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 current 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 noncoding 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 have been described AS exon 3 (ex3) with 2 tandem polyadenylation sequences (27,28). For analyses of neuronal synaptic connections, we (38). Oligonucleotide sequences are presented in cocultured iPSC-derived neurons with exogenous astrocytes Supplemental Table S2. For details, see Supplemental (9–11). For alpha-amino-3-hydroxy-5-methyl-4-isoxazole pro- Methods and Materials. pionic acid receptor (AMPAR)–miniature excitatory postsynaptic current (mEPSC) recordings, neural precursor cells Statistical Analyses (NPCs) were seeded on human astrocytes (ScienCell Research Statistical analyses were primarily performed using GraphPad Laboratories, Inc., Carlsbad, CA) or mouse astrocytes (29) and Prism software (GraphPad Software, San Diego, CA), with N allowed to mature for 12 to 16 weeks. For morphometric being the number of biological replicates from 2 to 4

140 Biological Psychiatry January 15, 2020; 87:139–149 www.sobp.org/journal Biological Functional Model of ASD-Associated PTCHD1-AS Deletions Psychiatry

Figure 1. Expression of PTCHD1 locus transcripts in induced pluripotent stem cell (iPSC)–derived neurons. (A) Schematic representation of the PTCHD1 locus, indicating the locations of transcripts and the autism spectrum disorder (ASD)–associated microdeletions of proband 1 (Prb1) and Prb2. (B) iPSCs were differentiated to neural precursor cells (NPCs) (left) and 6-week-old neurons (right), which were analyzed by immunocytochemistry with the indicated antibodies and counterstained with 40,6- diamidino-2-phenylindole (DAPI). Scale bars = 50 mm (left panel) and 20 mm (right panel). (C) Gene expression was analyzed in NPCs or 3-week-old neurons by quantitative reverse transcriptase polymerase chain reaction with the indicated primer sets (normalized to GAPDH). Data display the mean and SEM. N = biological-replicates/iPSC lines. *p , .05, **p , .01, ****p , .0001, t test. (D) Gene expression was analyzed by quantitative reverse transcriptase polymerase chain reaction in 2- to 8-week-old female control iPSC-derived neurons that were left unstimulated or were stimulated for 6 hours with 55 mmol/L potassium chloride (KCl) (expression was normalized to ACTB). *p , .05, t test. CNV, copy number variation; Ctrl, control; ex, exon; MAP2, microtubule-associated protein 2.

independent experiments. Biological replicates were defined and eliminates the conserved (15) third exon of PTCHD1-AS and as individual neurons (electrophysiology and/or dendrites), DDX53. As controls, we used iPSCs from the unaffected mother of coverslips (synapses), or cultures (quantitative reverse tran- Prb1 (28) and from an unaffected, unrelated male subject (39–41) scriptase polymerase chain reaction). N is typically displayed (Supplemental Table S4 and Supplemental Figure S1A). with the format A/B (A = biological replicates/B = iPSC lines). iPSCs were thoroughly characterized (Supplemental Normally distributed data were displayed as mean 6 SEM and Table S4). PTCHD1 was expressed in most iPSC lines analyzed using parametric statistical tests. Nonnormal data derived from the unaffected mother of Prb1 (Supplemental were displayed as median 6 95% confidence interval and Figure S1B), who is a carrier of the PTCHD1 microdeletion. analyzed using nonparametric statistical tests. For details, see Endonuclease accessibility analyses revealed clonal X chro- Supplemental Methods and Materials. mosome inactivation in PTCHD1-expressing female lines (Supplemental Figure S1C), suggesting that the X chromo- RESULTS some carrying the PTCHD1 deletion was inactivated. iPSCs synthesized pluripotency-associated proteins (Supplemental Generation of iPSCs for Functional Analyses of Figure S1D) and were functionally pluripotent in embryoid PTCHD1 Locus Deletions body and teratoma assays (Supplemental Figure S1E, F). To test the functional consequences of PTCHD1 locus deletions, Although PTCHD1-null iPSC lines tended to have abnormal we generated iPSCs from 2 male probands with ASD (Figure 1A karyotypes (Supplemental Table S5), analyses of karyotype and Supplemental Figure S1A). Proband 1 (Prb1) has a deletion (Supplemental Figure S2A) and CNVs (Supplemental that eliminates the promoters and first exons of PTCHD1 and Figure S2B) revealed that lines chosen for modeling ASD PTCHD1-AS (15). Prb2 has a deletion that is upstream of PTCHD1 were normal and exhibited no marked genetic instability.

Biological Psychiatry January 15, 2020; 87:139–149 www.sobp.org/journal 141 Biological Psychiatry Functional Model of ASD-Associated PTCHD1-AS Deletions

Figure 2. Synaptic function is impaired in neurons from subjects with autism spectrum disorder with PTCHD1-AS microdeletions. (A) Representative traces showing that induced pluripotent stem cell (iPSC)–derived neurons (14–16 weeks old) generate spontaneous action potentials. (B) Typical traces showing evoked action potentials in iPSC-derived neurons (8 weeks old). (C) Excitatory synapses were visualized in 8-week-old iPSC-derived neurons as overlapping punctae of SYN1 (presynaptic marker) (magenta) and HOMER1 (postsynaptic marker of excitatory synapses) (green) on microtubule-associated protein 2 (MAP2)–positive (blue) dendrites (scale bars = 5 mm). Overlapping magenta and green punctae are white in appear- ance. Images from the blue, green, and magenta channels are displayed individually in Supplemental Figure S4A. (D) Quantification of excitatory synapse density in iPSC-derived neurons. Excitatory synapses were detected by immunocytochemistry with anti-SYN1 (presynaptic marker) and anti-HOMER1 (postsynaptic marker of excitatory synapses). Graphs display median and 95% confidence interval. N = coverslips/iPSC lines (9 dendrite segments per coverslip, 2 coverslips per line, 2 replicate experiments). **p , .01, Kruskal-Wallis with Dunn’s post hoc test. Ctrl displays pooled data from the male and female control subjects, which are displayed separately in Supplemental Figure S4B. (E) Representa- tive traces showing alpha-amino-3-hydroxy-5- methyl-4-isoxazole propionic acid receptor (AMPAR)–miniature excitatory postsynaptic currents (mEPSCs) in 14- to 16-week-old neurons. Inset displays average mEPSCs in neurons from the left panel. (F) Scatter plots displaying all data points for the frequency (top panel) and amplitude (bottom panel) of AMPAR-mEPSCs. Graphs also display median and 95% confidence interval. N = neurons/ iPSC lines. ***p , .001, Kruskal-Wallis with Dunn’s post hoc test. Ctrl displays pooled data from the male and female control subjects, which are displayed separately in Supplemental Figure S4C. (G) Quantification of total dendrite length in neurons from control subjects and autism spectrum disorder probands. Graphs display mean, SEM, and all data points. N = neurons/iPSC lines. Ctrl displays pooled data from the male and female control subjects, which are displayed separately in Supplemental Figure S4E. (H) Dendrite complexity of iPSC- derived neurons was determined by Sholl analysis. Graphs display the mean number of dendrite cross- ings (and SEM) at the indicated distances from the soma of neurons that were analyzed in Figure 3G. Ctrl displays pooled data from the male and female control subjects, which are displayed separately in Supplemental Figure S4F. Prb, proband.

