Abstract
Accumulating evidence suggests that cerebellar dysfunction early in life is associated with autism spectrum disorder (ASD), but the molecular mechanisms underlying the cerebellar deficits at the cellular level are unclear. Tuberous sclerosis complex (TSC) is a neurocutaneous disorder that often presents with ASD. Here, we developed a cerebellar Purkinje cell (PC) model of TSC with patient-derived human induced pluripotent stem cells (hiPSCs) to characterize the molecular mechanisms underlying cerebellar abnormalities in ASD and TSC. Our results show that hiPSC-derived PCs from patients with pathogenic TSC2 mutations displayed mTORC1 pathway hyperactivation, defects in neuronal differentiation and RNA regulation, hypoexcitability and reduced synaptic activity when compared with those derived from controls. Our gene expression analyses revealed downregulation of several components of fragile X mental retardation protein (FMRP) targets in TSC2-deficient hiPSC-PCs. We detected decreased expression of FMRP, glutamate receptor δ2 (GRID2), and pre- and post-synaptic markers such as synaptophysin and PSD95 in the TSC2-deficient hiPSC-PCs. The mTOR inhibitor rapamycin rescued the deficits in differentiation, synaptic dysfunction, and hypoexcitability of TSC2 mutant hiPSC-PCs in vitro. Our findings suggest that these gene expression changes and cellular abnormalities contribute to aberrant PC function during development in TSC affected individuals.
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References
Casanova MF. The neuropathology of autism. Brain Pathol. 2007;17:422–33.
Whitney ER, Kemper TL, Bauman ML, Rosene DL, Blatt GJ. Cerebellar Purkinje cells are reduced in a subpopulation of autistic brains: a stereological experiment using calbindin-D28k. Cerebellum. 2008;7:406–16.
Limperopoulos C, Bassan H, Gauvreau K, Robertson RL Jr., Sullivan NR, Benson CB, et al. Does cerebellar injury in premature infants contribute to the high prevalence of long-term cognitive, learning, and behavioral disability in survivors? Pediatrics. 2007;120:584–93.
Lipton JO, Sahin M. The neurology of mTOR. Neuron. 2014;84:275–91.
Crino PB, Nathanson KL, Henske EP. The tuberous sclerosis complex. N Engl J Med. 2006;355:1345–56.
Dibble CC, Elis W, Menon S, Qin W, Klekota J, Asara JM, et al. TBC1D7 is a third subunit of the TSC1-TSC2 complex upstream of mTORC1. Mol Cell. 2012;47:535–46.
Inoki K, Li Y, Xu T, Guan KL. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 2003;17:1829–34.
Eluvathingal TJ, Behen ME, Chugani HT, Janisse J, Bernardi B, Chakraborty P, et al. Cerebellar lesions in tuberous sclerosis complex: neurobehavioral and neuroimaging correlates. J Child Neurol. 2006;21:846–51.
Weber AM, Egelhoff JC, McKellop JM, Franz DN. Autism and the cerebellum: evidence from tuberous sclerosis. J Autism Dev Disord. 2000;30:511–7.
Asano E, Chugani DC, Muzik O, Behen M, Janisse J, Rothermel R, et al. Autism in tuberous sclerosis complex is related to both cortical and subcortical dysfunction. Neurology. 2001;57:1269–77.
Ertan G, Arulrajah S, Tekes A, Jordan L, Huisman TA. Cerebellar abnormality in children and young adults with tuberous sclerosis complex: MR and diffusion weighted imaging findings. J Neuroradiol. 2010;37:231–8.
Tsai PT, Hull C, Chu Y, Greene-Colozzi E, Sadowski AR, Leech JM, et al. Autistic-like behaviour and cerebellar dysfunction in Purkinje cell Tsc1 mutant mice. Nature. 2012;488:647–51.
