Carvedilol increases seizure resistance in a mouse model of SCN8A-derived epilepsy.

Patients with mutations that alter the function of the sodium channel SCN8A present with a range of clinical features, including mild to severe seizures, developmental delay, intellectual disability, autism, feeding dysfunction, motor impairment, and hypotonia. In an effort to identify compounds that could be potentially beneficial in SCN8A-associated epilepsy, Atkin et al. conducted an in vitro screen which resulted in the identification of 90 compounds that effectively reduced sodium influx into the cells expressing the human SCN8A R1872Q mutation. The top compounds that emerged from this screen included amitriptyline, carvedilol, and nilvadipine. In the current study, we evaluated the ability of these three compounds to increase resistance to 6 Hz or pentylenetetrazole (PTZ)-induced seizures in wild-type CF1 mice and in a mouse line expressing the human SCN8A R1620L mutation. We also evaluated the effects of fenfluramine administration, which was recently associated with a 60%-90% decrease in seizure frequency in three patients with SCN8A-associated epilepsy. While amitriptyline, carvedilol, and fenfluramine provided robust protection against induced seizures in CF1 mice, only carvedilol was able to significantly increase resistance to 6 Hz- and PTZ-induced seizures in RL/+ mutants. These results provide support for further evaluation of carvedilol as a potential treatment for patients with SCN8A mutations.

Generation and initial characterization of mice lacking full-length BAI3 (ADGRB3) expression.

Brain-specific angiogenesis inhibitor 3 (ADGRB3/BAI3) belongs to the family of adhesion G protein-coupled receptors. It is most highly expressed in the brain where it plays a role in synaptogenesis and synapse maintenance. Genome-wide association studies have implicated ADGRB3 in disorders such as schizophrenia and epilepsy. Somatic mutations in ADGRB3 have also been identified in cancer. To better understand the in vivo physiological role of ADGRB3, we used CRISPR/Cas9 editing to generate a mouse line with a 7-base pair deletion in Adgrb3 exon 10. Western blot analysis confirmed that homozygous mutants (Adgrb3∆7/∆7 ) lack full-length ADGRB3 expression. The mutant mice were viable and reproduced in Mendelian ratios but demonstrated reduced brain and body weights and deficits in social interaction. Measurements of locomotor function, olfaction, anxiety levels and prepulse inhibition were comparable between heterozygous and homozygous mutants and wild-type littermates. Since ADGRB3 is also expressed in organs such as lung and pancreas, this new mouse model will facilitate elucidation of ADGRB3's role in non-central nervous system-related functions. Finally, since somatic mutations in ADGRB3 were identified in patients with several cancer types, these mice can be used to determine whether loss of ADGRB3 function contributes to tumour development.

Mutations in plasticity-related-gene-1 (PRG-1) protein contribute to hippocampal seizure susceptibility and modify epileptic phenotype.

The Phospholipid Phosphatase Related 4 gene (PLPPR4,  *607813) encodes the Plasticity-Related-Gene-1 (PRG-1) protein. This cerebral synaptic transmembrane-protein modulates cortical excitatory transmission on glutamatergic neurons. In mice, homozygous Prg-1 deficiency causes juvenile epilepsy. Its epileptogenic potential in humans was unknown. Thus, we screened 18 patients with infantile epileptic spasms syndrome (IESS) and 98 patients with benign familial neonatal/infantile seizures (BFNS/BFIS) for the presence of PLPPR4 variants. A girl with IESS had inherited a PLPPR4-mutation (c.896C > G, NM_014839; p.T299S) from her father and an SCN1A-mutation from her mother (c.1622A > G, NM_006920; p.N541S). The PLPPR4-mutation was located in the third extracellular lysophosphatidic acid-interacting domain and in-utero electroporation (IUE) of the Prg-1p.T300S construct into neurons of Prg-1 knockout embryos demonstrated its inability to rescue the electrophysiological knockout phenotype. Electrophysiology on the recombinant SCN1Ap.N541S channel revealed partial loss-of-function. Another PLPPR4 variant (c.1034C > G, NM_014839; p.R345T) that was shown to result in a loss-of-function aggravated a BFNS/BFIS phenotype and also failed to suppress glutamatergic neurotransmission after IUE. The aggravating effect of Plppr4-haploinsufficiency on epileptogenesis was further verified using the kainate-model of epilepsy: double heterozygous Plppr4-/+|Scn1awt|p.R1648H mice exhibited higher seizure susceptibility than either wild-type, Plppr4-/+, or Scn1awt|p.R1648H littermates. Our study shows that a heterozygous PLPPR4 loss-of-function mutation may have a modifying effect on BFNS/BFIS and on SCN1A-related epilepsy in mice and humans.

