Alzheimer's disease (AD), particularly late-onset AD (LOAD), affects millions worldwide, with the apolipoprotein ε 4 ( APOE4 ) allele being a significant genetic risk factor. Retinal abnormalities are a hallmark of LOAD, and our recent study demonstrated significant age-related retinal impairments in APOE4 -knock-in (KI) mice, highlighting that retinal impairments occur before the onset of cognitive decline in these mice. Müller cells (MCs), key retinal glia, are vital for retinal health, and their dysfunction may contribute to retinal impairments seen in AD. MCs maintain potassium balance via specialized inwardly rectifying K + channels 4.1 (Kir4.1). This study posits that Kir4.1 channels will be impaired in APOE4 -KI, resulting in MC dysfunction. Additionally, we demonstrate that MC dysfunction in APOE4 -KI stems from alterations in mitochondrial dynamics and oxidative stress. Kir4.1 expression and function were studied using immunofluorescence and through the whole-cell voltage clamp, respectively. In parallel, rat Müller cells (rMC-1) were used to create an in vitro model for further mechanistic studies. MitoQ was used to evaluate its potential to mitigate APOE4 -induced deficits. APOE4 retinas and APOE4 -transfected rMC-1 significantly reduced Kir4.1 expression, K+ buffering capacity, and increased mitochondrial damage. APOE4 -transfected rMC-1 showed reduced mitochondrial membrane potential (ΔΨm) and increased mitochondrial reactive oxygen species (ROS). MitoQ treatment significantly reduced mitochondrial ROS and restored Kir4.1 expression in APOE4 -expressing cells. Our results demonstrate that APOE4 causes mitochondrial dysfunction and MC impairment, which may contribute to retinal pathology in AD. MitoQ restored mitochondrial health and Kir4.1 expression in APOE4 -expressing rMC-1, suggesting targeting mitochondria may offer a promising therapeutic strategy for AD.

Pain is a serious medical condition with current treatments remaining limited by side effects. The Nav1.7 voltage-gated sodium channel is a crucial determinant of nociceptor excitability and a promising target for nonaddictive analgesics. However, development of blockers has been difficult. In this issue of the JCI, Singh, Bernabucci, and authors identify a strategy for reducing Nav1.7 currents. These findings identify fibroblast growth factor 13 (FGF13), a homologous factor distinct from typical growth factors (also known as FHF2B), which ramps up Nav1.7, nociceptor excitability, and pain. Compound PW164 was identified as a selective FGF13-Nav1.7 attenuator with analgesic activity. These findings highlight the power of targeting intrinsic modulators of Nav1.7 for pain management.

Voltage-gated sodium channels (VGSCs) play pivotal roles in cellular function, particularly in the regulation of electrical signaling. Structural defects in these channels cause deleterious effects in a myriad of cell types, leading to various diseases, like epilepsy, cardiac arrythmias, kidney disease, and certain cancers. Over the past decade, significant efforts have been geared toward developing drugs that target the pore domains of these channels, called pore-blocking agents. This approach has seen several setbacks, commonly due to the lack of isoform-specific binding. Alternative targeting strategies are being used to reduce or eliminate the side effects of pore-blocking agents. Transgenic mouse models have proven useful in such studies, and subtype-selective inhibitors were developed. The zebrafish model system was also used to explore neurological, cardiovascular, and metabolic diseases caused by voltage-gated sodium channel dysfunction. Here, we delve into the growing literature on the structure and function of voltage-gated sodium channels, their role in epilepsy and its comorbidities, and the advancement in the use of zebrafish as a model system to explore these channels as therapeutic targets.

Fibroblast growth factor homologous factors (FHFs) regulate the activity of several different voltage-gated sodium channels (Navs). However, more work is needed to determine how specific FHF isoforms and variants affect the properties of different Nav isoforms. In addition, it is not known if FHFs can differentially modulate the properties of Nav variants associated with disease. Here, we investigated the effects of FHF2A and FHF2B on Nav1.1 properties as well as on a familial hemiplegic migraine 3 (FHM3) causing mutation in this channel, F1774S. We found that FHF2A, but not 2B, induced prominent long-term inactivation (LTI) in the wild-type (WT) Nav1.1. Interestingly, FHF2A induced LTI in the F1774S FHM3 mutant channel to a greater extent than in the WT. Furthermore, persistent currents caused by the F1774S mutation were attenuated by the co-expression of FHF2A, leading to a possible rescue of the mutant channel phenotype. By contrast, the P1894L mutation, which is associated with epilepsy and mild intellectual disability, greatly attenuated the LTI induced by FHF2A. Overall, our data show for the first time that FHF2A might be a significant modulator of Nav1.1 that can differentially modulate the impact of Nav1.1 disease-associated mutations.

