Complex biophysical changes and reduced neuronal firing in an SCN8A variant associated with developmental delay and epilepsy.

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of NaV1.6G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of NaV1.6G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in NaV1.6G1625R. Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection.

Cardiac-Specific Deletion of Scn8a Mitigates Dravet Syndrome-Associated Sudden Death in Adults.

BACKGROUND: Sudden unexpected death in epilepsy (SUDEP) is a fatal complication experienced by otherwise healthy epilepsy patients. Dravet syndrome (DS) is an inherited epileptic disorder resulting from loss of function of the voltage-gated sodium channel, NaV 1.1, and is associated with particularly high SUDEP risk. Evidence is mounting that NaVs abundant in the brain also occur in the heart, suggesting that the very molecular mechanisms underlying epilepsy could also precipitate cardiac arrhythmias and sudden death. Despite marked reduction of NaV 1.1 functional expression in DS, pathogenic late sodium current (INa,L) is paradoxically increased in DS hearts. However, the mechanisms by which DS directly impacts the heart to promote sudden death remain unclear. OBJECTIVES: In this study, the authors sought to provide evidence implicating remodeling of Na+ - and Ca2+ -handling machinery, including NaV 1.6 and Na+/Ca2+exchanger (NCX) within transverse (T)-tubules in DS-associated arrhythmias. METHODS: The authors undertook scanning ion conductance microscopy (SICM)-guided patch clamp, super-resolution microscopy, confocal Ca2+ imaging, and in vivo electrocardiography studies in Scn1a haploinsufficient murine model of DS. RESULTS: DS promotes INa,L in T-tubular nanodomains, but not in other subcellular regions. Consistent with increased NaV activity in these regions, super-resolution microscopy revealed increased NaV 1.6 density near Ca2+release channels, the ryanodine receptors (RyR2) and NCX in DS relative to WT hearts. The resulting INa,L in these regions promoted aberrant Ca2+ release, leading to ventricular arrhythmias in vivo. Cardiac-specific deletion of NaV 1.6 protects adult DS mice from increased T-tubular late NaV activity and the resulting arrhythmias, as well as sudden death. CONCLUSIONS: These data demonstrate that NaV 1.6 undergoes remodeling within T-tubules of adult DS hearts serving as a substrate for Ca2+ -mediated cardiac arrhythmias and may be a druggable target for the prevention of SUDEP in adult DS subjects.

Sex differences in physiological response to increased neuronal excitability in a knockin mouse model of pediatric epilepsy.

BACKGROUND: Epilepsy is a common neurological disease; however, few if any of the currently marketed antiseizure medications prevent or cure epilepsy. Discovery of pathological processes in the early stages of epileptogenesis has been challenging given the common use of preclinical models that induce seizures in physiologically normal animals. Moreover, despite known sex dimorphism in neurological diseases, females are rarely included in preclinical epilepsy models. METHODS: We characterized sex differences in mice carrying a pathogenic knockin variant (p.N1768D) in the Scn8a gene that causes spontaneous tonic-clonic seizures (TCs) at ∼3 months of age and found that heterozygous females are more resilient than males in mortality and morbidity. To investigate the cellular mechanisms that underlie female resilience, we utilized blood-brain barrier (BBB) and hippocampal transcriptomic analyses in heterozygous mice before seizure onset (pre-TC) and in mice that experienced ∼20 TCs (post-TC). RESULTS: In the pre-TC latent phase, both sexes exhibited leaky BBB; however, patterns of gene expression were sexually dimorphic. Females exhibited enhanced oxidative phosphorylation and protein biogenesis, while males activated gliosis and CREB signaling. After seizure onset (chronic phase), females exhibited a metabolic switch to lipid metabolism, while males exhibited increased gliosis and BBB dysfunction and a strong activation of neuroinflammatory pathways. CONCLUSION: The results underscore the central role of oxidative stress and BBB permeability in the early stages of epileptogenesis, as well as sex dimorphism in response to increasing neuronal hyperexcitability. Our results also highlight the need to include both sexes in preclinical studies to effectively translate results of drug efficacy studies.