Novel Missense CNTNAP2 Variant Identified in Two Consanguineous Pakistani Families With Developmental Delay, Epilepsy, Intellectual Disability, and Aggressive Behavior.

We report the genetic analysis of two consanguineous pedigrees of Pakistani ancestry in which two siblings in each family exhibited developmental delay, epilepsy, intellectual disability and aggressive behavior. Whole-genome sequencing was performed in Family 1, and we identified ~80,000 variants located in regions of homozygosity. Of these, 615 variants had a minor allele frequency ≤ 0.001, and 21 variants had CADD scores ≥ 15. Four homozygous exonic variants were identified in both affected siblings: PDZD7 (c.1348_1350delGAG, p.Glu450del), ALG6 (c.1033G>C, p.Glu345Gln), RBM20 (c.1587C>G, p.Ser529Arg), and CNTNAP2 (c.785G>A, p.Gly228Arg). Sanger sequencing revealed co-segregation of the PDZD7, RBM20, and CNTNAP2 variants with disease in Family 1. Pathogenic variants in PDZD7 and RBM20 are associated with autosomal recessive non-syndromic hearing loss and autosomal dominant dilated cardiomyopathy, respectively, suggesting that these variants are unlikely likely to contribute to the clinical presentation. Gene panel analysis was performed on the two affected siblings in Family 2, and they were found to also be homozygous for the p.Gly228Arg CNTNAP2 variant. Together these families provide a LOD score 2.9 toward p.Gly228Arg CNTNAP2 being a completely penetrant recessive cause of this disease. The clinical presentation of the affected siblings in both families is also consistent with previous reports from individuals with homozygous CNTNAP2 variants where at least one allele was a nonsense variant, frameshift or small deletion. Our data suggests that homozygous CNTNAP2 missense variants can also contribute to disease, thereby expanding the genetic landscape of CNTNAP2 dysfunction.

Cannabidiol Increases Seizure Resistance and Improves Behavior in an Scn8a Mouse Model.

Voltage-gated sodium channel genes are an important family of human epilepsy genes. De novo missense mutations in SCN8A (encoding Nav1.6) are associated with a spectrum of clinical presentation, including multiple seizure types, movement disorders, intellectual disability, and behavioral abnormalities such as autism. Patients with SCN8A mutations are often treated with multiple antiepileptic drugs, the most common being sodium channel blockers. Cannabidiol (CBD) has been included as a component of treatment regimens for some SCN8A patients; however, to date, there are no clinical trials that have evaluated the therapeutic potential of CBD in patients with SCN8A mutations. In the current manuscript, we demonstrated a dose-dependent increase in seizure resistance following CBD treatment in mice expressing the human SCN8A mutation R1620L (RL/+). We also found that CBD treatment improved social behavior and reduced hyperactivity in the RL/+ mutants. Our findings suggest that CBD may be beneficial in patients with SCN8A-associated disease.

Mice lacking full length Adgrb1 (Bai1) exhibit social deficits, increased seizure susceptibility, and altered brain development.

