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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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).
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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The authors declare that they have no conflict of interest.
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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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