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DNA methylation is an important regulatory mechanism in the control of neuronal function. Both during development and following exposure to salient stimuli, plasticity in the methylation of cytosine residues leads to a change in neuron excitability that subsequently sculpts animal behavior. However, although the response of DNA methyltransferase enzymes in adult neurons to stimuli such as drugs of abuse have been described, less is known about how these enzymes regulate methylation at specific loci to change the drive to ingest natural rewards. Specifically, we do not understand how changes in methylation within important brain areas known to regulate palatable food intake can affect ingestion, while a detailed investigation of the neurophysiological and genomic effects of perturbing methyltransferase function has not been pursued. By deleting DNA methyltransferase 1 and 3a in the mouse prefrontal cortex, we observed the requirement for these enzymes in the regulation of nutrient rich food consumption in the absence of any effect on the intake of low fat and low sugar chow. We also determined that the deletion profoundly affected neuron excitability within pyramidal cells resident in superficial layers II/III of the cortex but had little effect in deep layer V neurons. Finally, reduced representation bisulfite sequencing revealed both hypo and hypermethylation in response to methyltransferase deletion, an effect that was observed in binding sites for retinoic acid receptor beta (RARβ) located within regulatory regions of genes known to affect neuronal function. Together, our data suggest that alterations in the actions of RARβ could shift neuronal activity to reduce palatable food intake.

Objective: Monoallelic de novo gain-of-function variants in the voltage-gated sodium channel SCN8A are one of the recurrent causes of severe developmental and epileptic encephalopathy (DEE). In addition, a small number of de novo or inherited monoallelic loss-of-function variants have been found in patients with intellectual disability, autism spectrum disorder, or movement disorders. Inherited monoallelic variants causing either gain or loss-of-function are also associated with less severe conditions such as benign familial infantile seizures and isolated movement disorders. In all three categories, the affected individuals are heterozygous for a SCN8A variant in combination with a wild-type allele. In the present study, we describe two unusual families with severely affected individuals who inherited biallelic variants of SCN8A. Methods: We identified two families with biallelic SCN8A variants by diagnostic gene panel sequencing. Functional analysis of the variants was performed using voltage clamp recordings from transfected ND7/23 cells. Results: We identified three probands from two unrelated families with DEE due to biallelic SCN8A variants. Each parent of an affected individual carried a single heterozygous SCN8A variant and exhibited mild cognitive impairment without seizures. In both families, functional analysis demonstrated segregation of one allele with complete loss-of-function, and one allele with altered biophysical properties consistent with partial loss-of-function. Significance: These studies demonstrate that SCN8A DEE may, in rare cases, result from inheritance of two variants, both of which exhibit reduced channel activity. In these families, heterozygosity for the dominant variants results in less severe disease than biallelic inheritance of two variant alleles. The clinical consequences of variants with partial and complete loss of SCN8A function are variable and likely to be influenced by genetic background.

SCN8A epileptic encephalopathy is a severe genetic epilepsy syndrome caused by de novo gain-of-function mutations of SCN8A encoding the voltage-gated sodium (Na) channel (VGSC) Na(V)1.6. Therapeutic management is difficult in many patients, leading to uncontrolled seizures and risk of sudden unexpected death in epilepsy (SUDEP). There is a need to develop novel anticonvulsants that can specifically target aberrant Na channel activity associated with SCN8A gain-of-function mutations. In this study, we investigate the effects of Prax330, a novel Na channel inhibitor, on the biophysical properties of WT Na(V)1.6 and the patient mutation p.Asn1768Asp (N1768D) in ND7/23 cells. The effects of Prax330 on persistent (I(NaP)) and resurgent (I(NaR)) Na currents and neuronal excitability in subiculum neurons from a knock-in mouse model of the Scn8a-N1768D mutation (Scn8a(D/+)) were also examined. In ND7/23 cells, Prax330 reduced I(NaP) currents recorded from cells expressing Scn8a-N1768D and hyperpolarized steady-state inactivation curves. Recordings from brain slices demonstrated elevated I(NaP) and I(NaR) in subiculum neurons from Scn8a(D/+) mutant mice and abnormally large action potential (AP) burst-firing events in a subset of neurons. Prax330 (1 μM) reduced both I(NaP) and I(NaR) and suppressed AP bursts, with smaller effect on AP waveforms that had similar morphology to WT neurons. Prax330 (1 μM) also reduced synaptically-evoked APs in Scn8a(D/+) subiculum neurons but not in WT neurons. Our results highlight the efficacy of targeting I(NaP) and I(NaR) and inactivation parameters in controlling subiculum excitability and suggest Prax330 as a promising novel therapy for SCN8A epileptic encephalopathy.