Search

Neuropsychiatric disorders (NPDs) are frequently co-morbid with epilepsy, but the biological basis of shared risk remains poorly understood. The 16p11.2 duplication is a copy number variant that confers risk for diverse NPDs including autism spectrum disorder, schizophrenia, intellectual disability and epilepsy. We used a mouse model of the 16p11.2 duplication (16p11.2dup/+) to uncover molecular and circuit properties associated with this broad phenotypic spectrum, and examined genes within the locus capable of phenotype reversal. Quantitative proteomics revealed alterations to synaptic networks and products of NPD risk genes. We identified an epilepsy-associated subnetwork that was dysregulated in 16p11.2dup/+ mice and altered in brain tissue from individuals with NPDs. Cortical circuits from 16p11.2dup/+ mice exhibited hypersynchronous activity and enhanced network glutamate release, which increased susceptibility to seizures. Using gene co-expression and interactome analysis, we show that PRRT2 is a major hub in the epilepsy subnetwork. Remarkably, correcting Prrt2 copy number rescued aberrant circuit properties, seizure susceptibility and social deficits in 16p11.2dup/+ mice. We show that proteomics and network biology can identify important disease hubs in multigenic disorders, and reveal mechanisms relevant to the complex symptomatology of 16p11.2 duplication carriers.

The genetic basis of many epilepsies is increasingly understood, giving rise to the possibility of precision treatments tailored to specific genetic etiologies. Despite this, current medical therapy for most epilepsies remains imprecise, aimed primarily at empirical seizure reduction rather than targeting specific disease processes. Intellectual and technological leaps in diagnosis over the past 10 years have not yet translated to routine changes in clinical practice. However, the epilepsy community is poised to make impressive gains in precision therapy, with continued innovation in gene discovery, diagnostic ability, and bioinformatics; increased access to genetic testing and counseling; fuller understanding of natural histories; agility and rigor in preclinical research, including strategic use of emerging model systems; and engagement of an evolving group of stakeholders (including patient advocates, governmental resources, and clinicians and scientists in academia and industry). In each of these areas, we highlight notable examples of recent progress, new or persistent challenges, and future directions. The future of precision medicine for genetic epilepsy looks bright if key opportunities on the horizon can be pursued with strategic and coordinated effort.

Genetic variants in SCN2A, encoding the NaV1.2 voltage-gated sodium channel, are associated with a range of neurodevelopmental disorders with overlapping phenotypes. Some variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to developmental and epileptic encephalopathy, while loss-of-function variants that reduce neuronal excitability lead to intellectual disability and/or autism spectrum disorder (ASD) with or without co-morbid seizures. One unique case less easily classified using this framework is the de novo missense variant SCN2A-p.K1422E, associated with infant-onset developmental delay, infantile spasms and features of ASD. Prior structure-function studies demonstrated that K1422E substitution alters ion selectivity of NaV1.2, conferring Ca2+ permeability, lowering overall conductance and conferring resistance to tetrodotoxin (TTX). Based on heterologous expression of K1422E, we developed a compartmental neuron model incorporating variant channels that predicted reductions in peak action potential (AP) speed. We generated Scn2aK1422E mice and characterized effects on neurons and neurological/neurobehavioral phenotypes. Cultured cortical neurons from heterozygous Scn2aK1422E/+ mice exhibited lower current density with a TTX-resistant component and reversal potential consistent with mixed ion permeation. Recordings from Scn2aK1442E/+ cortical slices demonstrated impaired AP initiation and larger Ca2+ transients at the axon initial segment during the rising phase of the AP, suggesting complex effects on channel function. Scn2aK1422E/+ mice exhibited rare spontaneous seizures, interictal electroencephalogram abnormalities, altered induced seizure thresholds, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Overall, Scn2aK1422E/+ mice present with phenotypes similar yet distinct from other Scn2a models, consistent with complex effects of K1422E on NaV1.2 channel function.