The adhesion G protein-coupled receptor BAI1/ADGRB1 plays an important role in suppressing angiogenesis, mediating phagocytosis, and acting as a brain tumor suppressor. BAI1 is also a critical regulator of dendritic spine and excitatory synapse development and interacts with several autism-relevant proteins. However, little is known about the relationship between altered BAI1 function and clinically relevant phenotypes. Therefore, we studied the effect of reduced expression of full length Bai1 on behavior, seizure susceptibility, and brain morphology in Adgrb1 mutant mice. We compared homozygous (Adgrb1-/-), heterozygous (Adgrb1+/-), and wild-type (WT) littermates using a battery of tests to assess social behavior, anxiety, repetitive behavior, locomotor function, and seizure susceptibility. We found that Adgrb1-/- mice showed significant social behavior deficits and increased vulnerability to seizures. Adgrb1-/- mice also showed delayed growth and reduced brain weight. Furthermore, reduced neuron density and increased apoptosis during brain development were observed in the hippocampus of Adgrb1-/- mice, while levels of astrogliosis and microgliosis were comparable to WT littermates. These results show that reduced levels of full length Bai1 is associated with a broader range of clinically relevant phenotypes than previously reported.

Pathogenic in-Frame Variants in SCN8A: Expanding the Genetic Landscape of SCN8A-Associated Disease.

Numerous SCN8A mutations have been identified, of which, the majority are de novo missense variants. Most mutations result in epileptic encephalopathy; however, some are associated with less severe phenotypes. Mouse models generated by knock-in of human missense SCN8A mutations exhibit seizures and a range of behavioral abnormalities. To date, there are only a few Scn8a mouse models with in-frame deletions or insertions, and notably, none of these mouse lines exhibit increased seizure susceptibility. In the current study, we report the generation and characterization of two Scn8a mouse models (ΔIRL/+ and ΔVIR/+) carrying overlapping in-frame deletions within the voltage sensor of domain 4 (DIVS4). Both mouse lines show increased seizure susceptibility and infrequent spontaneous seizures. We also describe two unrelated patients with the same in-frame SCN8A deletion in the DIV S5-S6 pore region, highlighting the clinical relevance of this class of mutations.

Extracellular vesicles in the treatment of neurological disorders.

Extracellular vesicles (EVs) are small, cell-derived membranous particles containing various nucleic acids, proteins, and lipids that play essential roles in intercellular communication. Evidence indicating that part of the regenerative benefit from stem cell therapy arises through EVs released from transplanted cells created interest in using EVs for clinical applications. EVs from various cellular sources, including mesenchymal stem cells, neural stem cells, and glia, are efficacious in models of neurological disease. In these models, EVs attenuate reactive gliosis, neuronal death, pro-inflammatory signaling, as well as reduce cognitive, behavioral, and motor deficits. EVs are naturally permeable to the blood-brain barrier and can be modified to contain molecules of interest, thereby also serving as a vehicle to transport therapeutics into the brain. This review summarizes the current state of research using EVs as a treatment in models of neurological disorders and highlights considerations for future research.

Autistic-like behavior, spontaneous seizures, and increased neuronal excitability in a Scn8a mouse model.

