Restricted accessResearch articleFirst published online 2020-12
Neuronal Differentiation of Induced Pluripotent Stem Cells from Schizophrenia Patients in Two-Dimensional and in Three-Dimensional Cultures Reveals Increased Expression of the Kv4.2 Subunit DPP6 That Contributes to Decreased Neuronal Activity
Although the molecular underpinnings of schizophrenia (SZ) are still incompletely understood, deficits in synaptic activity and neuronal connectivity have been identified as core pathomechanisms of SZ and other neuropsychiatric disorders. In this study, we generated induced pluripotent stem cell (iPSC) lines from skin fibroblasts from healthy donors and patients diagnosed with idiopathic SZ. We differentiated the human iPSC into cortical neurons both as adherent monolayers and as three-dimensional spheroids. RNA sequencing revealed little overlap in differentially expressed genes between 2D and 3D neuron cultures from SZ iPSC compared with controls. Notably, mRNA transcripts encoding dipeptidyl peptidase-like protein 6 (DPP6), an accessory subunit of Kv4.2 voltage-gated potassium channels, were massively increased in cortical neurons from SZ iPSC in the 2D and 3D model. Consistently, multielectrode array recordings and calcium imaging showed significantly decreased neuronal activity both in 2D and in 3D cultures from SZ neurons. To show a causal relationship, we treated iPSC-derived neurons in 2D cultures with lentiviral DPP6 shRNA vectors and the Kv4.2 channel blocker AmmTx3, respectively. Both treatments successfully reversed neuronal hypoexcitability and hypoactivity in cortical neurons from SZ iPSC. Our data highlight a contribution of DPP6 and Kv4.2 to the deficit in neurotransmission in an iPSC model for SZ, which may be of therapeutic relevance for a subset of SZ patients.
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References
1.
SarkarA, MarchettoMC and GageFH. (2017). Synaptic activity: an emerging player in schizophrenia. Brain Res, 1656:68–75.
2.
BrischR, SaniotisA, WolfR, BielauH, BernsteinHG, SteinerJ, BogertsB, BraunK, JankowskiZ, KumaratilakeJ, HennebergM and GosT. (2014). The role of dopamine in schizophrenia from a neurobiological and evolutionary perspective: old fashioned, but still in vogue. Front Psychiatry, 5:47.
3.
FromerM, PocklingtonAJ, KavanaghDH, WilliamsHJ, DwyerS, GormleyP, GeorgievaL, ReesE, PaltaP, et al. (2014). De novo mutations in schizophrenia implicate synaptic networks. Nature, 506:179–184.
4.
GlantzLA, GilmoreJH, LiebermanJA and JarskogLF. (2006). Apoptotic mechanisms and the synaptic pathology of schizophrenia. Schizophr Res, 81:47–63.
5.
GulsunerS, WalshT, WattsAC, LeeMK, ThorntonAM, CasadeiS, RippeyC, ShahinH, Consortium on the Genetics ofS, GroupPS, et al. (2013). Spatial and temporal mapping of de novo mutations in schizophrenia to a fetal prefrontal cortical network. Cell, 154:518–529.
6.
KennyEM, CormicanP, FurlongS, HeronE, KennyG, FaheyC, KelleherE, EnnisS, TropeaD, et al. (2014). Excess of rare novel loss-of-function variants in synaptic genes in schizophrenia and autism spectrum disorders. Mol Psychiatry, 19:872–879.
7.
LipsES, CornelisseLN, ToonenRF, MinJL, HultmanCM, International SchizophreniaC, HolmansPA, O'DonovanMC, PurcellSM, et al. (2012). Functional gene group analysis identifies synaptic gene groups as risk factor for schizophrenia. Mol Psychiatry, 17:996–1006.
8.
MirnicsK, MiddletonFA, LewisDA and LevittP. (2001). Analysis of complex brain disorders with gene expression microarrays: schizophrenia as a disease of the synapse. Trends Neurosci, 24:479–486.
9.
Schizophrenia Working Group of the Psychiatric GenomicsC. (2014). Biological insights from 108 schizophrenia-associated genetic loci. Nature, 511:421–427.
10.
SachsNA, SawaA, HolmesSE, RossCA, DeLisiLE and MargolisRL. (2005). A frameshift mutation in Disrupted in Schizophrenia 1 in an American family with schizophrenia and schizoaffective disorder. Mol Psychiatry, 10:758–764.