Reith RM, McKenna J, Wu H, Hashmi SS, Cho SH, Dash PK, et al. Loss of Tsc2 in Purkinje cells is associated with autistic-like behavior in a mouse model of tuberous sclerosis complex. Neurobiol Dis. 2013;51:93–103.
Schlaeger TM, Daheron L, Brickler TR, Entwisle S, Chan K, Cianci A, et al. A comparison of non-integrating reprogramming methods. Nat Biotechnol. 2015;33:58–63.
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–72.
Mussolino C, Morbitzer R, Lutge F, Dannemann N, Lahaye T, Cathomen T. A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity. Nucleic Acids Res. 2011;39:9283–93.
Horii T, Tamura D, Morita S, Kimura M, Hatada I. Generation of an ICF syndrome model by efficient genome editing of human induced pluripotent stem cells using the CRISPR system. Int J Mol Sci. 2013;14:19774–81.
Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci USA. 2013;110:15644–9.
Guo Q, Li K, Sunmonu NA, Li JY. Fgf8b-containing spliceforms, but not Fgf8a, are essential for Fgf8 function during development of the midbrain and cerebellum. Dev Biol. 2010;338:183–92.
Selvadurai HJ, Mason JO. Wnt/beta-catenin signalling is active in a highly dynamic pattern during development of the mouse cerebellum. PLoS ONE. 2011;6:e23012.
Sundberg M, Bogetofte H, Lawson T, Jansson J, Smith G, Astradsson A, et al. Improved cell therapy protocols for Parkinson’s disease based on differentiation efficiency and safety of hESC-, hiPSC-, and non-human primate iPSC-derived dopaminergic neurons. Stem Cells. 2013;31:1548–62.
Buchholz DE, Pennington BO, Croze RH, Hinman CR, Coffey PJ, Clegg DO. Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium. Stem Cells Transl Med. 2013;2:384–93.
Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;27:275–80.
Muguruma K, Nishiyama A, Kawakami H, Hashimoto K, Sasai Y. Self-organization of polarized cerebellar tissue in 3D culture of human pluripotent stem cells. Cell Rep. 2015;10:537–50.
Heuer H, Mason CA. Thyroid hormone induces cerebellar Purkinje cell dendritic development via the thyroid hormone receptor alpha1. J Neurosci. 2003;23:10604–12.
Wang S, Wang B, Pan N, Fu L, Wang C, Song G, et al. Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons. Sci Rep. 2015;5:9232.
Crino PB, Aronica E, Baltuch G, Nathanson KL. Biallelic TSC gene inactivation in tuberous sclerosis complex. Neurology. 2010;74:1716–23.
Costa V, Aigner S, Vukcevic M, Sauter E, Behr K, Ebeling M, et al. mTORC1 inhibition corrects neurodevelopmental and synaptic alterations in a human stem cell model of tuberous sclerosis. Cell Rep. 2016;15:86–95.
Huang J, Manning BD. The TSC1-TSC2 complex: a molecular switchboard controlling cell growth. Biochem J. 2008;412:179–90.
Brennand KJ, Simone A, Jou J, Gelboin-Burkhart C, Tran N, Sangar S, et al. Modelling schizophrenia using human induced pluripotent stem cells. Nature. 2011;473:221–5.
Grabole N, Zhang JD, Aigner S, Ruderisch N, Costa V, Weber FC, et al. Genomic analysis of the molecular neuropathology of tuberous sclerosis using a human stem cell model. Genome Med. 2016;8:94.
Ascano M Jr, Mukherjee N, Bandaru P, Miller JB, Nusbaum JD, Corcoran DL, et al. FMRP targets distinct mRNA sequence elements to regulate protein expression. Nature. 2012;492:382–6.
Darnell JC, Van Driesche SJ, Zhang C, Hung KY, Mele A, Fraser CE, et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell. 2011;146:247–61.
Cloetta D, Thomanetz V, Baranek C, Lustenberger RM, Lin S, Oliveri F, et al. Inactivation of mTORC1 in the developing brain causes microcephaly and affects gliogenesis. J Neurosci. 2013;33:7799–810.
