Voltage-gated Sodium Channel Subunit Research Articles

Cardiomyocyte-specific deletion of adhesion G protein-coupled receptor F5 (ADGRF5) increases susceptibility to stress and arrhythmia

G-Protein Coupled Receptors (GPCR) represent the largest family of cell surface receptors in the human body and serve as targets for more than 30% of all approved drugs. Adhesion GPCRs (AGPCR) are an understudied GPCR family containing numerous extracellular domains that interact with extracellular proteins, including many known mediators of cardiac remodeling responses to pathologic stress. However, their relative expression in the heart and impact on cardiac function normally or during disease has been largely unstudied. To address this, we evaluated RNASeq-attained AGPCR expression from left ventricular (LV) samples of adult mice, of which Adgrf5 was one of the most highly expressed. Notably, decreased ADGRF5 expression is observed across numerous failing human LV tissue GEO datasets, and C57BL6/J mice subjected to transaortic constriction (TAC) surgery to induce pressure overload-induced HF also displayed reduced LV Adgrf5 expression by 12 weeks post-TAC. Analysis of published single cell (sc)-RNASeq datasets confirmed Adgrf5 expression in cardiomyocytes (CM), which also decreased following TAC in mice and in CM from failing human hearts. To investigate the impact of CM-specific ADGRF5 on cardiac function and remodeling normally or during disease progression, we crossed floxed ADGRF5 (ADGRF5f/f) and αMHC-Cre mice to generate constitutive, CM-specific ADGRF5 knockout mice (F5cmKO). These mice display normal cardiac structure and function at 12 weeks of age versus αMHC-Cre mice, as assessed via echocardiography, gravimetrics and immunohistochemistry. However, F5cmKO mice develop cardiac dysfunction, maladaptive remodeling, and increased mortality over time, even in the absence of pathologic insult, suggesting a homeostatic function of CM-specific ADGRF5. Following TAC surgery, F5cmKO mice display similar outcomes including enhanced cardiac dysfunction with accelerated remodeling and increased mortality. To attain mechanistic insight into these outcomes, we performed RNA-sequencing on LV tissue of 12-week-old F5cmKO mice, identifying and subsequently validating Scn1b as significantly upregulated in F5cmKO CM. Scn1b encodes for the β subunits of voltage-gated sodium channels (Scnβ1/β1B), which regulate gating and kinetics of Nav1.5 as well as adhesive CM-CM interactions, disruption of which could lead to arrhythmias. Indeed, conscious electrocardiogram (ECG) readings via telemetry indicated F5cmKO mice had a high burden of premature ventricular contractions (PVC), including ventricular tachycardia associated with cardiac enlargement. Using a combination of luciferase and fluorescence resonance energy transfer (FRET) reporters and gene expression analyses in neonatal rat ventricular myocytes (NRVM), we confirmed that ADGRF5 controls Scn1b expression in a Gαq/11 protein-dependent manner. In all, we have discovered that loss of CM-specific ADGRF5 leads to increased Scn1b expression with enhanced susceptibility to stress, arrythmias and sudden death, thus may provide a novel GPCR target to promote the maintenance of cardiac rhythm. AHA Predoctoral Fellowship 834906 to J.M.E. P01 HL147841 to D.T. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Non-dravet syndrome epilepsy in 4 polish patients with SCN1A de novo mutations

Background: The spectrum of neurological disorders associated with mutations in SCN1A, which encodes the type 1 subunit of the neuronal voltage-gated sodium channel, is broad and can manifest as both epileptic (a heterogeneous group of epileptic syndromes) and non-epileptic conditions. Dravet syndrome (DRVT) and Genetic Epilepsy with Febrile Seizures plus (GEFS+) are the most frequent. However, there exist less common phenotypes with varying severity of clinical course that authors would like to highlight.

Sodium Channel β Subunits-An Additional Element in Animal Tetrodotoxin Resistance?