Chronic neuropathic pain associated with spinal cord injury (SCI) poses a significant medical challenge. Studies have shown that moderate thoracic (T10) SCI can substantially enhance the excitability of small diameter dorsal root ganglion (DRG) neurons, suggesting that pain resulting from SCI may arise from pathological alternations in peripheral neurons. However, the molecular mechanisms underlying these changes remain unclear. Here we show that contusive SCI significantly increases transient and resurgent sodium currents mainly through Nav1.8 in rat small DRG neurons. Additionally, SCI increases the proportion of small DRG neurons that produce tetrodotoxin-resistant resurgent currents. The SCI-induced increase of Nav1.8 currents can be reversed by ZL0177, a small peptidomimetic of the critical residues in fibroblast growth factor homologous factors 4 (FHF4) that binds to the C-terminal tail of sodium channels. ZL0177 not only decreases the transient and resurgent currents of Nav1.8 and Nav1.9 but also reduces the proportion of the neurons that generate Nav1.8 resurgent currents. We further show that ZL0177 greatly attenuates the hyperexcitability of small DRG neurons induced by SCI. Taken together, our results indicate Nav1.8 dysfunction following SCI plays a critical role in enhancing excitability of nociceptive neurons. Furthermore, the binding site of FHFs at the C-terminal tail of Nav1.8 and Nav1.9 can serve as a promising therapeutic target for the treatment of SCI pain. KEY POINTS: Traumatic spinal cord injury (SCI) often leads to chronic pain conditions. Recent clinical and experimental studies indicate that the pain induced by SCI may be attributed to abnormal peripheral neuron inputs; however, the molecular mechanisms underlying these changes remain unclear. Here we studied sodium currents and action potential firing in rat small DRG neurons following contusive SCI. Contusive SCI significantly increased Nav1.8 transient and resurgent sodium currents in the rat small DRG neurons. ZL0177, a peptidomimetic inhibitor of fibroblast growth factor homologous factor (FHF) binding to the C-terminus of sodium channels not only decreased Nav1.8/Nav1.9 currents, but also greatly attenuated the hyperexcitability of small DRG neurons induced by SCI. Thus, targeting FHF modulation of Nav1.8/Nav1.9 may serve as a promising treatment strategy for SCI pain.

Reactive astrocytes are known to exert detrimental effects upon neurons in several neurodegenerative diseases, yet our understanding of how astrocytes promote neurotoxicity remains incomplete, especially in human systems. In this study, we leveraged human pluripotent stem cell (hPSC) models to examine how reactivity alters astrocyte function and mediates neurodegeneration. hPSC-derived astrocytes were induced to a reactive phenotype, at which point they exhibited a hypertrophic profile and increased complement C3 expression. Functionally, reactive astrocytes displayed decreased intracellular calcium, elevated phagocytic capacity, and decreased contribution to the blood-brain barrier. Subsequently, co-culture of reactive astrocytes with a variety of neuronal cell types promoted morphological and functional alterations. Furthermore, when reactivity was induced in astrocytes from patient-specific hPSCs (glaucoma, Alzheimer's disease, and amyotrophic lateral sclerosis), the reactive state exacerbated astrocytic disease-associated phenotypes. These results demonstrate how reactive astrocytes modulate neurodegeneration, significantly contributing to our understanding of a role for reactive astrocytes in neurodegenerative diseases.

IntroductionResurgent current (INaR) generated by voltage-gated sodium channels (VGSCs) plays an essential role in maintaining high-frequency firing of many neurons and contributes to disease pathophysiology such as epilepsy and painful disorders. Targeting INaR may present a highly promising strategy in the treatment of these diseases. Navβ4 and A-type fibroblast growth factor homologous factors (FHFs) have been identified as two classes of important INaR mediators; however, their receptor sites in VGSCs remain unknown, which hinders the development of novel agents to effectively target INaR.MethodsNavβ4 and FHF4A can mediate INaR generation through the amino acid segment located in their C-terminus and N-terminus, respectively. We mainly employed site-directed mutagenesis, chimera construction and whole-cell patch-clamp recording to explore the receptor sites of Navβ4 peptide and FHF4A in Nav1.7 and Nav1.8.ResultsWe show that the receptor of Navβ4-peptide involves four residues, N395, N945, F1737 and Y1744, in Nav1.7 DI-S6, DII-S6, and DIV-S6. We show that A-type FHFs generating INaR depends on the segment located at the very beginning, not at the distal end, of the FHF4 N-terminus domain. We show that the receptor site of A-type FHFs also resides in VGSC inner pore region. We further show that an asparagine at DIIS6, N891 in Nav1.8, is a major determinant of INaR generated by A-type FHFs in VGSCs.DiscussionCryo-EM structures reveal that the side chains of the critical residues project into the VGSC channel pore. Our findings provide additional evidence that Navβ4 peptide and A-type FHFs function as open-channel pore blockers and highlight channel inner pore region as a hotspot for development of novel agents targeting INaR.

