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Autism spectrum disorder (ASD) is a genetically heterogeneous disorder. Sequencing studies have identified hundreds of risk genes for autism spectrum disorder (ASD), but the signaling networks of genes at the protein level remain largely unexplored, which can provide insight into previously unknown individual and convergent disease pathways in the brain. To address this gap, we used neuron-specific proximity-labeling proteomics (BioID) to identify protein-protein interaction (PPI) networks of 41 ASD-risk genes. Network analysis revealed the combined 41 risk gene PPI network map had more shared connectivity between unrelated ASD-risk genes than represented in existing public databases. We identified common pathways between established and uncharacterized risk genes, including synaptic transmission, mitochondrial/metabolic processes, Wnt signaling pathways, ion channel activity and MAPK signaling. Investigation of the mitochondrial and metabolic network using gene knockouts revealed a functional hub in neurons for multiple risk genes not previously associated with this pathway. Further, we identified that the uncharacterized ASD-risk gene PPP2R5D localizes to the synapse, which is disrupted by patient de novo missense mutations. Investigation of de novo missense variants of additional synaptic ASD-risk genes demonstrated that changes in PPI networks can capture synaptic transmission deficits. The neuronal 41 ASD-risk gene PPI network map also revealed enrichment for an additional 112 ASD-risk genes and human brain cell types implicated in ASD pathology. Interestingly, clustering of ASD-risk genes based on their PPI network connectivity identified multiple gene groups that correlate mutation-type to clinical behavior scores. Together, our data reveal that using PPI networks to map ASD risk genes can identify previously unknown individual and convergent neuronal signaling networks, provide a method to assess the impact of patient variants, and reveal new biological insight into disease mechanisms.

Background Pain is a complex neurobiological response with a multitude of causes; however, patients with autism spectrum disorder (ASD) often report chronic pain with no known etiology. Recent research has been aimed toward identifying the causal mechanisms of pain in mouse and human models of ASD. In recent years, efforts have been made to better document and explore secondary phenotypes observed in ASD patients in the clinic. As new sequencing studies have become more powered with larger cohorts within ASD, specific genes and their variants are often left uncharacterized or validated. In this review we highlight ASD risk genes often presented with pain comorbidities. Aims This mini-review bridges the gap between two fields of literature, neurodevelopmental disorders and pain research. We discuss the importance of the genetic landscape of ASD and its links to pain phenotypes. Results Among the numerous genes implicated in ASD, few have been implicated with varying severities of pain comorbidity. Mutations in these genes, such as SCN9A, SHANK3 , and CNTNAP2 , lead to altered neuronal function that produce different responses to pain, shown in both mouse and human models. Conclusion There is a necessity to use new technologies to advance the current understanding of ASD risk genes and their contributions to pain. Secondly, there is a need to power future ASD risk genes associated with pain with their own cohort, because a better understanding is needed of this subpopulation.

Copy number variations (CNVs) are associated with psychiatric and neurodevelopmental disorders (NDDs), and most, including the recurrent 15q13.3 microdeletion disorder, have unknown disease mechanisms. We used a heterozygous 15q13.3 microdeletion mouse model and patient iPSC-derived neurons to reveal developmental defects in neuronal maturation and network activity. To identify the underlying molecular dysfunction, we developed a neuron-specific proximity-labeling proteomics (BioID2) pipeline, combined with patient mutations, to target the 15q13.3 CNV genetic driver OTUD7A. OTUD7A is an emerging independent NDD risk gene with no known function in the brain, but has putative deubiquitinase function. The OTUD7A protein–protein interaction network included synaptic, axonal, and cytoskeletal proteins and was enriched for ASD and epilepsy risk genes (Ank3, Ank2, SPTAN1, SPTBN1). The interactions between OTUD7A and Ankyrin-G (Ank3) and Ankyrin-B (Ank2) were disrupted by an epilepsy-associated OTUD7A L233F variant. Further investigation of Ankyrin-G in mouse and human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed protein instability, increased polyubiquitination, and decreased levels in the axon initial segment, while structured illumination microscopy identified reduced Ankyrin-G nanodomains in dendritic spines. Functional analysis of human 15q13.3 microdeletion and OTUD7AL233F/L233F models revealed shared and distinct impairments to axonal growth and intrinsic excitability. Importantly, restoring OTUD7A or Ankyrin-G expression in 15q13.3 microdeletion neurons led to a reversal of abnormalities. These data reveal a critical OTUD7A-Ankyrin pathway in neuronal development, which is impaired in the 15q13.3 microdeletion syndrome, leading to neuronal dysfunction. Furthermore, our study highlights the utility of targeting CNV genes using cell type-specific proteomics to identify shared and unexplored disease mechanisms across NDDs.

SCN2A is an autism spectrum disorder (ASD) risk gene and encodes a voltage-gated sodium channel. However, the impact of autism-associated SCN2A de novo variants on human neuron development is unknown. We studied SCN2A using isogenic SCN2A-/- induced pluripotent stem cells (iPSCs), and patient-derived iPSCs harboring a p.R607* or a C-terminal p.G1744* de novo truncating variant. We used Neurogenin2 to generate excitatory glutamatergic neurons and found that SCN2A+/p.R607* and SCN2A-/- neurons displayed a reduction in synapse formation and excitatory synaptic activity using multielectrode arrays and electrophysiology. However, the p.G1744* variant, which leads to early-onset seizures in addition to ASD, altered action-potential dynamics but not synaptic activity. Proteomic and functional analysis of SCN2A+/p.R607* neurons revealed defects in neuronal morphology and bioenergetic pathways, which were not present in SCN2A+/p.G1744* neurons. Our study reveals that SCN2A de novo variants can have differential impact on human neuron function and signaling.

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