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, or PTCHD1-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.

Neuronal PAS domain protein 3 (NPAS3) and VGF (VGF Nerve Growth Factor (NGF) Inducible) are important for neurogenesis and psychiatric disorders. Previously, we have demonstrated that NPAS3 regulates VGF at the transcriptional level. In this study, VGF (non-acronymic) was found regulated by NPAS3 in neuronal stem cells. However, the underlying mechanism of this regulation remains unclear. The aim of this study was to explore the correlation of NPAS3 and VGF, and their roles in neural cell proliferation, in the context of psychiatric illnesses. First, we focused on the structure of NPAS3, to identify the functional domain of NPAS3. Truncated NPAS3 lacking transactivation domain was also found to activate VGF, which suggested that not only transactivation domain but other structural motifs were also involved in the regulation. Second, Mutated enhancer box (E-box) of VGF promoter showed a significant response to this basic helix-loop-helix (bHLH) transcription factor, which suggested an indirect regulatory mechanism for controlling VGF expression by NPAS3. κB site within VGF promoter was identified for VGF activation induced by NPAS3, apart from direct binding to E-box. Furthermore, ectopically expressed NPAS3 in PC12 cells produced parallel responses for nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB (P65)] expression, which specifies that NPAS3 regulates VGF through the NF-κB signaling pathway. Over-expression of NPAS3 also enhances the cell proliferation, which can be blocked by knockdown of VGF. Finally, NPAS3 was found to influence proliferation of neural cells through VGF. Therefore, downstream signaling pathways that are responsible for NPAS3-VGF induced proliferation via glutamate receptors were explored. Combining this work and published literature, a potential network composed by NPAS3, NF-κB, Brain-Derived Neurotrophic Factor (BDNF), NGF and VGF, was proposed. This network collectively detailed how NPAS3 connects with VGF and intersected neural cell proliferation, synaptic activity and psychiatric disorders.