Tetrodotoxin (TTX) is a neurotoxic molecule used by many animals for defense and/or predation, as well as an important biomedical tool. Its ubiquity as a defensive agent has led to repeated independent evolution of tetrodotoxin resistance in animals. TTX binds to voltage-gated sodium channels (VGSC) consisting of α and β subunits. Virtually all studies investigating the mechanisms behind TTX resistance have focused on the α subunit of voltage-gated sodium channels, where tetrodotoxin binds. However, the possibility of β subunits also contributing to tetrodotoxin resistance was never explored, though these subunits act in concert. In this study, we present preliminary evidence suggesting a potential role of β subunits in the evolution of TTX resistance. We gathered mRNA sequences for all β subunit types found in vertebrates across 12 species (three TTX-resistant and nine TTX-sensitive) and tested for signatures of positive selection with a maximum likelihood approach. Our results revealed several sites experiencing positive selection in TTX-resistant taxa, though none were exclusive to those species in subunit β1, which forms a complex with the main physiological target of TTX (VGSC Nav1.4). While experimental data validating these findings would be necessary, this work suggests that deeper investigation into β subunits as potential players in tetrodotoxin resistance may be worthwhile.

Sodium Channel Gene Variants in Fetuses with Abnormal Sonographic Findings: Expanding the Prenatal Phenotypic Spectrum of Sodium Channelopathies.

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in the brain and muscle. Pathogenic variants in genes encoding VGSCs have been associated with severe disorders including epileptic encephalopathies and congenital myopathies. In this study, we identified pathogenic variants in genes encoding the α subunit of VGSCs in the fetuses of two unrelated families with the use of trio-based whole exome sequencing, as part of a larger cohort study. Sanger sequencing was performed for variant confirmation as well as parental phasing. The fetus of the first family carried a known de novo heterozygous missense variant in the SCN2A gene (NM_001040143.2:c.751G>A p.(Val251Ile)) and presented intrauterine growth retardation, hand clenching and ventriculomegaly. Neonatally, the proband also exhibited refractory epilepsy, spasms and MRI abnormalities. The fetus of the second family was a compound heterozygote for two parentally inherited novel missense variants in the SCN4A gene (NM_000334.4:c.4340T>C, p.(Phe1447Ser), NM_000334.4:c.3798G>C, p.(Glu1266Asp)) and presented a severe prenatal phenotype including talipes, fetal hypokinesia, hypoplastic lungs, polyhydramnios, ear abnormalities and others. Both probands died soon after birth. In a subsequent pregnancy of the latter family, the fetus was also a compound heterozygote for the same parentally inherited variants. This pregnancy was terminated due to multiple ultrasound abnormalities similar to the first pregnancy. Our results suggest a potentially crucial role of the VGSC gene family in fetal development and early lethality.

Clinical and Genetic Features of Dravet Syndrome: A Prime Example of the Role of Precision Medicine in Genetic Epilepsy.

Dravet syndrome (DS), also known as severe myoclonic epilepsy of infancy, is a rare and drug-resistant form of developmental and epileptic encephalopathies, which is both debilitating and challenging to manage, typically arising during the first year of life, with seizures often triggered by fever, infections, or vaccinations. It is characterized by frequent and prolonged seizures, developmental delays, and various other neurological and behavioral impairments. Most cases result from pathogenic mutations in the sodium voltage-gated channel alpha subunit 1 (SCN1A) gene, which encodes a critical voltage-gated sodium channel subunit involved in neuronal excitability. Precision medicine offers significant potential for improving DS diagnosis and treatment. Early genetic testing enables timely and accurate diagnosis. Advances in our understanding of DS's underlying genetic mechanisms and neurobiology have enabled the development of targeted therapies, such as gene therapy, offering more effective and less invasive treatment options for patients with DS. Targeted and gene therapies provide hope for more effective and personalized treatments. However, research into novel approaches remains in its early stages, and their clinical application remains to be seen. This review addresses the current understanding of clinical DS features, genetic involvement in DS development, and outcomes of novel DS therapies.