Expression of PTCHD1 and PTCHD1-AS in expressed in neurons (Figure 1C). Neurons from Prb1 did not Functional Human Neurons express PTCHD1-AS, and neurons from Prb2 expressed the PTCHD1-AS2 PTCHD1 and PTCHD1-AS are expressed in the cortex and preserved ex1, but not the deleted ex3 cerebellum (15), and cortical PTCHD1 expression coincides (Figure 1C). In addition to steady-state gene expression, we with synaptogenesis and is enriched in deep layer excitatory also tested whether these iPSC-derived neurons could support neurons (42). iPSCs were differentiated into NPCs and cortical activity-dependent transcription (43). Neuronal depolarization neurons (Figure 1B) using our neural rosette-based protocol with potassium chloride resulted in an approximately 2-fold PTCHD1 (27). Subjects with ASD and control subjects exhibited similar induction of and an approximately 5-fold induction BDNF — expression of FOXG1 and PAX6 in NPCs (Figure 1C), and of the positive control (Figure 1D), suggesting that immunocytochemistry revealed no signiﬁcant change in the similar to other genes implicated in neurodevelopmental dis- —PTCHD1 number of PAX6-positive NPCs (Supplemental Figure S3A, B). orders (44) expression is stimulated by neuronal PTCHD1-AS was nearly undetectable in NPCs but was activity.

142 Biological Psychiatry January 15, 2020; 87:139–149 www.sobp.org/journal Biological Functional Model of ASD-Associated PTCHD1-AS Deletions Psychiatry

Figure 3. N-methyl-D-aspartate receptor (NMDAR) function is impaired in neurons from subjects with autism spectrum disorder with PTCHD1-AS microdeletions. (A) Representative traces displaying NMDA-evoked currents recorded at membrane potentials of 260 mV in induced pluripotent stem cell (iPSC)–derived neurons (8 weeks old) in the absence of extracellular Mg21 (left panel). Plot showing current-voltage relationship of NMDA-evoked currents in the 3 neurons displayed in the left panel, recorded in the absence of extracellular Mg21 (right panel). (B) Scatter plots displaying all data points for NMDA-evoked currents recorded at 260 mV in the absence of extracellular Mg21. Graphs also display mean and SEM. N = neurons/ iPSC lines. *p , .05; ***p , .001, one-way analysis of variance with Dunnett’s post hoc test. (C) Repre- sentative traces of NMDA-evoked currents from 8- week-old iPSC-derived neurons, recorded at membrane potentials from 260 mV to 160 mV in the presence of extracellular Mg21 (1 mmol/L). (D) Current-voltage relationship of NMDA-evoked currents recorded in the presence of extracellular Mg21 at 1 mmol/L. Graphs display mean and SEM. N = neurons/iPSC lines. *p , .05; **p , .01, one-way analysis of variance with Dunnett’s post hoc test, compared at 160 mV. Ctrl XX, female control; Prb, proband.

Having determined that neurons express PTCHD1 and compared with control subjects. To explore a potential mecha- PTCHD1-AS, we next verified that these neurons were func- nism for decreased synaptic activity, we examined dendrite tional. Neurons from control subjects and both subjects with morphology (Supplemental Figure S4D), which revealed no dif- ASD generated spontaneous (Figure 2A) or evoked (Figure 2B) ference in total dendrite length (Figure 2G and Supplemental action potentials, and electrophysiological recordings revealed Figure S4E) and only subtle changes in dendrite complexity no substantial differences in intrinsic membrane properties in (Figure 2H and Supplemental Figure S4F). Therefore, neurons proband neurons (Supplemental Table S6). These neurons also with deletions of the PTCHD1 locus exhibit decreased excitatory formed structural excitatory synapses (Figure 2C and synaptic activity without any change in dendrite morphology. Supplemental Figure S4A). Synapse quantification revealed no difference for Prb1 and an approximately 40% increase in the NMDAR Function Is Impaired in Neurons of number of excitatory synapses in Prb2 compared with the Subjects With ASD unaffected control subjects (Figure 2D and Supplemental Figure S4B). Generation of action potentials and formation of NMDARs play a key role in synaptic transmission and plasticity synapses indicate that these neurons are suitable for modeling (46), and they have also been implicated in ASD (47).To synaptic function (and dysfunction) in ASD. examine NMDAR function, we performed whole-cell voltage- clamp recordings, which revealed an approximately 30% to 50% decrease in NMDA-evoked current amplitude in proband Excitatory Synaptic Function Is Decreased in neurons compared with control neurons (Figure 3A, B), with no Neurons of Subjects With ASD change in the reversal potential of the currents. NMDARs have To examine functional excitatory synapses, we measured a property of voltage-dependent blockade by extracellular mEPSCs, which are primarily mediated by AMPARs and N- Mg21 (48), and loss of this blockade may contribute to neu- methyl-D-aspartate receptors (NMDARs) (45).Wefirst examined rodevelopmental disorders (49). Extracellular Mg21 led to a AMPAR-mEPSCs (Figure 2E) by blocking NMDARs with AP5. We voltage-dependent blockade in NMDA-evoked currents in observed an approximately 50% decrease in the frequency of control and proband neurons (Figure 3C), and NMDA-evoked AMPAR-mEPSCs in neurons from both Prb1 and Prb2 (Figure 2E currents were reduced by approximately 45% to 55% in pro- and Supplemental Figure S4C), with no change in amplitude band neurons held at 160 mV in the presence of extracellular

Biological Psychiatry January 15, 2020; 87:139–149 www.sobp.org/journal 143 Biological Psychiatry Functional Model of ASD-Associated PTCHD1-AS Deletions

