Karun K. Singh

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder caused by genetic or environmental perturbations during early development. Diagnoses are dependent on the identification of behavioral abnormalities that likely emerge well after the disorder is established, leaving critical developmental windows uncharacterized. This is further complicated by the incredible clinical and genetic heterogeneity of the disorder that is not captured in most mammalian models. In recent years, advancements in stem cell technology have created the opportunity to model ASD in a human context through the use of pluripotent stem cells (hPSCs), which can be used to generate 2D cellular models as well as 3D unguided- and region-specific neural organoids. These models produce profoundly intricate systems, capable of modeling the developing brain spatiotemporally to reproduce key developmental milestones throughout early development. When complemented with multi-omics, genome editing, and electrophysiology analysis, they can be used as a powerful tool to profile the neurobiological mechanisms underlying this complex disorder. In this review, we will explore the recent advancements in hPSC-based modeling, discuss present and future applications of the model to ASD research, and finally consider the limitations and future directions within the field to make this system more robust and broadly applicable.

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 driverOTUD7A.OTUD7Ais 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-associatedOTUD7AL233F variant. Further investigation of Ankyrin-G in mouse and human 15q13.3 microdeletion and OTUD7A^L233F/L233Fmodels 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 OTUD7A^L233F/L233Fmodels 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.

The precise development of the neocortex is a prerequisite for higher cognitive and associative functions. Despite numerous advances that have been made in understanding neuronal differentiation and cortex development, our knowledge regarding the impact of specific genes associated with neurodevelopmental disorders on these processes is still limited. Here, we show that Taok2, which is encoded in humans within the autism spectrum disorder (ASD) susceptibility locus 16p11.2, is essential for neuronal migration. Overexpression of de novo mutations or rare variants from ASD patients disrupts neuronal migration in an isoform-specific manner. The mutated TAOK2α variants but not the TAOK2β variants impaired neuronal migration. Moreover, the TAOK2α isoform colocalizes with microtubules. Consequently, neurons lacking Taok2 have unstable microtubules with reduced levels of acetylated tubulin and phosphorylated JNK1. Mice lacking Taok2 develop gross cortical and cortex layering abnormalities. Moreover, acute Taok2 downregulation or Taok2 knockout delayed the migration of upper-layer cortical neurons in mice, and the expression of a constitutively active form of JNK1 rescued these neuronal migration defects. Finally, we report that the brains of the Taok2 KO and 16p11.2 del Het mouse models show striking anatomical similarities and that the heterozygous 16p11.2 microdeletion mouse model displayed reduced levels of phosphorylated JNK1 and neuronal migration deficits, which were ameliorated upon the introduction of TAOK2α in cortical neurons and in the developing cortex of those mice. These results delineate the critical role of TAOK2 in cortical development and its contribution to neurodevelopmental disorders, including ASD.

Manuscript summary: There are hundreds of risk genes associated with autism spectrum disorder (ASD), but signaling networks at the protein level remain unexplored. We use neuron-specific proximity-labeling proteomics (BioID) to identify protein-protein interaction (PPI) networks for 41 ASD-risk genes. Neuron-specific PPI networks, including synaptic transmission proteins, are disrupted by de novo missense variants. The PPI network map reveals convergent pathways, including mitochondrial/metabolic processes, Wnt signaling, and MAPK signaling. CRISPR knockout reveal an association between mitochondrial activity and ASD-risk genes. The PPI network shows an enrichment of 112 additional ASD-risk genes and differentially expressed genes from post-mortem ASD patients. Clustering of risk genes based on PPI networks identifies gene groups corresponding to clinical behavior score severity. Our data reveal that cell type-specific PPI networks can identify individual and convergent ASD signaling networks, provide a method to assess patient variants, and reveal biological insight into disease mechanisms and sub-cohorts of ASD.

