ModellingNeuropsychiatricDisordersusinginvitromodels

FredH.GageSalkIns>tuteforBiologicalStudies

ICOSRO

3/28/17

Usinghumaninducedpluripotentstemcellsto

modelneurological/psychiatricdiseases

LV-SynP-HTG Rabies-EnvAΔG-mCherry

D0 D7 D12

NTP Co-twin SZ0

2

4

6

8P

resy

napt

ic /

Pos

tsyn

aptic

Rabies Virus Tracing

* ***

**

NTP:Neurotypical

Co-twin:Non-affectedsiblings

SZ:Schizophrenia-affectedsiblings

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

PathobiologyofSCZandBP

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

EndogenousWnt3ainearly

hippocampalpaVerning

Tole&Groveetal.JNeurosci,2001.MouseE10.5&E12.5

Insituhybridiza\on:

Brown-Wnt3aexpBlue-EphB1(a,b,c)

Blue-beta-tubulin(d)

Prox1-EGFP/Prox1/Dapi

Prox1isaspecificmarkerforhippocampal

DGgranuleneurons

An%-caudalizingfactors

Dissociaterose6es+FGF2

Day020264060

NPCdifferen%a%onAA+cAMP+Laminin+

Wnt3a+BDNF

PlateEBs NPCdifferen%a%onAA+cAMP+Laminin+BDNF

Differen%a%onofhumanembryonicstemcellsintohippocampalDGneuronsusingNPCs

0

20

40

60

80

100

120

0 10 20 30 40

Foldcha

ngerela%vetocontrol

Dayofdifferen%a%on

Emx2

NeuroD1

Pax6

0

5

10

15

20

25

30

35

40

45

0 10 20 30 40

Foldcha

ngerela%vetocontrol

Daysofdifferen%a%on

TBR1DCXFoxg1

HumanProx1-posi\vebipolargranuleneuronsarespontaneouslyac\ve

Func\onalassay:

hippocampalhNPCsgrafedinvivo

50k/ul

cellsuspension

Stereotaxic

injec\oninto

dentategyrus

P14NODSCIDmice

2-16wksinvivograf

D

A

B

C

Fig. 5

Hilus

GCL

Dapi

GFP/Prox1/Dapi

hNF-M/Dapi

CA3 CA3

Mor

phom

etric

s of

gra

fted

neur

ons

Mor

phom

etric

s of

gra

fted

neur

ons120

100

80

60

40

20

0Soma

Area (µm²)DendriteLength

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

50ms

20mV

25pA

10s

10pA

100ms

-70mV+CNQX

A. B.

C. D.

GFP/Prox1/DAPI50μm

Fig. 2. (A) An hiPSC-derivedGFP+ 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

BasalElectrophysiologyUnaffected

inSCZDhiPSCNeurons

BrennandetalNATURE,2011

induced

Na+/K+currents

inducedac\on

poten\alsspontaneousEPSCs spontaneousIPSCs

Prox1GranuleNeurons,DerivedfromSchizophrenics

arelessac\vethenControlNeurons

Schizophrenia:amajorpublichealthconcern

"Biologicalinsightsfrom108schizophrenia-associatedgene\cloci."Nature511.7510(2014):421.

Study how gene>c varia>on contributes to the development of the

disorder

Modelingpsychiatricdisordersinadish

AcohortincludesMonozygo\ctwinsthatarediscordantfor

schizophrenia(SZ)andhealthycontroltwins

Canweiden\fyaltera\onsthatarecommontotheSZpa\entsand

non-affectedsiblings?

Canweiden\fyaltera\onsthatarespecifictotheSZpa\ents?

Canweiden\fyaphenotypictraitthatisduetoasubsetofgenes?

HippocampalpaVerned(hp)NPCgiverisetoCA3neurons

Otx=cor\calhemmarker NeuN+Vglut1=matureexcitatoryneuron

hpNPCgiverisetohCA3neurons

ELAVL2=CA3marker;notpresentinDG

hCA3=ES/iPScellderivedCA3neurons

hDG=ES/iPScellderivedDGneurons

hpNPCgiverisetohCA3neurons

Selec\vely Comprehensively

SCGN+CA3neurons:Anovelsubtype

uniquetohuman

AlsopresentinhCA3invitro

hCA3neuronsarefunc\onallymature

andformsynap\cconnec\ons

Spontaneousac%vitybyMEArecording

Rabiestracingtoassaylevel

ofmonosynap%cconnec%vity

DGneuronsshowvlowlevelofintrinsicac\vityandlowconnec\vity(Gothard2001,Amaral

1990)