SCN1A Pathogenic Variants in a Turkish Cohort with GEFS+ Spectrum and Dravet Syndrome.

The α subunit of voltage-gated sodium channels in mammals is encoded by nine different genes, and the mutations in the SCN1A, SCN2A, SCN3A and SCN8A genes highly expressed in the CNS have been associated with epilepsy phenotypes. This study aimed at investigating the frequency of SCN1A gene mutations in DS and GEFS+ spectrum phenotype cases and discussing the molecular results in the context of genotype-phenotype correlation. 15 patients diagnosed with DS and 54 patients meeting the GEFS+ spectrum criteria were included in this study. All patients were evaluated by next-generation sequencing (NGS) method using a SCN1A gene commercial kit. A total of 17 different variants were detected in 18 index cases (26%) as a result of sequencing of the SCN1A gene. Note that 7 of the total 17 different variations (p.M1R, p.M147T, p.I767L, p.N1391Ifs *5, p.R1886G, p.E1915G, p.R1933Q) were novel variations. Of the 18 cases with variation in the SCN1A gene, 12 had DS and 6 had GEFS+ phenotype. The variations were de novo in all DS cases and in one case with a GEFS+ phenotype; in 5 GEFS+ cases, it was inherited from the affected parent. This study contributed to the mutation spectrum with the novel variations detected in cases with DS and GEFS+ phenotype. SCN1A genetic analysis can help in determining antiepileptic drugs to be selected or avoided in cases with mutations. The elucidation of the molecular etiology makes it possible to provide the family with effective genetic counseling for future pregnancies.

Expression changes of NaV channel subunits correlate with developmental maturation of electrophysiological characteristics of rat cerebellar Purkinje neurons

To investigate the variations in the expression of voltage-gated sodium (Nav) channel subunits during development of rat cerebellar Purkinje neurons and their correlation with maturation of electrophysiological characteristics of the neurons. We observed the changes in the expression levels of NaV1.1, 1.2, 1.3 and 1.6 during the development of Purkinje neurons using immunohistochemistry in neonatal (5-7 days after birth), juvenile (12-14 days), adolescent (21-24 days), and adult (42-60 days) SD rats. Using whole-cell patch-clamp technique, we recorded the spontaneous electrical activity of the neurons in ex vivo brain slices of rats of different ages to analyze the changes of electrophysiological characteristics of these neurons during development. The expression of NaV subunits in rat cerebellar Purkinje neurons showed significant variations during development. NaV1.1 subunit was highly expressed throughout the developmental stages and increased progressively with age (P < 0.05). NaV1.2 expression was not detected in the neurons in any of the developmental stages (P > 0.05). The expression level of NaV1.3 decreased with development and became undetectable after adolescence (P < 0.05). NaV1.6 expression was not detected during infancy, but increased with further development (P < 0.05). NaV1.1 and NaV1.3 were mainly expressed in the early stages of development. With the maturation of the rats, NaV1.3 expression disappeared and NaV1.6 expression increased in the neurons. NaV1.1 and NaV1.6 were mainly expressed after adolescence. The total NaV protein level increased gradually with development (P < 0.05) and tended to stabilize after adolescence. The spontaneous frequency and excitability of the Purkinje neurons increased gradually with development and reached the mature levels in adolescence. The developmental expression of NaV subunits was positively correlated with discharge frequency (r=0.9942, P < 0.05) and negatively correlated with the excitatory threshold of the neurons (r=0.9891, P < 0.05). The changes in the expression levels of NaV subunits are correlated with the maturation of high frequency electrophysiological properties of the neurons, suggesting thatmature NaV subunit expressions is the basis of maturation of electrophysiological characteristics of the neurons.

Voltage-gated sodium channels: from roles and mechanisms in the metastatic cell behavior to clinical potential as therapeutic targets.