CircFNTA promotes tumorigenesis and progression of gastric cancer via miR-604/miR-647/SCN8A axis.

Gastric cancer (GC) is a major contributor to cancer-related deaths and is characterized by high heterogeneity in epidemiology and histopathology worldwide. Increasing evidence indicates that circular RNAs (circRNAs) play multifaceted roles in cellular processes in human cancers. Here, we demonstrated that circFNTA high expression increases the proliferation, metastasis, and epithelial-mesenchymal transition process and tumorigenicity of GC cells. First, we found that circFNTA was upregulated in GC cells and tissues, and the high circFNTA levels were positively associated with the poor prognosis in GC patients. Using luciferase reporter and RNA-pull down assays, we elucidated that circFNTA sponged two microRNAs, miR-604 and miR-647. In addition, the proliferation and metastatic ability of GC cell reduction caused by silencing circFNTA was hindered by inhibitors of miR-604 and miR-647. Moreover, SCN8A was predicted by miRDB as a common target gene of miR-604 and miR-647, which was then verified by the luciferase reporter assay. Knockdown of circFNTA causes messenger RNA and protein levels in SCN8A to be downregulated in GC cells. However, this effect was overturned by cotransfection miR-604 and miR-647. Also, we identified that SCN8A was downregulated in GC tissues, which was positively correlated with circFNTA expression. In rescue experiments, the attenuated cell proliferation and metastatic ability caused by circFNTA knockdown was reversed by miR-604 and miR-647 inhibitors and SCN8A overexpression. Collectively, our findings suggest an oncogenic role of circFNTA in GC progression and elucidate that circFNTA exerts its function by modulating the miR-604/miR-647/SCN8A axis.

TNFR1 signaling converging on FGF14 controls neuronal hyperactivity and sickness behavior in experimental cerebral malaria.

BACKGROUND: Excess tumor necrosis factor (TNF) is implicated in the pathogenesis of hyperinflammatory experimental cerebral malaria (eCM), including gliosis, increased levels of fibrin(ogen) in the brain, behavioral changes, and mortality. However, the role of TNF in eCM within the brain parenchyma, particularly directly on neurons, remains underdefined. Here, we investigate electrophysiological consequences of eCM on neuronal excitability and cell signaling mechanisms that contribute to observed phenotypes. METHODS: The split-luciferase complementation assay (LCA) was used to investigate cell signaling mechanisms downstream of tumor necrosis factor receptor 1 (TNFR1) that could contribute to changes in neuronal excitability in eCM. Whole-cell patch-clamp electrophysiology was performed in brain slices from eCM mice to elucidate consequences of infection on CA1 pyramidal neuron excitability and cell signaling mechanisms that contribute to observed phenotypes. Involvement of identified signaling molecules in mediating behavioral changes and sickness behavior observed in eCM were investigated in vivo using genetic silencing. RESULTS: Exploring signaling mechanisms that underlie TNF-induced effects on neuronal excitability, we found that the complex assembly of fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ (Nav) channel 1.6 (Nav1.6) is increased upon tumor necrosis factor receptor 1 (TNFR1) stimulation via Janus Kinase 2 (JAK2). On account of the dependency of hyperinflammatory experimental cerebral malaria (eCM) on TNF, we performed patch-clamp studies in slices from eCM mice and showed that Plasmodium chabaudi infection augments Nav1.6 channel conductance of CA1 pyramidal neurons through the TNFR1-JAK2-FGF14-Nav1.6 signaling network, which leads to hyperexcitability. Hyperexcitability of CA1 pyramidal neurons caused by infection was mitigated via an anti-TNF antibody and genetic silencing of FGF14 in CA1. Furthermore, knockdown of FGF14 in CA1 reduced sickness behavior caused by infection. CONCLUSIONS: FGF14 may represent a therapeutic target for mitigating consequences of TNF-mediated neuroinflammation.

