Advertisement

Integrative Analysis Identifies Key Molecular Signatures Underlying Neurodevelopmental Deficits in Fragile X Syndrome

      Abstract

      Background

      Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by epigenetic silencing of FMR1 and loss of FMRP expression. Efforts to understand the molecular underpinnings of the disease have been largely performed in rodent or nonisogenic settings. A detailed examination of the impact of FMRP loss on cellular processes and neuronal properties in the context of isogenic human neurons remains lacking.

      Methods

      Using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 to introduce indels in exon 3 of FMR1, we generated an isogenic human pluripotent stem cell model of FXS that shows complete loss of FMRP expression. We generated neuronal cultures and performed genome-wide transcriptome and proteome profiling followed by functional validation of key dysregulated processes. We further analyzed neurodevelopmental and neuronal properties, including neurite length and neuronal activity, using multielectrode arrays and patch clamp electrophysiology.

      Results

      We showed that the transcriptome and proteome profiles of isogenic FMRP-deficient neurons demonstrate perturbations in synaptic transmission, neuron differentiation, cell proliferation and ion transmembrane transporter activity pathways, and autism spectrum disorder–associated gene sets. We uncovered key deficits in FMRP-deficient cells demonstrating abnormal neural rosette formation and neural progenitor cell proliferation. We further showed that FMRP-deficient neurons exhibit a number of additional phenotypic abnormalities, including neurite outgrowth and branching deficits and impaired electrophysiological network activity. These FMRP-deficient related impairments have also been validated in additional FXS patient–derived human-induced pluripotent stem cell neural cells.

      Conclusions

      Using isogenic human pluripotent stem cells as a model to investigate the pathophysiology of FXS in human neurons, we reveal key neural abnormalities arising from the loss of FMRP.