11.
MuirWJ, GosdenCM, BrookesAJ, FantesJ, EvansKL, MaguireSM, StevensonB, BoyleS, BlackwoodDH, St ClairDM, et al. (1995). Direct microdissection and microcloning of a translocation breakpoint region, t(1;11)(q42.2;q21), associated with schizophrenia. Cytogenet Cell Genet, 70:35–40.
12.
LichtensteinP, BjorkC, HultmanCM, ScolnickE, SklarP and SullivanPF. (2006). Recurrence risks for schizophrenia in a Swedish national cohort. Psychol Med, 36:1417–1425.
13.
SullivanPF, KendlerKS and NealeMC. (2003). Schizophrenia as a complex trait: evidence from a meta-analysis of twin studies. Arch Gen Psychiatry, 60:1187–1192.
14.
XuB, RoosJL, LevyS, van RensburgEJ, GogosJA and KarayiorgouM. (2008). Strong association of de novo copy number mutations with sporadic schizophrenia. Nat Genet, 40:880–885.
15.
TakahashiK, OkitaK, NakagawaM and YamanakaS. (2007). Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc, 2:3081–3089.
16.
TakahashiK and YamanakaS. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126:663–676.
17.
ShiY, InoueH, WuJC and YamanakaS. (2017). Induced pluripotent stem cell technology: a decade of progress. Nat Rev Drug Discov, 16:115–130.
18.
MarchettoMC, CarromeuC, AcabA, YuD, YeoGW, MuY, ChenG, GageFH and MuotriAR. (2010). A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell, 143:527–539.
19.
NageshappaS, CarromeuC, TrujilloCA, MesciP, Espuny-CamachoI, PasciutoE, VanderhaeghenP, VerfaillieCM, RaitanoS, et al. (2016). Altered neuronal network and rescue in a human MECP2 duplication model. Mol Psychiatry, 21:178–188.
20.
PakC, DankoT, ZhangY, AotoJ, AndersonG, MaxeinerS, YiF, WernigM and SudhofTC. (2015). Human neuropsychiatric disease modeling using conditional deletion reveals synaptic transmission defects caused by heterozygous mutations in NRXN1. Cell Stem Cell, 17:316–328.
21.
WenZ, NguyenHN, GuoZ, LalliMA, WangX, SuY, KimNS, YoonKJ, ShinJ, et al. (2014). Synaptic dysregulation in a human iPS cell model of mental disorders. Nature, 515:414–418.
22.
BrennandKJ, SimoneA, JouJ, Gelboin-BurkhartC, TranN, SangarS, LiY, MuY, ChenG, et al. (2011). Modelling schizophrenia using human induced pluripotent stem cells. Nature, 473:221–225.
23.
YuDX, Di GiorgioFP, YaoJ, MarchettoMC, BrennandK, WrightR, MeiA, McHenryL, LisukD, et al. (2014). Modeling hippocampal neurogenesis using human pluripotent stem cells. Stem Cell Reports, 2:295–310.
24.
MertensJ, WangQW, KimY, YuDX, PhamS, YangB, ZhengY, DiffenderferKE, ZhangJ, et al. (2015). Differential responses to lithium in hyperexcitable neurons from patients with bipolar disorder. Nature, 527:95–99.
25.
TopolA, ZhuS, HartleyBJ, EnglishJ, HaubergME, TranN, RittenhouseCA, SimoneA, RuderferDM, et al. (2016). Dysregulation of miRNA-9 in a subset of schizophrenia patient-derived neural progenitor cells. Cell Rep, 15:1024–1036.
26.
BrennandK, SavasJN, KimY, TranN, SimoneA, Hashimoto-ToriiK, BeaumontKG, KimHJ, TopolA, et al. (2015). Phenotypic differences in hiPSC NPCs derived from patients with schizophrenia. Mol Psychiatry, 20:361–368.
27.
TopolA, EnglishJA, FlahertyE, RajarajanP, HartleyBJ, GuptaS, DeslandF, ZhuS, GoffT, et al. (2015). Increased abundance of translation machinery in stem cell-derived neural progenitor cells from four schizophrenia patients. Transl Psychiatry, 5:e662.
28.
AminND and PascaSP. (2018). Building models of brain disorders with three-dimensional organoids. Neuron, 100:389–405.
