Advertisement

Modeling the Interplay Between Neurons and Astrocytes in Autism Using Human Induced Pluripotent Stem Cells

  • Author Footnotes
    1 FBR and BCF contributed equally to this work. ARM and PCBBB contributed equally to this work.
    Fabiele Baldino Russo
    Footnotes
    1 FBR and BCF contributed equally to this work. ARM and PCBBB contributed equally to this work.
    Affiliations
    Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil

    Department of Surgery, School of Veterinary Medicine, University of São Paulo, São Paulo, São Paulo, Brazil
    Search for articles by this author
  • Author Footnotes
    1 FBR and BCF contributed equally to this work. ARM and PCBBB contributed equally to this work.
    Beatriz Camille Freitas
    Footnotes
    1 FBR and BCF contributed equally to this work. ARM and PCBBB contributed equally to this work.
    Affiliations
    Department of Pediatrics, Rady Children’s Hospital San Diego, La Jolla, California

    Department of Cellular and Molecular Medicine, Stem Cell Program, University of California San Diego School of Medicine, Sanford Consortium for Regenerative Medicine, La Jolla, California
    Search for articles by this author
  • Graciela Conceição Pignatari
    Affiliations
    Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil
    Search for articles by this author
  • Isabella Rodrigues Fernandes
    Affiliations
    Department of Surgery, School of Veterinary Medicine, University of São Paulo, São Paulo, São Paulo, Brazil

    Department of Pediatrics, Rady Children’s Hospital San Diego, La Jolla, California

    Department of Cellular and Molecular Medicine, Stem Cell Program, University of California San Diego School of Medicine, Sanford Consortium for Regenerative Medicine, La Jolla, California
    Search for articles by this author
  • Jonathan Sebat
    Affiliations
    Department of Psychiatry, Cellular and Molecular Medicine, University of California San Diego, La Jolla, California
    Search for articles by this author
  • Alysson Renato Muotri
    Affiliations
    Department of Pediatrics, Rady Children’s Hospital San Diego, La Jolla, California

    Department of Cellular and Molecular Medicine, Stem Cell Program, University of California San Diego School of Medicine, Sanford Consortium for Regenerative Medicine, La Jolla, California
    Search for articles by this author
  • Patricia Cristina Baleeiro Beltrão-Braga
    Correspondence
    Address correspondence to Patricia Cristina Baleeiro Beltrão-Braga, Ph.D., Av Prof Lineu Prestes, 1374, room 227, Cidade Universitária, São Paulo, São Paulo 05508-900, Brazil.
    Affiliations
    Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil

    Department of Surgery, School of Veterinary Medicine, University of São Paulo, São Paulo, São Paulo, Brazil

    Department of Obstetrics, School of Arts, Sciences and Humanities, University of São Paulo, São Paulo, São Paulo, Brazil
    Search for articles by this author
  • Author Footnotes
    1 FBR and BCF contributed equally to this work. ARM and PCBBB contributed equally to this work.
Published:November 09, 2017DOI:https://doi.org/10.1016/j.biopsych.2017.09.021

      Abstract

      Background

      Autism spectrum disorder (ASD) is a neurodevelopmental disorder with unclear etiology and imprecise genetic causes. The main goal of this work was to investigate neuronal connectivity and the interplay between neurons and astrocytes from individuals with nonsyndromic ASD using induced pluripotent stem cells.

      Methods

      Induced pluripotent stem cells were derived from a clinically well-characterized cohort of three individuals with nonsyndromic ASD sharing common behaviors and three control subjects, two clones each. We generated mixed neural cultures analyzing synaptogenesis and neuronal activity using a multielectrode array platform. Furthermore, using an enriched astrocyte population, we investigated their role in neuronal maintenance.

