-specific Differentiation Homogeneity of characteristics Cell type relevant to the Disease Characterize developmental features not end point features Disadvantages of Lineage–specific Differentiation Wrong choice of Cell type or phenotype Does not provide adequate circuit properties
- 5. Pathobiology of SCZ and BP Prefrontal Cortex and Hippocampus linked to SCZ and BP Patients exhibit one or all of the following: • a reduced volume in non-pyramidal cell layers, including dentate gyrus • a reduced number of somatostatin-positive and paravalbumin-positive neurons • reduced somal volume of cornu ammonis sector 2/3 The dentate gyrus contributes to the formation of memories and plays a role in depression. Focus on lineage-specific cellular phenotypes: dentate granule neurons, the principal cells of the dentate gyrus
- 8. An%-caudalizing factors Dissociate rose6es + FGF2 Day 0 20 26 40 60 NPC differen%a%on AA+cAMP+Laminin+ Wnt3a +BDNF Plate EBs NPC differen%a%on AA+cAMP+Laminin+BDNF Differen%a%on of human embryonic stem cells into hippocampal DG neurons using NPCs 0 20 40 60 80 100 120 0 10 20 30 40 Fold change rela%ve to control Day of differen%a%on Emx2 NeuroD1 Pax6 0 5 10 15 20 25 30 35 40 45 0 10 20 30 40 Fold change rela%ve to control Days of differen%a%on TBR1 DCX Foxg1
- 11. D A B C Fig. 5 Hilus GCL Dapi GFP/Prox1/Dapi hNF-M/Dapi CA3 CA3 Morphometricsofgraftedneurons Morphometricsofgraftedneurons 120 100 80 60 40 20 0 Soma Area (µm²) Dendrite Length Total (µm) 0 1 2 3 4 5 6 Segments Tree Number 2wks 6wks * ** ** GFP/Dapi E F GFP Prox1 HuNu Transplant of Human iPS cells to adult NOD/Skid mouse Dentate Gyrus
- 12. 50ms 20mV 25pA 10s 10pA 100ms -70mV + CNQX A. B. C. D. GFP/Prox1/DAPI 50μm Fig. 2. (A) An hiPSC-derived GFP+ neuron transplanted into the granule cell layer (GCL) of the mouse DG. (B) Transplanted neurons are active and can generate action potential spikes in response to somatic current injection. They also functionally integrate into the endogenous hippocampal network, as evidenced by spontaneous post-synaptic currents (C), and by induced postsynaptic currents in response to stimulation of upstream presynaptic connections (D). These responses are attenuated by 10 µM CNQX, indicating that they are mainly generated by excitatory AMPA receptors. Human Neurons Functionally Connected in vivo
- 16. Study how gene>c varia>on contributes to the development of the disorder Modeling psychiatric disorders in a dish A cohort includes Monozygoc twins that are discordant for schizophrenia (SZ) and healthy control twins Can we idenfy alteraons that are common to the SZ paents and non-affected siblings? Can we idenfy alteraons that are specific to the SZ paents? Can we idenfy a phenotypic trait that is due to a subset of genes?
- 27. Summary • A novel protocol to generate hippocampal CA3 pyramidal neurons in vitro • hCA3 neurons represent a novel human specific subtype and demonstrate pyramidal neuronal diversity • CA3 neurons form circuitry with DG neurons in vitro • Spontaneous spike rate in CA3 and DG-CA3 populaon is impaired in Schizophrenia
- 28. Reprogramming patients in search of Cell autonomous and genetic mechanism for BP and Li+ responsiveness 4 - phenotypically normal 3 - Li+ responders 3 - Li+ non-responders Differentiate into Dentate Granule- Like Neurons Modeling Bipolar Disease with iPSC derived Neurons Mertens et al Nature 2015
- 30. 1- Differentially expressed genes in the BD derived DG neurons (456 genes). 2- Expression of representative genes involved in the PKA/PKC and AP firing systems revealed by RNA-seq – suggesting Hyper-Excitability
- 31. Patch clamp • Method invented late 70’s by Erwin Neher and Bert Sakmann , who received a Nobel prize for their work • Allowed to record currents of even single ion channels, and the study of the involvment of ion channel in neuronal func%on • Whole cell patch clamp in neurons marked by Prox1::eGFP lenviral vector
- 32. Patch clamp recording on Prox1::EGFP-expressing DG neurons (j) showed spontaneous postsynaptic currents (k) and Na+/K+ currents
- 33. Sample trace (s), frequency (t) and mean amplitude (u) of spontaneous APs.
- 34. Hippocampal dentate gyrus granule cells derived from BD patients show hyper-excitability.
- 35. Heatmaps (k) and MA plots (l) showing that Li significantly altered the gene expression profile of the LR neurons but not the NR neurons: 456 gene significantly changed in Li+ Rs while only 40 genes changed in NRs
- 36. Lithium rescues the hyperexcitability in hippocampal neurons derived from BD patient-iPSCs
- 38. Sample traces (a) and bar graph (b) showing that the Ca2+ transient events detected with Fluo 4-AM were abolished by TTX.