Figure 4. Genetic evidence for PTCHD1-AS as an autism spectrum disorder (ASD) candidate gene. Displayed are PTCHD1 locus transcripts and published, deposited, or novel genetic variants that disrupt genes within the locus. NR_073010.2 is the National Center for Biotechnology Information reference transcript for PTCHD1-AS. PTCHD1-AS1, PTCHD1-AS2, and PTCHD1-AS3 were previously described by Noor et al. (15). PTCHD1-AS2 and PTCHD1-AS3 transcriptional start sites are indicated as promoters. PTCHD1-AS1 is likely a truncated partial transcript, and the 50 end of the deposited sequence is too short to be unambiguously mapped (indicated by question mark). Hashed gray vertical lines correspond to exons of interest from PTCHD1 locus transcripts; hashed magenta lines correspond to novel exons of PTCHD1-AS reported in this study. The hashed red line indicates the location of the PTCHD1-AS2 transcriptional start site, which is 20 kb away from the nearest PTCHD1 deletion. Genetic variants that spare this transcriptional start site and all downstream exons are predicted to leave PTCHD1-AS transcripts preserved. All displayed genetic variants were identiﬁed in male subjects. Aside from frameshift mutations (indicated by asterisk) and 4 duplications, all displayed variants are deletions. NDD/ID indicates individuals with neurodevelopmental delays and/or intellectual disability in whom ASD was not diagnosed. ChrX, chromosome X; CNVs, copy number variations.

Mg21 (Figure 3D). Taken together, decreases in both the (Figure 4), although only ex1 through ex5 are deleted in ASD frequency of AMPAR-mEPSCs and the amplitude of NMDA- cases (15,17). evoked currents suggest pronounced impairments in excit- To gain insight into the consequences of PTCHD1-AS de- atory neurotransmission in neurons from subjects with ASD letions, we mined publicly available genomics data. Cap and with PTCHD1 locus deletions. analysis of gene expression sequencing data (50) revealed 2 transcriptional start sites (TSSs) for PTCHD1-AS, which are separated by approximately 40 kb and map to the ﬁrst exons of PTCHD1-AS Is an ASD Candidate Gene PTCHD1-AS2 and PTCHD1-AS3 (Figure 4 and Supplemental Our ﬁndings indicate that neurons from both subjects with Figure S5A). Chromatin immunoprecipitation sequencing data ASD displayed similar synaptic phenotypes, and PTCHD1-AS (51) from human neurons suggest that the PTCHD1-AS3 TSS is the only gene disrupted in both individuals (Figure 1A). shares a bidirectional promoter with PTCHD1 (Supplemental Therefore, we explored the structure and regulation of the Figure S5B) that bears histone markers of both promoters PTCHD1-AS gene. The PTCHD1-AS lncRNA is spliced into at (H3K4me3) and enhancers (H3K27Ac, H3K4me1) least 3 known variants (PTCHD1-AS1, PTCHD1-AS2, and (Supplemental Figure S5C). The second TSS corresponds to PTCHD1-AS3) (15) and is divergently transcribed away from PTCHD1-AS2 and is associated with enhancer markers. the PTCHD1 protein-coding gene (Figure 4). The National Together these data indicate that PTCHD1-AS transcription is Center for Biotechnology Information reference transcript for initiated at 2 spatially distinct sites (Figure 4). PTCHD1-AS (NR_073010.2, March 2016) includes all To survey the evidence for PTCHD1-AS as an ASD candidate PTCHD1-AS2 exons and additional downstream exons gene, we compiled 75 published and unpublished genomic

144 Biological Psychiatry January 15, 2020; 87:139–149 www.sobp.org/journal Biological Functional Model of ASD-Associated PTCHD1-AS Deletions Psychiatry