Likeinvivo,hCA3neuronsaredis\nctfromhDGneurons

hCA3neuronsformsynap\cconnec\onswith

hDGneurons

CoculturehCA3andhDG(1:1)

Rabiesvirusmarkspostsynap\candpresynap\cneurons;

Grik4=CA3marker;Grik4-GFPisspecificforCA3Calb1=matureDGneurons

DG!CA3mossyfibercircuitinvitro

~400um

Postsynap\cCA3neurons+rabiesvirus

SZ-DGneuronsshow

aVenuated

spontaneousac\vityYuDet.al.2014

SZ-iPSCsmakeCA3subtypes

SZ-CA3showreducedspontaneous

ac\vityCo-cultureDGand

CA3neuronsmixedReplatedat3week

Nochangeintotalcell#,subtype#,interneuron#sugges\ngintrinsicac\vitydeficits

hpNPC---------CA3neurons

Summary

•  AnovelprotocoltogeneratehippocampalCA3pyramidal

neuronsinvitro

•  hCA3neuronsrepresentanovelhumanspecificsubtypeand

demonstratepyramidalneuronaldiversity

•  CA3neuronsformcircuitrywithDGneuronsinvitro

•  SpontaneousspikerateinCA3andDG-CA3popula\onisimpairedinSchizophrenia

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

ModelingBipolarDiseasewithiPSCderivedNeurons

MertensetalNature2015

Reprogramming and Differentiation

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

Patchclamp

•  Methodinventedlate70’sbyErwinNeherandBertSakmann,whoreceivedaNobelprizefortheirwork

•  Allowedtorecordcurrentsofevensingleionchannels,andthestudyoftheinvolvmentofionchannelinneuronalfunc%on

•  Wholecellpatchclampinneurons

markedbyProx1::eGFPlen\viralvector

Patch clamp recording on Prox1::EGFP-expressing DG neurons (j) showed spontaneous postsynaptic currents (k) and Na+/K+ currents

Sample trace (s), frequency (t) and mean amplitude (u) of spontaneous APs.

Hippocampal dentate gyrus granule cells derived from BD patients show hyper-excitability.

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

Lithium rescues the hyperexcitability in hippocampal neurons derived from BD patient-iPSCs

hiPSDerivedNeuronsShow

SpontaneousCa2+Transients

Fluo-4AM,3montholdneurons,real\me

,JessicaJou,AnthonySimone,DianaYu

Sample traces (a) and bar graph (b) showing that the Ca2+ transient events detected with Fluo 4-AM were abolished by TTX.

Somatic Ca2+ imaging analysis reveals hyperactivity in the neural network formed by the BD iPSC-derived neurons

BD neurons show abnormalities in mitochondrial functions

Sample images of neurons expressing DsRed2-mito and Prox1::EGFP

Mitochondrial genes changed in BP Neuronsfl

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

TreatmentofLi+NR/BPpa\ents

•  Lamotrigineisanoraldrugthatisused

primarilyforseizures.Itischemically

unrelatedtootheran\-seizuredrugs.

•  Lamotriginealsoisusedforpreven\ngmood

episodesinindividuals18yearsorolderwith

BP.Thoughthemechanismisunknown,

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.

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.

BipolariPSCs

•  Cohortof4controlcelllines,

•  3Lithiumresponsive(LR)bipolarpa\ents

•  3non-responsive(NR)bipolarpa\ents

•  EBVallowshumanlymphocytestheabilitytoproliferate

indefinitely.

•  EBVgenesnotintegratedinthederivediPSCs•  Widelybankedcellsforstudyinghumandisease

Epstein-BarrVirus(EBV)immortalizedlymphocytes

BipolarNPCsandhippocampalDGgranulecellneurons

•  Wholecellpatch

clampinneurons

markedbyProx1::eGFPlen\viral

vector

Hyper-excitabilityandsustainedac%vity

EvokedAc\onpoten\alsatcurrentclampmode,neurons3.5weeksold

Evokedac%onpoten%als:Currentclampmode

•  Control:21.2±2.4,4celllines,95cells

•  LR:48±5.5,3celllines,84

cells

•  NR:48.7±8.3,3celllines,63

cellsp<0.0001forLRneuronsvs.

controlp=0.0002forNRneurons

vs.control

contr

ol

LR NR

Spontaneousac%vity:•  Control:0.24±0.06Hz

•  LR:0.71±0.14Hz•  NR:0.35±0.08Hz

P=0.0005LRvs.control,p=0.03LRvs.NR,p=0.0055

bipolarvs.control

v

TotalevokedAP

Spontaneousac%vity

Sodium/potassiumcurrentsSodiumcurrents

Fastpotassiumcurrents

Slowpotassiumcurrents

Sodiumtopotassium

ra\oin:Voltageclampmode

25%increaseinLR,not

significant

46%reducedinNR,

p=0.0006

Spikeshapecontr

ol

LR NR

CurrentclampmodeSpecialfeaturesofeachgrouparepreservedthroughouten\redifferen\a\on\mes