During the second half of the last century, the prevalent knowledge recognized the voltage-gated sodium channels (VGSCs) as the proteins responsible for the generation and propagation of action potentials in excitable cells. However, over the last 25years, new non-canonical roles of VGSCs in cancer hallmarks have been uncovered. Their dysregulated expression and activity have been associated with aggressive features and cancer progression towards metastatic stages, suggesting the potential use of VGSCs as cancer markers and prognostic factors. Recent work has elicited essential information about the signalling pathways modulated by these channels: coupling membrane activity to transcriptional regulation pathways, intracellular and extracellular pH regulation, invadopodia maturation, and proteolytic activity. In a promising scenario, the inhibition of VGSCs with FDA-approved drugs as well as with new synthetic compounds, reduces cancer cell invasion in vitro and cancer progression in vivo. The purpose of this review is to present an update regarding recent advances and ongoing efforts to have a better understanding of molecular and cellular mechanisms on the involvement of both pore-forming α and auxiliary β subunits of VGSCs in the metastatic processes, with the aim at proposing VGSCs as new oncological markers and targets for anticancer treatments.

Biallelic structural variations within FGF12 detected by long-read sequencing in epilepsy.

We discovered biallelic intragenic structural variations (SVs) in FGF12 by applying long-read whole genome sequencing to an exome-negative patient with developmental and epileptic encephalopathy (DEE). We also found another DEE patient carrying a biallelic (homozygous) single-nucleotide variant (SNV) in FGF12 that was detected by exome sequencing. FGF12 heterozygous recurrent missense variants with gain-of-function or heterozygous entire duplication of FGF12 are known causes of epilepsy, but biallelic SNVs/SVs have never been described. FGF12 encodes intracellular proteins interacting with the C-terminal domain of the alpha subunit of voltage-gated sodium channels 1.2, 1.5, and 1.6, promoting excitability by delaying fast inactivation of the channels. To validate the molecular pathomechanisms of these biallelic FGF12 SVs/SNV, highly sensitive gene expression analyses using lymphoblastoid cells from the patient with biallelic SVs, structural considerations, and Drosophila in vivo functional analysis of the SNV were performed, confirming loss-of-function. Our study highlights the importance of small SVs in Mendelian disorders, which may be overlooked by exome sequencing but can be detected efficiently by long-read whole genome sequencing, providing new insights into the pathomechanisms of human diseases.

VIP interneuron impairment promotes in vivo circuit dysfunction and autism-related behaviors in Dravet syndrome

Dravet syndrome (DS) is a severe neurodevelopmental disorder caused by loss-of-function variants in SCN1A, which encodes the voltage-gated sodium channel subunit Nav1.1. We recently showed that neocortical vasoactive intestinal peptide interneurons (VIP-INs) express Nav1.1 and are hypoexcitable in DS (Scn1a+/-) mice. Here, we investigate VIP-IN function at the circuit and behavioral level by performing invivo 2-photon calcium imaging in awake wild-type (WT) and Scn1a+/- mice. VIP-IN and pyramidal neuron activation during behavioral transition from quiet wakefulness to active running is diminished in Scn1a+/- mice, and optogenetic activation of VIP-INs restores pyramidal neuron activity to WT levels during locomotion. VIP-IN selective Scn1a deletion reproduces core autism-spectrum-disorder-related behaviors in addition to cellular- and circuit-level deficits in VIP-IN function, but without epilepsy, sudden death, or avoidance behaviors seen in the global model. Hence, VIP-INs are impaired invivo, which may underlie non-seizure cognitive and behavioral comorbidities in DS.

Voltage-Gated Sodium Channel Dysfunctions in Neurological Disorders.