This study reports that targeting intrinsically disordered regions (IDRs) of Na V 1.7 protein facilitated discovery of sodium channel inhibitory peptide aptamers (NaviPA) for adeno-associated virus (AAV)-mediated, sensory neuron-specific analgesia. A multipronged inhibition of I Na1.7 , I Na1.6 , and I Na1.3 , but not I Na1.5 and I Na1.8 was found for a prototype, named NaviPA1, which was derived from the Na V 1.7 intracellular loop 1 and is conserved among the TTXs Na V subtypes. NaviPA1 expression in primary sensory neurons (PSNs) of dorsal root ganglia (DRG) produced significant inhibition of TTXs I Na but not TTXr I Na . DRG injection of AAV6-encoded NaviPA1 significantly attenuated evoked and spontaneous pain behaviors in both male and female rats with neuropathic pain induced by tibial nerve injury (TNI). Whole-cell current-clamp of the PSNs showed that NaviPA1 expression normalized PSN excitability in TNI rats, suggesting that NaviPA1 attenuated pain by reversal of injury-induced neuronal hypersensitivity. Immunohistochemistry revealed efficient NaviPA1 expression restricted in PSNs and their central and peripheral terminals, indicating PSN-restricted AAV biodistribution. Inhibition of sodium channels by NaviPA1 was replicated in the human iPSC-derived sensory neurons. These results summate that NaviPA1 is a promising analgesic lead that, combined with AAV-mediated PSN-specific block of multiple TTXs Na V s has potential as peripheral nerve-restricted analgesic therapeutics.

Although the degeneration of retinal ganglion cells (RGCs) is a primary characteristic of glaucoma, astrocytes also contribute to their neurodegeneration in disease states. Although studies often explore cell-autonomous aspects of RGC neurodegeneration, a more comprehensive model of glaucoma should take into consideration interactions between astrocytes and RGCs. To explore this concept, RGCs and astrocytes were differentiated from human pluripotent stem cells (hPSCs) with a glaucoma-associated OPTN(E50K) mutation along with corresponding isogenic controls. Initial results indicated significant changes in OPTN(E50K) astrocytes, including evidence of autophagy dysfunction. Subsequently, co-culture experiments demonstrated that OPTN(E50K) astrocytes led to neurodegenerative properties in otherwise healthy RGCs, while healthy astrocytes rescued some neurodegenerative features in OPTN(E50K) RGCs. These results are the first to identify disease phenotypes in OPTN(E50K) astrocytes, including how their modulation of RGCs is affected. Moreover, these results support the concept that astrocytes could offer a promising target for therapeutic intervention in glaucoma.

Approximately 30% of patients with epilepsy do not achieve adequate seizure control through current anti-seizure drugs and treatment methods. Therefore, a critical need exists to efficiently screen anti-seizure drugs to enhance our ability to tailor treatment protocols and improve patient outcomes. The zebrafish pentylenetetrazol (PTZ) seizure model has become an increasingly popular screening paradigm for novel anti-seizure compounds. However, previous research using this model was variable due to differing experimental methods. Here, we present a method that was optimized to improve reliability and reproducibility in our laboratory using this PTZ model to develop a more robust screening of anti-seizure drugs comparing behavior and neural activity. Our behavior assay, spanning 90 min using 10 mM PTZ on 7 days post fertilization zebrafish, provides a broad window to observe anti-seizure drug efficacy. To compare our method with previously published data, we tested carbamazepine, lamotrigine, and topiramate, which have been tested in previous PTZ zebrafish assays. In addition, we assessed the candidate anti-seizure compound GS967, which has not been previously tested in the zebrafish seizure model. We examined the efficacy of anti-seizure drugs by acute administration concurrent with PTZ application and by pretreatment prior to exposure with PTZ. Pretreatment permitted us to examine potential neuroprotection and determine whether treatment time affects anti-seizure drugs' responses. As independent validation of anti-seizure drugs' effects, we evaluated whether the anti-seizure drug efficacy in the behavioral assay correlated with neural activity measurements, using electroencephalogram (EEG) and calcium signaling using GCaMP. There was no significant difference in the reduction of PTZ-induced seizure behavior activity between the pretreatment groups and acute treatment groups. Acute treatment with anti-seizure drugs in the EEG and GCaMP assays from 15 to 30 min post-anti-seizure drug exposure revealed consistent results between behavioral, EEG, and GCaMP assays for two of the three anti-seizure drugs. Lamotrigine only reduced neural activity (EEG and GCaMP assays). Carbamazepine, topiramate, and GS967 reduced activity in all three assays. The findings show that EEG and GCaMP assays largely correlate with the behavior findings, helping us connect physiological and behavior responses to anti-seizure drug and better assess anti-seizure drug efficacy.