variants of the PTCHD1 locus from male subjects (Figure 4 and PTCHD1-AS ex3 in Prb2 affected expression of the neigh- Supplemental Table S7), 71 of which are associated with ASD or boring protein-coding gene PTCHD1 (Figure 4). Quantitative NDD and/or intellectual disability (6,15,17,20,21).Wefound14 reverse transcriptase polymerase chain reaction analyses of variants that truncate or delete PTCHD1, while preserving MACS-enriched neurons revealed similar robust expression of PTCHD1-AS (i.e., at least one promoter and all downstream the neuronal marker TUBB3 and low levels of the astrocyte exons), and 2 of these individuals had ASD (Figure 4). However, marker GFAP (Figure 5C). PTCHD1-AS was not expressed in 51 deletions that eliminated both promoters or disrupted exons Prb1 and lacked ex3 in Prb2 (Figure 5C). DDX53 was detect- of PTCHD1-AS2 were found, and 35 of these individuals had able by quantitative reverse transcriptase polymerase chain ASD or ASD features (Figure 4). These findings show that 69% reaction in control neurons (Figure 5C). When we mined high- of variants that disrupt PTCHD1-AS are associated with ASD or coverage RNA-seq from MACS-enriched control iPSC-derived ASD features, in contrast to 14% of variants that exclusively neurons, we detected PTCHD1-AS ex3 in all replicates. interrupt PTCHD1.Thesefindings suggest that disruption of However, reads mapping to DDX53 did not span its full exon PTCHD1-AS is more likely to confer ASD risk than disruption of and had different distributions in each replicate (Supplemental PTCHD1 (p =.0005,Fisher’s exact test). Figure S8A), suggesting that DDX53 is expressed at low levels. We also identified several novel or unreported microdeletions. Genetic deletion of DDX53 in Prb2 eliminated its expression One noteworthy novel deletion disrupted PTCHD1-AS in 3 (Figure 5C), but the intact DDX53 gene was not expressed in brothers with ASD (8257_001, 8257_004, and 8257_005) Prb1 neurons, suggesting that transcription through the (Figure 4) and partially overlapped the deletion found in Prb2 PTCHD1-AS gene may promote DDX53 expression. Neurons (5298_3) (Figure 4). Both of these deletions encompass the third from Prb2 expressed PTCHD1 levels that were nearly identical exon of the annotated PTCHD1-AS transcript (Figure 4), which is to unaffected control neurons (Figure 5C). Similarly, we found evolutionarily conserved and is expressed in syntenic transcripts no difference in potassium chloride–induced expression of from rodents (15).Finally,wefoundPTCHD1-AS deletions in 4 PTCHD1 in Prb2 neurons (Supplemental Figure S8B). There- male control subjects from general population databases fore, synaptic phenotypes in Prb2 neurons are not mediated by (Figure 4), although none of these individuals would have been misregulation of PTCHD1 messenger RNA expression. assessed for ASD. Together these refined genetic data build on our previous findings (15) and strongly suggest that PTCHD1-AS Global Gene Expression Is Subtly Affected by deletions are ASD risk factors. PTCHD1 Locus Deletions trans PTCHD1-AS PTCHD1-AS To test for potential effects (54) of ,we Is Alternatively Spliced and Localized examined global gene expression in MACS-enriched neurons to the Nucleus from multiple iPSC lines of control subjects and subjects with To gain insights into the molecular basis of synaptic pheno- ASD (Supplemental Tables S1 and S4). Hierarchical clustering types in proband neurons, we characterized splicing and of microarray data showed that gene expression patterns localization of PTCHD1-AS RNA. Transcripts arising from ex1 segregated by clinical presentation, experimental subject, and through ex6 of the .1MbPTCHD1-AS gene were alternatively iPSC line (Figure 5D). Few genes were misregulated in com- spliced, ranged in size from 320 to 827 nucleotides mon in the 2 subjects with ASD: only 18 transcripts from 14 (Supplemental Figure S6A and Supplemental Table S8), and genes exhibited a fold change of .1.25 and achieved statis- included several undescribed exons (Figure 4 and tical significance in both subjects with ASD (Supplemental Supplemental Table S9). In control neurons, ex3 was detected Table S10), although none of these genes have known in most cloned splice variants from both PTCHD1-AS2 and neuronal functions. We also detected no consistent expression PTCHD1-AS3, but ex4 was absent (Figure 5A and changes in select genes with known roles in hedgehog Supplemental Figure S6A, B). In contrast, the only consistent signaling, regional specification of neurons, or neurotrans- difference in exon usage in Prb2 neurons (which lack ex3) was mitter subtypes (Supplemental Table S10). Therefore, gene inclusion of the fourth exon (Figure 5A). When analyzed by expression is only subtly affected in iPSC-derived neurons with subcellular fractionation (31), control transcripts OIP5-AS1 and PTCHD1 locus deletions. MALAT1 (52) were primarily localized in the cytoplasm and nucleus, respectively (Figure 5B). PTCHD1-AS was primarily Deletion of PTCHD1-AS ex3 Impairs Synaptic found in the nucleus—and mostly chromatin-associated—in Function neurons both from the control subject and from Prb2 Our genetic and electrophysiological data implicate PTCHD1- (Figure 5B). Enrichment in the chromatin fraction was not due AS in neuronal function. However, Prb1 has a deletion of to transcript instability (53),asPTCHD1-AS had a half-life of PTCHD1, and concurrent whole-genome sequencing of blood .5 hours (Supplemental Figure S7). Therefore, PTCHD1-AS is DNA from Prb2 revealed a de novo missense variant in the alternatively spliced and localizes to the nucleus, and loss of ASD risk gene SHANK2 (p.A1352T) (6), which may also ex3 leads to a shift in splicing patterns without a change in potentially contribute to the phenotype. subcellular localization. To further test the consequence of PTCHD1-AS disruption PTCHD1 cis in neurons, we used clustered regularly interspaced short Expression Is Not Affected in by palindromic repeat (CRISPR)/Cas9 genome editing in iPSCs PTCHD1-AS ex3 Deletion from the unaffected male subject. Guided by functional data Some nuclear lncRNAs act in cis to regulate expression of from Prb2 and supporting genetic data from a novel ASD- neighboring genes (54), so we tested whether deletion of associated microdeletion (8257_001, _004, and _005)

Biological Psychiatry January 15, 2020; 87:139–149 www.sobp.org/journal 145 Biological Psychiatry Functional Model of ASD-Associated PTCHD1-AS Deletions

Figure 5. PTCHD1-AS exon 3 (ex3) is essential for normal excitatory synaptic function in gene-edited induced pluripotent stem cell (iPSC)–derived neurons. (A) Heat map of exon usage in cloned PTCHD1-AS2 splice variants from the unaffected female control (Ctrl) subject and from proband 2 (Prb2). (B) Relative abundance of transcripts in the cytoplasm, nucleoplasm, and chromatin fractions of 7- to 8-week-old neurons from the unaffected female control subject and from Prb2. N = biological replicates from 1 line each. (C) Quantitative reverse transcriptase polymerase chain reaction analysis of gene expression (normalized to GAPDH/18s)in magnetic-activated cell sorting–enriched neurons (4 weeks old). The sample labeled Astro/NPC consisted of astrocytes and neural precursor cells enriched by magnetic-activated cell sorting with antibodies tar- geting CD44 and CD184. N = biological replicates/ iPSC lines. Graphs display mean and SEM. *p , .05, **p , .01, ***p , .001, ****p , .0001, analysis of variance with Tukey post hoc test. (D) Hierarchical clustering of differentially expressed genes in 4-week-old magnetic-activated cell sorting–enriched neurons from unaffected control subjects and subjects with autism spectrum disorder (ASD). Dis- played data are from multiple iPSC lines from 2 independent experiments (a, b). (E) Representative traces showing alpha-amino-3-hydroxy-5-methyl-4- isoxazole propionic acid receptor (AMPAR)–miniature excitatory postsynaptic currents (mEPSCs) from 14- to 16-week-old genome-edited iPSC-derived neurons and isogenic control (Iso ctrl) neurons. Inset displays average mEPSCs from the left panel. (F) Quantification of frequency (left panel) and amplitude (right panel) of AMPAR-mEPSCs from clustered regularly interspaced short palindromic repeat (CRISPR)–edited neurons and isogenic control neurons. All data points are displayed along with median and 95% confidence interval. N = total number of neurons analyzed from 2 independent experiments. *p , .05, ***p , .001, Mann-Whitney U test. (G) Representative traces showing AMPAR- mEPSCs from 12- to 13-week-old iPSC-derived neurons from ASD-70 and control subjects. Inset displays average mEPSCs from the left panel. (H) Quantification of frequency (left panel) and amplitude (right panel) of AMPAR-mEPSCs from iPSC-derived neurons from ASD-70 and unaffected control subjects 68 and 50. All data points are displayed along with median and 95% confidence interval. N = number of neurons/iPSC lines from 3 independent experiments. *p , .05, ns: p . .5, Mann-Whitney U test.