      Results

      ASD-derived neurons had a significant decrease in synaptic gene expression and protein levels, glutamate neurotransmitter release, and, consequently, reduced spontaneous firing rate. Based on co-culture experiments, we observed that ASD-derived astrocytes interfered with proper neuronal development. In contrast, control-derived astrocytes rescued the morphological neuronal phenotype and synaptogenesis defects from ASD neuronal co-cultures. Furthermore, after identifying interleukin-6 secretion from astrocytes in individuals with ASD as a possible culprit for neural defects, we were able to increase synaptogenesis by blocking interleukin-6 levels.

      Conclusions

      Our findings reveal the contribution of astrocytes to neuronal phenotype and confirm previous studies linking interleukin-6 and autism, suggesting potential novel therapeutic pathways for a subtype of individuals with ASD. This is the first report demonstrating that glial dysfunctions could contribute to nonsyndromic autism pathophysiology using induced pluripotent stem cells modeling disease technology.

      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

        • Quaak I.
        • Brouns M.R.
        • van de Bor M.
        The dynamics of autism spectrum disorders: How neurotoxic compounds and neurotransmitters interact.
        Int J Environ Res Public Health. 2013; 10: 3384-3408
        • Wing L.
        • Gould J.
        Severe impairment of social interactions and associated abnormalities in children: Epidemiology and classification.
        J Autism Dev Disord. 1979; 9: 11-29
        • Rutter M.
        Incidence of autism spectrum disorders: Changes over time and their meaning.
        Acta Paediatr. 2005; 94: 2-15
        • Blumberg S.J.
        • Bramlett M.D.
        • Kogan M.D.
        • Schieve L.A.
        • Jones J.R.
        Changes in prevalence of parent-reported autism spectrum disorder in school-aged U.S. children: 2007 to 2011–2012.
        Natl Health Stat Report. 2013; : 1-11
        • De Rubeis S.
        • He X.
        • Goldberg A.P.
        • Poultney C.S.
        • Samocha K.
        • Cicek A.E.
        • et al.
        Synaptic, transcriptional and chromatin genes disrupted in autism.
        Nature. 2014; 515: 209-215
        • Griesi-Oliveira K.
        • Acab A.
        • Gupta A.R.
        • Sunaga D.Y.
        • Chailangkarn T.
        • Nicol X.
        • et al.
        Modeling non-syndromic autism and the impact of TRPC6 disruption in human neurons.
        Mol Psychiatry. 2015; 20: 1350-1365
        • Mefford H.
        • Batshaw M.L.
        • Hoffman E.P.
        Genomics, intellectual disability, and autism.
        N Engl J Med. 2012; 366: 733-743
        • Phillips M.
        • Pozzo-Miller L.
        Dendritic spine dysgenesis in autism related disorders.
        Neurosci Lett. 2015; 601: 30-40
        • Edmonson C.
        • Ziats M.N.
        • Rennert O.M.
        Altered glial marker expression in autistic post-mortem prefrontal cortex and cerebellum.
        Mol Autism. 2014; 5: 3
        • Marchetto M.C.
        • Carromeu C.
        • Acab A.
        • Yu D.
        • Yeo G.W.
        • Mu Y.
        • et al.
        A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells.
        Cell. 2010; 143: 527-539
        • 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
        • Shcheglovitov A.
        • Shcheglovitova O.
        • Yazawa M.
        • Portmann T.
        • Shu R.
        • Sebastiano V.
        • et al.
        SHANK3 and IGF1 restore synaptic deficits in neurons from 22q13 deletion syndrome patients.
        Nature. 2013; 503: 267-271
        • Li H.L.
        • Fujimoto N.
        • Sasakawa N.
        • Shirai S.
        • Ohkame T.
        • Sakuma T.
        • et al.
