Modeling psychiatric disorders at the cellular and network …...Although psychiatric disorders such as autism spectrum disorders, schizophrenia and bipolar disorder affect a number

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Modeling psychiatric disorders at the cellular andnetwork levelsKJ Brennand, A Simone, N Tran and FH Gage

Laboratory of Genetics, Salk Institute for Biological Studies, La Jolla, CA, USA

Although psychiatric disorders such as autism spectrum disorders, schizophrenia and bipolardisorder affect a number of brain regions and produce a complex array of clinical symptoms,basic phenotypes likely exist at the level of single neurons and simple networks. Being highlyheritable, it is hypothesized that these disorders are amenable to cell-based studies in vitro.Using induced pluripotent stem cell-derived neurons and/or induced neurons from fibroblasts,limitless numbers of live human neurons can now be generated from patients with a geneticbackground permissive to the disease state. We predict that cell-based studies will ultimatelycontribute to our understanding of the initiation, progression and treatment of thesepsychiatric disorders.Molecular Psychiatry advance online publication, 3 April 2012; doi:10.1038/mp.2012.20

Keywords: autism spectrum disorders; bipolar disorder; neurons; schizophrenia; stem cells

Introduction

Autism spectrum disorders (ASDs), schizophrenia(SCZD) and bipolar disorder (BD) combine to affectnearly 1 in 30 adults throughout the global popula-tion.1 While these psychiatric disorders are character-ized by markedly different clinical phenotypes, recentgenetic studies have suggested that they may sharecommon underlying molecular causes. ASD, SCZDand BD are believed to be developmental in origin,resulting from events that occur in fetal developmentor early childhood. The molecular mechanism ofthese disorders is difficult to study in patients oranimal models because of the complex geneticetiologies and varying environmental effects contri-buting to disease.

Cell-based models produce live human neuronswith genetic backgrounds permissive to the diseasestate. Temporal analysis of disease initiation andprogression can be studied in the cell type relevantto disease. Human cell-based models can be idealexperimental paradigms with which to investigatedisease mechanisms; for example, studies of amyo-trophic lateral sclerosis have revealed a non-cellautonomous contribution of glial cells to this neuronaldisease.2,3 In order to be studied using an in vitromodel, a given disease must (1) be highly genetic,ensuring that cultured cells are afflicted by disease inthe absence of any potentially unresolved environ-mental factors and (2) affect a cell type that can survive

and, ideally, be robustly expanded when culturedin vitro. With respect to the first criterion, twin studieshave calculated the heritability of ASD, SCZD and BDto be between 70 and 90%.4–6 Our hypothesis is thatthis genetic predisposition to psychiatric illness issufficient that cultured neurons will consistentlyundergo disease initiation and progression. Regardingthe second criterion, while mature neurons are post-mitotic and cannot be expanded in culture, conditionsfor the survival of human neurons are well described,7

and robust quantities of neurons for study can begenerated through the growth and subsequent differ-entiation of proliferative neural progenitor cells.

The ability to compare cellular and network proper-ties of live human neurons in vitro represents animportant new approach with which to study psy-chiatric disease because live human neurons frompatients or controls are exceedingly rare. Recently,three new sources of live human neurons have beenreported: primary olfactory neural precursors, neuronsdifferentiated from human-derived induced pluripo-tent stem cells (hiPSC neurons) and induced neurons(iNeurons) generated from primary patient fibroblasts.Although olfactory neural precursors are capable ofself-renewal and differentiation to mature neurons,8,9

olfactory neural precursors cannot yield cells from theneural lineages specifically implicated in psychiatricdisorders, such as GABAergic or dopaminergicneurons. Because we believe it is critical that therelevant cell type affected in the disease state bestudied, we will therefore focus on in vitro models ofpsychiatric disease utilizing hiPSC neurons andiNeurons (Figure 1).

While generally considered to be whole-braindisorders, we suggest that ASD, SCZD and BD canbe broken down to component aberrations at the

Received 18 November 2011; revised 26 January 2012; accepted16 February 2012

Correspondence: FH Gage, PhD, Laboratory of Genetics, SalkInstitute for Biological Studies, 10010 North Torrey Pines Road,La Jolla, CA 92037, USA.E-mail: [email protected]

Molecular Psychiatry (2012), 1–15& 2012 Macmillan Publishers Limited All rights reserved 1359-4184/12

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cellular and/or network levels. For example, at thecellular level, the subtle synaptic defects that arebelieved to contribute to illness can be studied withcell-based models. Furthermore, while the cyclicalbehavioral swings of BD cannot be reproduced,patterns of spontaneous and stimulated neuronalnetwork activity can be measured in vitro. Usingcell-based models, one can study ASD, SCZD and BDby observing the abnormal development of neuronsand their circuitry in vitro.

In this review, we will discuss (1) post-mortem andanimal studies demonstrating that cellular pheno-types exist at the neuronal level in these disorders(Tables 1 and 2), (2) functional magnetic resonanceimaging (fMRI) and electrophysiological evidencefrom humans and rodents suggesting that networkdefects contribute to these disorders (Tables 1 and 2)and (3) the recent findings of novel in vitro models ofpsychiatric disorders (Table 3).

Evidence for cellular phenotypes in psychiatricdisorders

Aberrant neuronal connectivity, as assessed by den-dritic arborization and synaptic density, is a char-acteristic that appears to be shared between ASD,SCZD and BD. Perturbed neuronal migration has alsobeen linked to psychiatric disorders, althoughwhether this contributes to, or functions separatelyfrom, abnormal neuronal connectivity remains to bedemonstrated.