The pore-forming subunits (α subunits) of voltage-gated sodium channels (VGSC) are encoded in humans by a family of nine highly conserved genes. Among them, SCN1A, SCN2A, SCN3A, and SCN8A are primarily expressed in the central nervous system. The encoded proteins Nav1.1, Nav1.2, Nav1.3, and Nav1.6, respectively, are important players in the initiation and propagation of action potentials and in turn of the neural network activity. In the context of neurological diseases, mutations in the genes encoding Nav1.1, 1.2, 1.3 and 1.6 are responsible for many forms of genetic epilepsy and for Nav1.1 also of hemiplegic migraine. Several pharmacological therapeutic approaches targeting these channels are used or are under study. Mutations of genes encoding VGSCs are also involved in autism and in different types of even severe intellectual disability (ID). It is conceivable that in these conditions their dysfunction could indirectly cause a certain level of neurodegenerative processes; however, so far, these mechanisms have not been deeply investigated. Conversely, VGSCs seem to have a modulatory role in the most common neurodegenerative diseases such as Alzheimer's, where SCN8A expression has been shown to be negatively correlated with disease severity.

Comparing Mutability of Clinically Significant Voltage-Gated Sodium Channel Subunits (P4-1.003)

<h3>Objective:</h3> The objective of this project was to compare the mutability profile of the clinically significant beta1 and beta2 subunits of the voltage-gated sodium channel. <h3>Background:</h3> The voltage-gated sodium channel (VGSC) plays a role in generating action potentials in excitable tissues. Mutations in VGSC subunits, including the beta1 and beta3 subunits, are associated with diseases like epilepsy and cardiac arrhythmia. <h3>Design/Methods:</h3> We compared amino acid variants in the beta1 and beta2 subunits found in patients and the general population within the gnomAD database, and cancer cell variants from the cBioPortal Cancer Genomics database. We also reviewed variants for both subunits in cancer cells using the cBioPortal database. <h3>Results:</h3> Based on the data from gnomAD both subunits have variants reported in a similar percentage of the primary sequence (39.4% for beta1 and 39% for beta3), but pathogenic variants are more common in beta1 (5% of residues) than beta3 (1.86%, <i>p</i>=0.065). In 43789 patients with sequencing information for the genes SCN1B and SCN3B (coding for beta1 and beta3, respectively) 2.2% had non-synonymous variants in beta1 and 3.3% in beta3 (<i>p</i>=0.002). A minority of residues within each subunit (6.8% in beta1 and 9.76% in beta3) had a variant reported in cancer cells (<i>p</i>=.276). 4.58% of beta1 and 6.51% of beta3 residues have variants reported both in human and cancer cells. Of the residues reported with pathogenic variants in beta1, one (p.C21) had a variant found in cancer cells, and there were none for beta3. <h3>Conclusions:</h3> Our results suggest that the residues with pathogenic variants are a non-overlapping set between the two subunits, and might represent targets for their specialized function. In cancer cells, beta3 had a higher mutation load than beta1, suggesting different evolutionary pressures on both proteins. One limitation of our study is that we only focused on the main splice variant of <i>SCN1B</i> and <i>SCN3B</i> genes. <b>Disclosure:</b> Dr. Patino has received publishing royalties from a publication relating to health care. Dr. Patino has received personal compensation in the range of $0-$499 for serving as a project mentor with Manning Publications. Mr. Armstrong has nothing to disclose.

Distinct Etiologies of RANBP2-negative Acute Necrotizing Encephalopathy of Childhood (P10-9.008)