Patients with SCN8A epileptic encephalopathy exhibit a range of clinical features, including multiple seizure types, movement disorders, and behavioral abnormalities, such as developmental delay, mild-to-severe intellectual disability, and autism. Recently, the de novo heterozygous SCN8A R1620L mutation was identified in an individual with autism, intellectual disability, and behavioral seizures without accompanying electrographic seizure activity. To date, the effects of SCN8A mutations that are primarily associated with behavioral abnormalities have not been studied in a mouse model. To better understand the phenotypic and functional consequences of the R1620L mutation, we used CRISPR/Cas9 technology to generate mice expressing the corresponding SCN8A amino acid substitution. Homozygous mutants exhibit tremors and a maximum lifespan of 22 days, while heterozygous mutants (RL/+) exhibit autistic-like behaviors, such as hyperactivity and learning and social deficits, increased seizure susceptibility, and spontaneous seizures. Current clamp analyses revealed a reduced threshold for firing action potentials in heterozygous CA3 pyramidal neurons and reduced firing frequency, suggesting that the R1620L mutation has both gain- and loss-of-function effects. In vivo calcium imaging using miniscopes in freely moving RL/+ mutants showed hyperexcitability of cortical excitatory neurons that is likely to increase seizure susceptibility. Finally, we found that oxcarbazepine and Huperzine A, a sodium channel blocker and reversible acetylcholinesterase inhibitor, respectively, were capable of conferring robust protection against induced seizures in RL/+ mutants. This mouse line will provide the opportunity to better understand the range of clinical phenotypes associated with SCN8A mutations and to develop new therapeutic approaches.

Allosteric modulation of the cannabinoid 2 receptor confers seizure resistance in mice.

Mounting evidence suggests that modulation of cannabinoid 2 receptors (CB2Rs) is therapeutic in mouse models of neurological disorders, including neuropathic pain, neurodegenerative disease, and stroke. We previously showed that reducing CB2R activity increases seizure susceptibility in mice. In the present study, we evaluated the therapeutic potential of the CB2R positive allosteric modulator, Ec21a, against induced seizures in mice. The pharmacokinetic profile of Ec21 demonstrated a similar distribution in brain and plasma, with detection up to 12 h following injection. Ec21a increased resistance to induced seizures in CF1 wild-type mice and mice harboring the SCN1A R1648H human epilepsy mutation. A rotarod test provided evidence that Ec21a does not cause neurotoxicity-induced motor deficits at its therapeutic dose, and seizure protection was maintained with repeated drug administration. The selectivity of Ec21a for CB2R was supported by the ability of the CB2R antagonist AM630, but not the CB1R antagonist AM251, to block Ec21a-conferred seizure protection in mice, and a lack of significant binding of Ec21a to 34 brain-expressed receptors and transporters in vitro. These results identify allosteric modulation of CB2Rs as a promising therapeutic approach for the treatment of epilepsy.

Nanoparticle encapsulated oxytocin increases resistance to induced seizures and restores social behavior in Scn1a-derived epilepsy.

Oxytocin (OT) has broad effects in the brain and plays an important role in cognitive, social, and neuroendocrine function. OT has also been identified as potentially therapeutic in neuropsychiatric disorders such as autism and depression, which are often comorbid with epilepsy, raising the possibility that it might confer protection against the behavioral and seizure phenotypes in epilepsy. Dravet syndrome (DS) is an early-life encephalopathy associated with prolonged and recurrent early-life febrile seizures (FSs), treatment-resistant afebrile epilepsy, and cognitive and behavioral deficits. De novo loss-of-function mutations in the voltage-gated sodium channel SCN1A are the main cause of DS, while genetic epilepsy with febrile seizures plus (GEFS+), also characterized by early-life FSs and afebrile epilepsy, is typically caused by inherited mutations that alter the biophysical properties of SCN1A. Despite the wide range of available antiepileptic drugs, many patients with SCN1A mutations do not achieve adequate seizure control or the amelioration of associated behavioral comorbidities. In the current study, we demonstrate that nanoparticle encapsulation of OT conferred robust and sustained protection against induced seizures and restored more normal social behavior in a mouse model of Scn1a-derived epilepsy. These results demonstrate the ability of a nanotechnology formulation to significantly enhance the efficacy of OT. This approach will provide a general strategy to enhance the therapeutic potential of additional neuropeptides in epilepsy and other neurological disorders.

Transcriptomic and epigenomic dynamics associated with development of human iPSC-derived GABAergic interneurons.