29.
RoweRG and DaleyGQ. (2019). Induced pluripotent stem cells in disease modelling and drug discovery. Nat Rev Genet, 20:377–388.
30.
TrevinoAE, Sinnott-ArmstrongN, AndersenJ, YoonSJ, HuberN, PritchardJK, ChangHY, GreenleafWJ and PascaSP. (2020). Chromatin accessibility dynamics in a model of human forebrain development. Science, 367:eaay1645.
31.
KiznerV, NaujockM, FischerS, JagerS, ReichS, SchlotthauerI, ZuckschwerdtK, GeigerT, HildebrandtT, et al. (2020). CRISPR/Cas9-mediated Knockout of the Neuropsychiatric Risk Gene KCTD13 causes developmental deficits in human cortical neurons derived from induced pluripotent stem cells. Mol Neurobiol, 57:616–634.
32.
MarianiJ, CoppolaG, ZhangP, AbyzovA, ProviniL, TomasiniL, AmenduniM, SzekelyA, PalejevD, et al. (2015). FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell, 162:375–390.
33.
PascaAM, SloanSA, ClarkeLE, TianY, MakinsonCD, HuberN, KimCH, ParkJY, O'RourkeNA, et al. (2015). Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat Methods, 12:671–678.
34.
ShiY, KirwanP and LiveseyFJ. (2012). Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc, 7:1836–1846.
35.
CobosI, LongJE, ThwinMT and RubensteinJL. (2006). Cellular patterns of transcription factor expression in developing cortical interneurons. Cereb Cortex 16 Suppl, 1:i82–i88.
36.
ZhongS, ZhangS, FanX, WuQ, YanL, DongJ, ZhangH, LiL, SunL, et al. (2018). A single-cell RNA-seq survey of the developmental landscape of the human prefrontal cortex. Nature, 555:524–528.
37.
LewisDA and MoghaddamB. (2006). Cognitive dysfunction in schizophrenia: convergence of gamma-aminobutyric acid and glutamate alterations. Arch Neurol, 63:1372–1376.
38.
ElvevagB and GoldbergTE. (2000). Cognitive impairment in schizophrenia is the core of the disorder. Crit Rev Neurobiol, 14:1–21.
39.
Green MF. (2006). Cognitive impairment and functional outcome in schizophrenia and bipolar disorder. J Clin Psychiatry, 67:e12.
40.
KehrerC, MaziashviliN, DugladzeT and GloveliT. (2008). Altered Excitatory-Inhibitory Balance in the NMDA-Hypofunction Model of Schizophrenia. Front Mol Neurosci, 1:6.
41.
SunY, FarzanF, BarrMS, KiriharaK, FitzgeraldPB, LightGA and DaskalakisZJ. (2011). gamma oscillations in schizophrenia: mechanisms and clinical significance. Brain Res, 1413:98–114.
42.
GreenwoodTA, LazzeroniLC, MurraySS, CadenheadKS, CalkinsME, DobieDJ, GreenMF, GurRE, GurRC, et al. (2011). Analysis of 94 candidate genes and 12 endophenotypes for schizophrenia from the Consortium on the Genetics of Schizophrenia. Am J Psychiatry, 168:930–946.
43.
HauckeV, NeherE and SigristSJ. (2011). Protein scaffolds in the coupling of synaptic exocytosis and endocytosis. Nat Rev Neurosci, 12:127–138.
44.
CusterKL, AustinNS, SullivanJM and BajjaliehSM. (2006). Synaptic vesicle protein 2 enhances release probability at quiescent synapses. J Neurosci, 26:1303–1313.
45.
ChangWP and SudhofTC. (2009). SV2 renders primed synaptic vesicles competent for Ca2+ -induced exocytosis. J Neurosci, 29:883–897.
46.
LynchBA, LambengN, NockaK, Kensel-HammesP, BajjaliehSM, MatagneA and FuksB. (2004). The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam. Proc Natl Acad Sci U S A, 101:9861–9866.
47.
SnowAD, NochlinD, SekiguichiR and CarlsonSS. (1996). Identification in immunolocalization of a new class of proteoglycan (keratan sulfate) to the neuritic plaques of Alzheimer's disease. Exp Neurol, 138:305–317.
48.
