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Synaptic Dysfunction in Human Neurons with Autism-Associated Deletions in PTCHD1-AS

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Synaptic Dysfunction in Human Neurons with Autism-Associated Deletions in PTCHD1-AS Biological Archival Report Psychiatry Synaptic Dysfunction in Human Neurons With Autism-Associated Deletions in PTCHD1-AS P. Joel Ross, Wen-Bo Zhang, Rebecca S.F. Mok, Kirill Zaslavsky, Eric Deneault, Lia D’Abate, Deivid C. Rodrigues, Ryan K.C. Yuen, Muhammad Faheem, Marat Mufteev, Alina Piekna, Wei Wei, Peter Pasceri, Rebecca J. Landa, Andras Nagy, Balazs Varga, Michael W. Salter, Stephen W. Scherer, and James Ellis ABSTRACT BACKGROUND: The Xp22.11 locus that encompasses PTCHD1, DDX53, and the long noncoding RNA PTCHD1-AS is frequently disrupted in male subjects with autism spectrum disorder (ASD), but the functional consequences of these genetic risk factors for ASD are unknown. METHODS: To evaluate the functional consequences of PTCHD1 locus deletions, we generated induced pluripotent stem cells (iPSCs) from unaffected control subjects and 3 subjects with ASD with microdeletions affecting PTCHD1- AS/PTCHD1, PTCHD1-AS/DDX53,orPTCHD1-AS alone. Function of iPSC-derived cortical neurons was assessed using molecular approaches and electrophysiology. We also compiled novel and known genetic variants of the PTCHD1 locus to explore the roles of PTCHD1 and PTCHD1-AS in genetic risk for ASD and other neurodevelopmental disorders. Finally, genome editing was used to explore the functional consequences of deleting a single conserved exon of PTCHD1-AS. RESULTS: iPSC-derived neurons from subjects with ASD exhibited reduced miniature excitatory postsynaptic cur- rent frequency and N-methyl-D-aspartate receptor hypofunction. We found that 35 ASD-associated deletions mapping to the PTCHD1 locus disrupted exons of PTCHD1-AS. We also found a novel ASD-associated deletion of PTCHD1-AS exon 3 and showed that exon 3 loss altered PTCHD1-AS splicing without affecting expression of the neighboring PTCHD1 coding gene. Finally, targeted disruption of PTCHD1-AS exon 3 recapitulated diminished miniature excitatory postsynaptic current frequency, supporting a role for the long noncoding RNA in the etiology of ASD. CONCLUSIONS: Our genetic findings provide strong evidence that PTCHD1-AS deletions are risk factors for ASD, and human iPSC-derived neurons implicate these deletions in the neurophysiology of excitatory synapses and in ASD-associated synaptic impairment. Keywords: Autism spectrum disorder, Excitatory synapses, Genetics, Induced pluripotent stem cells, Long non- coding RNA, Neurons https://doi.org/10.1016/j.biopsych.2019.07.014 Autism spectrum disorder (ASD) is a common neuro- SHANK3 haploinsufficient neurons had impairments in developmental disorder that is characterized by impaired social dendrite complexity (10) and synaptic function (11). iPSC- interactions and repetitive, inflexible behaviors (1).Presentation derived neurons from subjects with ASD exhibited changes and severity of ASD features vary widely between individuals, in dendritic morphology and formed fewer synapses (12,13). suggesting etiological heterogeneity. Genetic factors play an iPSCs from people with idiopathic ASD and macrocephaly important role in the development of ASD, and rare genetic vari- overproduced inhibitory gamma-aminobutyric acidergic neu- ants in protein-coding genes have implicated altered synaptic rons (14). iPSC technology therefore enables functional sub- function in ASD development (2–7). However, much remains un- classification of ASD risk genes with respect to their effects on known regarding the functional consequences of ASD-associated synapse function and neuronal circuitry, which could facilitate genetic risk factors and their effects on neuronal circuitry. design and interpretation of clinical trials for ASD therapeutics. Induced pluripotent stem cell (iPSC) technology enables Genetic variants of the PTCHD1 locus on chromosome production of neurons that are genetically matched to people Xp22.11 are among the most common and penetrant genetic with ASD and can be used to identify ASD-associated neuronal risk factors for ASD and other neurodevelopmental disorders, phenotypes (8). Neurotransmitter release was reduced in hu- but the functional consequences of these variants remain un- man neurons with heterozygous mutation of NRXN1 (9). known (3,15). Although female subjects can carry PTCHD1 SEE COMMENTARY ON PAGE 95 ª 2019 Society of Biological Psychiatry. This is an open access article under the 139 CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). ISSN: 0006-3223 Biological Psychiatry January 15, 2020; 87:139–149 www.sobp.org/journal Biological Psychiatry Functional Model of ASD-Associated PTCHD1-AS Deletions locus microdeletions with no obvious damaging effects, these analyses, neurons were predifferentiated for 3 