Introduction Myocarditis is a life-threatening heart condition characterized by inflammation, cellular infiltrate, chest pain, reduced heart function, and arrhythmias (1). Myocarditis affects as many as 10-20 per 100,000 people, with [...], Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis. To model DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with heterozygous DSP truncating variants (DSPtvs) and a gene-edited homozygous deletion cell line ([DSP.sup.-/-]). At baseline, [DSP.sup.-/-] EHTs displayed a transcriptomic signature of innate immune activation, which was mirrored by cytokine release. Importantly, [DSP.sup.- /-] EHTs were hypersensitive to Toll-like receptor (TLR) stimulation, demonstrating more contractile dysfunction compared with isogenic controls. Relative to [DSP.sup.-/-] EHTs, heterozygous DSPtv EHTs had less functional impairment. DSPtv EHTs displayed heightened sensitivity to TLR stimulation, and when subjected to strain, DSPtv EHTs developed functional deficits, indicating reduced contractile reserve compared with healthy controls. Colchicine or NF-[kappa]B inhibitors improved straininduced force deficits in DSPtv EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Thus, EHTs replicate electrical and contractile phenotypes seen in human myocarditis, implicating cytokine release as a key part of the myogenic susceptibility to inflammation. The heightened innate immune activation and sensitivity are targets for clinical intervention.

Sudden cardiac death (SCD), the unexpected death due to acquired or genetic cardiovascular disease, follows distinct 24-hour patterns in occurrence. These 24-hour patterns likely reflect daily changes in arrhythmogenic triggers and the myocardial substrate caused by day/night rhythms in behavior, the environment, and endogenous circadian mechanisms. To better address fundamental questions regarding the circadian mechanisms, the National Heart, Lung, and Blood Institute convened a workshop, Understanding Circadian Mechanisms of Sudden Cardiac Death. We present a 2-part report of findings from this workshop. Part 1 summarizes the workshop and serves to identify research gaps and opportunities in the areas of basic and translational research. Among the gaps was the lack of standardization in animal studies for reporting environmental conditions (eg, timing of experiments relative to the light dark cycle or animal housing temperatures) that can impair rigor and reproducibility. Workshop participants also pointed to uncertainty regarding the importance of maintaining normal circadian rhythmic synchrony and the potential pathological impact of desynchrony on SCD risk. One related question raised was whether circadian mechanisms can be targeted to reduce SCD risk. Finally, the experts underscored the need for studies aimed at determining the physiological importance of circadian clocks in the many different cell types important to normal heart function and SCD. Addressing these gaps could lead to new therapeutic approaches/molecular targets that can mitigate the risk of SCD not only at certain times but over the entire 24-hour period.

Sudden cardiac death (SCD) is the sudden, unexpected death due to abrupt loss of heart function secondary to cardiovascular disease. In certain populations living with cardiovascular disease, SCD follows a distinct 24-hour pattern in occurrence, suggesting day/night rhythms in behavior, the environment, and endogenous circadian rhythms result in daily spans of increased vulnerability. The National Heart, Lung, and Blood Institute convened a workshop, Understanding Circadian Mechanisms of Sudden Cardiac Death to identify fundamental questions regarding the role of the circadian rhythms in SCD. Part 2 summarizes research gaps and opportunities in the areas of population and clinical research identified in the workshop. Established research supports a complex interaction between circadian rhythms and physiological responses that increase the risk for SCD. Moreover, these physiological responses themselves are influenced by several biological variables, including the type of cardiovascular disease, sex, age, and genetics, as well as environmental factors. The emergence of new noninvasive biotechnological tools that continuously measure key cardiovascular variables, as well as the identification of biomarkers to assess circadian rhythms, hold promise for generating large-scale human data sets that will delineate which subsets of individuals are most vulnerable to SCD. Additionally, these data will improve our understanding of how people who suffer from circadian disruptions develop cardiovascular diseases that increase the risk for SCD. Emerging strategies to identify new biomarkers that can quantify circadian health (eg, environmental, behavioral, and internal misalignment) may lead to new interventions and therapeutic targets to prevent the progression of cardiovascular diseases that cause SCD.

