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SCN2A loss-of-function variants cause a range of neurodevelopmental disorders. Here, we present a protocol to induce severe Scn2a insufficiency in mice. We describe steps for intracerebroventricular (ICV) antisense oligonucleotide (ASO) injection that causes a selective downregulation of Scn2a and ASO-mediated mRNA degradation. We then detail procedures for qPCR and western blot protocol to measure Scn2a mRNA and protein. This protocol can be used as a mouse model for behavioral and in vivo two-photon Ca 2+ imaging., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Children diagnosed with autism spectrum disorder (ASD) commonly present with sensory hypersensitivity or abnormally strong reactions to sensory stimuli. Such hypersensitivity can be overwhelming, causing high levels of distress that contribute markedly to the negative aspects of the disorder. Here, we identify a mechanism that underlies hypersensitivity in a sensorimotor reflex found to be altered in humans and in mice with loss of function in the ASD risk-factor gene SCN2A. The cerebellum-dependent vestibulo-ocular reflex (VOR), which helps maintain one's gaze during movement, was hypersensitized due to deficits in cerebellar synaptic plasticity. Heterozygous loss of SCN2A-encoded Na V 1.2 sodium channels in granule cells impaired high-frequency transmission to Purkinje cells and long-term potentiation, a form of synaptic plasticity important for modulating VOR gain. VOR plasticity could be rescued in mice via a CRISPR-activator approach that increases Scn2a expression, demonstrating that evaluation of a simple reflex can be used to assess and quantify successful therapeutic intervention., Competing Interests: Declaration of interests N.A. is the cofounder and on the scientific advisory board of Regel Tx. K.J.B. is on the scientific advisory board of Regel Tx. N.A. and K.J.B. receive funding from BioMarin Pharmaceutical Incorporated., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Dysfunction in sodium channels and their ankyrin scaffolding partners have both been implicated in neurodevelopmental disorders, including autism spectrum disorder (ASD). In particular, the genes SCN2A, which encodes the sodium channel Na V 1.2, and ANK2, which encodes ankyrin-B, have strong ASD association. Recent studies indicate that ASD-associated haploinsufficiency in Scn2a impairs dendritic excitability and synaptic function in neocortical pyramidal cells, but how Na V 1.2 is anchored within dendritic regions is unknown. Here, we show that ankyrin-B is essential for scaffolding Na V 1.2 to the dendritic membrane of mouse neocortical neurons and that haploinsufficiency of Ank2 phenocopies intrinsic dendritic excitability and synaptic deficits observed in Scn2a +/- conditions. These results establish a direct, convergent link between two major ASD risk genes and reinforce an emerging framework suggesting that neocortical pyramidal cell dendritic dysfunction can contribute to neurodevelopmental disorder pathophysiology., Competing Interests: Declaration of interests K.J.B. and S.J.S. receive research funding from BioMarin Pharmaceutical Inc. K.J.B. is on the SAB for Regel Tx and receives research support from Regel Tx., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Neuronal voltage-gated sodium channel Na V 1.2 C-terminal domain (CTD) binds calmodulin (CaM) constitutively at its IQ motif. A solution structure (6BUT) and other NMR evidence showed that the CaM N domain (CaM N ) is structurally independent of the C-domain (CaM C ) whether CaM is bound to the Na V 1.2 IQp (1,901-1,927) or Na V 1.2 CTD (1,777-1,937) with or without calcium. However, in the CaM + Na V 1.2 CTD complex, the Ca 2+ affinity of CaM N was more favorable than in free CaM, while Ca 2+ affinity for CaM C was weaker than in the CaM + Na V 1.2 IQp complex. The CTD EF-like (EFL) domain allosterically widened the energetic gap between CaM domains. Cardiomyopathy-associated CaM mutants (N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)) all bound the Na V 1.2 IQ motif favorably under resting (apo) conditions and bound calcium normally at CaM N sites. However, only N53I and A102V bound calcium at CaM C sites at [Ca 2+ ] < 100 μM. Thus, they are expected to respond like wild-type CaM to Ca 2+ spikes in excitable cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Alternative splicing plays an important role in brain development, but its global contribution to human neurodevelopmental diseases (NDDs) requires further investigation. Here we examine the relationships between splicing isoform expression in the brain and de novo loss-of-function mutations from individuals with NDDs. We analyze the full-length isoform transcriptome of the developing human brain and observe differentially expressed isoforms and isoform co-expression modules undetectable by gene-level analyses. These isoforms are enriched in loss-of-function mutations and microexons, are co-expressed with a unique set of partners, and have higher prenatal expression. We experimentally test the effect of splice-site mutations and demonstrate exon skipping in five NDD risk genes, including SCN2A, DYRK1A, and BTRC. Our results suggest that the splice site mutation in BTRC reduces translational efficiency, likely affecting Wnt signaling through impaired degradation of β-catenin. We propose that functional effects of mutations should be investigated at the isoform- rather than gene-level resolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Autism spectrum disorder (ASD) is strongly associated with de novo gene mutations. One of the most commonly affected genes is SCN2A. ASD-associated SCN2A mutations impair the encoded protein Na V 1.2, a sodium channel important for action potential initiation and propagation in developing excitatory cortical neurons. The link between an axonal sodium channel and ASD, a disorder typically attributed to synaptic or transcriptional dysfunction, is unclear. Here we show that Na V 1.2 is unexpectedly critical for dendritic excitability and synaptic function in mature pyramidal neurons in addition to regulating early developmental axonal excitability. Na V 1.2 loss reduced action potential backpropagation into dendrites, impairing synaptic plasticity and synaptic strength, even when Na V 1.2 expression was disrupted in a cell-autonomous fashion late in development. These results reveal a novel dendritic function for Na V 1.2, providing insight into cellular mechanisms probably underlying circuit and behavioral dysfunction in ASD., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Single nucleotide variants (SNVs), particularly loss-of-function mutations, are significant contributors to autism spectrum disorder (ASD) risk. Here we report the first systematic deep sequencing study of 55 postmortem ASD brains for SNVs in 78 known ASD candidate genes. Remarkably, even without parental samples, we find more ASD brains with mutations that are protein-altering (26/55 cases versus 12/50 controls, p = 0.015), deleterious (16/55 versus 5/50, p = 0.016), or loss-of-function (6/55 versus 0/50, p = 0.028) compared to controls, with recurrent deleterious mutations in ARID1B, SCN1A, SCN2A, and SETD2, suggesting these mutations contribute to ASD risk. In several cases, the identified mutations and medical records suggest syndromic ASD diagnoses. Two ASD and one Fragile X premutation case showed deleterious somatic mutations, providing evidence that somatic mutations occur in ASD cases, and supporting a model in which a combination of germline and/or somatic mutations may contribute to ASD risk on a case-by-case basis., (Copyright © 2015 Elsevier Inc. All rights reserved.)