Altered dendritic arborizationAt onset, ASD is often characterized by excessive brainvolume; MRI studies observed increased cerebral whitematter volume in 2- to 4-year-old autistic children,10–12

which has been correlated with an excess number ofneurons in the prefrontal cortex.13 Over the lifetime ofthe ASD patient, however, the brain overgrowthphenotype is typically reversed; studies of adults withASD have observed cortical thinning14,15 and reducedfrontal lobe10,16,17 and corpus callosum18 volumes.While we found few anatomical post-mortem studies

of ASD, there are reports of reduced dendritic arboriza-tion in hippocampal neurons in two cases of ASD.19 InRett syndrome (RTT), a severe and rare ASD caused bythe mutation of the MECP2 gene,20,21 post-mortemstudies report reduced neuronal cell size and dendriticarborization throughout the cortex.22 Reports of fragileX syndrome (FX), another monogenetic form of ASD,have been less conclusive: while one study reporteddifferences in dendritic arborization following in vitrodifferentiation of neurospheres derived from post-mortem human FXS brain tissue,23 a second groupfailed to observe significant differences in a similarstudy.24

Decreased whole-brain volume is consistently ob-served in SCZD,25–27 particularly in the gray matter of thefrontal cortex, temporal lobe (particularly the hippo-campus and amygdala) and the basal ganglia,27–29 andlongitudinal studies report that progressive brainvolume declines for at least 20 years after the onsetof symptoms. Post-mortem studies of brains frompatients with SCZD have not found evidence ofneuronal loss; instead, they observe smaller neuronalsomas30,31 reduced dendritic arborizations31,32 andincreased neuronal density without changes in abso-lute cell number in the cortex and hippocampus.30,33

Decreased brain volumes in the limbic system,particularly the amygdala and hippocampus, and inthe frontal cortex are associated with BD.34,35 Itremains unclear, however, whether brain volumechanges are a preexisting factor contributing to thedevelopment of BD or a consequence of prolongedillness; one recent study suggests that brain volumechanges are more tightly correlated to active psycho-sis than BD.36 Despite observations of diminishedbrain volumes and reduced neuronal density in BDpatients,37,38 we found no report of altered dendriticarborization or synaptic density in post-mortemstudies of BD patient brains.

Mouse models of a number of psychiatric disordershave been developed. For the most part, these micehave reduced expression of rare, highly penetrantgenes implicated in ASD, SCZD or BD. RTT (Mecp2null) mouse brains show a reduction in neuronal size;39

Figure 1 Cell-based modeling of psychiatric disorders. Fibroblast cells obtained from patients can be used to generate livehuman neurons with a genetic background known to produce the disease state. Fibroblasts can be reprogrammed to human-derived induced pluripotent stem cells (hiPSCs) by transient expression of OCT4, SOX2, KLF4 and cMYC and thensubsequently differentiated into mature neurons. Alternately, fibroblasts can be directly converted into a neuronal fate bytransient expression of ASCL1, BRN2, MYT1L and NEUROD.

iPSC modeling of psychiatric disordersKJ Brennand et al

2

Molecular Psychiatry

Table

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3


and abnormalities in dendritic arborization.39–41 Con-versely, dendritic arborization defects have not beenreported in FX (Fmr1 null) mice. Many SCZD mousemodels, including Disc1 and heterozygous-null Nrg1and Erbb4 mice, have reduced neurite outgrowthand reduced dendritic complexity.42–45 Although fewgenetic mouse models of BD have been reported,neurons from mice with a point mutation in thecircadian Clock gene display complex changes indendritic morphology,46 which can be ameliorated withlithium, a drug routinely used in the treatment of BD.46

Altered synaptic densityA number of genes implicated in ASD, SCZD and BDhave been associated with synaptic maturation andfunction.47 Post-mortem synaptic spine density has notbeen adequately explored in human patients withASD. One report found that relative to controls, spinedensities on cortical pyramidal cells were greater inASD subjects, and highest spine densities were mostcommonly found in ASD subjects with lower levels ofcognitive functioning.48 Conversely, the number ofspines in dendrites of neurons from post-mortem RTTbrains is reduced.49 In FX patients, post-mortemstudies have identified abnormalities in dendriticspine shape in cortical pyramidal cells, which tendto be both longer and more slender than controls.50

Post-mortem studies of SCZD patient brains foundreduced dendritic spine density in the cortex51,52 andhippocampus.31,53 What post-mortem analysis of ASDand SCZD has failed to resolve is whether diseaseprogression reflects developmental aberrations duringneuronal differentiation or activity-dependent atrophyof neuronal dendrites or synapses in mature neurons.33

Animal studies recapitulate these synaptic defects.Using mouse models of RTT, decreased Mecp2levels have been implicated in defects of synapticcontact formation and synaptic transmission.41,54–56

Comparably, inherited mutations of Shank3, whichalso model ASD, result in reduced dendritic synapticspine induction and maturation.57 Similar to post-mortem observations of FX patient brains, Fmr1 micehave abnormally thin and elongated dendritic spinemorphology and greater spine density.58 Fmr1, thegene affected in FX, regulates the translation ofmessages important for activity-dependent synapticmodulation.59 Although a number of animal models ofASD recapitulate defects in synaptic maturation, thedirection of the change varies depending uponthe gene under investigation, which is consistent withthe hypothesis that ASD is a spectrum of complexgenetic disorders involving impaired developmentalsynaptic maturation, stabilization, elimination orpruning. Studies of mouse models of SCZD have alsoobserved synaptic defects; there is reduced hippo-campal synaptic transmission in Disc1 mice,42,43

impaired synaptic maturation and function in Nrg1mice60–62 and fewer cortical neurons with slightlysmaller spines in mouse models of the human SCZDcopy number variant at 22q11.2.63–65 In mice withreduced Reelin (Reln) expression (putative models ofT

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ral

dif

fere

nti

ati

on

,re

du

ced

cort

ical

mig

rati

on

,d

imin

ish

ed

resp

on

seto

cA

MP

-sen

siti

ve

rep

uls

ive

cu

es,

red

uced

neu

rite

ou

tgro

wth

,sy

nap

tic

tran

smis

sion

an

dalt

ere

dd

istr

ibu

tion

of

hip

pocam

pal

neu

ron

s

Mao

et

al.

,67

Kam

iya

et

al.

,74

Kvajo

et

al.

,42

Li

et

al.

43

SC

ZD

Dis

c1

kn

ockd

ow

nH

ipp

ocam

pal

ad

ult

-born

neu

ron

sA

du

ltn

ew

born

neu

ron

ssh

ow

accele

rate

dd

en

dri

tic

develo

pm

en

tan

dsy

nap

sefo

rmati

on

,d

efe

cts

inaxon

al

targ

eti

ng,

en

han

ced

excit

abil

ity

Fau

lker

et

al.

,70

Du

an

et

al.

69

SC

ZD

Nrg

1C

ort

ex,

peri

ph

era

ln

erv

es

Aberr

an

tta

ngen

tial

mig

rati

on

of

neu

ron

sd

eri

ved

from

the

ven

tral

tele

ncep

halo

n,

imp

air

ed

syn

ap

tic

matu

rati

on

an

dfu

ncti

on

,h

yp

om

yeli

nati

on

Lop

ez-B

en

dit

oet

al.