Voltage-gated sodium channel NaV1.8 regulates transmission of pain signals to the brain. While NaV1.8 has the potential to serve as a drug target, the molecular mechanisms that shape NaV1.8 gating are not completely understood, particularly mechanisms that couple activation to inactivation. Interactions between toxin producing animals and their predators provide a novel approach for investigating NaV structure-function relationships. Arizona bark scorpions produce Na+ channel toxins that initiate pain signaling. However, in predatory grasshopper mice, toxins inhibit NaV1.8 currents and block pain signals. A screen of synthetic peptide toxins predicted from bark scorpion venom showed that peptide NaTx36 inhibited Na+ current recorded from a recombinant grasshopper mouse NaV1.8 channel (OtNaV1.8). Toxin NaTx36 hyperpolarized OtNaV1.8 activation, steady-state fast inactivation, and slow inactivation. Mutagenesis revealed that the first gating charge in the domain I (DI) S4 voltage sensor and an acidic amino acid (E) in the DII SS2 – S6 pore loop are critical for the inhibitory effects of NaTx36. Computational modeling showed that a DI S1 – S2 asparagine (N) stabilizes the NaTx36 – OtNaV1.8 complex while residues in the DI S3 – S4 linker and S4 voltage sensor form electrostatic interactions that allow a toxin glutamine (Q) to contact the first S4 gating charge. Surprisingly, the models predicted that NaTx36 contacts amino acids in the DII S5 – SS1 pore loop instead of the SS2 – S6 loop; the DII SS2 – S6 loop motif (QVSE) alters the conformation of the DII S5 – SS1 pore loop, enhancing allosteric interactions between toxin and the DII S5 – SS1 pore loop. Few toxins have been identified that modify NaV1.8 gating. Moreover, few toxins have been described that modify sodium channel gating via the DI S4 voltage sensor. Thus, NaTx36 and OtNaV1.8 provide tools for investigating the structure-activity relationship between channel activation and inactivation gating, and the connection to alternative pain phenotypes.

Resurgent currents (INaR) produced by voltage-gated sodium channels are required for many neurons to maintain high-frequency firing and contribute to neuronal hyperexcitability and disease pathophysiology. Here, we show, for the first time, that INaR can be reconstituted in a heterologous system by coexpression of sodium channel α-subunits and A-type fibroblast growth factor homologous factors (FHFs). Specifically, A-type FHFs induces INaR from Nav1.8, Nav1.9 tetrodotoxin (TTX)-resistant neuronal channels, and, to a lesser extent, neuronal Nav1.7 and cardiac Nav1.5 channels. Moreover, we identified the N-terminus of FHF as the critical molecule responsible for A-type FHFs-mediated INaR. Among the FHFs, FHF4A is the most important isoform for mediating Nav1.8 and Nav1.9 INaR. In nociceptive sensory neurons, FHF4A knockdown significantly reduces INaR amplitude and the percentage of neurons that generate INaR, substantially suppressing excitability. Thus, our work reveals a novel molecular mechanism underlying TTX-resistant INaR generation and provides important potential targets for pain treatment.