Responsetochroniclithiumtreatment•  Animportantvalida\onoftheseneuronsasmodelneuronsforbipolar

isdifferen\alresponsetoneuronsdependingifthepa\entrespondedtolithiumtreatment

TotalevokedAP

TotalevokedAP

Bioinforma%cs:Neuronsderivedfrombipolarpa%entsdivideintotwoveryintrinsicallydifferentgroups,butisitpredic%veofdrugresponse?

AUC0.98

NeuronsfromBipolarDisorderpa%entsarecharacterizedbyintrinsicallydifferentsubpopula%onsofneurons

•  LRneuronsspikesarenarrowandhighamplitude,NRneuronsspikesarewidewithalowamplitude,andwithamoredepolarizedthreshold

•  Animportantfeatureis

sharedbyboththebipolar

disordersgroups:alargefastafer-hyperpolariza\on

(AHP)

•  Thisfeatureisknownfrom

fastspikinginterneurons

Summary

•  DGneuronsderivedfrombipolarpa\entsarehyperexcitable

•  EBVimmortalizedlymphocytescanbewelldifferen\atedintoDGgranule

neurons,maintainingpa\entbypa\entdifferences.Thesecellsarehighlybanked,andtheircollec\oninducesminimalstresstothepa\ent

•  Bipolardisorderneuronssub-divideintosubgroupsofintrinsicallydifferentneurons

•  Predic\veofthepa\ent’sresponsetoLi

•  Thesesubpopula\onsofneuronsshareafeatureofalargefastAfer-hyperpolariza\on,probablyamaincontributortotheirfast,sustainedspiking

abili\es

•  Anumericalmodelreproducestheexperimentalresults

•  ChronicLitreatmentreduceshyperexcitabilityinneuronsderivedfromthe

pa\entswhorespondtoLi

Acknowledgments

CarolMarche+oShaniSternLynneMooreAniSarkarMeiyanWangSaraLinkerRenataSantosSimonSchaferPatrickReedJunYaoYongsungKim

ReneKahnUtrectandMt.Sinai-NYCJohnKelsoeUCSDMarEnAlda,CanadaAnneBang,SBP

RuthKeithleyAnnaMendesAriannaMei

IsabelleGuimont

Lithiumtreatmenteffectsonspikeshape

•  fastAHPamplitudeis

reduced,andthespikebroadens.

•  Twofeatureswhich

contributetoreduced

excitability

•  fastAHPamplitudeis

reduced

•  Thethresholdbecomeslessdepolarized

FastAHP

FastAHP

threshold

Spikewidth

Spikewidth

threshold

Spikeheight

Spikeheight

Inputconductance

Inputconductance

Summary

•  DGneuronsderivedfrombipolarpa\entsarehyperexcitable

•  EBVimmortalizedlymphocytescanbewelldifferen\atedinto

DGgranuleneurons,maintainingpa\entbypa\entdifferences.

Thesecellsarehighlybanked,andtheircollec\oninduces

minimalstresstothepa\ent

•  Bipolardisorderneuronssub-divideintosubgroupsofintrinsicallydifferentneurons

•  Predic\veofthepa\ent’sresponsetoLi

•  Thesesubpopula\onsofneuronsshareafeatureofalargefastAfer-hyperpolariza\on,probablyamaincontributortotheir

fast,sustainedspikingabili\es

•  Anumericalmodelreproducestheexperimentalresults

•  ChronicLitreatmentreduceshyperexcitabilityinneurons

Nextsteps

•  CA3andinterneurons

•  Bipolarastrocytesandco-culturing

•  Patch-seq

FredGage•  LeahBoyer•  MikeMcConnell•  YanLi•  YanglingMu•  DianaYu

GongChenJonathanSebatShaneMcCarthyEdwardCookGeneYeo•  MichaelLovci•  Jus%nArnoldEricCourchesne&ACEJohnKelsoe&PGBD

MarkLawson

MyStudents:• AnthonySimone• JessicaJou• ChelseaGelboin-Burkhart• NgocTran• SarahSangar• SamLarkin• LisaJohnson

EdCallaway• NicholasWall

KonradHochedlinger

SalkStemCellCoreTravisBerggeronMargaretLutz

CIRMFellow