<h3>Objective:</h3> We sought to determine the etiology of <i>RANBP2</i>-negative pediatric patients presenting with acute necrotizing encephalopathy of childhood (ANEC). <h3>Background:</h3> ANEC is a rare childhood syndrome associated with fulminant encephalopathy and characteristic neuroimaging typically precipitated by acute infection and often associated with heterozygous mutation in <i>RANBP2</i>. Additional genes implicated in ANEC include <i>CPT2, MC1DN1</i> and <i>ILAE4</i>, yet many patients who meet clinical criteria for ANEC do not have mutations in any known genes and the cause of these cases has remained cryptic. <h3>Design/Methods:</h3> We performed a multicenter trio-based whole exome analysis of 12 <i>RANBP2</i>-negative pediatric patients diagnosed with ANEC using our established pipeline and filtering criteria. Whole exome sequencing was performed using a TruSeq exome capture kit and run on an Illumina NovaSeq 6000. Fastq files were analyzed using our established GATK-based pipeline and filtered for rare variants using our published approach. We prioritized rare candidate genes to determine plausible candidates based on the existing literature. <h3>Results:</h3> 32 candidate genes survived filtering and were considered in our analysis. We identified three high-confidence candidate genes. The first was <i>SCN1A</i>, segregating a heterozygous <i>de novo</i> (p.F1765Cfs*28). SCN1A encodes a voltage-gated sodium channel subunit. Mutations in SCN1A are well-known causes of Dravet Syndrome and other forms of epilepsy, but have also been associated with a fulminant encephalopathy phenotype independent of seizure burden. The second was <i>TNFAIP3</i> (heterozygous <i>de novo</i> p.R728C), previously shown to lead to an autoinflammatory Behcet-like syndrome. The third was <i>PRF1</i> (homozygous p.R225W), associated with a hemophagocytic lymphocystiocytosis-like phenotype with encephalopathy, ataxia, deep white matter lesions, and increased intracranial pressure. <h3>Conclusions:</h3> Our study indicates that ANEC is genetically heterogeneous and highlights three additional genes that should be considered in individuals presenting with ANEC. Our results also showcase how monogenic conditions can impact immune system regulation and lead to neuroimmunologic disorders. <b>Disclosure:</b> Dr. Vaidyanathan has nothing to disclose. Somayeh Bakhtiari has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for Xilis Inc. Somayeh Bakhtiari has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant (Bioinformatics) with Phoenix Childrens Hospital. Dr. Appavu has received personal compensation in the range of $0-$499 for serving on a Speakers Bureau for Natus. The institution of Dr. Appavu has received research support from American Heart Association. The institution of Dr. Appavu has received research support from United States Department of Defense. Derek Neilson has nothing to disclose. The institution of Dr. Kruer has received research support from NIH NINDs. Dr. Kruer has received research support from Cure CP. Dr. Kruer has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant with NHRSA.

Drug repositioning for idiopathic epilepsy using gene expression signature data.

Epilepsy is one of the most common neurological disorders, affecting millions of patients with a substantial economic and human burden. About 30-40% of epileptic patients remain un-treated after the therapeutic option. Genetic or idiopathic epilepsy count about 40% of total epilepsy patients, showing a maximum percentage for drug-resistant epilepsy. Since the last century basic approach to understanding disease progression and drug discovery has been through the prism, exploring all possible causes and treatment options. Here we report about the gene expression-based drug repositioning study for epilepsy. Epilepsy gene expression data was retrieved from the Gene Expression Omnibus database, while drugs-associated gene expression data was retrieved from the Connectivity map (CMAP). The study predicted309 drug compounds which can alter genetic epilepsy-mediated gene signature using an in-house developed R-script. These compounds were docked against identified epilepsy targets- Voltage-gated sodium channel subunit α2 (Nav1.2); GABA receptor α1-β1; and Voltage-gated calcium channel α1G (Cav3.1)using Carbamazepine, Clonazepam, and Pregabalin as standard drugs, respectively. Twenty-one predicted drug compounds showed better binding affinity than respective standards against the selected epileptic receptors. Among these drug compounds, Ergocalciferol, Oxaprozin, Flunarizine, Triprolidine and Cyproheptadine have been previously reported for anti-epileptic activities and can be potential hits to target idiopathic epilepsy.

Therapeutic Potential of Targeting Regulated Intramembrane Proteolysis Mechanisms of Voltage-Gated Ion Channel Subunits and Cell Adhesion Molecules.

