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.

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.

Revisions to Limits for Toluene in Spacecraft Air.

INTRODUCTION: The original Spacecraft Maximal Allowable Concentrations (SMACs) for toluene (set for 1 h, 24 h, 7 d, 30 d, and 180 d) were first established by NASA in 1996 based on a human study in which no irritation or neurotoxicity was reported following 6-h exposure to 40 ppm toluene vapors. While the toluene SMACs were updated in 2008 to account for auditory, visual, and hormonal effects (for 7 d, 30 d, and 180 d) and to include a long-term SMAC (1000 d) in anticipation of longer spaceflight exploration missions, the short-term SMAC limits (1 h and 24 h) remained unchanged. Acute toluene exposure is reported to result in ocular and nasal irritation, although it is not a primary irritant, as well as central nervous system effects including headaches and dizziness. Long-term exposure to toluene can elicit hepatotoxicity, nephrotoxicity, neurotoxicity, and endocrine toxicity.RESULTS AND DISCUSSION: Since publication of the original and revised toluene SMACs, the National Academy of Sciences developed interim Acute Exposure Guideline Limits reviewed by the National Research Council Committee. Based on these data, we have increased the limits for toluene in crewed spacecraft to 40 ppm for 1 h, 24 h, 7 d, and 30 d. SMACs for durations of 180 and 1000 d will remain unchanged.changed.Tapia CM, Langford SD, Ryder VE. Revisions to limits for toluene in spacecraft air. Aerosp Med Hum Perform. 2024; 95(7):399-402.

Differential Modulation of the Voltage-Gated Na+ Channel 1.6 by Peptides Derived From Fibroblast Growth Factor 14.

The voltage-gated Na+ (Nav) channel is a primary molecular determinant of the initiation and propagation of the action potential. Despite the central role of the pore-forming α subunit in conferring this functionality, protein:protein interactions (PPI) between the α subunit and auxiliary proteins are necessary for the full physiological activity of Nav channels. In the central nervous system (CNS), one such PPI occurs between the C-terminal domain of the Nav1.6 channel and fibroblast growth factor 14 (FGF14). Given the primacy of this PPI in regulating the excitability of neurons in clinically relevant brain regions, peptides targeting the FGF14:Nav1.6 PPI interface could be of pre-clinical value. In this work, we pharmacologically evaluated peptides derived from FGF14 that correspond to residues that are at FGF14's PPI interface with the CTD of Nav1.6. These peptides, Pro-Leu-Glu-Val (PLEV) and Glu-Tyr-Tyr-Val (EYYV), which correspond to residues of the ß12 sheet and ß8-ß9 loop of FGF14, respectively, were shown to inhibit FGF14:Nav1.6 complex assembly. In functional studies using whole-cell patch-clamp electrophysiology, PLEV and EYYV were shown to confer differential modulation of Nav1.6-mediated currents through mechanisms dependent upon the presence of FGF14. Crucially, these FGF14-dependent effects of PLEV and EYYV on Nav1.6-mediated currents were further shown to be dependent on the N-terminal domain of FGF14. Overall, these data suggest that the PLEV and EYYV peptides represent scaffolds to interrogate the Nav1.6 channel macromolecular complex in an effort to develop targeted pharmacological modulators.

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.

Topographic transcriptomics of the nucleus accumbens shell: Identification and validation of fatty acid binding protein 5 as target for cocaine addiction.