Increased expression of Nav1.6 of reactive astrocytes in the globus pallidus is closely associated with motor deficits in a model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease in elderly people, which is characterized by motor disabilities in PD patients. Nav1.6 is the most abundant subtype of voltage-gated sodium channels (VGSCs) in the brain of adult mammals and rodents. Here we investigated the role of Nav1.6 in the external globus pallidus (GP) involved in the pathogenesis of motor deficits in unilateral 6-OHDA(6-hydroxydopamine)lesioned rats. The results show that Nav1.6 is dramatically increased in reactive astrocytes of the ipsilateral GP in the middle stage, but not different from the control rats in the later stage of the pathological process in 6-OHDA lesioned rats. Furthermore, the down-regulation of Nav1.6 expression in the ipsilateral GP can significantly improve motor deficits in 6-OHDA lesioned rats in the middle stage of the pathological process. The electrophysiological experiments show that the down-regulation of Nav1.6 expression in the ipsilateral GP significantly decreases the abnormal high synchronization between the ipsilateral M1 (the primary motor cortex) and GP in 6-OHDA lesioned rats. Ca2+ imaging reveals that the down-regulation of Nav1.6 expression reduces the intracellular concentration of Ca2+ ([Ca2+ ]i) in primary cultured astrocytes. These findings suggest that the increased Nav1.6 expression of reactive astrocytes in the GP play an important role in the pathogenesis of motor dysfunction in the middle stage in 6-OHDA lesioned rats, which may participate in astrocyte-neuron communication by regulating [Ca2+ ]i of astrocytes, thereby contributing to the formation of abnormal electrical signals of the basal ganglia (BG) in 6-OHDA lesioned rats.

Upregulation of Nav1.6 Mediated by the p38 MAPK Pathway in the Dorsal Root Ganglia Contributes to Cancer-Induced Bone Pain in Rats.

Cancer-induced bone pain (CIBP) occurs frequently among advanced cancer patients. Voltage-gated sodium channels (VGSCs) have been associated with chronic pain, but how VGSCs function in CIBP is poorly understood. Here, we aimed to investigate the specific role of VGSCs in the dorsal root ganglia (DRGs) in CIBP. A CIBP rat model was generated by the intratibial inoculation of MRMT-1 breast carcinoma cells. Transcriptome sequencing was conducted to assess the gene expression profiles. The expression levels of key genes and differentiated genes related to activated pathways were measured by Western blotting and qPCR. We implanted a catheter intrathecally for the administration of lentivirus and drugs. Then, the changes in the mechanical withdrawal threshold (MWT) were measured. We identified 149 differentially expressed mRNAs (DEmRNAs) in the DRGs of CIBP model rats. The expression of Nav1.6, which was among these DEmRNAs, was significantly upregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEmRNAs showed that they were mainly enriched in the mitogen-activated protein kinase (MAPK) pathway. The decrease in MWT induced by bone cancer was attenuated by Nav1.6 knockdown. Western blot analysis revealed that a p38 inhibitor decreased the expression of Nav1.6 and attenuated pain behavior. Our study shows that the upregulation of Nav1.6 expression by p38 MAPK in the DRGs of rats contributes to CIBP.

De novo Y1460C missense variant in NaV1.1 impedes the pore region and results in epileptic encephalopathy.

Epilepsy is a common neurological disorder characterized by recurrent unprovoked seizures. SCN1A encodes NaV1.1, a neuronal voltage-gated Na+ channel that is highly expressed throughout the central nervous system. NaV1.1 is localized within the axon initial segment where it plays a critical role in the initiation and propagation of action potentials and neuronal firing, predominantly in γ-amino-butyric-acid (GABA)ergic neurons of the hippocampus. The objective of this study was to characterize a de novo missense variant of uncertain significance in the SCN1A gene of a proband presented with febrile status epilepticus characterized by generalized tonic clonic movements associated with ictal emesis and an abnormal breathing pattern. Screening a gene panel revealed a heterozygous missense variant of uncertain significance in the SCN1A gene, designated c.4379A>G, p.(Tyr1460Cys). The NaV1.1 wild-type (WT) and mutant channel reproduced in vivo and were transfected in HEK 293 cells. Na+ currents were recorded using the whole-cell configuration of the patch-clamp technique. This NaV1.1 variant (Tyr1460Cys) failed to express functional Na+ currents when expressed in HEK293 cells, most probably due to a pore defect of the channel given that the cell surface expression of the channel was normal. Currents generated after co-transfection with functional WT channels exhibited biophysical properties comparable to those of WT channels, which was mainly due to the functional WT channels at the cell surface. The NaV1.1 variant failed to express functional Na+ currents, most probably due to pore impairment and exhibited a well-established loss of function mechanism. The present study highlights the added-value of functional testing for understanding the pathophysiology and potential treatment decisions for patients with undiagnosed developmental epileptic encephalopathy.