Several integral membrane proteins undergo regulated intramembrane proteolysis (RIP), a tightly controlled process through which cells transmit information across and between intracellular compartments. RIP generates biologically active peptides by a series of proteolytic cleavage events carried out by two primary groups of enzymes: sheddases and intramembrane-cleaving proteases (iCLiPs). Following RIP, fragments of both pore-forming and non-pore-forming ion channel subunits, as well as immunoglobulin super family (IgSF) members, have been shown to translocate to the nucleus to function in transcriptional regulation. As an example, the voltage-gated sodium channel β1 subunit, which is also an IgSF-cell adhesion molecule (CAM), is a substrate for RIP. β1 RIP results in generation of a soluble intracellular domain, which can regulate gene expression in the nucleus. In this review, we discuss the proposed RIP mechanisms of voltage-gated sodium, potassium, and calcium channel subunits as well as the roles of their generated proteolytic products in the nucleus. We also discuss other RIP substrates that are cleaved by similar sheddases and iCLiPs, such as IgSF macromolecules, including CAMs, whose proteolytically generated fragments function in the nucleus. Importantly, dysfunctional RIP mechanisms are linked to human disease. Thus, we will also review how understanding RIP events and subsequent signaling processes involving ion channel subunits and IgSF proteins may lead to the discovery of novel therapeutic targets. SIGNIFICANCE STATEMENT: Several ion channel subunits and immunoglobulin superfamily molecules have been identified as substrates of regulated intramembrane proteolysis (RIP). This signal transduction mechanism, which generates polypeptide fragments that translocate to the nucleus, is an important regulator of gene transcription. RIP may impact diseases of excitability, including epilepsy, cardiac arrhythmia, and sudden death syndromes. A thorough understanding of the role of RIP in gene regulation is critical as it may reveal novel therapeutic strategies for the treatment of previously intractable diseases.

Error-prone protein synthesis recapitulates early symptoms of Alzheimer disease in aging mice.

Age-related neurodegenerative diseases (NDDs) are associated with the aggregation and propagation of specific pathogenic protein species (e.g., Aβ, α-synuclein). However, whether disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation, with its frequent outcome of random protein misfolding, is sufficient to recapitulate many early features of NDDs, including perturbed Ca2+ signaling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in behavioral changes reminiscent of early Alzheimer disease (AD), such as learning and memory deficits, maladaptive emotional responses, epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation, accompanied by hippocampal NPY expression and cerebral glucose hypometabolism. Collectively, our findings suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs, providing an alternative framework for understanding the initiation of AD.

Genetic Landscape of SCN1A Variants in a Turkish Cohort with GEFS+ Spectrum and Dravet Syndrome

Introduction: The α subunit of voltage-gated sodium channels in mammals is encoded by 9 different genes, and variations in the SCN1A, SCN2A, SCN3A, and SCN8A genes highly expressed in the CNS have been associated with epilepsy phenotypes. This study aimed at investigating the frequency of SCN1A gene variations in Dravet syndrome (DS) and GEFS+ spectrum phenotype cases and discussing the molecular results in the context of genotype-phenotype correlation. Methods: Fifteen patients diagnosed with DS and 54 patients meeting the GEFS+ spectrum criteria were included in this study. All patients were evaluated by next-generation sequencing and multiplex ligation-dependent probe amplification using an SCN1A gene commercial kit. Results: A total of 17 different variants were detected in 18 index cases (26%), of which 7 were novel variations (p.M1R, p.M147T, p.I767L, p.N1391Ifs*5, p.R1886G, p.E1915G, p.R1933Q). Of the 18 cases with variation in the SCN1A gene, 12 had DS and 6 had GEFS+ phenotype. The variations were de novo in all DS cases and in 1 case with a GEFS+ phenotype; in 5 GEFS+ cases, the variant was inherited from the affected parent. Discussion: This study contributes to the variation spectrum in cases with DS and GEFS+ phenotype with the novel variants detected. SCN1A genetic analysis can help in determining whether antiseizure medication should be selected or avoided in cases with variations. The elucidation of the molecular etiology makes it possible to provide the family with effective genetic counseling for future pregnancies.

A SCN8A variant associated with severe early onset epilepsy and developmental delay: Loss- or gain-of-function?