,44

Barr

os

et

al.

,60

Ch

en

et

al.

62

SC

ZD

Erb

B4

Hip

pocam

pu

s,cort

ex

Aberr

an

tn

eu

rite

ou

tgro

wth

an

dsy

nap

sem

atu

rati

on

,re

du

ced

lon

g-t

erm

pote

nti

ati

on

,su

pp

ress

ed

Src

-dep

en

den

ten

han

cem

en

tof

NM

DA

Rre

spon

ses

du

rin

gth

eta

-bu

rst

stim

ula

tion

,re

du

ced

excit

ato

ryin

pu

ton

toG

AB

Aerg

icn

eu

ron

s,P

PI

defi

cit

s

Kri

vosh

eya

et

al.

,45

Pit

ch

er

et

al.

,61

Pit

ch

er

et

al.

,101

Barr

os

et

al.

,60

Li

et

al.

,95

Ch

en

et

al.

62

SC

ZD

22q11.2

Cort

ex,

hip

pocam

pu

sF

ew

er

cort

ical

neu

ron

sw

ith

small

er

spin

es,

alt

ere

dsh

ort

-an

dlo

ng-

term

syn

ap

tic

pla

stic

ity

an

dcalc

ium

kin

eti

cs,

imp

air

ed

hip

pocam

pal-

pre

fron

tal

syn

ch

ron

y

Fen

elo

net

al.

63

Earl

set

al.

,64

Sig

urd

sson

et

al.

65

SC

ZD

Reln

Cort

ex,

hip

pocam

pu

sD

ecre

ase

dd

en

dri

tic

spin

em

atu

rati

on

,d

en

sity

an

dp

last

icit

yP

ap

pas

et

al.

66

SC

ZD

Dtn

bp

1G

AB

AD

ecre

ase

dP

PI,

red

uced

evoked

gam

ma

acti

vit

yC

arl

son

et

al.

104

BD

Clo

ck

Str

iatu

m(n

ucle

us

accu

mben

s)In

cre

ase

dle

ngth

an

dcom

ple

xit

yof

den

dri

tes,

norm

al

syn

ap

tic

den

sity

,d

ysf

un

cti

on

al

gam

ma

acti

vit

yacro

ssli

mbic

cir

cu

its,

imp

roved

by

lith

ium

treatm

en

t

Dzir

asa

et

al.

46

Abbre

via

tion

s:A

SD

,au

tism

spectr

um

dis

ord

er;

BD

,bip

ola

rd

isord

er;

FX

,fr

agil

eX

syn

dro

me;

PP

I,p

rep

uls

ein

hib

itio

n;

RT

T,

Rett

syn

dro

me;

SC

ZD

,sc

hiz

op

hre

nia

dis

ord

er.


5


Table

3S

um

mary

of

pu

bli

shed

rep

ort

sof

hiP

SC

-base

dm

od

els

of

AS

D,

SC

ZD

an

dB

D

Dis

ease

Refe

ren

ce

Gen

eti

cm

uta

tion

Neu

ron

al

ph

en

oty

pe

hiP

SC

meth

od

Sou

rce

of

cell

sP

ati

en

tse

x;

age

at

bio

psy

(years

);avail

able

ph

en

oty

pic

info

rmati

on

RT

TC

heu

ng

et

al.

122

MeC

P2

(D3–4,

T158

M,

R306C

)D

ecre

ase

dso

ma

size

Retr

ovir

us:

fou

rfa

cto

rs(O

CT

4,

SO

X2,

KLF

4,

c-M

YC

)

Fib

robla

st:

pati

en

tbio

psy

(1)

an

dC

ori

ell

GM

11270

(2),

GM

17880

(3)

(1)

Fem

ale

;6

gro

wth

an

dd

evelo

pm

en

tal

dela

y,in

abil

ity

tow

alk

wit

hou

tass

ista

nce,

ata

xia

,n

on

verb

al,

has

no

han

du

sean

dcon

stan

tre

peti

tive

han

dm

oti

on

s,so

me

trem

or,

has

had

ep

ilep

tic

seiz

ure

san

dsi

gn

ific

an

tabn

orm

al

ele

ctr

oen

cep

halo

gra

m,

teeth

gri

nd

ing,

som

esl

eep

dif

ficu

ltie

s,an

dbre

ath

hold

ing

an

dh

yp

erv

en

tila

tion

(2)

Fem

ale

;8;

norm

al

lyso

som

al

en

zym

es,

cli

nic

all

yaff

ecte

d,

cla

ssic

al

sym

pto

ms

(3)

Fem

ale

;5;ass

ista

nce

requ

ired

for

walk

ing,d

ela

yin

gro

wth

an

dd

evelo

pm

en

t,sl

eep

pro

ble

ms,

abn

orm

al

EE

Gw

ith

no

sym

pto

ms

of

seiz

ure

s,gri

nd

ing

of

teeth

,bre

ath

hold

ing,

hyp

erv

en

tila

tion

,n

on

verb

al,

lack

of

han

du

sage,

rep

eti

tive

han

dm

oti

on

s,d

iffi

cu

lty

eati

ng

an

dsl

igh

tre

flu

xes,

slig

ht

trem

or,

small

feet

RT

TM

arc

hett

oet

al.

123

MeC

P2

(1155d

el3

,Q

244X

,T

158

M,

R306C

)

Red

uced

som

asi

ze

an

dsp

ine

den

sity

,fe

wer

syn

ap

ses,

alt

ere

dcalc

ium

sign

ali

ng,

ele

ctr

op

hysi

olo

gic

al

abn

orm

ali

ties

Retr

ovir

us:

fou

rfa

cto

rs(O

CT

4,

SO

X2,

KLF

4,

c-M

YC

)

Fib

robla

st:

Cori

ell

GM

11272

(1),

GM

16548

(2),

GM

17880

(3),

GM

11270

(4)

(1)

Fem

ale

;3;

norm

al

lyso

som

al

en

zym

es,

cli

nic

all

yaff

ecte

d,

cla

ssic

al

sym

pto

ms

(2)