Modulating effects of FGF12 variants on NaV1.2 and NaV1.6 being associated with developmental and epileptic encephalopathy and Autism spectrum disorder: A case series.

OBJECTIVE: Fibroblast Growth Factor 12 (FGF12) may represent an important modulator of neuronal network activity and has been associated with developmental and epileptic encephalopathy (DEE). We sought to identify the underlying pathomechanism of FGF12-related disorders. METHODS: Patients with pathogenic variants in FGF12 were identified through published case reports, GeneMatcher and whole exome sequencing of own case collections. The functional consequences of two missense and two copy number variants (CNVs) were studied by co-expression of wildtype and mutant FGF12 in neuronal-like cells (ND7/23) with the sodium channels NaV1.2 or NaV1.6, including their beta-1 and beta-2 sodium channel subunits (SCN1B and SCN2B). RESULTS: Four variants in FGF12 were identified for functional analysis: one novel FGF12 variant in a patient with autism spectrum disorder and three variants from previously published patients affected by DEE. We demonstrate the differential regulating effects of wildtype and mutant FGF12 on NaV1.2 and NaV1.6 channels. Here, FGF12 variants lead to a complex kinetic influence on NaV1.2 and NaV1.6, including loss- as well as gain-of function changes in fast and slow inactivation. INTERPRETATION: We could demonstrate the detailed regulating effect of FGF12 on NaV1.2 and NaV1.6 and confirmed the complex effect of FGF12 on neuronal network activity. Our findings expand the phenotypic spectrum related to FGF12 variants and elucidate the underlying pathomechanism. Specific variants in FGF12-associated disorders may be amenable to precision treatment with sodium channel blockers. FUNDING: DFG, BMBF, Hartwell Foundation, National Institute for Neurological Disorders and Stroke, IDDRC, ENGIN, NIH, ITMAT, ILAE, RES and GRIN.

Generation of a human induced pluripotent stem cell line (CIPi002-A) from an early infantile epileptic encephalopathy patient with a heterozygous mutation in SCN8A.

SCN8A is linked to early infantile epileptic encephalopathy type 13 (EIEE13). We generate a human induced pluripotent stem cell (iPSC) line from a child diagnosed with EIEE, caused by SCN8A variation. The iPSC line expresses high pluripotency markers, retains SCN8A variation and is able to differentiate into three germ layers in vitro. The iPSC lines will provide useful resources for studying the pathogenesis and drug screening of SCN8A-related epilepsy.

Nav1.6 promotes the progression of human follicular thyroid carcinoma cells via JAK-STAT signaling pathway.

Follicular thyroid carcinoma (FTC) is one of the most common malignant tumors of the endocrine system. Recent studies have shown that voltage-gated sodium channels (VGSCs) affect the proliferation, migration, and invasion of tumor cells. However, the expression and functions of VGSCs, and the molecular pathways activated by VGSCs in FTC cells remain unclear. Our studies revealed that the expression of Nav1.6, encoded by SCN8A, was the predominantly upregulated subtype of VGSCs in FTC tissues. Knockdown of Nav1.6 significantly inhibited the proliferation, epithelial-mesenchymal transition and invasiveness of FTC cells. Using gene set enrichment analysis and Kyoto Encyclopedia of Genes and Genomics, SCN8A was predicted to be related to the JAK-STAT signaling pathway. Hence, we targeted the JAK-STAT pathway and demonstrated that Nav1.6 enhanced FTC cell proliferation, epithelial-mesenchymal transition, and invasion by phosphorylating JAK2 to activate STAT3. Furthermore, downregulating the expression of Nav1.6 improve the susceptibility of FTC cells to ubenimex in vitro. These results suggest Nav1.6 accelerates FTC progression through JAK/STAT signaling and may be a potential target for FTC therapy.