        Precise correction of the dystrophin gene in duchenne muscular dystrophy patient induced pluripotent stem cells by TALEN and CRISPR-Cas9.
        Stem Cell Rep. 2015; 4: 143-154
        • Russo F.B.
        • Cugola F.R.
        • Fernandes I.R.
        • Pignatari G.C.
        • Beltrão-Braga P.C.B.
        Induced pluripotent stem cells for modeling neurological disorders.
        World J Transplant. 2015; 5: 209-222
        • Beltrão-Braga P.C.B.
        • Pignatari G.C.
        • Russo F.B.
        • Fernandes I.R.
        • Muotri A.R.
        In-a-dish: Induced pluripotent stem cells as a novel model for human diseases.
        Cytometry A. 2013; 83A: 11-17
        • Beltrão-Braga P.C.
        • Muotri A.R.
        Modeling autism spectrum disorders with human neurons.
        Brain Res. 2017; 1656: 49-54
        • Marchetto M.C.
        • Belinson H.
        • Tian Y.
        • Freitas B.C.
        • Fu C.
        • Vadodaria K.C.
        • et al.
        Altered proliferation and networks in neural cells derived from non-syndromic autistic individuals.
        Mol Psychiatry. 2017; 22: 820-835
        • Mariani J.
        • Coppola G.
        • Zhang P.
        • Abyzov A.
        • Provini L.
        • Amenduni M.
        • et al.
        FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders.
        Cell. 2015; 162: 375-390
        • DeRosa B.A.
        • Van Baaren J.M.
        • Dubey G.K.
        • Lee J.M.
        • Cuccaro M.L.
        • Vance J.M.
        • et al.
        Derivation of autism spectrum disorder-specific induced pluripotent stem cells from peripheral blood mononuclear cells.
        Neurosci Lett. 2012; 516: 9-14
        • Li H.
        • Durbin R.
        Fast and accurate short read alignment with Burrows-Wheeler transform.
        Bioinformatics. 2009; 25: 1754-1760
        • Li H.
        • Handsaker B.
        • Wysoker A.
        • Fennell T.
        • Ruan J.
        • Homer N.
        • et al.
        The Sequence Alignment/Map format and SAMtools.
        Bioinformatics. 2009; 25: 2078-2079
        • McKenna A.
        • Hanna M.
        • Banks E.
        • Sivachenko A.
        • Cibulskis K.
        • Kernytsky A.
        • et al.
        The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data.
        Genome Res. 2010; 20: 1297-1303
        • DePristo M.A.
        • Banks E.
        • Poplin R.
        • Garimella K.V.
        • Maguire J.R.
        • Hartl C.
        • et al.
        A framework for variation discovery and genotyping using next-generation DNA sequencing data.
        Nat Genet. 2011; 43: 491-498
        • Michaelson J.J.
        • Shi Y.
        • Gujral M.
        • Zheng H.
        • Malhotra D.
        • Jin X.
        • et al.
        Whole-genome sequencing in autism identifies hot spots for de novo germline mutation.
        Cell. 2012; 151: 1431-1442
        • Narzisi G.
        • O’Rawe J.A.
        • Iossifov I.
        • Fang H.
        • Lee Y.-H.
        • Wang Z.
        • et al.
        Accurate de novo and transmitted indel detection in exome-capture data using microassembly.
        Nat Methods. 2014; 11: 1033-1036
        • Rimmer A.
        • Phan H.
        • Mathieson I.
        • Iqbal Z.
        • Twigg S.R.F.
        • WGS500 Consortium
        • et al.
        Integrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications.
        Nat Genet. 2014; 46: 912-918
        • Chailangkarn T.
        • Trujillo C.A.
        • Freitas B.C.
        • Hrvoj-Mihic B.
        • Herai R.H.
        • Yu D.X.
        • et al.
        A human neurodevelopmental model for Williams syndrome.
        Nature. 2016; 536: 338-343
        • Grozeva D.
        • Carss K.
        • Spasic-Boskovic O.
        • Parker M.J.
        • Archer H.
        • Firth H.V.
        • et al.
        De novo loss-of-function mutations in SETD5, encoding a methyltransferase in a 3p25 microdeletion syndrome critical region, cause intellectual disability.