Fem

ale

;5;

cli

nic

all

yaff

ecte

d,

slig

htl

ycu

rved

spin

e,

am

bu

lato

ry,

slig

ht

rigid

ity

an

dsp

ast

icit

y,d

ecre

asi

ng

head

cir

cu

mfe

ren

ce,

aberr

an

tsl

eep

patt

ern

s,d

ecre

ase

dh

an

du

sage,

rep

eti

tive

han

dm

oti

on

s,bre

ath

hold

ing,

non

verb

al,

con

stip

ati

on

,d

ecre

ase

dh

an

dan

dfe

et

cir

cu

lati

on

,ra

rese

lf-

inju

riou

sbeh

avio

r,sl

igh

teati

ng

pro

ble

ms

an

dre

flu

zes,

teeth

gri

nd

ing,

slig

ht

EE

Gabn

orm

ali

ties,

trem

ors

(3)

Fem

ale

;5;ass

ista

nce

requ

ired

for

walk

ing,d

ela

yin

gro

wth

an

dd

evelo

pm

en

t,sl

eep

pro

ble

ms,

abn

orm

al

EE

Gw

ith

no

sym

pto

ms

of

seiz

ure

s,gri

nd

ing

of

teeth

,bre

ath

hold

ing,

hyp

erv

en

tila

tion

,n

on

verb

al,

lack

of

han

du

sage,

rep

eti

tive

han

dm

oti

on

s,d

iffi

cu

lty

eati

ng

an

dsl

igh

tre

flu

xes,

slig

ht

trem

or,

small

feet

(4)

Fem

ale

;8;

norm

al

lyso

som

al

en

zym

es,

cli

nic

all

yaff

ecte

d,

cla

ssic

al

sym

pto

ms

RT

TA

nan

iev

et

al.

124

MeC

P2

(T158

M,

V247X

,R

306C

)D

ecre

ase

inn

ucle

ar

an

dn

eu

ron

size

Len

tivir

us:

fou

rfa

cto

rs(O

CT

4,N

AN

OG

,S

OX

2,

LIN

28),

Retr

ovir

us:

4fa

cto

rs(O

CT

4,

SO

X2,

KLF

4,

c-M

YC

)

Fib

robla

st:

Cori

ell

GM

17880

(1),

GM

07982

(2),

GM

11270

(3)

(1)

Fem

ale

;5;ass

ista

nce

requ

ired

for

walk

ing,d

ela

yin

gro

wth

an

dd

evelo

pm

en

t,sl

eep

pro

ble

ms,

abn

orm

al

EE

Gw

ith

no

sym

pto

ms

of

seiz

ure

s,gri

nd

ing

of

teeth

,bre

ath

hold

ing,

hyp

erv

en

tila

tion

,n

on

verb

al,

lack

of

han

du

sage,

rep

eti

tive

han

dm

oti

on

s,d

iffi

cu

lty

eati

ng

an

dsl

igh

tre

flu

xes,

slig

ht

trem

or,

small

feet

(2)

Fem

ale

;25;

cli

nic

all

yaff

ecte

d,

mic

rocep

haly

,se

vere

lyre

tard

ed

,h

an

dw

rin

gin

gst

art

ing

at

age

2,

scoli

osi

sat

age

12,

kyp

hosc

oli

osi

sat

age

25,

start

ed

tolo

sesk

ills

at

2years

old

,C

Tsc

an

at

25

show

ed

atr

op

hy,

slow

,abn

orm

al

EE

G,

no

sleep

pro

ble

ms

(3)

Fem

ale

;8;

norm

al

lyso

som

al

en

zym

es,

cli

nic

all

yaff

ecte

d,

cla

ssic

al

sym

pto

ms


6


Table

3C

on

tin

ued

Dis

ease

Refe

ren

ce

Gen

eti

cm

uta

tion

Neu

ron

al

ph

en

oty

pe

hiP

SC

meth

od

Sou

rce

of

cell

sP

ati

en

tse

x;

age

at

bio

psy

(years

);avail

able

ph

en

oty

pic

info

rmati

on

Tim

oth

ysy

nd

rom

eP

asc

aet

al.

129

CA

CN

A1C

Defe

cts

incalc

ium

sign

ali

ng,

decre

ase

dexp

ress

ion

of

cort

ical

gen

es,

incre

ase

dp

rod

ucti

on

of

nore

pin

ep

hri

ne

an

dd

op

am

ine

Retr

ovir

us:

fou

rfa

cto

rs(O

CT

4,

SO

X2,

KLF

4,

c-M

YC

)

Fib

robla

st:

pati

en

tbio

psy

(1)

Fem

ale

;n

ot

state

d;

not

state

d(2

)N

ot

state

d;

not

state

d;

not

state

d

FX

SU

rbach

et

al.

127

FM

R1

No

neu

ron

sgen

era

ted

Retr

ovir

us:

fou

rfa

cto

rs(O

CT

4,

SO

X2,

KLF

4,

c-M

YC

)

Fib

robla

st:

Cori

ell

GM

05848

(1),

GM

07072

(2),

GM

09497

(3)

(1)

Male

;4;in

cre

ase

dear

size,elo

ngate

dfa

ce,ap

pears

pro

gn

ath

ic,

men

tal

reta

rdati

on

,u

nd

efi

ned

con

necti

ve

tiss

ue

dysp

lasi

a(2

)M

ale

;22;

9/5

0cord

blo

od

lym

ph

ocyte

ssh

ow

ed

fra(X

),m

oth

er

isan

obli

gate

carr

ier

for

fra(x

)(3

)M

ale

;28;

aff

ecte

dbro

ther,

larg

eears

,m

en

tal

reta

rdati

on

,m

acro

-orc

hid

ism

,h

yp

era

cti

ve,

20%

of

PB

Lp

osi

tive

for

fra(x

)

FX

SS

heri

dan

et

al.

128

FM

R1

Few

er

an

dsh

ort

er

neu

ral

pro

cess

es,

incre

ase

dgli

al

cell

sw

ith

more

com

pact

morp

holo

gy

Retr

ovir

us:

fou

rfa

cto

rs(O

CT

4,

SO

X2,

KLF

4,

c-M

YC

)

Fib

robla

st:

Cori

ell

GM

05848

(1),

GM

05131

(2),

GM

05185

(3)

(1)

Male

;4;in

cre

ase

dear

size,elo

ngate

dfa

ce,ap

pears

pro

gn

ath

ic,

men

tal

reta

rdati

on

,u

nd

efi

ned

con

necti

ve

tiss

ue

dysp

lasi

a(2

)M

ale

;3;

aff

ecte

dbro

ther

an

du

ncle

,(3

)M

ale

;26;

46,f

ra(X

),Y

pre

sen

tin

30–50%

of

PB

L

SC

ZD

Ch

ian

get

al.