Reducing Nav1.6 expression attenuates the pathogenesis of Alzheimer's disease by suppressing BACE1 transcription.

Aberrant increases in neuronal network excitability may contribute to cognitive deficits in Alzheimer's disease (AD). However, the mechanisms underlying hyperexcitability of neurons are not fully understood. Voltage-gated sodium channels (VGSC or Nav), which are involved in the formation of excitable cell's action potential and can directly influence the excitability of neural networks, have been implicated in AD-related abnormal neuronal hyperactivity and higher incidence of spontaneous non-convulsive seizures. Here, we have shown that the reduction of VGSC α-subunit Nav1.6 (by injecting adeno-associated virus (AAV) with short hairpin RNA (shRNA) into the hippocampus) rescues cognitive impairments and attenuates synaptic deficits in APP/PS1 transgenic mice. Concurrently, amyloid plaques in the hippocampus and levels of soluble Aß are significantly reduced. Interfering with Nav1.6 reduces the transcription level of ß-site APP-cleaving enzyme 1 (BACE1), which is Aß-dependent. In the presence of Aß oligomers, knockdown of Nav1.6 reduces intracellular calcium overload by suppressing reverse sodium-calcium exchange channel, consequently increasing inactive NFAT1 (the nuclear factor of activated T cells) levels and thus reducing BACE1 transcription. This mechanism leads to a reduction in the levels of Aß in APP/PS1 transgenic mice, alleviates synaptic loss, improves learning and memory disorders in APP/PS1 mice after downregulating Nav1.6 in the hippocampus. Our study offers a new potential therapeutic strategy to counteract hippocampal hyperexcitability and subsequently rescue cognitive deficits in AD by selective blockade of Nav1.6 overexpression and/or hyperactivity.

A Novel SCN8A mutation in a case of early-onset infantile epileptic encephalopathy: A Case Report.

SCN8A gene encodes sodium channel alpha subunit Nav1.6, and its mutation is associated with Early Infantile Epileptic Encephalopathy-13 (EIEE-13). The mean age of onset is 4-5 months. The phenotype of SCN8A mutation varies from benign epilepsy syndromes, movement disorder, intellectual disability to severe epileptic syndromes with different types of seizures. We hereby report a case of a one-year old female who had an onset of infantile spasms on the seventeenth day of life, which gradually progressed to focal, multifocal, GTCS, and epileptic encephalopathy by one year of age associated with global developmental delay and hypotonia. All metabolic workup, TMS, GCMS, and MRI brain were normal. EEG at 2.5 months was suggestive of epileptic discharge arising from the left frontal region, evolving into generalized discharges. Whole exome sequencing revealed a heterozygous mutation in the SCN8A gene at exon 16 (p.Val892Ala) suggestive of Early Infantile Epileptic Encephalopathy-13 (EIEE-13). This is a novel mutation in the SCN8A gene which has not been reported previously in the literature.

Inhibition of the Akt/PKB Kinase Increases Nav1.6-Mediated Currents and Neuronal Excitability in CA1 Hippocampal Pyramidal Neurons.

In neurons, changes in Akt activity have been detected in response to the stimulation of transmembrane receptors. However, the mechanisms that lead to changes in neuronal function upon Akt inhibition are still poorly understood. In the present study, we interrogate how Akt inhibition could affect the activity of the neuronal Nav channels with while impacting intrinsic excitability. To that end, we employed voltage-clamp electrophysiological recordings in heterologous cells expressing the Nav1.6 channel isoform and in hippocampal CA1 pyramidal neurons in the presence of triciribine, an inhibitor of Akt. We showed that in both systems, Akt inhibition resulted in a potentiation of peak transient Na+ current (INa) density. Akt inhibition correspondingly led to an increase in the action potential firing of the CA1 pyramidal neurons that was accompanied by a decrease in the action potential current threshold. Complementary confocal analysis in the CA1 pyramidal neurons showed that the inhibition of Akt is associated with the lengthening of Nav1.6 fluorescent intensity along the axonal initial segment (AIS), providing a mechanism for augmented neuronal excitability. Taken together, these findings provide evidence that Akt-mediated signal transduction might affect neuronal excitability in a Nav1.6-dependent manner.