(Figure 4), we focused on PTCHD1-AS ex3 (Supplemental ASD-70 and control subjects (Ctrl 68 and Ctrl 50B, an unre- Figure S9A). We designed CRISPR guide RNAs to cut within lated unaffected male subject) were differentiated using an ex3 (Supplemental Figure S9B), facilitating homologous adherent neuronal differentiation protocol, and neuron identity recombination to replace ex3 with two tandem polyadenylation was conﬁrmed by RNA-seq detection of cortical layer markers sequences (Supplemental Figure S9C) with the intent of (Supplemental Figure S9E). ASD-70 neurons did not express prematurely terminating transcription of PTCHD1-AS.To PTCHD1-AS ex3 and expressed other PTCHD1 locus tran- complement this isogenic pair of lines, we also obtained blood scripts at similar levels to control neurons (Supplemental for reprogramming from the 8257 family with the ex3 micro- Figure S8F). deletion that leaves DDX53 intact (Figure 4). iPSCs were Synaptic function was examined in neurons with PTCHD1- generated and characterized from this subject with ASD AS ex3 deletions by recording AMPAR-mEPSCs. We found a (8257_005, ASD-70) and his unaffected father (8257_001, Ctrl decrease in the frequency of AMPAR-mEPSCs in the CRISPR- 68) (Supplemental Table S11). edited neurons compared with the isogenic control neurons To examine expression of genes in the PTCHD1 locus, we (Figure 5E, F) and in neurons from ASD-70 compared with differentiated the CRISPR-edited line into neurons. These control neurons (Figure 5G, H). mEPSC amplitude was neurons did not express PTCHD1-AS ex3 as expected, but decreased in CRISPR-edited neurons (Figure 5F), but not in instead exhibited decreased expression of other PTCHD1 ASD-70 neurons (Figure 5H). These data reveal that targeted locus transcripts (Supplemental Figure S9D). iPSCs from mutation of PTCHD1-AS ex3 impairs synaptic function,

146 Biological Psychiatry January 15, 2020; 87:139–149 www.sobp.org/journal Biological Functional Model of ASD-Associated PTCHD1-AS Deletions Psychiatry

consistent with our results from Prb1 and Prb2, thereby therapeutic target in some ASD cases, including individuals implicating this lncRNA in the etiology of ASD. with deletions of PTCHD1-AS.

DISCUSSION Role of PTCHD1-AS in ASD We describe genetic and functional data that strengthen the The accumulating data indicate that the PTCHD1-AS lncRNA link between the PTCHD1-AS lncRNA and ASD. Our genetic is an important target for early and/or confirmatory diagnosis of findings, including the identification of novel ASD-associated ASD (58). In fact, two of the most frequently observed CNVs in microdeletions, strongly suggest a role for PTCHD1-AS in ASD cases are microdeletions of the PTCHD1-AS locus in conferring ASD risk in male subjects. We also used a combi- male subjects and chromosome 16p11.2 in both male and nation of cellular reprogramming and genome editing to show female subjects (3,6). Combined data from published reports that disruption of PTCHD1-AS impairs excitatory and new subjects described here indicate that 69% of male neurotransmission. carriers of PTCHD1-AS microdeletions present with ASD, PTCHD1 Disruption of PTCHD1 Locus Impairs Excitatory whereas likely has an independent role in suscepti- Synaptic Function bility to NDD and/or intellectual disability. Further resolving the relative contributions of PTCHD1-AS and PTCHD1 in ASD- PTCHD1-AS deletions led to decreased AMPAR-mEPSC fre- associated and NDD-associated synaptic phenotypes will be quency in Prb1 and Prb2 with no consistent morphometric a priority for future experiments. changes. Prb2 had more structural excitatory synapses than Another future priority is elucidating the molecular functions SHANK2 did the control neurons, which may be due to the of PTCHD1-AS. Owing to its low abundance, PTCHD1-AS is p.A1352T missense mutation, consistent with our recent report not efficiently detected in conventional low-coverage RNA-seq SHANK2 fi of increased synapse numbers in -haploinsuf cient studies, and this has hindered efforts to define exon use in neurons (41). The apparent discordance between synapse naturally occurring splice variants. It is therefore not clear numbers and mEPSC frequency may be caused by structural which of the many PTCHD1-AS transcript variants is most synapses that are postsynaptically silent (55,56). It is also appropriate for transgene rescue experiments. Moreover, possible that coculturing neurons with exogenous normal as- rescue of a cis-acting nuclear lncRNA may require synthesis at trocytes may have obscured potential morphometric pheno- the specific genomic locus (59). Finally, the low abundance and types, which could be revealed by using only endogenous inherent diversity of PTCHD1-AS transcripts makes them fi mutant astrocytes produced at de ned ratios during differen- challenging for biochemical enrichment to identify bound tiation (34). Despite the increased synapse number in Prb2, we proteins or target genes (60). Identification of the molecular observed a reduction in AMPAR-mEPSC frequency. Overall, functions of PTCHD1-AS will likely benefit from studies of reduced mEPSC frequency was consistently observed in all 4 knockout mouse models, where brain regions that express the PTCHD1-AS mutations studied, being corroborated in the ex3 transcripts can be identified and neurons collected in abun- deleted CRISPR isogenic pair and in the ASD-70 neurons dance for biochemistry experiments. fi compared with control neurons. Our ndings show that Heterogeneity in genetic and functional underpinnings of PTCHD1-AS hypoactivity is cell autonomous in neurons ASD represents a major challenge for identifying novel thera- because it is detected even when control human astrocytes peutics, as this likely leads to inconsistent treatment are present in the cultures. It would be interesting to explore responses in clinical trials (61). By identifying neurophysio- PTCHD1-AS potential effects of in inhibitory neurons or non- logical deficits associated with specific ASD risk loci, human cell autonomous effects by astrocytes in future work. Such iPSC models will facilitate stratification of treatment groups for impaired excitatory neurotransmission has also been observed improved trial design. Our findings therefore help to highlight – in other human pluripotent stem cell derived neurons with new therapeutic targets to the physiological pathways affected SHANK3 fi haploinsuf ciency (10,11) or with targeted mutations by PTCHD1-AS deletions and suggest that individuals with NRXN1 fi of (9). Together these ndings suggest that reduced these deletions may benefit from treatments that enhance function of excitatory synapses plays an important role in the excitatory synaptic function. development of ASD. In addition to decreased AMPAR-mEPSC frequency, we also found that neurons from Prb1 and Prb2 had diminished ACKNOWLEDGMENTS AND DISCLOSURES responses to NMDA, implicating hypofunction of NMDARs, This study was supported by the National Institutes of Health (Grant No. R33 which should be verified in future studies of neurons with MH087908 [to JE and SWS] and Grant No. R01 MH059630 [to RJL]), PTCHD1-AS deletions. Future studies will also address the Canadian Institutes of Health Research (CIHR) (Grant No. EPS-129129 [to JE and AN] and Grant Nos. MOP-102649 and MOP-133423 [to JE and mechanism of NMDAR dysfunction, which could result from MWS]), Genome Canada (to SWS), Autism Speaks MSSNG Project, Cana- changes in NMDAR subunit expression, localization, or phos- dian Institute for Advanced Research, Ontario Brain Institute (to JE and phorylation (28,47). NMDAR function was impaired in mice with SWS), Simons Foundation for Autism Research (Grant No. 569293 [to SWS heterozygous deletion of the ASD candidate gene Tbr1, likely and MWS]), Ontario Stem Cell Initiative Fellowship (to PJR), Ontario Ministry via altered expression of its target Grin2B (57), which is also an of Research and Innovation Fellowship (to PJR), CIHR Banting Postdoctoral ASD candidate gene (47). Interestingly, ASD-associated Fellowship (to ED), CIHR Vanier Scholarship (to KZ), International Rett fi Tbr11/2 Syndrome Foundation Fellowship (to DCR), and CIHR Postdoctoral behavioral de cits in mice were rescued by treat- Fellowship (to RKCY). SWS is the GlaxoSmithKline-CIHR Endowed Chair in ment with D-cycloserine (57), which is a partial agonist of Genome Sciences at The Hospital for Sick Children. MWS is the Northbridge NMDARs. NMDAR hypofunction may therefore represent a Chair in Paediatric Research at the Hospital for Sick Children.