131

DIS

C1

No

neu

ron

sgen

era

ted

Ep

isom

e:

fou

rfa

cto

rs(O

CT

4,

SO

X2,

KLF

4,

c-M

YC

)

Fib

robla

st:

pati

en

tbio

psy

(1)

Male

;N

A;

dia

gn

ose

dw

ith

ch

ron

icu

nd

iffe

ren

tiate

dsc

hiz

op

hre

nia

,au

dit

ory

an

dvis

ual

hall

ucin

ati

on

s,m

ult

iple

delu

sion

san

dh

ad

form

al

thou

gh

td

isord

er

(2)

Fem

ale

;N

A;

dia

gn

ose

dch

ron

icp

ara

noid

sch

izop

hre

nia

,au

dit

ory

an

dvis

ual

hall

ucin

ati

on

s,m

ult

iple

delu

sion

san

dh

ad

form

al

thou

gh

td

isord

er

SC

ZD

Bre

nn

an

det

al.

132

Sp

ora

dic

Red

uced

neu

ron

al

con

necti

vit

y,fe

wer

neu

rite

s,d

ecre

ase

dP

SD

95

an

dglu

tam

ate

recep

tor

exp

ress

ion

levels

Tetr

acycli

ne-i

nd

ucib

lele

nti

vir

us:

five

facto

rs(O

CT

4,

SO

X2,

KLF

4,

c-M

YC

,LIN

28)

Fib

robla

st:

Cori

ell

GM

02038

(1),

GM

01792

(2),

GM

01835

(3),

GM

02497

(4)

(1)

Male

;22;

on

set

at

age

6,

com

mit

ted

suic

ide

(2)

Male

;26;

recu

rren

ces

of

agit

ati

on

,d

elu

sion

sof

pers

ecu

tion

,fe

ar

of

ass

ass

inati

on

,fa

ther

an

dsi

ster

aff

ecte

d(3

)F

em

ale

;27;

sch

izoaff

ecti

ve

dis

ord

er,

pro

ble

ms

of

dru

gabu

se,

hosp

itali

zed

,fa

ther

aff

ecte

d(4

)M

ale

;23;

para

logic

al

thin

kin

g,

spli

ttin

gof

eff

ect

from

con

ten

t,su

spic

iou

sness

,aff

ecti

ve

shie

ldin

g,

on

set

at

age

15,

hosp

itali

zed

,p

osi

tive

fam

ily

his

tory

SC

ZD

Pau

lsen

et

al.

133

Sp

ora

dic

Ele

vate

dextr

a-

mit

och

on

dri

al

oxygen

con

sum

pti

on

,in

cre

ase

dle

vels

of

reacti

ve

oxygen

specie

s

Retr

ovir

us:

fou

rfa

cto

rs(O

CT

4,

SO

X2,

KLF

4,

c-M

YC

)

Fib

robla

st:

pati

en

tbio

psy

Fem

ale

;48;

clo

zap

ine-r

esi

stan

t

Abbre

via

tion

s:A

SD

,au

tism

spectr

um

dis

ord

er;

BD

,bip

ola

rd

isord

er;

EE

G,

ele

ctr

oen

cep

halo

gra

mte

st;

FX

,fr

agil

eX

syn

dro

me;

hiP

SC

,h

um

an

-deri

ved

ind

uced

plu

rip

ote

nt

stem

cell

;N

A,

not

ap

pli

cable

;P

BL

,p

eri

ph

era

lblo

od

lym

ph

ocyte

;R

TT

,R

ett

syn

dro

me;

SC

ZD

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SCZD and BD), there is decreased dendritic spinematuration and plasticity, leading to decreased spinedensity,66 whereas Clock mice, a model of mania inBD, appear to have normal synaptic density.46

Particularly with respect to SCZD, aberrations insynaptic activity have also been observed in adultneurogenesis in the hippocampus. Similar to corticalembryonic development, where downregulation ofDisc1 results in premature cell cycle exit of neuralprogenitor cells,67,68 adult-born neurons with reducedDisc1 have hastened neural development. Disc1knockdown results in accelerated dendritic develop-ment, soma hypertrophy, aberrant positioning andincreased neural excitability.69–71 It remains unknownif aberrant adult neurogenesis contributes to psychia-tric disease in humans.

Aberrant neuronal migrationIn ASD patients, defects of migration can lead to avariety of morphological outcomes, particularly hetero-topias and dysplastic changes. One recent pathologicalstudy identified a number of abnormalities andlesions in most of the ASD brains studied, includinga loss of vertical and horizontal organization of corticallayers in some patients.72 It has been hypothesizedthat altered expression of cytoskeletal proteins and lossof neuronal polarity contribute to these corticalmigration defects.73

Animal models also show phenotypes consistentwith abnormal neuronal migration: mice with re-duced Disc1 activity have reduced cortical migra-tion,74 Nrg1 mutant mice have reduced tangentialneural migration from the ventral telencephalon44,45

and Cntnap2-null mice have impaired migration ofcortical projection neurons.75 Disc1 mutant mice havealtered distribution of hippocampal mossy fiberterminals on CA3,42 and axons with Disc1 knockdownmiss CA3 altogether and project onto CA1.70 dnDISC1neurites have deficits in neurite repulsionin vitro.42 Although DISC1 is a rare SCZD allele, anunderstanding of the downstream targets or bindingpartners through which it mediates its cellulareffects may identify drug targets relevant to thebroader SCZD population. One putative downstreamtarget of DISC1 is Glycogen Synthase 3-beta (Gsk3b),67

Gsk3b functions within several central path-ways (including cAMP and Wnt) is a direct targetof lithium (a drug commonly used to treat BD)76,77

and mounting evidence indicates that Gsk3bmay be a central mediator of axon outgrowthdynamics.78 Cell-based assays will allow the studyof the effects of Gsk3b, cAMP and WNT levels onneurite outgrowths and axon migration of live humanneurons.

Evidence for network phenotypes in psychiatricdisorders

While comparable neuronal phenotypes, particularlyaberrant dendritic arborization, synaptic density andneuronal migration, are shared between ASD, SCZD

and BD, these cellular phenotypes likely result invastly different network effects in each disorder.Functional imaging facilitates the study of the abnor-mal neural circuitry behind cognitive dysfunction.