Astrocyte reactivity in a mouse model of SCN8A epileptic encephalopathy.

OBJECTIVE: SCN8A epileptic encephalopathy is caused predominantly by de novo gain-of-function mutations in the voltage-gated sodium channel Nav 1.6. The disorder is characterized by early onset of seizures and developmental delay. Most patients with SCN8A epileptic encephalopathy are refractory to current anti-seizure medications. Previous studies determining the mechanisms of this disease have focused on neuronal dysfunction as Nav 1.6 is expressed by neurons and plays a critical role in controlling neuronal excitability. However, glial dysfunction has been implicated in epilepsy and alterations in glial physiology could contribute to the pathology of SCN8A encephalopathy. In the current study, we examined alterations in astrocyte and microglia physiology in the development of seizures in a mouse model of SCN8A epileptic encephalopathy. METHODS: Using immunohistochemistry, we assessed microglia and astrocyte reactivity before and after the onset of spontaneous seizures. Expression of glutamine synthetase and Nav 1.6, and Kir 4.1 channel currents were assessed in astrocytes in wild-type (WT) mice and mice carrying the N1768D SCN8A mutation (D/+). RESULTS: Astrocytes in spontaneously seizing D/+ mice become reactive and increase expression of glial fibrillary acidic protein (GFAP), a marker of astrocyte reactivity. These same astrocytes exhibited reduced barium-sensitive Kir 4.1 currents compared to age-matched WT mice and decreased expression of glutamine synthetase. These alterations were only observed in spontaneously seizing mice and not before the onset of seizures. In contrast, microglial morphology remained unchanged before and after the onset of seizures. SIGNIFICANCE: Astrocytes, but not microglia, become reactive only after the onset of spontaneous seizures in a mouse model of SCN8A encephalopathy. Reactive astrocytes have reduced Kir 4.1-mediated currents, which would impair their ability to buffer potassium. Reduced expression of glutamine synthetase would modulate the availability of neurotransmitters to excitatory and inhibitory neurons. These deficits in potassium and glutamate handling by astrocytes could exacerbate seizures in SCN8A epileptic encephalopathy. Targeting astrocytes may provide a new therapeutic approach to seizure suppression.

Pharmacologically Targeting the Fibroblast Growth Factor 14 Interaction Site on the Voltage-Gated Na+ Channel 1.6 Enables Isoform-Selective Modulation.

Voltage-gated Na+ (Nav) channels are the primary molecular determinant of the action potential. Among the nine isoforms of the Nav channel α subunit that have been described (Nav1.1-Nav1.9), Nav1.1, Nav1.2, and Nav1.6 are the primary isoforms expressed in the central nervous system (CNS). Crucially, these three CNS Nav channel isoforms display differential expression across neuronal cell types and diverge with respect to their subcellular distributions. Considering these differences in terms of their localization, the CNS Nav channel isoforms could represent promising targets for the development of targeted neuromodulators. However, current therapeutics that target Nav channels lack selectivity, which results in deleterious side effects due to modulation of off-target Nav channel isoforms. Among the structural components of the Nav channel α subunit that could be pharmacologically targeted to achieve isoform selectivity, the C-terminal domains (CTD) of Nav channels represent promising candidates on account of displaying appreciable amino acid sequence divergence that enables functionally unique protein-protein interactions (PPIs) with Nav channel auxiliary proteins. In medium spiny neurons (MSNs) of the nucleus accumbens (NAc), a critical brain region of the mesocorticolimbic circuit, the PPI between the CTD of the Nav1.6 channel and its auxiliary protein fibroblast growth factor 14 (FGF14) is central to the generation of electrical outputs, underscoring its potential value as a site for targeted neuromodulation. Focusing on this PPI, we previously developed a peptidomimetic derived from residues of FGF14 that have an interaction site on the CTD of the Nav1.6 channel. In this work, we show that whereas the compound displays dose-dependent effects on the activity of Nav1.6 channels in heterologous cells, the compound does not affect Nav1.1 or Nav1.2 channels at comparable concentrations. In addition, we show that the compound correspondingly modulates the action potential discharge and the transient Na+ of MSNs of the NAc. Overall, these results demonstrate that pharmacologically targeting the FGF14 interaction site on the CTD of the Nav1.6 channel is a strategy to achieve isoform-selective modulation, and, more broadly, that sites on the CTDs of Nav channels interacted with by auxiliary proteins could represent candidates for the development of targeted therapeutics.