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.

Calmodulin (CaM) plays a critical role in intracellular signaling and regulation of Ca2+-dependent proteins and ion channels. Mutations in CaM cause life-threatening cardiac arrhythmias. Among the known CaM targets, small-conductance Ca2+-activated K+ (SK) channels are unique, since they are gated solely by beat-to-beat changes in intracellular Ca2+. However, the molecular mechanisms of how CaM mutations may affect the function of SK channels remain incompletely understood. To address the structural and functional effects of these mutations, we introduced prototypical human CaM mutations in human induced pluripotent stem cell-derived cardiomyocyte-like cells (hiPSC-CMs). Using structural modeling and molecular dynamics simulation, we demonstrate that human calmodulinopathy-associated CaM mutations disrupt cardiac SK channel function via distinct mechanisms. CaMD96V and CaMD130G mutants reduce SK currents through a dominant-negative fashion. By contrast, specific mutations replacing phenylalanine with leucine result in conformational changes that affect helix packing in the C-lobe, which disengage the interactions between apo-CaM and the CaM-binding domain of SK channels. Distinct mutant CaMs may result in a significant reduction in the activation of the SK channels, leading to a decrease in the key Ca2+-dependent repolarization currents these channels mediate. The findings in this study may be generalizable to other interactions of mutant CaMs with Ca2+-dependent proteins within cardiac myocytes.

Epileptic encephalopathies are severe forms of infantile-onset epilepsy often complicated by severe neurodevelopmental impairments. Some forms of early-onset epileptic encephalopathy (EOEE) have been associated with variants in SCN2A, which encodes the brain voltage-gated sodium channel NaV1.2. Many voltage-gated sodium channel genes, including SCN2A, undergo developmentally regulated mRNA splicing. The early onset of these disorders suggests that developmentally regulated alternative splicing of NaV1.2 may be an important consideration when elucidating the pathophysiological consequences of epilepsy-associated variants. We hypothesized that EOEE-associated NaV1.2 variants would exhibit greater dysfunction in a splice isoform that is prominently expressed during early development. We engineered five EOEE-associated NaV1.2 variants (T236S, E999K, S1336Y, T1623N, and R1882Q) into the adult and neonatal splice isoforms of NaV1.2 and performed whole-cell voltage clamp to elucidate their functional properties. All variants exhibited functional defects that could enhance neuronal excitability. Three of the five variants (T236S, E999K, and S1336Y) exhibited greater dysfunction in the neonatal isoform compared with those observed in the adult isoform. Computational modeling of a developing cortical pyramidal neuron indicated that T236S, E999K, S1336Y, and R1882Q showed hyperexcitability preferentially in immature neurons. These results suggest that both splice isoform and neuronal developmental stage influence how EOEE-associated NaV1.2 variants affect neuronal excitability.

Advances in gene discovery for neurodevelopmental disorders have identified SCN2A dysfunction as a leading cause of infantile seizures, autism spectrum disorder, and intellectual disability. SCN2A encodes the neuronal sodium channel NaV1.2. Functional assays demonstrate strong correlation between genotype and phenotype. This insight can help guide therapeutic decisions and raises the possibility that ligands that selectively enhance or diminish channel function may improve symptoms. The well-defined function of sodium channels makes SCN2A an important test case for investigating the neurobiology of neurodevelopmental disorders more generally. Here, we discuss the progress made, through the concerted efforts of a diverse group of academic and industry scientists as well as policy advocates, in understanding and treating SCN2A-related disorders.