GABAergic interneurons (GINs) are a heterogeneous population of inhibitory neurons that collectively contribute to the maintenance of normal neuronal excitability and network activity. Identification of the genetic regulatory elements and transcription factors that contribute toward GIN function may provide new insight into the pathways underlying proper GIN activity while also indicating potential therapeutic targets for GIN-associated disorders, such as schizophrenia and epilepsy. In this study, we examined the temporal changes in gene expression and chromatin accessibility during GIN development by performing transcriptomic and epigenomic analyses on human induced pluripotent stem cell-derived neurons at 22, 50 and 78 days (D) post-differentiation. We observed 13 221 differentially accessible regions (DARs) of chromatin that associate with temporal changes in gene expression at D78 and D50, relative to D22. We also classified families of transcription factors that are increasingly enriched at DARs during differentiation, indicating regulatory networks that likely drive GIN development. Collectively, these data provide a resource for examining the molecular networks regulating GIN functionality.

Mutations in the Scn8a DIIS4 voltage sensor reveal new distinctions among hypomorphic and null Nav 1.6 sodium channels.

Mutations in the voltage-gated sodium channel gene SCN8A cause a broad range of human diseases, including epilepsy, intellectual disability, and ataxia. Here we describe three mouse lines on the C57BL/6J background with novel, overlapping mutations in the Scn8a DIIS4 voltage sensor: an in-frame 9 bp deletion (Δ9), an in-frame 3 bp insertion (∇3) and a 35 bp deletion that results in a frameshift and the generation of a null allele (Δ35). Scn8a Δ9/+ and Scn8a ∇3/+ heterozygous mutants display subtle motor deficits, reduced acoustic startle response, and are resistant to induced seizures, suggesting that these mutations reduce activity of the Scn8a channel protein, Nav 1.6. Heterozygous Scn8a Δ35/+ mutants show no alterations in motor function or acoustic startle response, but are resistant to induced seizures. Homozygous mutants from each line exhibit premature lethality and severe motor impairments, ranging from uncoordinated gait with tremor (Δ9 and ∇3) to loss of hindlimb control (Δ35). Scn8a Δ9/Δ9 and Scn8a ∇3/∇3 homozygous mutants also exhibit impaired nerve conduction velocity, while normal nerve conduction was observed in Scn8a Δ35/Δ35 homozygous mice. Our results suggest that hypomorphic mutations that reduce Nav 1.6 activity will likely result in different clinical phenotypes compared to null alleles. These three mouse lines represent a valuable opportunity to examine the phenotypic impacts of hypomorphic and null Scn8a mutations without the confound of strain-specific differences.

Reduced cannabinoid 2 receptor activity increases susceptibility to induced seizures in mice.

OBJECTIVE: The endocannabinoid system (ECS) is comprised of cannabinoid receptors 1 and 2 (CB1R and CB2R), endogenous ligands, and regulatory enzymes, and serves to regulate several important physiological functions throughout the brain and body. Recent evidence suggests that the ECS may be a promising target for the treatment of epilepsy, including epilepsy subtypes that arise from mutations in the voltage-gated sodium channel SCN1A. The objective of this study was to explore the effects of modulating CB2R activity on seizure susceptibility. METHODS: We examined susceptibility to induced seizures using a number of paradigms in CB2R knockout mice (Cnr2-/- ), and determined the effects of the CB2R agonist, JWH-133, and the CB2R antagonist, SR144528, on seizure susceptibility in wild-type mice. We also examined seizure susceptibility in Cnr2 mutants harboring the human SCN1A R1648H (RH) epilepsy mutation and performed Electroencephalography (EEG) analysis to determine whether the loss of CB2Rs would increase spontaneous seizure frequency in Scn1a RH mutant mice. RESULTS: Both heterozygous (Cnr2+/- ) and homozygous (Cnr2-/- ) knockout mice exhibited increased susceptibility to pentylenetetrazole (PTZ)-induced seizures. The CB2R agonist JWH-133 did not significantly alter seizure susceptibility in wild-type mice; however, administration of the CB2R antagonist SR144528 resulted in increased susceptibility to PTZ-induced seizures. In offspring from a cross between the Cnr2 × RH lines, both Cnr2 and RH mutants were susceptible to PTZ-induced seizures; however, seizure susceptibility was not significantly increased in mutants expressing both mutations. No spontaneous seizures were observed in either RH or Cnr2/RH mutants during 336-504 hours of continuous EEG recordings. SIGNIFICANCE: Our results demonstrate that reduced CB2R activity is associated with increased seizure susceptibility. CB2Rs might therefore provide a therapeutic target for the treatment of some forms of epilepsy.