One hypothesis concerning ASD is that short-distance over-connectivity in the cortex leads to afailure of long-distance coupling.79 This hypothesispredicts that impaired long-distance connectivity inthe cortex impedes information integration acrossdiverse functional systems (emotional, sensory, auto-nomic, memory). Consistent with this prediction,fMRI studies of resting state brain activity haveobserved increased connectivity between proximalregions, such as the posterior cingulate and theparahippocampal gyrus,80 and decreased connectivitybetween the distal regions, such as the frontal cortexand the parietal lobe,81 the insular cortices and thesomatosensory cortices or amygdala,82 the frontalcortex and the posterior cingulate,80 as well asdecreased interhemispheric synchronization.83 Com-parable defects in long-distance connectivity werefound when ASD patients performed social andintrospective tasks.84 Among ASD patients, a negativecorrelation exists between functional connectivity inthese regions and severity of social and communica-tion impairment.80,82

Just as pathological studies of SCZD reporteddecreased frontal and temporal lobe volumes, earlyfMRI studies of SCZD patients revealed brain activityabnormalities in the frontal and temporal lobes.85,86

More recent studies have further shown that SCZDpatients exhibit cortical hyper-activity and hyper-connectivity of the prefrontal cortex at rest, butreduced activation of the medial prefrontal cortexduring working memory tasks.87 While functionalconnectivity of the parietal cortex to the ventralprefrontal cortex is greater in SCZD, it is reduced tothe dorsal prefrontal cortex.88 This is consistent withanatomical neuronal network maps, which reveal aloss of network ‘hubs’ in the frontal cortex, andincreased connection distance. These network aberra-tions are thought to result from neurodevelopmentalabnormalities impacting cortical organization.89

Although fMRI studies can reveal regions of thebrain with aberrant activity in the disease state, theycannot elucidate the specific neuronal cell typesaffected. Therefore, pharmacological and post-mor-tem studies have generated hypotheses concerningthe cell types affected by SCZD. Similar studies ofASD and BD have been less successful in identifyingthe specific cell types relevant to disease.

Good evidence now links aberrant neurotransmittersignaling to SCZD. Dopamine receptor antagonistsreduce the symptoms of SCZD and evidence nowlinks SCZD with increased dopamine receptor levelsand sensitivity.90,91 Comparably, glutamate-blockingdrugs such as ketamine produce symptoms generallyassociated with SCZD,92 whereas the glutamatereceptor2/3 agonist LY2140023 may ameliorate thesymptoms of SCZD.93 Post-mortem studies of SCZDbrains have found decreased glutamate receptor


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expression,94 whereas among GABAergic interneur-ons, a decrease in GAD67 and calcium-bindingproteins was found. Changes in GABAergic neuronsare particularly relevant as they are thought toproduce gamma oscillations, which synchronizepyramidal neuron firing, an activity that is impairedin SCZD. Evidence in mice suggests that SCZD results,at least in part, from reduced excitatory glutamatergicinput onto GABAergic inhibitory neurons.60,95,96 Itremains unclear whether aberrant dopamine, gluta-mate or GABA signaling is the primary cause of SCZD,as aberrant activity of any neuronal cell type couldaffect neurotransmitter activity of the remaining celltypes in the disease state.

In model organisms from Drosophila to mice, genesassociated with ASD, SCZD and BD have been shownto regulate synaptic activity and plasticity. Forexample, a screen in Drosophila for genes critical inmaintaining homeostatic modulation of synaptictransmission identified the SCZD gene Dysbindin(DTNBP1). Dtnbp1 acts presynaptically, in a dose-dependent manner, to regulate adaptive neuralplasticity.97 In Mecp2 mice, although synapse forma-tion, elimination and strengthening are normal, theexperience-dependent phase of synapse remodelingis impaired98 and Mecp2 mice show altered activity-dependent neural gene expression.99 Cntnap2-nullmice, lacking a gene associated with ASD, havereduced GABAergic neurons and decreased neuronalsynchrony.100 Disc1 mice have reduced hippocampalsynaptic transmission.42,43 Nrg1 mice have impairedsynaptic maturation and function58,60–62,101 and22q11.2 mice show altered short- and long-termsynaptic plasticity as well as calcium kinetics inCA3 presynaptic terminals. Defects in synapticplasticity at the cellular level likely contributeto the network aberrations observed in psychiatricdisorders.

One characteristic network defect observed inSCZD is prepulse inhibition (PPI). PPI is a measureof sensory gating, in which a weaker prestimulus(prepulse) inhibits the reaction of an organism to asubsequent strong startling stimulus (pulse). Deficitsin PPI are observed in Nrg1 mice60,95 and are reversedby dopamine receptor antagonists.102,103 Dtnbp1 micedisplay not only decreased PPI but also reducedevoked g-activity, a second pattern seen in patientswith SCZD.104 In humans, polymorphisms in circa-dian genes such as CLOCK convey risk for BD; mutantClock mice also have dysfunctional g-activity acrosslimbic circuits, which can be improved by chroniclithium treatment.46

While PPI is attributed to glutamatergic activity,reduced g-activity indicates abnormal GABAergicneurotransmission. Therefore, although pharmacolo-gical evidence implicates dopaminergic and glutama-tergic neurons in SCZD, network analysis revealsdefects in both glutamatergic and GABAergic activityin SCZD and BD. Aberrations originating in any oneneuronal subtype would ultimately be expected toaffect activity in other types of neurons and in a

variety of brain regions. The ability to testsynaptic activity in defined populations of humanglutamatergic, GABAergic and dopaminergicneurons affected by ASD, SCZD or BD might help toelucidate the neuronal subtypes at the core of eachdisorder.

Introduction to hiPSCs and iNeurons

The transient expression of four factors (OCT3/4,KLF4, SOX2 and c-MYC) is sufficient to directlyreprogram adult somatic cells into an iPSC state.105–107

Because hiPSCs can be derived from adult patientsafter the development of disease, hiPSCs represent apotentially limitless source of human cells withwhich to study disease, even without knowing whichgenes are interacting to produce the disease state inan individual patient. Methods to efficiently differ-entiate pluripotent stem cells to neurons were devel-oped initially in studies using human embryonic stemcells.108 Through the addition of various morphogensto recapitulate the cues of embryonic development,ESCs and iPSCs can be directed to differentiate toregional identities including forebrain,109 midbrain/hindbrain110,111 and spinal cord.112,113 It is generallythought that every cell type present in vivo can bedifferentiated in vitro using hiPSCs, although meth-ods for many remain unexplored or inefficient.