Hagan N, Guarente J, Ellisor D, Zervas M. The temporal contribution of the Gbx2 lineage to cerebellar neurons. Front Neuroanat. 2017;11:50.
Kassai H, Sugaya Y, Noda S, Nakao K, Maeda T, Kano M, et al. Selective activation of mTORC1 signaling recapitulates microcephaly, tuberous sclerosis, and neurodegenerative diseases. Cell Rep. 2014;7:1626–39.
Magri L, Cambiaghi M, Cominelli M, Alfaro-Cervello C, Cursi M, Pala M, et al. Sustained activation of mTOR pathway in embryonic neural stem cells leads to development of tuberous sclerosis complex-associated lesions. Cell Stem Cell. 2011;9:447–62.
Normand EA, Crandall SR, Thorn CA, Murphy EM, Voelcker B, Browning C, et al. Temporal and mosaic Tsc1 deletion in the developing thalamus disrupts thalamocortical circuitry, neural function, and behavior. Neuron. 2013;78:895–909.
Grajkowska W, Kotulska K, Jurkiewicz E, Matyja E. Brain lesions in tuberous sclerosis complex. Review. Folia Neuropathol. 2010;48:139–49.
Jay V, Edwards V, Musharbash A, Rutka JT. Cerebellar pathology in tuberous sclerosis. Ultrastruct Pathol. 1998;22:331–9.
Way SW, McKenna J 3rd, Mietzsch U, Reith RM, Wu HC, Gambello MJ. Loss of Tsc2 in radial glia models the brain pathology of tuberous sclerosis complex in the mouse. Hum Mol Genet. 2009;18:1252–65.
Mietzsch U, McKenna J 3rd, Reith RM, Way SW, Gambello MJ. Comparative analysis of Tsc1 and Tsc2 single and double radial glial cell mutants. J Comp Neurol. 2013;521:3817–31.
Carson RP, Kelm ND, West KL, Does MD, Fu C, Weaver G, et al. Hypomyelination following deletion of Tsc2 in oligodendrocyte precursors. Ann Clin Transl Neurol. 2015;2:1041–54.
Lebrun-Julien F, Bachmann L, Norrmen C, Trotzmuller M, Kofeler H, Ruegg MA, et al. Balanced mTORC1 activity in oligodendrocytes is required for accurate CNS myelination. J Neurosci. 2014;34:8432–48.
Ercan E, Han JM, Di Nardo A, Winden K, Han MJ, Hoyo L, et al. Neuronal CTGF/CCN2 negatively regulates myelination in a mouse model of tuberous sclerosis complex. J Exp Med. 2017;214:681–97.
Meikle L, Talos DM, Onda H, Pollizzi K, Rotenberg A, Sahin M, et al. A mouse model of tuberous sclerosis: neuronal loss of Tsc1 causes dysplastic and ectopic neurons, reduced myelination, seizure activity, and limited survival. J Neurosci. 2007;27:5546–58.
Makki MI, Chugani DC, Janisse J, Chugani HT. Characteristics of abnormal diffusivity in normal-appearing white matter investigated with diffusion tensor MR imaging in tuberous sclerosis complex. Am J Neuroradiol. 2007;28:1662–7.
Arulrajah S, Ertan G, Jordan L, Tekes A, Khaykin E, Izbudak I, et al. Magnetic resonance imaging and diffusion-weighted imaging of normal-appearing white matter in children and young adults with tuberous sclerosis complex. Neuroradiology. 2009;51:781–6.
Peters JM, Sahin M, Vogel-Farley VK, Jeste SS, Nelson CA 3rd, Gregas MC, et al. Loss of white matter microstructural integrity is associated with adverse neurological outcome in tuberous sclerosis complex. Acad Radiol. 2012;19:17–25.