Donepezil increases resistance to induced seizures in a mouse model of Dravet syndrome.

De novo loss-of-function mutations in SCN1A are the main cause of Dravet syndrome, a catastrophic encephalopathy characterized by recurrent early-life febrile seizures, a number of other afebrile seizure types that are often refractory to treatment, and behavioral abnormalities including social deficits, motor dysfunction, and cognitive impairment. We previously demonstrated that the reversible acetylcholinesterase inhibitor, Huperzine A, increases seizure resistance in Scn1a mutants. In the present study, we evaluated the therapeutic potential of donepezil, a reversible acetylcholinesterase inhibitor approved by the Food and Drug Administration, in a mouse model of Dravet syndrome (Scn1a+/- ). We found that donepezil conferred robust protection against induced seizures in Scn1a+/- mutants.

From DREADD to Treatment in Temporal Lobe Epilepsy.

Pharmaco-Genetic Therapeutics Targeting Parvalbumin Neurons Attenuate Temporal Lobe Epilepsy Wang Y, Liang J, Chen L, Shen Y, Zhao J, Xu C, Wu X, Cheng H, Ying X, Guo Y, Wang S, Zhou Y, Wang Y, Chen Z. Neurobiol Dis. 2018;117:149-60. Epub 2018/06/13. doi: 10.1016/j.nbd.2018.06.006. PubMed PMID: 29894753. Temporal lobe epilepsy (TLE) is the most common type of epilepsy and is often medically refractory. Previous studies suggest that selective pharmaco-genetic inhibition of pyramidal neurons has therapeutic value for the treatment of epilepsy; however, there is a risk of disrupting normal physical functions. Here, we test whether pharmaco-genetic activation of parvalbumin neurons, which are transgenetically transduced with the modified muscarinic receptor hM3Dq, can attenuate TLE. We found that pharmaco-genetic activation of hippocampal parvalbumin neurons in epileptogenic zone not only significantly extends the latency to different seizure stages and attenuates seizure activities in acute seizure model but also greatly alleviates the severity of seizure onsets in 2 chronic epilepsy models. This manipulation did not affect the normal physical function evaluated in various cognitive tasks. Further, the activation of parvalbumin neurons produced an inhibition on parts of surrounding pyramidal neurons, and the direct inactivation of pyramidal neurons via the viral expression of a modified muscarinic receptor hM4Di produced a similar anti-ictogenic effect. Interestingly, pharmacogenetic inactivation of pyramidal neurons was more sensitive to impair cognitive function. Those data demonstrated that pharmaco-genetic seizure attenuation through targeting parvalbumin neurons rather than pyramidal neurons may be a novel and relatively safe approach for treating refractory TLE.

A two-hit story: Seizures and genetic mutation interaction sets phenotype severity in SCN1A epilepsies.