        Am J Hum Genet. 2014; 94: 618-624
        • Kuechler A.
        • Zink A.M.
        • Wieland T.
        • Lüdecke H.-J.
        • Cremer K.
        • Salviati L.
        • et al.
        Loss-of-function variants of SETD5 cause intellectual disability and the core phenotype of microdeletion 3p25.3 syndrome.
        Eur J Hum Genet. 2015; 23: 753-760
        • Kellogg G.
        • Sum J.
        • Wallerstein R.
        Deletion of 3p25.3 in a patient with intellectual disability and dysmorphic features with further definition of a critical region.
        Am J Med Genet A. 2013; 161A: 1405-1408
        • Pinto D.
        • Delaby E.
        • Merico D.
        • Barbosa M.
        • Merikangas A.
        • Klei L.
        • et al.
        Convergence of genes and cellular pathways dysregulated in autism spectrum disorders.
        Am J Hum Genet. 2014; 94: 677-694
        • Szczałuba K.
        • Brzezinska M.
        • Kot J.
        • Rydzanicz M.
        • Płoski R.
        • Walczak A.
        • et al.
        SETD5 loss-of-function mutation as a likely cause of a familial syndromic intellectual disability with variable phenotypic expression.
        Am J Med Genet A. 2016; 170: 2322-2327
        • Chauhan A.
        • Chauhan V.
        Oxidative stress in autism.
        Pathophysiology. 2006; 13: 171-181
        • Maezawa I.
        • Jin L.W.
        Rett syndrome microglia damage dendrites and synapses by the elevated release of glutamate.
        J Neurosci. 2010; 30: 5346-5356
        • Deverman B.E.
        • Patterson P.H.
        Cytokines and CNS development.
        Neuron. 2009; 64: 61-78
        • Williams E.C.
        • Zhong X.
        • Mohamed A.
        • Li R.
        • Liu Y.
        • Dong Q.
        • et al.
        Mutant astrocytes differentiated from Rett syndrome patients-specific iPSCs have adverse effects on wildtype neurons.
        Hum Mol Genet. 2014; 23: 2968-2980
        • Sajdel-Sulkowska E.M.
        • Xu M.
        • McGinnis W.
        • Koibuchi N.
        Brain region-specific changes in oxidative stress and neurotrophin levels in autism spectrum disorders (ASD).
        Cerebellum. 2011; 10: 43-48
        • Spooren W.
        • Lindemann L.
        • Ghosh A.
        • Santarelli L.
        Synapse dysfunction in autism: A molecular medicine approach to drug discovery in neurodevelopmental disorders.
        Trends Pharmacol Sci. 2012; 33: 669-684
        • Sanders S.J.
        • Murtha M.T.
        • Gupta A.R.
        • Murdoch J.D.
        • Raubeson M.J.
        • Willsey A.J.
        • et al.
        De novo mutations revealed by whole-exome sequencing are strongly associated with autism.
        Nature. 2012; 485: 237-241
        • Bemben M.A.
        • Nguyen Q.A.
        • Wang T.
        • Li Y.
        • Nicoll R.A.
        • Roche K.W.
        Autism-associated mutation inhibits protein kinase C-mediated neuroligin-4X enhancement of excitatory synapses.
        Proc Natl Acad Sci U S A. 2015; 112: 2551-2556
        • Chen J.
        • Lin M.
        • Foxe J.J.
        • Pedrosa E.
        • Hrabovsky A.
        • Carroll R.
        • et al.
        Transcriptome comparison of human neurons generated using induced pluripotent stem cells derived from dental pulp and skin fibroblasts.
        PLoS One. 2013; 8: e75682
        • Beltrão-Braga P.C.
        • Pignatari G.C.
        • Maiorka P.C.
        • Oliveira N.A.J.
        • Lizier N.F.
        • Wenceslau C.V.
        • et al.
        Feeder-free derivation of induced pluripotent stem cells from human immature dental pulp stem cells.
        Cell Transplant. 2011; 20: 1707-1719
        • Ullian E.M.
        • Christopherson K.S.
        • Barres B.A.
        Role for glia in synaptogenesis.
        Glia. 2004; 47: 209-216