Douvaras P, Wang J, Zimmer M, Hanchuk S, O’Bara MA, Sadiq S, et al. Efficient generation of myelinating oligodendrocytes from primary progressive multiple sclerosis patients by induced pluripotent stem cells. Stem Cell Rep. 2014;3:250–9.
Wang S, Bates J, Li X, Schanz S, Chandler-Militello D, Levine C, et al. Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell. 2013;12:252–64.
Stacpoole SR, Spitzer S, Bilican B, Compston A, Karadottir R, Chandran S, et al. High yields of oligodendrocyte lineage cells from human embryonic stem cells at physiological oxygen tensions for evaluation of translational biology. Stem Cell Rep. 2013;1:437–50.
Sundberg M, Hyysalo A, Skottman H, Shin S, Vemuri M, Suuronen R, et al. A xeno-free culturing protocol for pluripotent stem cell-derived oligodendrocyte precursor cell production. Regen Med. 2011;6:449–60.
Nistor GI, Totoiu MO, Haque N, Carpenter MK, Keirstead HS. Human embryonic stem cells differentiate into oligodendrocytes in high purity and myelinate after spinal cord transplantation. Glia. 2005;49:385–96.
Bauman ML, Kemper TL. Neuroanatomic observations of the brain in autism: a review and future directions. Int J Dev Neurosci. 2005;23:183–7.
Bailey A, Luthert P, Dean A, Harding B, Janota I, Montgomery M, et al. A clinicopathological study of autism. Brain. 1998;121:889–905.
Vargas DL, Nascimbene C, Krishnan C, Zimmerman AW, Pardo CA. Neuroglial activation and neuroinflammation in the brain of patients with autism. Ann Neurol. 2005;57:67–81.
Weisenfeld NI, Peters JM, Tsai PT, Prabhu SP, Dies KA, Sahin M, et al. A magnetic resonance imaging study of cerebellar volume in tuberous sclerosis complex. Pediatr Neurol. 2013;48:105–10.
Boer K, Troost D, Jansen F, Nellist M, van den Ouweland AM, Geurts JJ, et al. Clinicopathological and immunohistochemical findings in an autopsy case of tuberous sclerosis complex. Neuropathology. 2008;28:577–90.
Crino PB. Molecular pathogenesis of tuber formation in tuberous sclerosis complex. J Child Neurol. 2004;19:716–25.
Miyata H, Chiang AC, Vinters HV. Insulin signaling pathways in cortical dysplasia and TSC-tubers: tissue microarray analysis. Ann Neurol. 2004;56:510–9.
Tavazoie SF, Alvarez VA, Ridenour DA, Kwiatkowski DJ, Sabatini BL. Regulation of neuronal morphology and function by the tumor suppressors Tsc1 and Tsc2. Nat Neurosci. 2005;8:1727–34.
Uhlmann EJ, Wong M, Baldwin RL, Bajenaru ML, Onda H, Kwiatkowski DJ, et al. Astrocyte-specific TSC1 conditional knockout mice exhibit abnormal neuronal organization and seizures. Ann Neurol. 2002;52:285–96.
Jaworski J, Spangler S, Seeburg DP, Hoogenraad CC, Sheng M. Control of dendritic arborization by the phosphoinositide-3′-kinase-Akt-mammalian target of rapamycin pathway. J Neurosci. 2005;25:11300–12.
Kumar V, Zhang MX, Swank MW, Kunz J, Wu GY. Regulation of dendritic morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK signaling pathways. J Neurosci. 2005;25:11288–99.
Mirzaa GM, Parry DA, Fry AE, Giamanco KA, Schwartzentruber J, Vanstone M, et al. De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome. Nat Genet. 2014;46:510–5.
Mirzaa G, Dodge NN, Glass I, Day C, Gripp K, Nicholson L, et al. Megalencephaly and perisylvian polymicrogyria with postaxial polydactyly and hydrocephalus: a rare brain malformation syndrome associated with mental retardation and seizures. Neuropediatrics. 2004;35:353–9.