Pharmacological Inhibition of Wee1 Kinase Selectively Modulates the Voltage-Gated Na+ Channel 1.2 Macromolecular Complex.

Voltage-gated Na+ (Nav) channels are a primary molecular determinant of the action potential (AP). Despite the canonical role of the pore-forming α subunit in conferring this function, protein-protein interactions (PPI) between the Nav channel α subunit and its auxiliary proteins are necessary to reconstitute the full physiological activity of the channel and to fine-tune neuronal excitability. In the brain, the Nav channel isoforms 1.2 (Nav1.2) and 1.6 (Nav1.6) are enriched, and their activities are differentially regulated by the Nav channel auxiliary protein fibroblast growth factor 14 (FGF14). Despite the known regulation of neuronal Nav channel activity by FGF14, less is known about cellular signaling molecules that might modulate these regulatory effects of FGF14. To that end, and building upon our previous investigations suggesting that neuronal Nav channel activity is regulated by a kinase network involving GSK3, AKT, and Wee1, we interrogate in our current investigation how pharmacological inhibition of Wee1 kinase, a serine/threonine and tyrosine kinase that is a crucial component of the G2-M cell cycle checkpoint, affects the Nav1.2 and Nav1.6 channel macromolecular complexes. Our results show that the highly selective inhibitor of Wee1 kinase, called Wee1 inhibitor II, modulates FGF14:Nav1.2 complex assembly, but does not significantly affect FGF14:Nav1.6 complex assembly. These results are functionally recapitulated, as Wee1 inhibitor II entirely alters FGF14-mediated regulation of the Nav1.2 channel, but displays no effects on the Nav1.6 channel. At the molecular level, these effects of Wee1 inhibitor II on FGF14:Nav1.2 complex assembly and FGF14-mediated regulation of Nav1.2-mediated Na+ currents are shown to be dependent upon the presence of Y158 of FGF14, a residue known to be a prominent site for phosphorylation-mediated regulation of the protein. Overall, our data suggest that pharmacological inhibition of Wee1 confers selective modulatory effects on Nav1.2 channel activity, which has important implications for unraveling cellular signaling pathways that fine-tune neuronal excitability.

Soluble tumor necrosis factor-alpha-induced hyperexcitability contributes to retinal ganglion cell apoptosis by enhancing Nav1.6 in experimental glaucoma.

BACKGROUND: Neuroinflammation plays an important role in the pathogenesis of glaucoma. Tumor necrosis factor-alpha (TNF-α) is a major pro-inflammatory cytokine released from activated retinal glial cells in glaucoma. Here, we investigated how TNF-α induces retinal ganglion cell (RGC) hyperexcitability and injury. METHODS: Whole-cell patch-clamp techniques were performed to explore changes in spontaneous firing and evoked action potentials, and Na+ currents in RGCs. Both intravitreal injection of TNF-α and chronic ocular hypertension (COH) models were used. Western blotting, immunofluorescence, quantitative real-time polymerase chain reaction (q-PCR), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) techniques were employed to investigate the molecular mechanisms of TNF-α effects on RGCs. RESULTS: Intravitreal injection of soluble TNF-α significantly increased the spontaneous firing frequencies of RGCs in retinal slices. When the synaptic transmissions were blocked, more than 90% of RGCs still showed spontaneous firing; both the percentage of cells and firing frequency were higher than the controls. Furthermore, the frequency of evoked action potentials was also higher than the controls. Co-injection of the TNF-α receptor 1 (TNFR1) inhibitor R7050 eliminated the TNF-α-induced effects, suggesting that TNF-α may directly act on RGCs to induce cell hyperexcitability through activating TNFR1. In RGCs acutely isolated from TNF-α-injected retinas, Na+ current densities were upregulated. Perfusing TNF-α in RGCs of normal rats mimicked this effect, and the activation curve of Na+ currents shifted toward hyperpolarization direction, which was mediated through p38 MAPK and STAT3 signaling pathways. Further analysis revealed that TNF-α selectively upregulated Nav1.6 subtype of Na+ currents in RGCs. Similar to observations in retinas of rats with COH, intravitreal injection of TNF-α upregulated the expression of Nav1.6 proteins in both total cell and membrane components, which was reversed by the NF-κB inhibitor BAY 11-7082. Inhibition of TNFR1 blocked TNF-α-induced RGC apoptosis. CONCLUSIONS: TNF-α/TNFR1 signaling induces RGC hyperexcitability by selectively upregulating Nav1.6 Na+ channels, thus contributing to RGC apoptosis in glaucoma.