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Biological Psychiatry
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Hagerman R.J.
        • Berry-Kravis E.
        • Hazlett H.C.
        • Bailey D.B.
        • Moine H.
        • Kooy R.F.
        • et al.
        Fragile X syndrome.
        Nat Rev Dis Primers. 2017; 3: 17065
        • Bagni C.
        • Tassone F.
        • Neri G.
        • Hagerman R.
        Fragile X syndrome: Causes, diagnosis, mechanisms, and therapeutics.
        J Clin Invest. 2012; 122: 4314-4322
        • Verkerk A.J.
        • Pieretti M.
        • Sutcliffe J.S.
        • Fu Y.H.
        • Kuhl D.P.
        • Pizzuti A.
        • et al.
        Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome.
        Cell. 1991; 65: 905-914
        • Darnell J.C.
        • Van Driesche S.J.
        • Zhang C.
        • Hung K.Y.S.
        • Mele A.
        • Fraser C.E.
        • et al.
        FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism.
        Cell. 2011; 146: 247-261
        • Darnell J.C.
        • Jensen K.B.
        • Jin P.
        • Brown V.
        • Warren S.T.
        • Darnell R.B.
        Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function.
        Cell. 2001; 107: 489-499
        • Berry-Kravis E.M.
        • Lindemann L.
        • Jønch A.E.
        • Apostol G.
        • Bear M.F.
        • Carpenter R.L.
        • et al.
        Drug development for neurodevelopmental disorders: Lessons learned from fragile X syndrome.
        Nat Rev Drug Discov. 2018; 17: 280-299
        • Kazdoba T.M.
        • Leach P.T.
        • Silverman J.L.
        • Crawley J.N.
        Modeling fragile X syndrome in the Fmr1 knockout mouse.
        Intractable Rare Dis Res. 2014; 3: 118-133
        • Davis J.K.
        • Broadie K.
        Multifarious functions of the fragile X mental retardation protein.
        Trends Genet. 2017; 33: 703-714
        • Comery T.A.
        • Harris J.B.
        • Willems P.J.
        • Oostra B.A.
        • Irwin S.A.
        • Weiler I.J.
        • Greenough W.T.
        Abnormal dendritic spines in fragile X knockout mice: Maturation and pruning deficits.
        Proc Natl Acad Sci U S A. 1997; 94: 5401-5404
        • Bhakar A.L.
        • Dölen G.
        • Bear M.F.
        The pathophysiology of fragile X (and what it teaches us about synapses).
        Annu Rev Neurosci. 2012; 35: 417-443
        • Contractor A.
        • Klyachko V.A.
        • Portera-Cailliau C.
        Altered neuronal and circuit excitability in fragile X syndrome.
        Neuron. 2015; 87: 699-715
        • Chamberlain S.J.
        Disease modelling using human iPSCs.
        Hum Mol Genet. 2016; 25: R173-R181
        • Sheridan S.D.
        • Theriault K.M.
        • Reis S.A.
        • Zhou F.
        • Madison J.M.
        • Daheron L.
        • et al.
        Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome.
        PLoS One. 2011; 6e26203
        • Telias M.
        • Segal M.
        • Ben-Yosef D.
        Neural differentiation of fragile X human embryonic stem cells reveals abnormal patterns of development despite successful neurogenesis.
        Dev Biol. 2013; 374: 32-45
        • Doers M.E.
        • Musser M.T.
        • Nichol R.
        • Berndt E.R.
        • Baker M.
        • Gomez T.M.
        • et al.
        iPSC-derived forebrain neurons from FXS individuals show defects in initial neurite outgrowth.
        Stem Cells Dev. 2014; 23: 1777-1787
        • Halevy T.
        • Czech C.
        • Benvenisty N.
        Molecular mechanisms regulating the defects in fragile X syndrome neurons derived from human pluripotent stem cells.
        Stem Cell Rep. 2015; 4: 37-46
        • Telias M.
        • Kuznitsov-Yanovsky L.
        • Segal M.
        • Ben-Yosef D.
        Functional deficiencies in fragile X neurons derived from human embryonic stem cells.
        J Neurosci. 2015; 35: 15295-15306
        • Telias M.
        • Mayshar Y.
        • Amit A.
        • Ben-Yosef D.
        Molecular mechanisms regulating impaired neurogenesis of fragile X syndrome human embryonic stem cells.
        Stem Cells Dev. 2015; 24: 2353-2365
        • Zhou Y.
        • Kumari D.
        • Sciascia N.
        • Usdin K.
        CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons.
        Mol Autism. 2016; 7: 42
        • Park C.-Y.
        • Halevy T.
        • Lee D.R.
        • Sung J.J.
        • Lee J.S.
        • Yanuka O.
        • et al.
        Reversion of FMR1 methylation and silencing by editing the triplet repeats in fragile X iPSC-derived neurons.
        Cell Rep. 2015; 13: 234-241
        • Liu X.S.
        • Wu H.
        • Krzisch M.
        • Wu X.
        • Graef J.
        • Muffat J.
        • et al.
        Rescue of fragile X syndrome neurons by DNA methylation editing of the FMR1 gene.
        Cell. 2018; 172: 979-992.e6
        • Zhang Z.
        • Marro S.G.
        • Zhang Y.
        • Arendt K.L.
        • Patzke C.
        • Zhou B.
        • et al.
        The fragile X mutation impairs homeostatic plasticity in human neurons by blocking synaptic retinoic acid signaling.
        Sci Transl Med. 2018; 10: eaar4338
        • Thomson J.A.
        • Itskovitz-Eldor J.
        • Shapiro S.S.
        • Waknitz M.A.
        • Swiergiel J.J.
        • Marshall V.S.
        • Jones J.M.
        Embryonic stem cell lines derived from human blastocysts.
