SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons

Research Articles: Neurobiology of Disease SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, and Network Activity in Developing Human Neurons https://doi.org/10.1523/JNEUROSCI.1367-20.2020 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1367-20.2020 Received: 31 May 2020 Revised: 26 July 2020 Accepted: 24 August 2020 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 SYNGAP1 Controls the Maturation of Dendrites, Synaptic Function, 2 and Network Activity in Developing Human Neurons 3 4 Nerea Llamosas1, Vineet Arora1, Ridhima Vij2,3,Murat Kilinc1,Lukasz Bijoch4, Camilo 5 Rojas1, Adrian Reich5, BanuPriya Sridharan6∞, Erik Willems7, David R. Piper7, Louis 6 Scampavia6, Timothy P. Spicer6, Courtney A. Miller1,6, J. Lloyd Holder Jr2,3, Gavin 7 Rumbaugh1* 8 9 1Department of Neuroscience, Scripps Research, Jupiter, Florida 33458, USA 10 2Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, Houston 11 Texas 77030, USA 12 3Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030, USA 13 4Laboratory of Neurobiology, BRAINCITY, Nencki Institute of Experimental Biology, Polish 14 Academy of Sciences, Warsaw, Poland 15 5Center for Computational Biology and Bioinformatics, Scripps Research, Jupiter, Florida 33458, 16 USA 17 6Department of Molecular Medicine, Scripps Research, Jupiter, Florida 33458, USA 18 7Cell Biology, Thermo Fisher Scientific, Carlsbad, California 92008, USA 19 ∞Current Address: GSK 1250 S Collegeville Rd., Collegeville, PA 19426, 20 21 *Corresponding Author: 22 Gavin Rumbaugh 23 Department of Neuroscience 24 130 Scripps Way #3B3 25 Jupiter, FL 33458 26 [email protected] 27 28 Abbreviated Title: SYNGAP1 function in human neurons 29 30 Figures = 7; Tables = 1; Abstract = 243; Introduction = 649; Discussion = 1428 31 Acknowledgments 32 Funding: This work was supported in part by NIH grants from the National Institute of Mental 33 Health (MH096847 and MH108408 to G.R.) and the National Institute for Neurological Disorders 34 and Stroke (NS064079 and NS110307 to G.R. and NS091381 to J.H.). N.L. was supported by a 35 generous postdoctoral training fellowship and J.H. by a grant from the SynGAP Research Fund 36 (SRF; https://syngapresearchfund.org). This work was supported by Grant 201763 from the 37 Doris Duke Charitable Foundation to J.H. V.A. was supported by a generous postdoctoral 38 training fellowship from Leon and Friends e.V (https://leonandfriends.org). L.B. was partially 39 supported by a travel grant for junior researchers from Boehringer Ingelheim Fonds (BIF). J.H. 40 also received generous support from the Robbins Foundation and Mr. Charif Souki. 41 Author Contributions: N.L. performed experiments, designed experiments, analyzed data, co- 42 wrote and edited the manuscript. V.A, R. V., M.K., L.B., C.R., A.R., C.H., B.S., and E.W. 43 performed experiments, analyzed data and interpreted data. G.R. conceived project, designed 44 experiments, interpreted data, and co-wrote the manuscript. D.R.P., L.S., T.P.S., C.A.M., and 45 J.L.H. designed and interpreted experiments and edited the manuscript. 46 Competing interests: E.W. and D.R.P. are employed by Thermo Fisher Scientific. The authors 47 declare no competing interests. 48 Data and materials availability: hiPSC clones and data supporting the findings of this study 49 are available from the corresponding author upon reasonable request. 1 50 Abstract 51 SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum 52 disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a 53 neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, 54 and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that 55 Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function 56 variants driving formation of larger dendritic spines and stronger glutamatergic transmission. 57 However, studies to date have been limited to mouse and rat neurons. Therefore, it remains 58 unknown how SYNGAP1 loss-of-function impacts the development and function of human 59 neurons. To address this, we employed CRISPR/Cas9 technology to ablate SYNGAP1 protein 60 expression in neurons derived from a commercially available induced pluripotent stem cell line 61 (hiPSC) obtained from a human female donor. Reducing SynGAP protein expression in 62 developing hiPSC-derived neurons enhanced dendritic morphogenesis, leading to larger 63 neurons compared to those derived from isogenic controls. Consistent with larger dendritic 64 fields, we also observed a greater number of morphologically defined excitatory synapses in 65 cultures containing these neurons. Moreover, neurons with reduced SynGAP protein had 66 stronger excitatory synapses and expressed synaptic activity earlier in development. Finally, 67 distributed network spiking activity appeared earlier, was substantially elevated, and exhibited 68 greater bursting behavior in SYNGAP1 null neurons. We conclude that SYNGAP1 regulates the 69 postmitotic maturation of human neurons made from hiPSCs, which influences how activity 70 develops within nascent neural networks. Alterations to this fundamental neurodevelopmental 71 process may contribute to the etiology of SYNGAP1-related disorders. 