SCN1A (NaV1.1 sodium channel) mutations cause Dravet syndrome (DS) and GEFS+ (which is in general milder), and are risk factors in other epilepsies. Phenotypic variability limits precision medicine in epilepsy, and it is important to identify factors that set phenotype severity and their mechanisms. It is not yet clear whether SCN1A mutations are necessary for the development of severe phenotypes or just for promoting seizures. A relevant example is the pleiotropic R1648H mutation that can cause either mild GEFS+ or severe DS. We used a R1648H knock-in mouse model (Scn1aRH/+) with mild/asymptomatic phenotype to dissociate the effects of seizures and of the mutation per se. The induction of short repeated seizures, at the age of disease onset for Scn1a mouse models (P21), had no effect in WT mice, but transformed the mild/asymptomatic phenotype of Scn1aRH/+ mice into a severe DS-like phenotype, including frequent spontaneous seizures and cognitive/behavioral deficits. In these mice, we found no major modifications in cytoarchitecture or neuronal death, but increased excitability of hippocampal granule cells, consistent with a pathological remodeling. Therefore, we demonstrate for our model that an SCN1A mutation is a prerequisite for a long term deleterious effect of seizures on the brain, indicating a clear interaction between seizures and the mutation for the development of a severe phenotype generated by pathological remodeling. Applied to humans, this result suggests that genetic alterations, even if mild per se, may increase the risk of second hits to develop severe phenotypes.

Noradrenergic Transmission at Alpha1-Adrenergic Receptors in the Ventral Periaqueductal Gray Modulates Arousal.

BACKGROUND: Dysregulation of arousal is symptomatic of numerous psychiatric disorders. Previous research has shown that the activity of dopamine (DA) neurons in the ventral periaqueductal gray (vPAG) tracks with arousal state, and lesions of vPAGDA cells increase sleep. However, the circuitry controlling these wake-promoting DA neurons is unknown. METHODS: This study combined designer receptors exclusively activated by designer drugs (DREADDs), behavioral pharmacology, electrophysiology, and immunoelectron microscopy in male and female mice to elucidate mechanisms in the vPAG that promote arousal. RESULTS: Activation of locus coeruleus projections to the vPAG or vPAGDA neurons induced by DREADDs promoted arousal. Similarly, agonist stimulation of vPAG alpha1-adrenergic receptors (α1ARs) increased latency to fall asleep, whereas α1AR blockade had the opposite effect. α1AR stimulation drove vPAGDA activity in a glutamate-dependent, action potential-independent manner. Compared with other dopaminergic brain regions, α1ARs were enriched on astrocytes in the vPAG, and mimicking α1AR transmission specifically in vPAG astrocytes via Gq-DREADDS was sufficient to increase arousal. In general, the wake-promoting effects observed were not accompanied by hyperactivity. CONCLUSIONS: These experiments revealed that vPAG α1ARs increase arousal, promote glutamatergic input onto vPAGDA neurons, and are abundantly expressed on astrocytes. Activation of locus coeruleus inputs, vPAG astrocytes, or vPAGDA neurons increase sleep latency but do not produce hyperactivity. Together, these results support an arousal circuit whereby noradrenergic transmission at astrocytic α1ARs activates wake-promoting vPAGDA neurons via glutamate transmission.

Epileptic Encephalopathy and Cerebellar Atrophy Resulting from Compound Heterozygous CACNA2D2 Variants.

CACNA2D2 encodes an auxiliary subunit of the voltage-dependent calcium channel. To date, there have only been two reports of individuals with early-infantile epileptic encephalopathy due to CACNA2D2 mutations. In both reports, patients were homozygous for the identified variants. Here, we report a patient with epileptic encephalopathy and cerebellar atrophy who was found to have two novel variants in the CACNA2D2 gene: c.782C>T (p.Pro261Leu) and c.3137T>C (p.Leu1046Pro), by whole-exome sequencing. The variants were shown to be inherited in trans and the unaffected parents were confirmed to be heterozygous carriers. This is the third report of recessive CACNA2D2 variants associated with disease and the first report of compound heterozygous variants. The clinical description of this new case highlights the phenotypic similarities amongst individuals with CACNA2D2-related disease and suggests that CACNA2D2 should be considered as a differential diagnosis in individuals with cerebellar dysfunction and multiple seizure types that begin in the first year of life.