Di Nardo A, Wertz MH, Kwiatkowski E, Tsai PT, Leech JD, Greene-Colozzi E, et al. Neuronal Tsc1/2 complex controls autophagy through AMPK-dependent regulation of ULK1. Hum Mol Genet. 2014;23:3865–74.
Ebrahimi-Fakhari D, Saffari A, Wahlster L, Di Nardo A, Turner D, Lewis TL Jr, et al. Impaired mitochondrial dynamics and mitophagy in neuronal models of tuberous sclerosis complex. Cell Rep. 2016;17:2162.
Adusei DC, Pacey LK, Chen D, Hampson DR. Early developmental alterations in GABAergic protein expression in fragile X knockout mice. Neuropharmacology. 2010;59:167–71.
Curia G, Papouin T, Seguela P, Avoli M. Downregulation of tonic GABAergic inhibition in a mouse model of fragile X syndrome. Cereb Cortex. 2009;19:1515–20.
Sabanov V, Braat S, D’Andrea L, Willemsen R, Zeidler S, Rooms L, et al. Impaired GABAergic inhibition in the hippocampus of Fmr1 knockout mice. Neuropharmacology. 2017;116:71–81.
Narayanan U, Nalavadi V, Nakamoto M, Thomas G, Ceman S, Bassell GJ, et al. S6K1 phosphorylates and regulates fragile X mental retardation protein (FMRP) with the neuronal protein synthesis-dependent mammalian target of rapamycin (mTOR) signaling cascade. J Biol Chem. 2008;283:18478–82.
Sarbassov DD, Ali SM, Sengupta S, Sheen JH, Hsu PP, Bagley AF, et al. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell. 2006;22:159–68.
Sundberg M, Sahin M. Cerebellar development and autism spectrum disorder in tuberous sclerosis complex. J Child Neurol. 2015;30:1954–62.
Acknowledgements
We thank the individuals affected by TSC who took part in this study. We are grateful to Dr. Kleiman, Dr. Chen, and Dr. Buttermore for helpful discussion, and Kerem Muslu for assistance in qRT-PCR analyses. We would like to thank research associate Christopher Stoddard (UConn Health, Human Genome Editing Core, US) for assistance with CRISPR-gene modification of hiPSCs. The hiPSC line F628 was provided by Dr. George Daley (Boston Children’s Hospital). We thank BCH IDDRC Cellular Imaging Core for help with confocal imaging (U54 HD090255). This study was funded by U.S. Army Medical Research Tuberous Sclerosis Complex Research Program (W81XWH-15-1-0189), Nancy Lurie Marks Family Foundation, Harvard Stem Cell Institute, and the Children’s Hospital Boston Translational Research Program (to MS). KW is supported by R25 NS07068207S1. The project was co-sponsored by the Iris and Jumming Le Foundation and the Rockefeller University Center for Clinical and Translational Science #UL1 TR000043 (NCATS, NIH), CTSA and NIH R21 NS093540-01 (to MEH).
Author contributions
MS: PC differentiation protocol development for hiPSC, experimental design, PC differentiation, phenotyping in vitro, FC, transcriptional profiling, data analyses, writing of manuscript. IT: electrophysiology, data analyses, writing of manuscript. DEB: PC differentiation protocol development for hESC. KW: transcriptional expression analyses, writing of manuscript. VK: confocal microscopy. KK: statistical analyses. DC: cell analyses. DT: technical assistance. M-JH: hiPSC line derivation, CJW and MEH: discussion of data. MS and MEH: experimental design, writing of manuscript.
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Sundberg, M., Tochitsky, I., Buchholz, D.E. et al. Purkinje cells derived from TSC patients display hypoexcitability and synaptic deficits associated with reduced FMRP levels and reversed by rapamycin. Mol Psychiatry 23, 2167–2183 (2018). https://doi.org/10.1038/s41380-018-0018-4
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DOI: https://doi.org/10.1038/s41380-018-0018-4
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