72 2 73 Significance Statement 74 SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum 75 disorder, and epileptic encephalopathy. While this gene is well studied in rodent neurons, its 76 function in human neurons remains unknown. We employed CRISPR/Cas9 technology to 77 disrupt SYNGAP1 protein expression in neurons derived from an induced pluripotent stem cell 78 line. We found that iNeurons lacking SynGAP expression exhibited accelerated dendritic 79 morphogenesis, increased accumulation of postsynaptic markers, early expression of synapse 80 activity, enhanced excitatory synaptic strength, and early onset of neural network activity. We 81 conclude that SYNGAP1 regulates the postmitotic differentiation rate of developing human 82 neurons and disrupting this process impacts the function of nascent neural networks. These 83 altered developmental processes may contribute to the etiology of SYNGAP1 disorders. 84 3 85 Introduction 86 Pathogenic loss-of-function variants in the SYNGAP1 gene are causally-linked to global 87 developmental delay (GDD)/intellectual disability (ID) (Hamdan et al., 2009; Rauch et al., 2012; 88 Deciphering Developmental Disorders, 2015, 2017) and severe epilepsy (Carvill et al., 2013; 89 von Stulpnagel et al., 2015; Vlaskamp et al., 2019). SYNGAP1 is also strongly implicated in 90 autism spectrum disorders (Rauch et al., 2012; O'Roak et al., 2014; Satterstrom et al., 2020). 91 While pathogenic variants in SYNAGP1 are overall rare, they are common relative to the pool of 92 genes capable of causing sporadic neurodevelopmental disorders, explaining up to ~1% of 93 GDD/ID cases (Berryer et al., 2013; Parker et al., 2015), which is in the range of other 94 monogenic disorders that are more extensively studied by the scientific community. Causality of 95 SYNGAP1 pathogenicity is now clear because of its high intolerance to loss-of-function 96 mutations. The constraint metric loss-of-function observed/expected upper bound fraction 97 (LOEUF) is 0.05 derived from the >141,000 individuals from the v.2.1.1 gnomAD database 98 (Karczewski et al., 2020), demonstrating its extreme loss-of-function mutation intolerance. 99 Moreover, the non-neuro dataset of exomes from >114,000 individuals from gnomAD reveals 100 only three frameshift variants with two of these lying in the extreme 5 or 3 prime regions of the 101 gene, an area known to undergo extensive alternative splicing. Based on substantial clinical 102 evidence, proper SYNGAP1 expression is required for normal human brain development and 103 function. 104 105 Syngap1 gene function has been studied in rodent neurons (Kilinc et al., 2018; Gamache et al., 106 2020), where it is a potent regulator of Hebbian plasticity at excitatory synapses. Heterozygous 107 knockout mice exhibit deficits in hippocampal LTP evoked through a variety of synaptic 108 stimulation protocols (Komiyama et al., 2002; Kim et al., 2003). Genetic re-expression of 109 Syngap1 in adult mutant mice rescues hippocampal LTP and associated downstream signaling 110 pathways (Ozkan et al., 2014). Thus, SynGAP regulation of synapse plasticity is a dynamic 4 111 function of the protein that is retained throughout life. Hundreds of genes regulate synaptic 112 plasticity as referenced by the Gene Ontology Browser 113 (http://www.informatics.jax.org/vocab/gene_ontology/GO:0048167). However, most of them do 114 not cause disease when heterozygously expressed, as is the case for SYNGAP1. Therefore, 115 SYNGAP1 likely has additional functions beyond regulation of synapse plasticity that contribute 116 to disease etiology. Indeed, SynGAP expression in developing mouse neurons acts to regulate 117 the maturation rate of excitatory synapse strength and this function is independent from its role 118 in plasticity. SynGAP protein expression rises quickly during

Load more