An alternative approach for generating patient-specific neurons to study complex psychiatric dis-orders is now possible. Expression of four factors(ASCL1, BRN2, MYT1L and NEUROD) can convertfibroblasts into functional iNeurons in vitro.114,115 Theprocess is rapid, generating electrophysiologicallymature neurons with functional synapses within 14days, and it is efficient, yielding up to 8% neurons. Todate, methods exist to transform fibroblasts directly toglutamatergic115 and dopaminergic neurons,116 butmethods to generate GABAergic iNeurons have notyet been reported. The regional identity of eachneurotransmitter subtype remains unclear.

Both hiPSC neurons and iNeurons have thecapacity to generate vast numbers of live humanneurons for the study of psychiatric disorders.Because iNeuron generation bypasses neuronal differ-entiation and maturation, hiPSC neurons are likelythe best method by which to model developmentalfacets of disease. For example, if SCZD ultimatelyresults from abnormal synaptic maturation, it ispossible that direct reprogramming would bypassthe developmental window in which the SCZDcellular phenotype can be observed in vitro. Addi-tionally, as aberrant ASCL1, BRN2 and MYT1L haveall been linked to neurological disease,117–120 it is notunreasonable to predict that overexpression of one ormore of these key neuronal genes might affect theinitiation or progression of a psychiatric disorder invitro. Conversely, the rapid experimental timeframe ofiNeuron generation makes it an ideal system withwhich to study phenotypic effects in mature neurons.If ASD is indeed a disease of activity-dependent


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synaptic modulation rather than synaptic maturation,iNeurons represent a more direct cell type with whichto assay network properties. As the efficiency ofiNeuron generation increases, and spontaneous neu-ronal networks result, this method may facilitaterobust and swift network analysis of ASD, SCZD andBD neurons. It is important to note that both strategiesfacilitate novel experiments of innate neuron-specificdeficits in psychiatric disease that are not confoundedby environmental factors, such as treatment history,drug and alcohol abuse or poverty, that typicallyplague clinical studies.

Psychiatric disorders result in hiPSC neuronalphenotypes in vitro

While no reported studies have yet characterizediNeurons from patients with psychiatric disorders, anumber of groups, including ours, have now pub-lished studies of hiPSC neurons derived from patientswith ASD and SCZD. During neuronal differentiationof hiPSCs, a number of neuronal genes alreadyimplicated in ASD, SCZD and BD, such as transcrip-tion factors and chromatin modifiers like POU3F2and ZNF804A and cell adhesion genes like NRXN1and NLGN1,121 are upregulated, permitting compar-isons of expression levels in diseased and healthy livehuman neurons.

Three groups have now generated hiPSCs from atotal of seven RTT patients, representing a number ofunique point mutations and deletions in the MeCP2gene (Q244X, 1155del 32, T158 M, R306C,D3-4,R294X, V247X).122–124 None of the groups observedaltered replication or differentiation of RTT hiPSCs orneural progenitor cells. Rather, consistent withanimal and post-mortem patient studies, all threegroups reported that neuronal soma size of RTThiPSC neurons is reduced by approximately 10–20%compared with controls.122–124 Furthermore, we ob-served that RTT hiPSC neurons have reduced spinedensity, decreased neuronal spontaneous calciumsignaling and decreased spontaneous excitatory andinhibitory postsynaptic currents. The reproducibilityof these findings across three independent reportsvalidates the use of hiPSC-based models. Further-more, by demonstrating the ability to test drugs torescue synaptic deficiency in RTT neurons, thesestudies hint at future uses of hiPSC neurons for high-throughout drug screening to identify new therapeu-tic drugs for psychiatric disorders.

FX is caused by the absence of expression of thefragile X mental retardation 1 (FMR1) gene,125 whichis believed to result from transcriptional silencingduring embryonic development, owing to a CGGtriplet-repeat expansion in the 50 untranslated regionof the gene.126 Although the somatic cells of three FXpatients were successfully reprogrammed to pluripo-tency, the FMR1 gene remained inactive in all FXhiPSC lines, unlike FX embryonic stem cell lines.Consequently, the authors of this first report of FXhiPSCs concluded that ‘FX-iPSCs do not model the

differentiation-dependent silencing of the FMR1gene,’ and therefore chose not to assess their FX-hiPSC neurons for phenotypic abormalities.127 Morerecently, a second group generated hiPSCs from threepatients (including one patient common to the firstgroup) via nearly identical methods. They noted thata number of hiPSC lines had FMR1 CGG-repeatlengths that were clearly different from the originalfibroblasts, and they also failed to detect FMR1 geneexpression in the original FX fibroblasts or their FXhiPSCs.128 Despite also not observing reactivatedFMR1 expression in FX hiPSCs, this group comparedneural differentiation of FX and control hiPSCs. Theyobserved that neural cultures generated from FXhiPSCs consisted of neurons with fewer and shorterprocesses, as well as a larger number of glial cellswith more compact morphology, suggesting thatdecreased FMR1 expression levels, rather than theslow silencing of FMR1 during neuronal differentia-tion, are sufficient to produce the disease state in FX.

Timothy syndrome is caused by a mutation in theL-type calcium channel Ca(v)1.2 and is associatedwith heart arrhythmias and ASD. From two patientswith Timothy syndrome, hiPSC-derived corticalneural progenitor cells (NPCs) and neurons weregenerated. Neurons from these individuals wereshown to have aberrant calcium signaling,129 whilean earlier publication by this same group demon-strated that hiPSC-derived cardiomyocytes from thesesame patients had irregular contraction, abnormalcalcium transients and irregular electrical activity.130

Timothy syndrome hiPSC neurons underwent abnor-mal cortical differentiation, showing decreased ex-pression of cortical genes and increased production ofnorepinephrine and DA. Notably, treatment withroscovitine, a cyclin-dependent kinase inhibitor andatypical L-type-channel blocker, was sufficient toameliorate many characteristics of Timothy syndromeneurons in vitro.129

DISC1 mutations cause a rare monogenic form ofSCZD. The generation of hiPSCs from SCZD patientswith a DISC1 mutation have now been reported,131

although neurons differentiated from these hiPSCshave not yet been characterized. We predict thatDISC1–hiPSC-derived neurons will ultimately beshown to recapitulate the cellular phenotypes ob-served in dnDISC mice, just as RTT and FX-hiPSCneurons have replicated findings from mouse studies.

