Biology subjects

Skarnes, W. C.

Publications and source records attributed to Skarnes, W. C..

4 recordsLinked to original sources

bioRxiv2021

Severe deficiency of voltage-gated sodium channel NaV1.2 elevates neuronal excitability in adult mice

Scn2a encodes voltage-gated sodium channel NaV1.2, which mediates neuronal firing. The current paradigm suggests that NaV1.2 gain-of-function variants enhance neuronal excitability resulting in epilepsy, whereas NaV1.2 deficiency impairs neuronal excitability contributing to autism. In this paradigm, however, why about a third of patients with NaV1.2 deficiency still develop seizures remains a mystery. Here we challenge the conventional wisdom, reporting that neuronal excitability is increased with severe NaV1.2 deficiency. Using a unique gene-trap knockout mouse model of Scn2a, we found enhanced intrinsic excitabilities of principal neurons in the cortico-striatal circuit, known to be involved in Scn2a-related seizures. This increased excitability is autonomous, and is reversible by genetic restoration of Scn2a expression in adult mice. Mechanistic investigation reveals a compensatory downregulation of potassium channels including KV1.1, which could be targeted to alleviate neuronal hyperexcitability. Our unexpected findings may explain NaV1.2 deficiency-related epileptic seizures in humans and provide molecular targets for potential interventions. TEASERSevere NaV1.2 deficiency results in neuronal hyperexcitability via the compensatory downregulation of potassium channels. HIGHLIGHTSO_LISevere NaV1.2 deficiency results in enhanced excitability of medium spiny neurons (MSNs) and pyramidal neurons in adult mice; C_LIO_LIIncreased neuronal excitability in MSNs is accompanied by elevated voltage threshold; C_LIO_LINaV1.2 deficiency-related hyperexcitability is reversible with the restoration of Scn2a expression, and is autonomous; C_LIO_LIThe expression of the KV1.1 channel has a compensatory reduction in neurons with NaV1.2 deficiency, and KV channels openers normalize the neuronal excitability; C_LIO_LIThe enhanced excitability in brain slices translates to elevated in vivo firing commonly associated with seizures. C_LI

neuroscience↗

bioRxiv2021

Sodium channel Nav1.2-L1342P variant displaying complex biophysical properties renders hyperexcitability of cortical neurons derived fromhuman iPSCs

With the wide adoption of whole-exome sequencing in children having seizures, an increasing number of SCN2A variants has been revealed as possible genetic causes of epilepsy. Voltage-gated sodium channel Nav1.2, encoded by gene SCN2A, is strongly expressed in the pyramidal excitatory neurons and supports action potential firing. One recurrent SCN2A variant is L1342P, which was identified in multiple patients with early-onset encephalopathy and intractable seizures. Our biophysical analysis and computational modeling predicted gain-of-function features of this epilepsy-associated Nav1.2 variant. However, the mechanism underlying L1342P mediated seizures and the pharmacogenetics of this variant in human neurons remain unknown. To understand the core phenotypes of the L1342P variant in human neurons, we took advantage of a reference human induced pluripotent stem cell (hiPSC) line, in which L1342P was engineered by CRISPR/Cas9 mediated genome-editing. Using patch-clamping and micro-electrode array (MEA) recording, we found that the cortical neurons derived from hiPSCs carrying heterozygous L1342P variant presented significantly increased intrinsic excitability, higher sodium current density, and enhanced bursting and synchronous network firing, showing clear hyperexcitability phenotypes. Interestingly, the L1342P neuronal culture displayed a degree of resistance to the anti-seizure medication (phenytoin), which likely recapitulated aspects of clinical observation of patients carrying the L1342P variant. In contrast, phrixotoxin-3 (PTx3), a Nav1.2 isoform-specific blocker, was able to potently alleviate spontaneous and chemical-induced hyperexcitability of neurons carrying the L1342P variant. Our results reveal a possible pathogenic underpinning of Nav1.2-L1342P mediated epileptic seizures, and demonstrate the utility of genome-edited hiPSCs as an in vitro platform to advance personalized phenotyping and drug discovery.

physiology↗

bioRxiv2020

Dissecting mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage

Mutational signatures are imprints of pathophysiological processes arising through tumorigenesis. Here, we generate isogenic CRISPR-Cas9 knockouts ({Delta}) of 43 genes in human induced pluripotent stem cells, culture them in the absence of added DNA damage, and perform wholegenome sequencing of 173 daughter subclones. {Delta}OGG1, {Delta}UNG, {Delta}EXO1, {Delta}RNF168, {Delta}MLH1, {Delta}MSH2, {Delta}MSH6, {Delta}PMS1, and {Delta}PMS2 produce marked mutational signatures indicative of being critical mitigators of endogenous DNA changes. Detailed analyses reveal that 8-oxo-dG removal by different repair proteins is sequence-context-specific while uracil clearance is sequencecontext-independent. Signatures of mismatch repair (MMR) deficiency show components of C>A transversions due to oxidative damage, T>C and C>T transitions due to differential misincorporation by replicative polymerases, and T>A transversions for which we propose a reverse template slippage model. {Delta}MLH1, {Delta}MSH6, and {Delta}MSH2 signatures are similar to each other but distinct from {Delta}PMS2. We validate these gene-specificities in cells from patients with Constitutive Mismatch Repair Deficiency Syndrome. Based on these experimental insights, we develop a classifier, MMRDetect, for improved clinical detection of MMR-deficient tumors.

genomics↗

bioRxiv2020

Characterization of a gene-trap knockout mouse model of Scn2a encoding voltage-gated sodium channel Nav1.2

Recent large-scale genomic studies have revealed SCN2A as one of the most frequently mutated gene in patients with neurodevelopmental disorders including autism spectrum disorder and intellectual disability. SCN2A encodes for voltage-gated sodium channel isoform 1.2 (Nav1.2), which is mainly expressed in the central nervous system and responsible for the propagation of neuronal action potentials. Homozygous knockout (null) of Scn2a is perinatal lethal, whereas heterozygous knockout of Scn2a results in mild behavior abnormalities. To achieve a more substantial, but not complete, reduction of Scn2a expression, we characterized a Scn2a deficient mouse model using a targeted gene trap knockout (gtKO) strategy to recapitulate loss-of-function SCN2A disorders. This model produces viable homozygous mice (Scn2agtKO/gtKO) that can survive to adulthood, with markedly low but detectable Nav1.2 expression. Although Scn2agtKO/gtKO adult mice possess normal olfactory, taste, hearing, and mechanical sensitivity, they have decreased thermal and cold tolerance. Innate behaviors are profoundly impaired including impaired nesting, marble burying, and mating. These mice also have increased food and water intake with subsequent increases in fecal excretion of more but smaller fecal boli. This novel Scn2a gene trap knockout mouse thus provides a unique model to study pathophysiology associated with Scn2a deficiency.

neuroscience↗