        • Christopherson K.S.
        • Ullian E.M.
        • Stokes C.C.A.
        • Mullowney C.E.
        • Hell J.W.
        • Agah A.
        • et al.
        Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis.
        Cell. 2005; 120: 421-433
        • Johnson M.A.
        • Weick J.P.
        • Pearce R.A.
        • Zhang S.C.
        Functional neural development from human embryonic stem cells: accelerated synaptic activity via astrocyte coculture.
        J Neurosci. 2007; 27: 3069-3077
        • Eroglu C.
        • Barres B.A.
        Regulation of synaptic connectivity by glia.
        Nature. 2010; 468: 223-231
        • Zhang Y.
        • Barres B.A.
        Astrocyte heterogeneity: An underappreciated topic in neurobiology.
        Curr Opin Neurobiol. 2010; 20: 588-594
        • Colangelo A.M.
        • Alberghina L.
        • Papa M.
        Astrogliosis as a therapeutic target for neurodegenerative diseases.
        Neurosci Lett. 2014; 565: 59-64
        • Pekny M.
        • Wilhelmsson U.
        • Pekna M.
        The dual role of astrocyte activation and reactive gliosis.
        Neurosci Lett. 2014; 565: 30-38
        • Yang C.J.
        • Liu C.L.
        • Sang B.
        • Zhu X.M.
        • Du Y.J.
        The combined role of serotonin and interleukin-6 as biomarker for autism.
        Neuroscience. 2015; 284: 290-296
        • Emanuele E.
        • Orsi P.
        • Boso M.
        • Broglia D.
        • Brondino N.
        • Barale F.
        • et al.
        Low-grade endotoxemia in patients with severe autism.
        Neurosci Lett. 2010; 471: 162-165
        • Ashwood P.
        • Enstrom A.
        • Krakowiak P.
        • Hertz-Picciotto I.
        • Hansen R.L.
        • Croen L.A.
        • et al.
        Decreased transforming growth factor beta1 in autism: A potential link between immune dysregulation and impairment in clinical behavioral outcomes.
        J Neuroimmunol. 2008; 204: 149-153
        • Malik M.
        • Sheikh A.M.
        • Wen G.
        • Spivack W.
        • Brown W.T.
        • Li X.
        Expression of inflammatory cytokines, Bcl2 and cathepsin D are altered in lymphoblasts of autistic subjects.
        Immunobiology. 2011; 216: 80-85
        • Li X.
        • Chauhan A.
        • Sheikh A.M.
        • Patil S.
        • Chauhan V.
        • Li X.M.
        • et al.
        Elevated immune response in the brain of autistic patients.
        J Neuroimmunol. 2009; 207: 111-116
        • Wei H.
        • Zou H.
        • Sheikh A.M.
        • Malik M.
        • Dobkin C.
        • Brown W.T.
        • Li X.
        IL-6 is increased in the cerebellum of autistic brain and alters neural cell adhesion, migration and synaptic formation.
        J Neuroinflammation. 2011; 8: 52
        • Vargas D.L.
        • Nascimbene C.
        • Krishnan C.
        • Zimmerman A.W.
        • Pardo C.A.
        Neuroglial activation and neuroinflammation in the brain of patients with autism.
        Ann Neurol. 2005; 57: 67-81
        • Wei H.
        • Alberts I.
        • Li X.
        Brain IL-6 and autism.
        Neuroscience. 2013; 252: 320-325
        • Parenti I.
        • Teresa-Rodrigo M.E.
        • Pozojevic J.
        • Ruiz Gil S.
        • Bader I.
        • Braunholz D.
        • et al.
        Mutations in chromatin regulators functionally link Cornelia de Lange syndrome and clinically overlapping phenotypes.
        Hum Genet. 2017; 136: 307-320
        • Beauchemin P.
        • Carruthers R.
        MS arising during Tocilizumab therapy for rheumatoid arthritis.
        Mult Scler. 2016; 22: 254-256
        • Lehtimäki K.A.
        • Liimatainen S.
        • Peltola J.
        • Arvio M.
        The serum level of interleukin-6 in patients with intellectual disability and refractory epilepsy.
        Epilepsy Res. 2011; 95: 184-187