Substance use disorders for cocaine are major public health concerns with few effective treatment options. Therefore, identification of novel pharmacotherapeutic targets is critical for future therapeutic development. Evolution has ensured that genes are expressed largely only where they are needed. Therefore, examining the gene expression landscape of the nucleus accumbens shell (NAcSh), a brain region important for reward related behaviors, may lead to the identification of novel targets for cocaine use disorder. In this study, we conducted a novel two-step topographic transcriptomic analysis using five seed transcripts with enhanced expression in the NAcSh to identify transcripts with similarly enhanced expression utilizing the correlation feature to search the more than 20,000 in situ hybridization experiments of the Allen Mouse Brain Atlas. Transcripts that correlated with at least three seed transcripts were analyzed with Ingenuity Pathway Analysis (IPA). We identified 7-fold more NAcSh-enhanced transcripts than our previous analysis using single voxels in the NAcSh as the seed. Analysis of the resulting transcripts with IPA identified many previously identified signaling pathways such as retinoic acid signaling as well as novel pathways. Manipulation of the retinoic acid pathway specifically in the NAcSh of male rats via viral vector-mediated RNA interference targeting fatty acid binding protein 5 (FABP5) decreased cocaine self-administration and modulates excitability of medium spiny neurons in the NAcSh. These results not only validate the prospective strategy of conducting a topographic transcriptomic analysis, but also further validate retinoic acid signaling as a promising pathway for pharmacotherapeutic development against cocaine use disorder.

Effects of Deltamethrin Acute Exposure on Nav1.6 Channels and Medium Spiny Neurons of the Nucleus Accumbens.

Exposure to pyrethroids, a popular insecticide class that targets voltage-gated Na+ (Nav) channels, has been correlated to an increase in diagnosis of neurodevelopmental disorders, such as attention deficit hyperactive disorder (ADHD), in children. Dysregulation of medium spiny neurons (MSNs) firing in the nucleus accumbens (NAc) is thought to play a critical role in the pathophysiology of ADHD and other neurodevelopmental disorders. The Nav1.6 channel is the primary molecular determinant of MSN firing and is sensitive to modification by pyrethroids. Building on previous studies demonstrating that deltamethrin (DM), a commonly used pyrethroid, leads to use-dependent enhancement of sodium currents, we characterized the effect of the toxin on long-term inactivation (LTI) of the Nav1.6 channel, a parameter known to affect neuronal firing, and characterized changes in MSN intrinsic excitability. We employed whole-cell patch-clamp electrophysiology to measure sodium currents in HEK-293 cells stably expressing Nav1.6 channels and intrinsic excitability of MSNs in the brain slice preparation. We found that in response to repetitive stimulation acute exposure to 10 µM DM potentiated a build-up of residual sodium currents and modified availability of Nav1.6 by inducing LTI. In the NAc, DM modified MSN intrinsic excitability increasing evoked action potential firing frequency and inducing aberrant action potentials with low amplitude and depolarized voltage threshold, phenotypes that could be explained by DM induced changes on the Nav1.6 channel. These results provide a potential initial mechanism of toxicity of DM that could lead to disruption of the NAc circuitry overtime, increasing the risk of ADHD and other neurodevelopmental disorders.

JAK2 regulates Nav1.6 channel function via FGF14Y158 phosphorylation.

BACKGROUND: Protein interactions between voltage-gated sodium (Nav) channels and accessory proteins play an essential role in neuronal firing and plasticity. However, a surprisingly limited number of kinases have been identified as regulators of these molecular complexes. We hypothesized that numerous as-of-yet unidentified kinases indirectly regulate the Nav channel via modulation of the intracellular fibroblast growth factor 14 (FGF14), an accessory protein with numerous unexplored phosphomotifs and required for channel function in neurons. METHODS: Here we present results from an in-cell high-throughput screening (HTS) against the FGF14: Nav1.6 complex using >3000 diverse compounds targeting an extensive range of signaling pathways. Regulation by top kinase targets was then explored using in vitro phosphorylation, biophysics, mass-spectrometry and patch-clamp electrophysiology. RESULTS: Compounds targeting Janus kinase 2 (JAK2) were over-represented among HTS hits. Phosphomotif scans supported by mass spectrometry revealed FGF14Y158, a site previously shown to mediate both FGF14 homodimerization and interactions with Nav1.6, as a JAK2 phosphorylation site. Following inhibition of JAK2, FGF14 homodimerization increased in a manner directly inverse to FGF14:Nav1.6 complex formation, but not in the presence of the FGF14Y158A mutant. Patch-clamp electrophysiology revealed that through Y158, JAK2 controls FGF14-dependent modulation of Nav1.6 channels. In hippocampal CA1 pyramidal neurons, the JAK2 inhibitor Fedratinib reduced firing by a mechanism that is dependent upon expression of FGF14. CONCLUSIONS: These studies point toward a novel mechanism by which levels of JAK2 in neurons could directly influence firing and plasticity by controlling the FGF14 dimerization equilibrium, and thereby the availability of monomeric species for interaction with Nav1.6.