We recently reported neuronal phenotypes ofhiPSC neurons from four patients with complexgenetic forms of SCZD. When assayed by retrogradetransmission of rabies virus neuronal labeling, SCZD-hiPSC neurons showed reduced neuronal connectiv-ity and altered gene expression profiles.132 Whilenearly 25% of genes with altered expression had beenpreviously implicated in SCZD, we also identified anumber of new pathways that may contribute toSCZD. A second group has now reported an oxygenmetabolism phenotype associated with SCZD;133 theyobserved a twofold increase in extra-mitochondrialoxygen consumption as well as elevated levels of


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reactive oxygen species in neural progenitor cellsderived from hiPSCs from one SCZD patient relativeto controls. Although a small study, this observationis consistent with animal studies134,135 and deservesattention. Oxygen metabolism defects have not beenwell demonstrated in human neurons, owing to a lackof live human cells for study. This is an excellentexample of the type of hiPSC study that can inve-stigate hypotheses not testable in human patients.

Limitations of hiPSC-based modeling

A number of major limitations currently restricthiPSC-based studies, particularly concerning thescalability of hiPSC generation, neural differentiationand phenotypic characterization of derived neuronsand neural networks. These technical limitationshave made it hard to accurately address the inherentvariability of cell-based studies, which exist in threemajor forms: (1) neuron-to-neuron (intra-patient), (2)hiPSC-to-hiPSC (intra-patient) and (3) patient-to-patient (inter-patient). To produce meaningful data,each cell-based experiment should ideally comparemultiple neuronal differentiations from multipleindependent hiPSC lines from multiple patients.Owing to cost and time constraints, such largeexperiments have not yet been completed. Conse-quently, the hiPSC studies reported to date mayultimately prove to be proof-of-concept demonstra-tions until methods to compare derived neurons fromhundred or thousands of patients and controls arerefined.

Intra-patient variability results from differencesbetween neurons and iPSCs generated from a singlepatient; it is the major constraint on signal to noise incell-based experiments. Differences between indivi-dual hiPSC neurons derived from a single patientproduce neuron-to-neuron variability. To some extent,this variability may be unavoidable, although it iscurrently exacerbated by the heterogeneity of cellularsubtypes in hiPSC neural populations; none of thereports described in this review compare pureneuronal populations of a specific subtype. At theexperimental level, neural subtype heterogeneityresults because current neuronal differentiation pro-tocols are not 100% efficient and, in contrast to thehematopoietic system, cell surface markers by whichspecific subtypes of neurons might be purified havenot been developed. It is well established thatindividual hiPSC lines vary genetically, epigeneti-cally and in terms of neural differentiation propen-sities to produce hiPSC-to-hiPSC variability. Geneticdifferences include the location and number of viralintegrations produced during the reprogrammingprocess and spontaneous mutations that have beenobserved during hiPSC generation and expansion.136

Epigenetic differences reflect the somatic cell typeused for reprogramming and the completeness of itschromatin remodeling.137 Differences in developmen-tal potential exist among human embryonic stem celllines138 and between individual hiPSC lines.139

Inter-patient variability reflects the heterogeneity inclinical outcomes between patients with ASD, SCZDor BD. Consequently, given the small sample size(typically 1–4 patients) of the current hiPSC-basedstudies discussed in this review, a major concern iswhether their findings are representative of the largerpatient population. In the short term, this has beenaddressed by recruiting patients with well-definedclinical or genetic characteristics as well as matchedhealthy controls. Ultimately, methods will have to bedeveloped to permit comparisons of thousands ofpatients.

Future directions of cell-based studies

Whole-brain disorders should be studied at the levelof component aberrations of cells and neural net-works. Neuroimaging, post-mortem anatomical andpharmacological studies of patients may be measuringconsequences of the disease state, rather than itsorigin. Cell-based studies will lead to the discernmentand characterization of the molecular causes of ASD,SCZD and BD and facilitate studies of the cellular andnetwork phenotypes that serve as neuronal predis-positions to disease. Furthermore, these studiesconfer the ability to test various neuron non-cell-autonomous effects, such as inflammation, oxidativestress, activity-dependent modulations and the influ-ence of stress hormones in psychiatric disorders.High-throughput screening of new classes of com-pounds capable of pharmacological amelioration ofneuronal and/or network phenotypes for treatment ofthese disorders is possible.

Small defects at the cellular level could ultimatelymanifest as complex psychiatric disorders with anarray of symptoms in patients. For example, ifneurons derived from psychiatric patients show adecrease in the absolute number of connectionsbetween cells, and if this phenotype is restricted toa specific subtype of neurons, this finding might hintat the central cell type relevant to the disease state.Because synaptic strength is highly modulated bysynaptic activity, a decrease in the strength ofindividual connections between neurons in psychia-tric patients could indicate aberrant synaptic activityor plasticity in patient brains. Finally, perturbedneuronal migration or axon targeting in vitro mightsuggest that mis-targeted neuronal connections,rather than decreased neuronal connectivity, iscentral to disease. Cellular phenotypes hint at theneuronal predispositions contributing to psychiatricdisorders and may help to unlock the complexities ofpsychiatric illness.

While overlapping genetic susceptibilities mightproduce a common cellular phenotype, or predis-position, to psychiatric illness, clinical outcome maybe determined by activity at the network level.Synaptic pruning (either whole brain or in specificregions) is an activity-dependent process that couldgenerate the clinical differences distinguishing ASD,SCZD and BD. Cell-based studies of neuronal and


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network aberrations in psychiatric disorders may leadto predictions of activity-dependent environmentalinfluences that contribute to disease progression.

Human iPSC- and iNeuron-based methods have thepotential to simplify whole-brain disorders like ASD,SCZD and BD to their cellular and network compo-nents, contributing to our understanding of theseconditions. Although many technical issues, particu-larly, concerning the scalability of hiPSC generation,neuronal differentiation and neural assays remain, webelieve that studies of neuronal networks constructedfrom defined neuronal populations are feasible. Byrecapitulating and monitoring healthy and diseasenetworks in a dish, it is likely that new methods ofin vitro modeling of psychiatric disorders willresult in new insights into the mechanism of diseaseinitiation, progression and, ultimately, treatment.

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments

The Gage Laboratory is partially funded by CIRMGrant RL1-00649-1, The Lookout and Mathers Foun-dation, the Helmsley Foundation as well as Sanofi-Aventis. We thank J Simon for illustrations and MLGage for editorial comments.

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Modeling psychiatric disorders at the cellular and network …...Although psychiatric disorders such as autism spectrum disorders, schizophrenia and bipolar disorder affect a number

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