MudgeJ, MillerNA, KhrebtukovaI, LindquistIE, MayGD, HuntleyJJ, LuoS, ZhangL, van VelkinburghJC, et al. (2008). Genomic convergence analysis of schizophrenia: mRNA sequencing reveals altered synaptic vesicular transport in post-mortem cerebellum. PLoS One, 3:e3625.
49.
MattheisenM, MuhleisenTW, StrohmaierJ, TreutleinJ, NenadicI, AlblasM, MeierS, DegenhardtF, HermsS, et al. (2012). Genetic variation at the synaptic vesicle gene SV2A is associated with schizophrenia. Schizophr Res, 141:262–265.
50.
NadalMS, OzaitaA, AmarilloY, Vega-Saenz de MieraE, MaY, MoW, GoldbergEM, MisumiY, IkeharaY, NeubertTA and RudyB. (2003). The CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels. Neuron, 37:449–461.
51.
BirnbaumSG, VargaAW, YuanLL, AndersonAE, SweattJD and SchraderLA. (2004). Structure and function of Kv4-family transient potassium channels. Physiol Rev, 84:803–833.
52.
SunW, MaffieJK, LinL, PetraliaRS, RudyB and HoffmanDA. (2011). DPP6 establishes the A-type K(+) current gradient critical for the regulation of dendritic excitability in CA1 hippocampal neurons. Neuron, 71:1102–1115.
53.
PisciottaM, CoronasFI, BlochC, PrestipinoG and PossaniLD. (2000). Fast K(+) currents from cerebellum granular cells are completely blocked by a peptide purified from Androctonus australis Garzoni scorpion venom. Biochim Biophys Acta, 1468:203–212.
54.
VacherH, Romi-LebrunR, MourreC, LebrunB, KourrichS, MasmejeanF, NakajimaT, LegrosC, CrestM, BougisPE and Martin-EauclaireMF. (2001). A new class of scorpion toxin binding sites related to an A-type K+ channel: pharmacological characterization and localization in rat brain. FEBS Lett, 501:31–36.
55.
MaffieJK, DvoretskovaE, BougisPE, Martin-EauclaireMF and RudyB. (2013). Dipeptidyl-peptidase-like-proteins confer high sensitivity to the scorpion toxin AmmTX3 to Kv4-mediated A-type K+ channels. J Physiol, 591:2419–2427.
56.
GantzSC and BeanBP. (2017). Cell-autonomous excitation of midbrain dopamine neurons by endocannabinoid-dependent lipid signaling. Neuron, 93:1375–1387 e2.
57.
TruchetB, ManriqueC, SrengL, ChaillanFA, RomanFS and MourreC. (2012). Kv4 potassium channels modulate hippocampal EPSP-spike potentiation and spatial memory in rats. Learn Mem, 19:282–293.
58.
JohnstoneM, VasisthaNA, BarbuMC, DandoO, BurrK, ChristopherE, GlenS, RobertC, FetitR, et al. (2019). Reversal of proliferation deficits caused by chromosome 16p13.11 microduplication through targeting NFkappaB signaling: an integrated study of patient-derived neuronal precursor cells, cerebral organoids and in vivo brain imaging. Mol Psychiatry, 24:294–311.
59.
ReifA, FritzenS, FingerM, StrobelA, LauerM, SchmittA and LeschKP. (2006). Neural stem cell proliferation is decreased in schizophrenia, but not in depression. Mol Psychiatry, 11:514–522.
YeF, KangE, YuC, QianX, JacobF, YuC, MaoM, PoonRYC, KimJ, et al. (2017). DISC1 regulates neurogenesis via modulating kinetochore attachment of Ndel1/Nde1 during Mitosis. Neuron, 96:1204.
62.
Spitzer NC. (2006). Electrical activity in early neuronal development. Nature, 444:707–712.
63.
WangP, MokhtariR, PedrosaE, KirschenbaumM, BayrakC, ZhengD and LachmanHM. (2017). CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in cerebral organoids derived from iPS cells. Mol Autism, 8:11.
64.
CacaceR, HeemanB, Van MosseveldeS, De RoeckA, HoogmartensJ, De RijkP, GossyeH, De VosK, De CosterW, et al. (2019). Loss of DPP6 in neurodegenerative dementia: a genetic player in the dysfunction of neuronal excitability. Acta Neuropathol, 137:901–918.
65.