Biological Psychiatry January 15, 2020; 87:139–149 www.sobp.org/journal 147 Biological Psychiatry Functional Model of ASD-Associated PTCHD1-AS Deletions

Author responsibilities were as follows: conceptualization—JE, SWS, 18 new candidate genes for autism spectrum disorder. Nat Neurosci PJR, MWS, and W-BZ; methodology—PJR, JE, W-BZ, MWS, RSFM, KZ, 20:602–611. ED, BV, AN, DCR, LD, and SWS; formal analysis—PJR, JE, W-BZ, MWS, 7. Bourgeron T (2015): From the genetic architecture to synaptic plas- KZ, RSFM, ED, LD, MF, MM, and RKCY; investigation—PJR, W-BZ, RSFM, ticity in autism spectrum disorder. Nat Rev Neurosci 16:551–563. KZ, ED, LD, DCR, AP, WW, PP, and RKCY; resources—RL; writing of 8. Kim D-S, Ross PJ, Zaslavsky K, Ellis J (2014): Optimizing neuronal original manuscript draft—PJR, JE, W-BZ, and KZ; review and editing of differentiation from induced pluripotent stem cells to model ASD. Front final manuscript—PJR, JE, W-BZ, MWS, SWS, KZ, ED, RSFM, LD, DCR, Cell Neurosci 8:109. and PP; visualization—PJR, W-BZ, KZ, LD, ED, MM, and MF; supervision— 9. Pak C, Danko T, Zhang Y, Aoto J, Anderson G, Maxeiner S, et al. JE, SWS, and MWS. (2015): Human neuropsychiatric disease modeling using conditional We thank Dr. John Vincent for helpful discussions; Zhanna Konovalova, deletion reveals synaptic transmission defects caused by heterozy- Tadeo Thompson, Attey Rostami, and Janice Hicks for technical support; gous mutations in NRXN1. Cell Stem Cell 17:316–328. Dr. Andrea Vaags for preliminary copy number variation analyses; and Drs. 10. Yi F, Danko T, Botelho SC, Patzke C, Pak C, Wernig M, Südhof TC Wendy Roberts, Rosanna Weksberg, Brian Chung, and Melissa Carter for (2016): Autism-associated SHANK3 haploinsufficiency causes Ih obtaining skin biopsy specimens. We also thank the participants and their channelopathy in human neurons. Science 352:aaf2669. family members for their contributions to this study. 11. Shcheglovitov A, Shcheglovitova O, Yazawa M, Portmann T, Shu R, SWS is on the Scientific Advisory Committees of Population Bio and Sebastiano V, et al. (2013): SHANK3 and IGF1 restore synaptic deficits in Deep Genomics, and the intellectual property from aspects of his research neurons from 22q13 deletion syndrome patients. Nature 503:267–271. performed at the Hospital for Sick Children is licensed to Athena Diagnostics 12. Griesi-Oliveira K, Acab A, Gupta AR, Sunaga DY, Chailangkarn T, and Lineagen and co-held with Population Bio. These relationships did not Nicol X, et al. (2015): Modeling non-syndromic autism and the impact influence data interpretation or presentation during this study, but are being of TRPC6 disruption in human neurons. Mol Psychiatry 20:1350–1365. disclosed for potential future considerations. The other authors report no 13. Deshpande A, Yadav S, Dao DQ, Wu Z-YY, Hokanson KC, Cahill MK, relevant biomedical financial interests or potential conflicts of interest. et al. (2017): Cellular phenotypes in human iPSC-derived neurons from a genetic model of autism spectrum disorder. Cell Rep 21:2678–2687. 14. Mariani J, Coppola G, Zhang P, Abyzov A, Provini L, Tomasini L, et al. ARTICLE INFORMATION (2015): FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 162:375–390. From the Developmental and Stem Cell Biology Program (PJR, RSFM, KZ, 15. Noor A, Whibley A, Marshall CR, Gianakopoulos PJ, Piton A, Carson AR, DCR, MM, AP, WW, PP, JE), Neurosciences and Mental Health Program (W- et al. (2010): Disruption at the PTCHD1 locus on Xp22.11 in autism BZ, MWS), Genetics and Genome Biology Program (ED, LD, RKCY, MF, spectrum disorder and intellectual disability. Sci Transl Med 2:49ra68. SWS), and The Centre for Applied Genomics (ED, LD, RKCY, MF, SWS), The 16. Marshall CR, Noor A, Vincent JB, Lionel AC, Feuk L, Skaug J, et al. Hospital for Sick Children; Department of Molecular Genetics (RSFM, KZ, (2008): Structural variation of chromosomes in autism spectrum dis- LD, RKCY, MM, SWS, JE), Department of Physiology (MWS), McLaughlin order. Am J Hum Genet 82:477–488. Centre (SWS), Institute of Medical Science (AN), and Department of 17. Chaudhry A, Noor A, Degagne B, Baker K, Bok L, Brady A, et al. Obstetrics and Gynecology (AN), University of Toronto; Lunenfeld-Ten- (2015): Phenotypic spectrum associated with PTCHD1 deletions and enbaum Research Institute (AN, BV), Mount Sinai Hospital, Toronto, Ontario, truncating mutations includes intellectual disability and autism spec- Canada; Center for Autism and Related Disorders (RJL), Kennedy Krieger trum disorder. Clin Genet 88:224–233. Institute; and Department of Psychiatry and Behavioral Sciences (RJL), 18. Whibley AC, Plagnol V, Tarpey PS, Abidi F, Fullston T, Choma MK, et al. Johns Hopkins University School of Medicine, Baltimore, Maryland. (2010): Fine-scale survey of X chromosome copy number variants and fi PJR is currently af liated with the Department of Biology, University of indels underlying intellectual disability. Am J Hum Genet 87:173–188. Prince Edward Island, Charlottetown, Prince Edward Island, Canada. 19. Pinto D, Pagnamenta AT, Klei L, Anney R, Merico D, Regan R, et al. fi BV is currently af liated with the Wellcome - MRC Cambridge Stem Cell (2010): Functional impact of global rare copy number variation in Institute, University of Cambridge, Cambridge, United Kingdom. autism spectrum disorders. Nature 466:368–372. PJR and W-BZ contributed equally to this work. 20. Levy D, Ronemus M, Yamrom B, Lee YH, Leotta A, Kendall J, et al. Address correspondence to James Ellis, Ph.D., Developmental and Stem (2011): Rare de novo and transmitted copy-number variation in autistic Cell Biology Program, The Hospital for Sick Children, Peter Gilgan Centre for spectrum disorders. Neuron 70:886–897. Research and Learning, 686 Bay Street, Room 16-9715, Toronto, ON, 21. Gambin T, Yuan B, Bi W, Liu P, Rosenfeld JA, Coban-Akdemir Z, et al. Canada M5G 0A4; E-mail: [email protected]. (2017): Identification of novel candidate disease genes from de novo Received Jul 24, 2018; revised Jun 23, 2019; accepted Jul 16, 2019. exonic copy number variants. Genome Med 9:83. Supplementary material cited in this article is available online at https:// 22. Ung DC, Iacono G, Méziane H, Blanchard E, Papon M-AA, Selten M, doi.org/10.1016/j.biopsych.2019.07.014. et al. (2017): Ptchd1 deficiency induces excitatory synaptic and cognitive dysfunctions in mouse. Mol Psychiatry 23:1356–1367. 23. Tora D, Gomez AM, Michaud J-FF, Yam PT, Charron F, Scheiffele P REFERENCES (2017): Cellular functions of the autism risk factor PTCHD1 in mice. 1. Anagnostou E, Zwaigenbaum L, Szatmari P, Fombonne E, J Neurosci 37:11993–12005. Fernandez BA, Woodbury-Smith M, et al. (2014): Autism spectrum 24. Wells MF, Wimmer RD, Schmitt LI, Feng G, Halassa MM (2016): disorder: Advances in evidence-based practice. CMAJ 186:509–519. Thalamic reticular impairment underlies attention deficit in Ptchd1(Y/-) 2. Sanders SJ, He X, Willsey AJ, Ercan-Sencicek AG, Samocha KE, mice. Nature 532:58–63. Cicek AE, et al. (2015): Insights into autism spectrum disorder genomic 25. Hotta A, Cheung AY, Farra N, Garcha K, Chang WY, Pasceri P, et al. architecture and biology from 71 risk loci. Neuron 87:1215–1233. (2009): EOS lentiviral vector selection system for human induced 3. Devlin B, Scherer SW (2012): Genetic architecture in autism spectrum pluripotent stem cells. Nat Protoc 4:1828–1844. disorder. Curr Opin Genet Dev 22:229–237. 26. Cheung AY, Horvath LM, Grafodatskaya D, Pasceri P, Weksberg R, 4. Pinto D, Delaby E, Merico D, Barbosa M, Merikangas A, Klei L, et al. Hotta A, et al. (2011): Isolation of MECP2-null Rett syndrome patient (2014): Convergence of genes and cellular pathways dysregulated in hiPS cells and isogenic controls through X-chromosome inactivation. autism spectrum disorders. Am J Hum Genet 94:677–694. Hum Mol Genet 20:2103–2115. 5. De Rubeis S, He X, Goldberg AP, Poultney CS, Samocha K, Cicek A, 27. Djuric U, Cheung AY, Zhang W, Mok RS, Lai W, Piekna A, et al. (2015): et al. (2014): Synaptic, transcriptional and chromatin genes disrupted MECP2e1 isoform mutation affects the form and function of neurons in autism. Nature 515:209–215. derived from Rett syndrome patient iPS cells. Neurobiol Dis 76:37–45. 6. C Yuen RK, Merico D, Bookman M, L Howe J, Thiruvahindrapuram B, 28. Zhang W-B, Ross PJ, Tu Y, Wang Y, Beggs S, Sengar AS, et al. (2016): Patel RV, et al. (2017): Whole genome sequencing resource identifies Fyn kinase regulates GluN2B subunit-dominant NMDA receptors in

148 Biological Psychiatry January 15, 2020; 87:139–149 www.sobp.org/journal Biological Functional Model of ASD-Associated PTCHD1-AS Deletions Psychiatry