weeks, disso- deletions are highly penetrant ASD risk factors in male subjects ciated, reseeded on coverslips with mouse astrocytes, and (15,16) and account for ,1% of ASD cases (3). PTCHD1 en- analyzed 5 weeks later. For some gene expression analyses, codes a transmembrane protein with a patched domain, and NPCs and astrocytes were depleted by magnetic-activated its involvement in neurodevelopmental disorders is supported cell sorting (MACS) as described elsewhere (30), and neurons by microdeletions and frameshift mutations in individuals with were reseeded on Matrigel (Corning Life Sciences, Oneonta, neurodevelopmental delay (NDD), intellectual disability, and NY). For details, see Supplemental Methods and Materials and ASD (15–21). However, recently described Ptchd1 mutant mice Supplemental Table S1. had impairments in attention and cognition (22–24) but did not overtly exhibit ASD-associated behaviors. Also, many ASD- RNA Analyses associated PTCHD1 locus microdeletions are upstream of RNA was harvested using TRIzol (Thermo Fisher Scientific, the PTCHD1 protein-coding gene and disrupt exons of the Waltham, MA), and reverse transcription was performed using neighboring brain-enriched long noncoding RNA (lncRNA) SuperScript II or III (Thermo Fisher Scientific). Primers are listed PTCHD1-AS (15). Some upstream deletions also encompass in Supplemental Table S1. RNA fractionation (31,32) and half- the protein-coding gene DDX53, but this gene reportedly has life assays (33) were performed using published protocols. For limited expression in the brain (15,17). The goal of this study details, see Supplemental Methods and Materials. was to evaluate the effects of PTCHD1 locus deletions on neuronal circuitry and to explore the roles of PTCHD1 locus Immunocytochemistry and Imaging genes in mediating the cellular phenotypes that we observed. Antibodies for immunocytochemistry are listed in PTCHD1 We link upstream genomic rearrangement of the lo- Supplemental Table S3. Excitatory synapses were quantified cus with ASD and employ iPSCs and genome editing to determine as overlapping SYN1 and/or HOMER1 punctae/10 mmof PTCHD1 the functional consequences of locus deletions in human microtubule-associated protein 2–positive dendrite (13,34) in neurons. We generated iPSCs from control subjects and 3 male confocal Z stacks. Dendrites in individual neurons were labeled PTCHD1 subjects with ASD and deletions of the locus. iPSC- by low-efficiency transfection of the plasmid pL-SIN-EF1a- derived neurons from the subjects with ASD exhibited similar im- eGFP using Lipofectamine 2000 (Thermo Fisher Scientific). pairments in excitatory synaptic function, and synaptic impairment Measurements of total dendrite length and complexity were was also observed in neurons with engineered disruption of performed using the Simple Neurite Tracer plugin for ImageJ PTCHD1-AS fi .Our ndings strengthen the connection between (National Institute of Mental Health, Bethesda, MD). For details, synaptic dysfunction and ASD and argue that disruption of see Supplemental Methods and Materials. PTCHD1-AS is a compelling ASD risk factor. Microarrays and RNA Sequencing METHODS AND MATERIALS Copy number variations (CNVs) (35) and RNA expression (36) were analyzed using microarrays as described. Data are Induced Pluripotent Stem Cells deposited in Gene Expression Omnibus (www.ncbi.nlm.nih. iPSC work was approved by the Canadian Institutes of Health gov/geo/): accession GSE83089 (CNVs), GSE81624 (expres- Research Stem Cell Oversight Committee. iPSCs were sion microarray), GSE123753 (high coverage RNA sequencing generated from dermal fibroblasts or from CD341 blood cells, [RNA-seq]), and GSE129808 (ASD-70 RNA-seq). For details, which were obtained at The Hospital for Sick Children with see Supplemental Methods and Materials. informed consent and SickKids Research Ethics Board approval. Fibroblasts were reprogrammed with retrovirus Patch-Clamp Recordings in Human iPSC-Derived vectors (25) and characterized and/or cultured (26,27) as Neurons described elsewhere. Blood cells were reprogrammed with Whole-cell patch-clamp recordings were performed at room Sendai virus and characterized at the Centre for the temperature in human iPSC-derived neurons, cultured with human Commercialization of Regenerative Medicine. Teratoma ex- or mouse astrocytes (12–16 weeks old) or without astrocytes periments were approved by the SickKids Animal Care Com- (8 weeks old), as described elsewhere (28). For details, see mittee and complied with guidelines of the Canadian Council Supplemental Methods and Materials and Supplemental Table S1. on Animal Care. For details, see Supplemental Methods and Materials. iPSC Genome Editing Genome editing was performed as described elsewhere (37) in Neuronal Differentiation iPSCs from the unaffected male subject to replace PTCHD1- Neuronal differentiation procedures
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