Mapping of the FGF14:Nav1.6 complex interface reveals FLPK as a functionally active peptide modulating excitability.

The voltage-gated sodium (Nav) channel complex is comprised of pore-forming α subunits (Nav1.1-1.9) and accessory regulatory proteins such as the intracellular fibroblast growth factor 14 (FGF14). The cytosolic Nav1.6 C-terminal tail binds directly to FGF14 and this interaction modifies Nav1.6-mediated currents with effects on intrinsic excitability in the brain. Previous studies have identified the FGF14V160 residue within the FGF14 core domain as a hotspot for the FGF14:Nav1.6 complex formation. Here, we used three short amino acid peptides around FGF14V160 to probe for the FGF14 interaction with the Nav1.6 C-terminal tail and to evaluate the activity of the peptide on Nav1.6-mediated currents. In silico docking predicts FLPK to bind to FGF14V160 with the expectation of interfering with the FGF14:Nav1.6 complex formation, a phenotype that was confirmed by the split-luciferase assay (LCA) and surface plasmon resonance (SPR), respectively. Whole-cell patch-clamp electrophysiology studies demonstrate that FLPK is able to prevent previously reported FGF14-dependent phenotypes of Nav1.6 currents, but that its activity requires the FGF14 N-terminal tail, a domain that has been shown to contribute to Nav1.6 inactivation independently from the FGF14 core domain. In medium spiny neurons in the nucleus accumbens, where both FGF14 and Nav1.6 are abundantly expressed, FLPK significantly increased firing frequency by a mechanism consistent with the ability of the tetrapeptide to interfere with Nav1.6 inactivation and potentiate persistent Na+ currents. Taken together, these results indicate that FLPK might serve as a probe for characterizing molecular determinants of neuronal excitability and a peptide scaffold to develop allosteric modulators of Nav channels.

Consequences of acute Nav1.1 exposure to deltamethrin.

BACKGROUND: Pyrethroid insecticides are the most popular class of insecticides in the world, despite their near-ubiquity, their effects of delaying the onset of inactivation of voltage-gated sodium (Nav) channels have not been well-evaluated in all the mammalian Nav isoforms. OBJECTIVE: Here we compare the well-studied Nav1.6 isoforms to the less-understood Nav1.1 in their responses to acute deltamethrin exposure. METHODS: We used patch-clamp electrophysiology to record sodium currents encoded by either Nav1.1 or Nav1.6 channels stably expressed in HEK293 cells. Protocols evaluating both resting and use-dependent modification were employed. RESULTS: We found that exposure of both isoforms to 10µM deltamethrin significantly potentiated persistent and tail current densities without affecting peak transient current densities, and only Nav1.1 maintained these significant effects at 1µM deltamethrin. Window currents increased for both as well, and while only Nav1.6 displayed changes in activation slope and V1/2 of steady-state inactivation for peak currents, V1/2 of persistent current activation was hyperpolarized of ∼10mV by deltamethrin in Nav1.1 cells. Evaluating use-dependence, we found that deltamethrin again potentiated persistent and tail current densities in both isoforms, but only Nav1.6 demonstrated use-dependent enhancement, indicating the primary deltamethrin-induced effects on Nav1.1 channels are not use-dependent. CONCLUSION: Collectively, these data provide evidence that Nav1.1 is indeed vulnerable to deltamethrin modification at lower concentrations than Nav1.6, and this effect is primarily mediated during the resting state. GENERAL SIGNIFICANCE: These findings identify Nav1.1 as a novel target of pyrethroid exposure, which has major implications for the etiology of neuropsychiatric disorders associated with loss of Nav1.1-expressing inhibitory neurons.