ObjectiveATP1A3-related neurologic disorders encompass a broad range of phenotypes that extend well beyond initial phenotypic criteria associated with alternating hemiplegia of childhood (AHC) and rapid-onset dystonia parkinsonism.MethodsIn 2014, the Alternating Hemiplegia of Childhood Foundation hosted a multidisciplinary workshop intended to address fundamental challenges surrounding the diagnosis and management of individuals with ATP1A3-related disorders.ResultsWorkshop attendees were charged with the following: (1) to achieve consensus on expanded diagnostic criteria to facilitate the identification of additional patients, intended to supplement existing syndrome-specific diagnostic paradigms; (2) to standardize definitions for the broad range of paroxysmal manifestations associated with AHC to disseminate to families; (3) to create clinical recommendations for common recurrent issues facing families and medical care providers; (4) to review data related to the death of individuals in the Alternating Hemiplegia of Childhood Foundation database to guide future efforts in identifying at-risk subjects and potential preventative measures; and (5) to identify critical gaps where we most need to focus national and international research efforts.ConclusionsThis report summarizes recommendations of the workshop committee, highlighting the key phenotypic features to facilitate the diagnosis of possible ATP1A3 mutations, providing recommendations for genetic testing, and outlining initial acute management for common recurrent clinical conditions, including epilepsy.

Introduction Parkinson's disease (PD) is the second most common neurodegenerative disease (1, 2). The cardinal motor symptoms of PD are caused by the degeneration of dopaminergic neurons in the substantia [...], Levodopa-induced dyskinesia (LID) poses a significant health care challenge for Parkinson's disease (PD) patients. Amantadine is currently the only drug proven to alleviate LID. Although its efficacy in treating LID is widely assumed to be mediated by blockade of N-methyl-D-aspartate (NMDA) glutamate receptors, our experiments demonstrate that at therapeutically relevant concentrations, amantadine preferentially blocks inward-rectifying 1C channel type 2 (Kir2) channels in striatal spiny projection neurons (SPNs)--not NMDA receptors. In so doing, amantadine enhances dendritic integration of excitatory synaptic potentials in SPNs and enhances--not antagonizes--the induction of long-term potentiation (LTP) at excitatory, axospinous synapses. Taken together, our studies suggest that the alleviation of LID in PD patients is mediated by diminishing the disparity in the excitability of direct- and indirect-pathway SPNs in the on state, rather than by disrupting LTP induction. This insight points to a pharmacological approach that could be used to effectively ameliorate LID and improve the quality of life for PD patients.

RationaleCalmodulin (CaM) mutations are associated with an autosomal dominant syndrome of ventricular arrhythmia and sudden death that can present with divergent clinical features of catecholaminergic polymorphic ventricular tachycardia (CPVT) or long QT syndrome (LQTS). CaM binds to and inhibits ryanodine receptor (RyR2) Ca release channels in the heart, but whether arrhythmogenic CaM mutants alter RyR2 function is not known.ObjectiveTo gain mechanistic insight into how human CaM mutations affect RyR2 Ca channels.Methods and resultsWe studied recombinant CaM mutants associated with CPVT (N54I and N98S) or LQTS (D96V, D130G, and F142L). As a group, all LQTS-associated CaM mutants (LQTS-CaMs) exhibited reduced Ca affinity, whereas CPVT-associated CaM mutants (CPVT-CaMs) had either normal or modestly lower Ca affinity. In permeabilized ventricular myocytes, CPVT-CaMs at a physiological intracellular concentration (100 nmol/L) promoted significantly higher spontaneous Ca wave and spark activity, a typical cellular phenotype of CPVT. Compared with wild-type CaM, CPVT-CaMs caused greater RyR2 single-channel open probability and showed enhanced binding affinity to RyR2. Even a 1:8 mixture of CPVT-CaM:wild-type-CaM activated Ca waves, demonstrating functional dominance. In contrast, LQTS-CaMs did not promote Ca waves and exhibited either normal regulation of RyR2 single channels (D96V) or lower RyR2-binding affinity (D130G and F142L). None of the CaM mutants altered Ca/CaM binding to CaM-kinase II.ConclusionsA small proportion of CPVT-CaM is sufficient to evoke arrhythmogenic Ca disturbances, whereas LQTS-CaMs do not. Our findings explain the clinical presentation and autosomal dominant inheritance of CPVT-CaM mutations and suggest that RyR2 interactions are unlikely to explain arrhythmogenicity of LQTS-CaM mutations.