Abstract
SCN2A, encoding the neuronal voltage-gated Na+ channel NaV1.2, is one of the most commonly affected loci linked to autism spectrum disorders (ASDs). Most ASD-associated mutations in SCN2A are loss-of-function mutations, but studies examining how such mutations affect neuronal function and whether Scn2a mutant mice display ASD endophenotypes have been inconsistent. We generated a protein truncation variant Scn2a mouse model (Scn2aΔ1898/+) by CRISPR that eliminates the NaV1.2 channel’s distal intracellular C-terminal domain, and we analyzed the molecular and cellular consequences of this variant in a heterologous expression system, in neuronal culture, in brain slices, and in vivo. We also analyzed multiple behaviors in WT and Scn2aΔ1898/+ mice and correlated behaviors with clinical data obtained in human subjects with SCN2A variants. Expression of the NaV1.2 mutant in a heterologous expression system revealed decreased NaV1.2 channel function, and cultured pyramidal neurons isolated from Scn2aΔ1898/+ forebrain showed correspondingly reduced voltage-gated Na+ channel currents without compensation from other CNS voltage-gated Na+ channels. Na+ currents in inhibitory neurons were unaffected. Consistent with loss of voltage-gated Na+ channel currents, Scn2aΔ1898/+ pyramidal neurons displayed reduced excitability in forebrain neuronal culture and reduced excitatory synaptic input onto the pyramidal neurons in brain slices. Scn2aΔ1898/+ mice displayed several behavioral abnormalities, including abnormal social interactions that reflect behavior observed in humans with ASD and with harboring loss-of-function SCN2A variants. This model and its cellular electrophysiological characterizations provide a framework for tracing how a SCN2A loss-of-function variant leads to cellular defects that result in ASD-associated behaviors.
Highlights
Within the CNS, voltage-gated Na+ (NaV) channels such as the SCN2A-encoded NaV1.2 initiate action potentials (APs) and are, fundamental to defining neuronal excitability
We found that severe reduction of NaV1.2 function reduced neuronal excitability in cultured forebrain pyramidal neurons isolated from Scn2aΔ1898/+ forebrains, and it decreased excitatory synaptic input to pyramidal neurons in the medial prefrontal cortex and basolateral amygdala (BLA) in acute brain slice from adult Scn2aΔ1898/+ mice
As Scn2a is almost exclusively expressed in excitatory neurons, and since Na+ channels drive APs, we characterized APs elicited in the cultured forebrain pyramidal neurons in which we identified the reduction in total Na+ currents for Scn2aΔ1898/+ mice
Summary
Within the CNS, voltage-gated Na+ (NaV) channels such as the SCN2A-encoded NaV1.2 initiate action potentials (APs) and are, fundamental to defining neuronal excitability. Most mature excitatory CNS neurons express NaV1.2 and NaV1.6, and these NaV channels confer distinct features that tweak electrical activity and contribute to the defining features of AP initiation and conduction in different types of neurons. NaV1.2 is the dominant Na+ channel expressed in excitatory neurons, where it predominantly localizes to the axon initial segment [2, 3]. Because of this preferential expression of NaV1.2 during the vulnerable developmental period when most autism spectrum disorder–associated (ASD-associated) mutations exert their influence [4], NaV1.2 is well positioned to exert a potent effect on AP initiation and conduction, thereby influencing neuronal excitability and activity-dependent development in the maturing brain. Analyses in heterologous expression systems of several disease-associated SCN2A variants found that ASD-associated mutations generally cause channel loss-of-function effects (often because of protein truncation variants), while epilepsyassociated mutations reveal various biophysical gain-of-function effects (increased Na+ influx) [10]
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