human induced pluripotent stem cell-derived neurons. Sci Rep 45. Kerchner GA, Nicoll RA (2008): Silent synapses and the emergence of 6:23837. a postsynaptic mechanism for LTP [published correction appears in 29. Kim HJ, Magrané J (2011): Isolation and culture of neurons and as- Nat Rev Neurosci 2009; 10:242]. Nat Rev Neurosci 9:813–825. trocytes from the mouse brain cortex. Methods Mol Biol 793:63–75. 46. Kalia LV, Kalia SK, Salter MW (2008): NMDA receptors in clinical 30. Djuric U, Rodrigues DC, Batruch I, Ellis J, Shannon P, Diamandis P neurology: Excitatory times ahead. Lancet Neurol 7:742–755. (2017): Spatiotemporal proteomic profiling of human cerebral devel- 47. Burnashev N, Szepetowski P (2015): NMDA receptor subunit muta- opment. Mol Cell Proteomics 16:1548–1562. tions in neurodevelopmental disorders. Curr Opin Pharmacol 20:73– 31. Gagnon KT, Li L, Chu Y, Janowski BA, Corey DR (2014): RNAi factors 82. are present and active in human cell nuclei. Cell Rep 6:211–221. 48. MacDonald JF, Porietis AV, Wojtowicz JM (1982): L-Aspartic acid 32. Pandya-Jones A, Bhatt DM, Lin C-HH, Tong A-JJ, Smale ST, Black DL induces a region of negative slope conductance in the current- (2013): Splicing kinetics and transcript release from the chromatin voltage relationship of cultured spinal cord neurons. Brain Res compartment limit the rate of lipid A-induced gene expression. RNA 237:248–253. 19:811–827. 49. Endele S, Rosenberger G, Geider K, Popp B, Tamer C, Stefanova I, 33. Rodrigues DC, Kim D-SS, Yang G, Zaslavsky K, Ha KC, Mok RS, et al. et al. (2010): Mutations in GRIN2A and GRIN2B encoding regulatory (2016): MECP2 is post-transcriptionally regulated during human neu- subunits of NMDA receptors cause variable neurodevelopmental rodevelopment by combinatorial action of RNA-binding proteins and phenotypes. Nat Genet 42:1021–1026. miRNAs. Cell Rep 17:720–734. 50. Forrest AR, Kawaji H, Rehli M, Baillie KJ, de Hoon MJ, Haberle V, et al. 34. Russo FB, Freitas BC, Pignatari GC, Fernandes IR, Sebat J, Muotri AR, (2014): A promoter-level mammalian expression atlas. Nature Beltrão-Braga PCBC (2018): Modeling the interplay between neurons 507:462–470. and astrocytes in autism using human induced pluripotent stem cells. 51. Zhou X, Maricque B, Xie M, Li D, Sundaram V, Martin EA, et al. (2011): Biol Psychiatry 83:569–578. The Human Epigenome Browser at Washington University. Nat 35. Marshall CR, Scherer SW (2012): Detection and characterization of Methods 8:989–990. copy number variation in autism spectrum disorder. Methods Mol Biol 52. Lennox KA, Behlke MA (2016): Cellular localization of long non-coding 838:115–135. RNAs affects silencing by RNAi more than by antisense oligonucleo- 36. Yuen RK, Merico D, Cao H, Pellecchia G, Alipanahi B, tides. Nucleic Acids Res 44:863–877. Thiruvahindrapuram B, et al. (2016): Genome-wide characteristics of 53. Bhatt DM, Pandya-Jones A, Tong A-JJ, Barozzi I, Lissner MM, de novo mutations in autism. NPJ Genom Med 1:160271–1602710. Natoli G, et al. (2012): Transcript dynamics of proinflammatory genes 37. Miyaoka Y, Chan AH, Judge LM, Yoo J, Huang M, Nguyen TD, et al. revealed by sequence analysis of subcellular RNA fractions. Cell (2014): Isolation of single-base genome-edited human iPS cells 150:279–290. without antibiotic selection. Nat Methods 11:291–293. 54. Briggs JA, Wolvetang EJ, Mattick JS, Rinn JL, Barry G (2015): 38. McFarland TJ, Zhang Y, Atchaneeyaskul LO, Francis P, Stout JT, Mechanisms of long non-coding RNAs in mammalian nervous system Appukuttan B (2006): Evaluation of a novel short polyadenylation development, plasticity, disease, and evolution. Neuron 88:861–877. signal as an alternative to the SV40 polyadenylation signal. Plasmid 55. Christopherson KS, Ullian EM, Stokes CC, Mullowney CE, Hell JW, 56:62–67. Agah A, et al. (2005): Thrombospondins are astrocyte-secreted pro- 39. Deneault E, Faheem M, White SH, Rodrigues DC, Sun S, Wei W, et al. teins that promote CNS synaptogenesis. Cell 120:421–433. (2019): CNTN5-/1 or EHMT2-/1 human iPSC-derived neurons from 56. Kucukdereli H, Allen NJ, Lee AT, Feng A, Ozlu MI, Conatser LM, et al. individuals with autism develop hyperactive neuronal networks. Elife 8: (2011): Control of excitatory CNS synaptogenesis by astrocyte- e40092. secreted proteins Hevin and SPARC. Proc Natl Acad Sci U S A 40. Deneault E, White SH, Rodrigues DC, Ross PJ, Faheem M, 108:E440–E449. Zaslavsky K, et al. (2018): Complete disruption of autism-susceptibility 57. Huang T-NN, Chuang H-CC, Chou W-HH, Chen C-YY, Wang H-FF, genes by gene editing predominantly reduces functional connectivity Chou S-JJ, Hsueh Y-PP (2014): Tbr1 haploinsufficiency impairs of isogenic human neurons. Stem Cell Rep 11:1211–1225. amygdalar axonal projections and results in cognitive abnormality. Nat 41. Zaslavsky K, Zhang W-BB, McCready FP, Rodrigues DC, Deneault E, Neurosci 17:240–247. Loo C, et al. (2019): SHANK2 mutations associated with autism 58. Carter MT, Scherer SW (2013): Autism spectrum disorder in the ge- spectrum disorder cause hyperconnectivity of human neurons. Nat netics clinic: A review. Clin Genet 83:399–407. Neurosci 22:556–564. 59. Bassett AR, Akhtar A, Barlow DP, Bird AP, Brockdorff N, Duboule D, 42. Parikshak NN, Luo R, Zhang A, Won H, Lowe JK, Chandran V, et al. et al. (2014): Considerations when investigating lncRNA function (2013): Integrative functional genomic analyses implicate specific in vivo. Elife 3:e03058. molecular pathways and circuits in autism. Cell 155:1008–1021. 60. Quinn JJ, Chang HY (2015): In situ dissection of RNA functional 43. Hardingham GE, Pruunsild P, Greenberg ME, Bading H (2018): Line- subunits by domain-specific chromatin isolation by RNA purification age divergence of activity-driven transcription and evolution of (dChIRP). Methods Mol Biol 1262:199–213. cognitive ability. Nat Rev Neurosci 19:9–15. 61. Delorme R, Ey E, Toro R, Leboyer M, Gillberg C, Bourgeron T (2013): 44. Ebert DH, Greenberg ME (2013): Activity-dependent neuronal signal- Progress toward treatments for synaptic defects in autism. Nat Med ling and autism spectrum disorder. Nature 493:327–337. 19:685–694.

Biological Psychiatry January 15, 2020; 87:139–149 www.sobp.org/journal 149