- 39. Somatic Ca2+ imaging analysis reveals hyperactivity in the neural network formed by the BD iPSC-derived neurons
- 40. BD neurons show abnormalities in mitochondrial functions Sample images of neurons expressing DsRed2-mito and Prox1::EGFP Mitochondrial genes changed in BP Neuronsfl
- 43. JC-1 is a cationic dye that can accumulate in energized mitochondria, the ratio of red to green fluorescence reflects the functional efficiency of mitochondria
- 46. Representative traces of APs evoked during 300 ms step-wise depolarization periods in the normal and NR neurons with and without 100 µM lamotrigine (LTG) treatment.
- 47. Summary • BP patient derived hippocampal Dentate Gyrus neurons are molecularly and functionally hyper-excitability. • Li+ can reverse the excitability BUT only in neurons from patients that responded behavioral to Li+. • BP patients have impaired mitochondria, likely required to supply energy to impaired synapses. This impairment is reversed in Li+ responding patient neurons, but not the NRs. –we do not know if the energy deficit is primary or secondary to the disease.
- 48. Bipolar iPSCs • Cohort of 4 control cell lines, • 3 Lithium responsive (LR) bipolar paents • 3 non-responsive (NR) bipolar paents • EBV allows human lymphocytes the ability to proliferate indefinitely. • EBV genes not integrated in the derived iPSCs • Widely banked cells for studying human disease Epstein-Barr Virus (EBV) immortalized lymphocytes
- 50. Hyper-excitability and sustained ac%vity Evoked Acon potenals at current clamp mode, neurons 3.5 weeks old Evoked ac%on poten%als : Current clamp mode • Control:21.2±2.4 , 4 cell lines, 95 cells • LR: 48±5.5, 3 cell lines, 84 cells • NR: 48.7±8.3 , 3 cell lines, 63 cells p<0.0001 for LR neurons vs. control p=0.0002 for NR neurons vs. control contr ol LR NR Spontaneous ac%vity: • Control:0.24±0.06 Hz • LR: 0.71±0.14 Hz • NR: 0.35±0.08 Hz P=0.0005 LR vs. control, p=0.03 LR vs. NR, p=0.0055 bipolar vs. control v Total evoked AP Spontaneous ac%vity
- 54. Neurons from Bipolar Disorder pa%ents are characterized by intrinsically different sub popula%ons of neurons • LR neurons spikes are narrow and high amplitude, NR neurons spikes are wide with a low amplitude, and with a more depolarized threshold • An important feature is shared by both the bipolar disorders groups: a large fast afer-hyperpolarizaon (AHP) • This feature is known from fast spiking interneurons
- 55. Summary • DG neurons derived from bipolar paents are hyperexcitable • EBV immortalized lymphocytes can be well differenated into DG granule neurons, maintaining paent by paent differences. These cells are highly banked, and their collecon induces minimal stress to the paent • Bipolar disorder neurons sub-divide into subgroups of intrinsically different neurons • Predicve of the paent’s response to Li • These sub populaons of neurons share a feature of a large fast Afer- hyperpolarizaon, probably a main contributor to their fast, sustained spiking abilies • A numerical model reproduces the experimental results • Chronic Li treatment reduces hyperexcitability in neurons derived from the paents who respond to Li
- 57. Lithium treatment effects on spike shape • fast AHP amplitude is reduced, and the spike broadens. • Two features which contribute to reduced excitability • fast AHP amplitude is reduced • The threshold becomes less depolarized Fast AHP Fast AHP thresh old Spike width Spike width thresh old Spike height Spike height Input conductance Input conductance
- 58. Summary • DG neurons derived from bipolar paents are hyperexcitable • EBV immortalized lymphocytes can be well differenated into DG granule neurons, maintaining paent by paent differences. These cells are highly banked, and their collecon induces minimal stress to the paent • Bipolar disorder neurons sub-divide into subgroups of intrinsically different neurons • Predicve of the paent’s response to Li • These sub populaons of neurons share a feature of a large fast Afer-hyperpolarizaon, probably a main contributor to their fast, sustained spiking abilies • A numerical model reproduces the experimental results
- 60. Fred Gage • Leah Boyer • Mike McConnell • Yan Li • Yangling Mu • Diana Yu Gong Chen Jonathan Sebat Shane McCarthy Edward Cook Gene Yeo • Michael Lovci • Jus%n Arnold Eric Courchesne & ACE John Kelsoe & PGBD Mark Lawson My Students: • Anthony Simone • Jessica Jou • Chelsea Gelboin-Burkhart • Ngoc Tran • Sarah Sangar • Sam Larkin • Lisa Johnson Ed Callaway • Nicholas Wall Konrad Hochedlinger Salk Stem Cell Core Travis Berggeron Margaret Lutz CIRM Fellow