SCN8A, encoding the voltage-gated sodium channel subunit NaV1.6, has been associated with a wide spectrum of neuropsychiatric disorders. Missense variants in SCN8A which increase the channel activity can cause a severe developmental and epileptic encephalopathy (DEE). One DEE variant (p.(Arg223Gly)) was described to cause a predominant loss-of-function (LOF) mechanism when expressed in neuroblastoma cells, which is not consistent with the genotype-phenotype correlations in this gene. To resolve this discrepancy and understand the pathophysiological mechanism of this variant, we performed comprehensive electrophysiological studies in both neuroblastoma cells and primary hippocampal neuronal cultures. Although we also found that p.(Arg223Gly) significantly decreased Na+ current density and enhanced fast inactivation compared to the wild type (WT) channel in transfected neuroblastoma cells (both LOF mechanisms), it also caused a strong hyperpolarizing shift of steady-state activation and accelerated the recovery from fast inactivation (both gain-of-function (GOF) mechanisms). In cultured neurons transfected with mutant vs. WT NaV1.6 channels, we found more depolarized resting membrane potentials and a decreased rheobase leading to enhanced action potential firing. We conclude that SCN8A p.(Arg223Gly) leads to a net GOF resulting in neuronal hyperexcitability and a higher firing rate, fitting with the central role of GOF mechanisms in DEE.

Congenital insensitivity to pain: a novel mutation affecting a U12-type intron causes multiple aberrant splicing of SCN9A.

Mutations in the alpha subunit of voltage-gated sodium channel 1.7 (NaV1.7), encoded by SCN9A gene, play an important role in the regulation of nociception and can lead to a wide range of clinical outcomes, ranging from extreme pain syndromes to congenital inability to experience pain. To expand the phenotypic and genotypic spectrum of SCN9A-related channelopathies, we describe the proband, a daughter born from consanguineous parents, who had pain insensitivity, diminished temperature sensation, foot burns, and severe loss of nociceptive nerve fibers in the epidermis. Next-generation sequencing of SCN9A (NM_002977.3) revealed a novel homozygous substitution (c.377+7T>G) in the donor splice site of intron 3. As the RNA functional testing is challenging, the in silico analysis is the first approach to predict possible alterations. In this case, the computational analysis was unable to identify the splicing consensus and could not provide any prediction for splicing defects. The affected intron indeed belongs to the U12 type, a family of introns characterised by noncanonical consensus at the splice sites, accounting only for 0.35% of all human introns, and is not included in most of the training sets for splicing prediction. A functional study on proband RNA showed different aberrant transcripts, where exon 3 was missing and an intron fragment was included. A quantification study using real-time polymerase chain reaction showed a significant reduction of the NaV1.7 canonical transcript. Collectively, these data widen the spectrum of SCN9A-related insensitivity to pain by describing a mutation causing NaV1.7 deficiency, underlying the nociceptor dysfunction, and highlight the importance of molecular investigation of U12 introns' mutations despite the silent prediction.

The Voltage-Gated Sodium Channel Beta4 Subunit Maintains Epithelial Phenotype in Mammary Cells.

The SCN4B gene, coding for the NaVβ4 subunit of voltage-gated sodium channels, was recently found to be expressed in normal epithelial cells and down-regulated in several cancers. However, its function in normal epithelial cells has not been characterized. In this study, we demonstrated that reducing NaVβ4 expression in MCF10A non-cancer mammary epithelial cells generated important morphological changes observed both in two-dimensional cultures and in three-dimensional cysts. Most notably, the loss of NaVβ4 induced a complete loss of epithelial organisation in cysts and increased proteolytic activity towards the extracellular matrix. Loss of epithelial morphology was associated with an increased degradation of β-catenin, reduced E-cadherin expression and induction of mesenchymal markers N-cadherin, vimentin, and α-SMA expression. Overall, our results suggest that Navβ4 may participate in the maintenance of the epithelial phenotype in mammary cells and that its downregulation might be a determining step in early carcinogenesis.