        Science. 1998; 282: 1145-1147
        • Gerhardt J.
        • Tomishima M.J.
        • Zaninovic N.
        • Colak D.
        • Yan Z.
        • Zhan Q.
        • et al.
        The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells.
        Mol Cell. 2014; 53: 19-31
        • Achuta V.S.
        • Grym H.
        • Putkonen N.
        • Louhivuori V.
        • Kärkkäinen V.
        • Koistinaho J.
        • et al.
        Metabotropic glutamate receptor 5 responses dictate differentiation of neural progenitors to NMDA-responsive cells in fragile X syndrome.
        Dev Neurobiol. 2017; 77: 438-453
        • Li W.
        • Sun W.
        • Zhang Y.
        • Wei W.
        • Ambasudhan R.
        • Xia P.
        • et al.
        Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors.
        Proc Natl Acad Sci U S A. 2011; 108: 8299-8304
        • Brennand K.J.
        • Simone A.
        • Jou J.
        • Gelboin-Burkhart C.
        • Tran N.
        • Sangar S.
        • et al.
        Modelling schizophrenia using human induced pluripotent stem cells.
        Nature. 2011; 473: 221-225
        • Ooi J.
        • Langley S.R.
        • Xu X.
        • Utami K.H.
        • Sim B.
        • Huang Y.
        • et al.
        Unbiased profiling of isogenic Huntington disease hPSC-derived CNS and peripheral cells reveals strong cell-type specificity of CAG length effects.
        Cell Rep. 2019; 26: 2494-2508.e7
        • Geyer P.E.
        • Kulak N.A.
        • Pichler G.
        • Holdt L.M.
        • Teupser D.
        • Mann M.
        Plasma proteome profiling to assess human health and disease.
        Cell Systems. 2016; 2: 185-195
        • Kulak N.A.
        • Pichler G.
        • Paron I.
        • Nagaraj N.
        • Mann M.
        Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells.
        Nat Methods. 2014; 11: 319-324
        • Eiges R.
        • Urbach A.
        • Malcov M.
        • Frumkin T.
        • Schwartz T.
        • Amit A.
        • et al.
        Developmental study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos.
        Cell Stem Cell. 2007; 1: 568-577
        • Colak D.
        • Zaninovic N.
        • Cohen M.S.
        • Rosenwaks Z.
        • Yang W.-Y.
        • Gerhardt J.
        • et al.
        Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome.
        Science. 2014; 343: 1002-1005
        • Avitzour M.
        • Mor-Shaked H.
        • Yanovsky-Dagan S.
        • Aharoni S.
        • Altarescu G.
        • Renbaum P.
        • et al.
        FMR1 epigenetic silencing commonly occurs in undifferentiated fragile X-affected embryonic stem cells.
        Stem Cell Rep. 2014; 3: 699-706
        • Boland M.J.
        • Nazor K.L.
        • Tran H.T.
        • Szücs A.
        • Lynch C.L.
        • Paredes R.
        • et al.
        Molecular analyses of neurogenic defects in a human pluripotent stem cell model of fragile X syndrome.
        Brain. 2017; 140: 582-598
        • Abrahams B.S.
        • Arking D.E.
        • Campbell D.B.
        • Mefford H.C.
        • Morrow E.M.
        • Weiss L.A.
        • et al.
        SFARI Gene 2.0: A community-driven knowledgebase for the autism spectrum disorders (ASDs).
        Mol Autism. 2013; 4: 36
        • Zerbi V.
        • Ielacqua G.D.
        • Markicevic M.
        • Haberl M.G.
        • Ellisman M.H.
        • A-Bhaskaran A
        • et al.
        Dysfunctional autism risk genes cause circuit-specific connectivity deficits with distinct developmental trajectories.
        Cereb Cortex. 2018; 28: 2495-2506
        • Brumback A.C.
        • Ellwood I.T.
        • Kjaerby C.
        • Iafrati J.
        • Robinson S.
        • Lee A.T.
        • et al.
        Identifying specific prefrontal neurons that contribute to autism-associated abnormalities in physiology and social behavior.
        Mol Psychiatry. 2018; 23: 2078-2089
        • Willemsen M.H.
        • Ba W.
        • Wissink-Lindhout W.M.
        • de Brouwer A.P.M.
        • Haas S.A.
        • Bienek M.
        • et al.
        Involvement of the kinesin family members KIF4A and KIF5C in intellectual disability and synaptic function.
        J Med Genet. 2014; 51: 487-494
        • Dunn R.
        • Dudbridge F.
        • Sanderson C.M.
        The use of edge-betweenness clustering to investigate biological function in protein interaction networks.
        BMC Bioinformatics. 2005; 6: 39
        • Houlden H.
        • Singleton A.B.
        The genetics and neuropathology of Parkinson’s disease.
        Acta Neuropathol. 2012; 124: 325-338
        • Loy C.T.
        • Schofield P.R.
        • Turner A.M.
        • Kwok J.B.J.
        Genetics of dementia.
        Lancet. 2014; 383: 828-840
        • Peñagarikano O.
        • Abrahams B.S.
        • Herman E.I.
        • Winden K.D.
        • Gdalyahu A.
        • Dong H.
        • et al.
        Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities, and core autism-related deficits.
        Cell. 2011; 147: 235-246
        • Maglorius Renkilaraj M.R.L.
        • Baudouin L.
        • Wells C.M.
        • Doulazmi M.
        • Wehrlé R.
        • Cannaya V.
        • et al.
        The intellectual disability protein PAK3 regulates oligodendrocyte precursor cell differentiation.
        Neurobiol Dis. 2017; 98: 137-148
        • Craddock N.
        • Owen M.J.
        • O’Donovan M.C.