Distinctive Properties and Powerful Neuromodulation of Nav1.6 Sodium Channels Regulates Neuronal Excitability.

Voltage-gated sodium channels (Navs) are critical determinants of cellular excitability. These ion channels exist as large heteromultimeric structures and their activity is tightly controlled. In neurons, the isoform Nav1.6 is highly enriched at the axon initial segment and nodes, making it critical for the initiation and propagation of neuronal impulses. Changes in Nav1.6 expression and function profoundly impact the input-output properties of neurons in normal and pathological conditions. While mutations in Nav1.6 may cause channel dysfunction, aberrant changes may also be the result of complex modes of regulation, including various protein-protein interactions and post-translational modifications, which can alter membrane excitability and neuronal firing properties. Despite decades of research, the complexities of Nav1.6 modulation in health and disease are still being determined. While some modulatory mechanisms have similar effects on other Nav isoforms, others are isoform-specific. Additionally, considerable progress has been made toward understanding how individual protein interactions and/or modifications affect Nav1.6 function. However, there is still more to be learned about how these different modes of modulation interact. Here, we examine the role of Nav1.6 in neuronal function and provide a thorough review of this channel's complex regulatory mechanisms and how they may contribute to neuromodulation.

Mice Heterozygous for the Sodium Channel Scn8a (Nav1.6) Have Reduced Inflammatory Responses During EAE and Following LPS Challenge.

Voltage gated sodium (Nav) channels contribute to axonal damage following demyelination in experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). The Nav1.6 isoform has been implicated as a primary contributor in this process. However, the role of Nav1.6 in immune processes, critical to the pathology of both MS and EAE, has not been extensively studied. EAE was induced with myelin oligodendrocyte (MOG35-55) peptide in Scn8admu/+ mice, which have reduced Nav1.6 levels. Scn8admu/+ mice demonstrated improved motor capacity during the recovery and early chronic phases of EAE relative to wild-type animals. In the optic nerve, myeloid cell infiltration and the effects of EAE on the axonal ultrastructure were also significantly reduced in Scn8admu/+ mice. Analysis of innate immune parameters revealed reduced plasma IL-6 levels and decreased percentages of Gr-1high/CD11b+ and Gr-1int/CD11b+ myeloid cells in the blood during the chronic phase of EAE in Scn8admu/+ mice. Elevated levels of the anti-inflammatory cytokines IL-10, IL-13, and TGF-ß1 were also observed in the brains of untreated Scn8admu/+ mice. A lipopolysaccharide (LPS) model was used to further evaluate inflammatory responses. Scn8admu/+ mice displayed reduced inflammation in response to LPS challenge. To further evaluate if this was an immune cell-intrinsic difference or the result of changes in the immune or hormonal environment, mast cells were derived from the bone marrow of Scn8admu/+ mice. These mast cells also produced lower levels of IL-6, in response to LPS, compared with those from wild type mice. Our results demonstrate that in addition to its recognized impact on axonal damage, Nav1.6 impacts multiple aspects of the innate inflammatory response.