PurcellSM, MoranJL, FromerM, RuderferD, SolovieffN, RoussosP, O'DushlaineC, ChambertK, BergenSE, et al. (2014). A polygenic burden of rare disruptive mutations in schizophrenia. Nature, 506:185–190.
66.
RuzzoEK, Perez-CanoL, JungJY, WangLK, Kashef-HaghighiD, HartlC, SinghC, XuJ, HoekstraJN, et al. (2019). Inherited and de novo genetic risk for autism impacts shared networks. Cell, 178:850–866 e26.
67.
MukaiJ, CannavoE, CrabtreeGW, SunZ, DiamantopoulouA, ThakurP, ChangCY, CaiY, LomvardasS, et al. (2019). Recapitulation and reversal of schizophrenia-related phenotypes in setd1a-deficient mice. Neuron, 104:471–487 e12.
68.
GandalMJ, ZhangP, HadjimichaelE, WalkerRL, ChenC, LiuS, WonH, van BakelH, VargheseM, et al. (2018). Transcriptome-wide isoform-level dysregulation in ASD, schizophrenia, and bipolar disorder. Science, 362:eaat8127.
69.
IchidaJK and KiskinisE. (2015). Probing disorders of the nervous system using reprogramming approaches. EMBO J, 34:1456–1477.
70.
SeshadriM, BanerjeeD, ViswanathB, RamakrishnanK, PurushottamM, VenkatasubramanianG and JainS. (2017). Cellular models to study schizophrenia: a systematic review. Asian J Psychiatr, 25:46–53.
71.
WenZ, ChristianKM, SongH and MingGL. (2016). Modeling psychiatric disorders with patient-derived iPSCs. Curr Opin Neurobiol, 36:118–127.
72.
BreenMS, DobbynA, LiQ, RoussosP, HoffmanGE, StahlE, ChessA, SklarP, LiJB, et al. (2019). Global landscape and genetic regulation of RNA editing in cortical samples from individuals with schizophrenia. Nat Neurosci, 22:1402–1412.
73.
VacherH, AlamiM, CrestM, PossaniLD, BougisPE and Martin-EauclaireMF. (2002). Expanding the scorpion toxin alpha-KTX 15 family with AmmTX3 from Androctonus mauretanicus. Eur J Biochem, 269:6037–6041.
74.
ForrestMP, ZhangH, MoyW, McGowanH, LeitesC, DionisioLE, XuZ, ShiJ, SandersAR, et al. (2017). Open Chromatin Profiling in hiPSC-derived neurons prioritizes functional noncoding psychiatric risk variants and highlights neurodevelopmental Loci. Cell Stem Cell, 21:305–318 e8.
75.
LeeHY, GeWP, HuangW, HeY, WangGX, Rowson-BaldwinA, SmithSJ, JanYN and JanLY. (2011). Bidirectional regulation of dendritic voltage-gated potassium channels by the fragile X mental retardation protein. Neuron, 72:630–642.
76.
FenelonK, XuB, LaiCS, MukaiJ, MarkxS, StarkKL, HsuPK, GanWB, FischbachGD, MacDermottAB, KarayiorgouM and GogosJA. (2013). The pattern of cortical dysfunction in a mouse model of a schizophrenia-related microdeletion. J Neurosci, 33:14825–14839.
77.
ZhouY, KaiserT, MonteiroP, ZhangX, Van der GoesMS, WangD, BarakB, ZengM, LiC, et al. (2016). Mice with Shank3 mutations associated with ASD and Schizophrenia display both shared and distinct defects. Neuron, 89:147–162.
78.
KrystalJH, AnticevicA, YangGJ, DragoiG, DriesenNR, WangXJ and MurrayJD. (2017). Impaired tuning of neural ensembles and the pathophysiology of Schizophrenia: a translational and computational neuroscience perspective. Biol Psychiatry, 81:874–885.
79.
HoffmanGE, HartleyBJ, FlahertyE, LadranI, GochmanP, RuderferDM, StahlEA, RapoportJ, SklarP and BrennandKJ. (2017). Transcriptional signatures of schizophrenia in hiPSC-derived NPCs and neurons are concordant with post-mortem adult brains. Nat Commun, 8:2225.
80.
LinL, LongLK, HatchMM and HoffmanDA. (2014). DPP6 domains responsible for its localization and function. J Biol Chem, 289:32153–32165.
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