        The catechol-O-methyl transferase (COMT) gene as a candidate for psychiatric phenotypes: Evidence and lessons.
        Mol Psychiatry. 2006; 11: 446-458
        • Xu X.
        • Tay Y.
        • Sim B.
        • Yoon S.-I.
        • Huang Y.
        • Ooi J.
        • et al.
        Reversal of phenotypic abnormalities by CRISPR/Cas9-mediated gene correction in Huntington disease patient-derived induced pluripotent stem cells.
        Stem Cell Rep. 2017; 8: 619-633
        • Kathuria A.
        • Nowosiad P.
        • Jagasia R.
        • Aigner S.
        • Taylor R.D.
        • Andreae L.C.
        • et al.
        Stem cell-derived neurons from autistic individuals with SHANK3 mutation show morphogenetic abnormalities during early development.
        Mol Psychiatry. 2018; 23: 735-746
        • Obien M.E.J.
        • Deligkaris K.
        • Bullmann T.
        • Bakkum D.J.
        • Frey U.
        Revealing neuronal function through microelectrode array recordings.
        Front Neurosci. 2014; 8: 423
        • Sandoe J.
        • Eggan K.
        Opportunities and challenges of pluripotent stem cell neurodegenerative disease models.
        Nat Neurosci. 2013; 16: 780-789
        • Bhattacharyya A.
        • McMillan E.
        • Wallace K.
        • Tubon Jr., T.C.
        • Capowski E.E.
        • Svendsen C.N.
        Normal neurogenesis but abnormal gene expression in human fragile X cortical progenitor cells.
        Stem Cells Dev. 2008; 17: 107-118
        • Castrén M.
        • Tervonen T.
        • Kärkkäinen V.
        • Heinonen S.
        • Castrén E.
        • Larsson K.
        • et al.
        Altered differentiation of neural stem cells in fragile X syndrome.
        Proc Natl Acad Sci U S A. 2005; 102: 17834-17839
        • Antar L.N.
        • Li C.
        • Zhang H.
        • Carroll R.C.
        • Bassell G.J.
        Local functions for FMRP in axon growth cone motility and activity-dependent regulation of filopodia and spine synapses.
        Mol Cell Neurosci. 2006; 32: 37-48
        • Ferron L.
        Fragile X mental retardation protein controls ion channel expression and activity.
        J Physiol. 2016; 594: 5861-5867
        • Brager D.H.
        • Akhavan A.R.
        • Johnston D.
        Impaired dendritic expression and plasticity of h-channels in the fmr1(-/y) mouse model of fragile X syndrome.
        Cell Rep. 2012; 1: 225-233
        • Zhang Y.
        • Brown M.R.
        • Hyland C.
        • Chen Y.
        • Kronengold J.
        • Fleming M.R.
        • et al.
        Regulation of neuronal excitability by interaction of fragile X mental retardation protein with slack potassium channels.
        J Neurosci. 2012; 32: 15318-15327
        • Routh B.N.
        • Johnston D.
        • Brager D.H.
        Loss of functional A-type potassium channels in the dendrites of CA1 pyramidal neurons from a mouse model of fragile X syndrome.
        J Neurosci. 2013; 33: 19442-19450
        • Kalmbach B.E.
        • Johnston D.
        • Brager D.H.
        Cell-type specific channelopathies in the prefrontal cortex of the fmr1-/y mouse model of fragile X syndrome.
        eNeuro. 2015; 2 (ENEURO.0114-15.2015)
        • Deng P.-Y.
        • Klyachko V.A.
        Increased persistent sodium current causes neuronal hyperexcitability in the entorhinal cortex of Fmr1 knockout mice.
        Cell Rep. 2016; 16: 3157-3166
        • Deng P.-Y.
        • Carlin D.
        • Oh Y.M.
        • Myrick L.K.
        • Warren S.T.
        • Cavalli V.
        • Klyachko V.A.
        Voltage-independent SK-channel dysfunction causes neuronal hyperexcitability in the hippocampus of Fmr1 knock-out mice.
        J Neurosci. 2019; 39: 28-43
        • Wilson P.G.
        • Stice S.S.
        Development and differentiation of neural rosettes derived from human embryonic stem cells.
        Stem Cell Rev. 2006; 2: 67-77
        • Elkabetz Y.
        • Panagiotakos G.
        • Al Shamy G.
        • Socci N.D.
        • Tabar V.
        • Studer L.
        Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage.
        Genes Dev. 2008; 22: 152-165
        • Luo Y.
        • Shan G.
        • Guo W.
        • Smrt R.D.
        • Johnson E.B.
        • Li X.
        • et al.
        Fragile X mental retardation protein regulates proliferation and differentiation of adult neural stem/progenitor cells.
        PLoS Genet. 2010; 6e1000898
        • Callan M.A.
        • Cabernard C.
        • Heck J.
        • Luois S.
        • Doe C.Q.
        • Zarnescu D.C.
        Fragile X protein controls neural stem cell proliferation in the Drosophila brain.
        . 2010; 19: 3068-3079

      Linked Article

      • Using an Isogenic Human Pluripotent Stem Cell Model for Better Understanding Neurodevelopmental Defects in Fragile X Syndrome
        Biological PsychiatryVol. 88Issue 6
        • Preview
          FMR1 has a dynamic trinucleotide CGG repeat element in the promotor region, and when the CGG repeat expands to a repeat length above 200 during maternal transmission, FMR1 becomes methylated and its gene and protein expressions are silenced. Deficiency of the FMR1 protein product FMRP causes fragile X syndrome (FXS), the most commonly inherited form of intellectual disability and autism spectrum disorder. FXS is associated with a wide spectrum of comorbidities, including seizures, sensory hypersensitivity, hyperactivity, impulsivity, anxiety, and impaired learning (1).
        • Full-Text
        • PDF