Aberrant Nav1.6 activity can induce hyperexcitability associated with epilepsy. Gain-of-function mutations in the SCN8A gene encoding Nav1.6 are linked to epilepsy development; however, the molecular mechanisms mediating these changes are remarkably heterogeneous and may involve post-translational regulation of Nav1.6. Because calcium/calmodulin-dependent protein kinase II (CaMKII) is a powerful modulator of Nav1.6 channels, we investigated whether CaMKII modulates disease-linked Nav1.6 mutants. Whole-cell voltage clamp recordings in ND7/23 cells show that CaMKII inhibition of the epilepsy-related mutation R850Q largely recapitulates the effects previously observed for WT Nav1.6. We also characterized a rare missense variant, R639C, located within a regulatory hotspot for CaMKII modulation of Nav1.6. Prediction software algorithms and electrophysiological recordings revealed gain-of-function effects for R639C mutant channel activity, including increased sodium currents and hyperpolarized activation compared to WT Nav1.6. Importantly, the R639C mutation ablates CaMKII phosphorylation at a key regulatory site, T642, and, in contrast to WT and R850Q channels, displays a distinct response to CaMKII inhibition. Computational simulations demonstrate that modeled neurons harboring the R639C or R850Q mutations are hyperexcitable, and simulating the effects of CaMKII inhibition on Nav1.6 activity in modeled neurons differentially reduced hyperexcitability. Acute CaMKII inhibition may represent a promising mechanism to attenuate gain-of-function effects produced by Nav1.6 mutations.

Objective Family members carrying the same SCN1A variant often exhibit differences in the clinical severity of epilepsy. This variable expressivity suggests that other factors aside from the primary sodium channel variant influence the clinical manifestation. However, identifying such factors has proven challenging in humans. Methods We perform whole exome sequencing (WES) in a large family in which an SCN1A variant (p.K1372E) is segregating that is associated with a broad spectrum of phenotypes ranging from lack of epilepsy, to febrile seizures and absence seizures, to Dravet syndrome. We assessed the hypothesis that the severity of the SCN1A-related phenotype was affected by alternate alleles at a modifier locus (or loci). Results One of our top candidates identified by WES was a second variant in the SCN1A gene (p.L375S) that was shared exclusively by unaffected carriers of the K1372E allele. To test the hypothesized that L375S variant nullifies the loss-of-function effect of K1372E, we transiently expressed Nav1.1 carrying the two variants in HEK293T cells and compared their biophysical properties with the wild-type (WT) variant, and then co-expressed WT with K1372E or L375S with K1372E in equal quantity and tested the functional consequence. The data demonstrated that co-expression of the L375S and K1372E alleles reversed the loss-of-function property brought by the K1372E variant, whereas WT-K1372E co-expression remained partial loss-of-function. Significance These results support the hypothesis that L375S counteracts the loss-of-function effect of K1372E such that individuals carrying both alleles in trans do not present epilepsy-related symptoms. We demonstrate that monogenic epilepsies with wide expressivity can be modified by additional variants in the disease gene, providing a novel framework for the gene-phenotype relationship in genetic epilepsies.

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure–activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a “CXCE” motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

Abstract Puberty and reproduction are initiated and controlled through the hypothalamic-pituitary-gonadal (HPG) axis. A critical surge of luteinizing hormone (LH) and follicle stimulating hormone (FSH) are released from the anterior pituitary upon release of gonadotrophins from gonadotrophin releasing hormone (GnRH) neurons. Thus, GnRH neurons are key regulators of the HPG axis. GnRH neurons become active when kisspeptin (Kiss1) neuropeptides are released from neurons in the arcuate nucleus. Kiss1 binds to the Kiss1 receptor (Kiss1R), a G-protein coupled receptor (GPCR) which localizes to the primary cilia of GnRH neurons. Loss-of-function mutations of Kiss1R cause hypogonadism in mouse and human models while gain-of-function mutations are associated with precocious puberty. Interestingly, the subset of GnRH neurons that express Kiss1R are observed to be polyciliated, possessing more than one primary cilia, an uncommon property as most neurons only possess a single, primary cilium. The mechanism and conditions leading to GnRH neuron polyciliation are unknown. It is also unclear if multiple cilia impact Kiss1R or other GPCR signaling in these neurons. Here, we utilize cultured mouse primary hypothalamic neurons to begin addressing some of these questions. We have confirmed with qPCR that the ligands GnRH and Kiss1, as well as Kiss1R, are all expressed in these cultures. Surprisingly, when treated with Kiss1 and GnRH ligands we observed a small subset of polyciliated neurons compared to vehicle treated neurons. These observations mirror what is seen during sexual maturation in vivo and suggest that our model system may help elucidate fundamental questions about how ciliary localization of Kiss1r and other GPCRs participate in initiation of puberty and regulation of reproduction. Future studies will focus on the mechanisms of polyciliation and the conditions needed to induce the formation of new cilia in GnRH neurons. Investigating neuronal polyciliation could provide insights into new signaling paradigm in hypogonadism and HPG signaling.