Copy number variations (CNV) 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 (DUB) function. The OTUD7A protein-protein interaction (PPI) network revealed interactions with synaptic, axonal, and cytoskeletal proteins and was enriched for known ASD and epilepsy risk genes. The interactions between OTUD7A and the NDD risk genes 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 OTUD7A ^L233F/L233F models revealed protein instability, increased polyubiquitination, and decreased levels in the axon initial segment (AIS), while structured illumination microscopy identified reduced Ankyrin-G nanodomains in dendritic spines. Functional analysis of human 15q13.3 microdeletion and OTUD7A ^L233F/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. Further, our study highlights the utility of targeting CNV genes using cell-type specific proteomics to identify shared and unexplored disease mechanisms across NDDs.

SUMMARY: 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. HIGHTLIGHTS: - Isogenic SCN2A ^-/- neurons display intrinsic hyperexcitability and impaired excitatory synapse function - SCN2A ^{+/ p . R607 *} variant reduces excitatory synapse function in patient neurons - C-terminal SCN2A ^{+/ p . G1744 *} variant enhances action potential properties but not synaptic transmission in patient neurons - SCN2A ^{+/ p . R607 *} variant display impacts on morphological and bioenergetic signaling networks through proteomic and functional analysis eTOC: - Brown et al. examined Autism-associated SCN2A variants using patient-derived iPSC NGN2-neurons. They discover that genetic variants differentially impact neuronal development and synaptic function, and highlight neuronal and bioenergetic signaling networks underlying SCN2A loss-of-function.

Background: Pain is a complex neurobiological response with a multitude of causes, however, patients with ASD often report chronic pain with no known etiology. Recent research has been aimed towards 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, as a better understanding is needed of this subpopulation.

DIX-domain containing 1 (Dixdc1) is an important regulator of neuronal development including cortical neurogenesis, neuronal migration and synaptic connectivity, and sequence variants in the gene have been linked to autism spectrum disorders (ASDs). Previous studies indicate that Dixdc1 controls neurogenesis through Wnt signaling, whereas its regulation of dendrite and synapse development requires Wnt and cytoskeletal signaling. However, the prediction of these signaling pathways is primarily based on the structure of Dixdc1. Given the role of Dixdc1 in neural development and brain disorders, we hypothesized that Dixdc1 may regulate additional signaling pathways in the brain. We performed transcriptomic and proteomic analyses of Dixdc1 KO mouse cortices to reveal such alterations. We found that transcriptomic approaches do not yield any novel findings about the downstream impacts of Dixdc1. In comparison, our proteomic approach reveals that several important mitochondrial proteins are significantly dysregulated in the absence of Dixdc1, suggesting a novel function of Dixdc1.

Proteomics is the large-scale study of the total protein content and their overall function within a cell through multiple facets of research. Advancements in proteomic methods have moved past the simple quantification of proteins to the identification of post-translational modifications (PTMs) and the ability to probe interactions between these proteins, spatially and temporally. Increased sensitivity and resolution of mass spectrometers and sample preparation protocols have drastically reduced the large amount of cells required and the experimental variability that had previously hindered its use in studying human neurological disorders. Proteomics offers a new perspective to study the altered molecular pathways and networks that are associated with autism spectrum disorders (ASD). The differences between the transcriptome and proteome, combined with the various types of post-translation modifications that regulate protein function and localization, highlight a novel level of research that has not been appropriately investigated. In this review, we will discuss strategies using proteomics to study ASD and other neurological disorders, with a focus on how these approaches can be combined with induced pluripotent stem cell (iPSC) studies. Proteomic analysis of iPSC-derived neurons have already been used to measure changes in the proteome caused by patient mutations, analyze changes in PTMs that resulted in altered biological pathways, and identify potential biomarkers. Further advancements in both proteomic techniques and human iPSC differentiation protocols will continue to push the field towards better understanding ASD disease pathophysiology. Proteomics using iPSC-derived neurons from individuals with ASD offers a window for observing the altered proteome, which is necessary in the future development of therapeutics against specific targets.