Dataset Information

Cannabidiol counters the effects of a dominant-negative pathogenic Kv7.2 variant.

ABSTRACT: Epilepsy and neurodevelopmental disorders can arise from pathogenic variants of KCNQ (Kv7) channels. A patient with developmental and epileptic encephalopathy exhibited an in-frame deletion of histidine 260 on Kv7.2. Coexpression of Kv7.2 mutant (mut) subunits with Kv7.3 invoked a decrease in current density, a depolarizing shift in voltage for activation, and a decrease in membrane conductance. Biotinylation revealed an increased level of surface Kv7.2mut compared to Kv7.3 with no change in total membrane protein expression. Super-resolution and FRET imaging confirmed heteromeric channel formation and a higher expression density of Kv7.2mut. Cannabidiol (1 μM) offset the effects of Kv7.2mut by inducing a hyperpolarizing shift in voltage for activation independent of CB1 or CB2 receptors. These data reveal that the ability for cannabidiol to reduce the effects of a pathogenic Kv7.2 variant supports its use as a potential therapeutic to reduce seizure activity.

SUBMITTER: Zhan X

PROVIDER: S-EPMC9490039 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Cannabidiol counters the effects of a dominant-negative pathogenic Kv7.2 variant.

Zhan Xiaoqin X Drummond-Main Chris C Greening Dylan D Yao Jinjing J Chen S W R SWR Appendino J P JP Au P Y Billie PYB Turner Ray W RW

iScience 20220906 10

Epilepsy and neurodevelopmental disorders can arise from pathogenic variants of KCNQ (Kv7) channels. A patient with developmental and epileptic encephalopathy exhibited an in-frame deletion of histidine 260 on Kv7.2. Coexpression of Kv7.2 mutant (mut) subunits with Kv7.3 invoked a decrease in current density, a depolarizing shift in voltage for activation, and a decrease in membrane conductance. Biotinylation revealed an increased level of surface Kv7.2mut compared to Kv7.3 with no change in tot ...[more]

PMID: 36157585

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Project description:Developmental and epileptic encephalopathies (DEE) are a group of severe epilepsies that usually present with intractable seizures, developmental delay, and often have elevated risk for premature mortality. Numerous genes have been identified as a monogenic cause of DEE, including KCNB1. The voltage-gated potassium channel KV2.1, encoded by KCNB1, is primarily responsible for delayed rectifier potassium currents that are important regulators of excitability in electrically excitable cells, including neurons. In addition to its canonical role as a voltage-gated potassium conductance, KV2.1 also serves a highly conserved structural function organizing endoplasmic reticulum-plasma membrane junctions clustered in the soma and proximal dendrites of neurons. The de novo pathogenic variant KCNB1-p.G379R was identified in an infant with epileptic spasms, and atonic, focal and tonic-clonic seizures that were refractory to treatment with standard antiepileptic drugs. Previous work demonstrated deficits in potassium conductance, but did not assess non-conducting functions. To determine if the G379R variant affected KV2.1 clustering at endoplasmic reticulum-plasma membrane junctions, KV2.1-G379R was expressed in HEK293T cells. KV2.1-G379R expression did not induce formation of endoplasmic reticulum-plasma membrane junctions, and co-expression of KV2.1-G379R with KV2.1-wild-type lowered induction of these structures relative to KV2.1-WT alone, consistent with a dominant negative effect. To model this variant in vivo, we introduced Kcnb1G379R into mice using CRISPR/Cas9 genome editing. We characterized neuronal expression, neurological and neurobehavioral phenotypes of Kcnb1G379R/+ (Kcnb1R/+) and Kcnb1G379R/G379R (Kcnb1R/R) mice. Immunohistochemistry studies on brains from Kcnb1+/+, Kcnb1R/+ and Kcnb1R/R mice revealed genotype-dependent differences in the expression levels of KV2.1 protein, as well as associated KV2.2 and AMIGO-1 proteins. Kcnb1R/+ and Kcnb1R/R mice displayed profound hyperactivity, repetitive behaviors, impulsivity and reduced anxiety. Spontaneous seizures were observed in Kcnb1R/R mice, as well as seizures induced by exposure to novel environments and/or handling. Both Kcnb1R/+ and Kcnb1R/R mutants were more susceptible to proconvulsant-induced seizures. In addition, both Kcnb1R/+ and Kcnb1R/R mice exhibited abnormal interictal EEG activity, including isolated spike and slow waves. Overall, the Kcnb1G379R mice recapitulate many features observed in individuals with DEE due to pathogenic variants in KCNB1. This new mouse model of KCNB1-associated DEE will be valuable for improving the understanding of the underlying pathophysiology and will provide a valuable tool for the development of therapies to treat this pharmacoresistant DEE.

Project description:BackgroundParkinson's disease (PD) is a progressive neurodegenerative disorder. Mendelian forms have revealed multiple genes, with a notable emphasis on membrane trafficking; RAB GTPases play an important role in PD as a subset are both regulators and substrates of LRRK2 protein kinase. To explore the role of RAB GTPases in PD, we undertook a comprehensive examination of their genetic variability in familial PD.MethodsAffected probands from 130 multi-incident PD families underwent whole-exome sequencing and genotyping, Potential pathogenic variants in 61 RAB GTPases were genotyped in relatives to assess disease segregation. These variants were also genotyped in a larger case-control series, totaling 3,078 individuals (2,734 with PD). The single most significant finding was subsequently validated within genetic data (6,043 with PD). Clinical and pathologic findings were summarized for gene-identified patients, and haplotypes were constructed. In parallel, wild-type and mutant RAB GTPase structural variation, protein interactions, and resultant enzyme activities were assessed.FindingsWe found RAB32 c.213C>G (Ser71Arg) to co-segregate with autosomal dominant parkinsonism in three multi-incident families. RAB32 Ser71Arg was also significantly associated with PD in case-control samples: genotyping and database searches identified thirteen more patients with the same variant that was absent in unaffected controls. Notably, RAB32 Ser71Arg heterozygotes share a common haplotype. At autopsy, one patient had sparse neurofibrillary tangle pathology in the midbrain and thalamus, without Lewy body pathology. In transfected cells the RAB32 Arg71 was twice as potent as Ser71 wild type to activate LRRK2 kinase.InterpretationOur study provides unequivocal evidence to implicate RAB32 Ser71Arg in PD. Functional analysis demonstrates LRRK2 kinase activation. We provide a mechanistic explanation to expand and unify the etiopathogenesis of monogenic PD.FundingNational Institutes of Health, the Canada Excellence Research Chairs program, Aligning Science Across Parkinson's, the Michael J. Fox Foundation for Parkinson's Research, and the UK Medical Research Council.

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Cannabidiol counters the effects of a dominant-negative pathogenic Kv7.2 variant.

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Cannabidiol counters the effects of a dominant-negative pathogenic Kv7.2 variant.

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