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.

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.

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.

Bidirectional Modulation of the Voltage-Gated Sodium (Nav1.6) Channel by Rationally Designed Peptidomimetics.

Disruption of protein:protein interactions (PPIs) that regulate the function of voltage-gated Na+ (Nav) channels leads to neural circuitry aberrations that have been implicated in numerous channelopathies. One example of this pathophysiology is mediated by dysfunction of the PPI between Nav1.6 and its regulatory protein fibroblast growth factor 14 (FGF14). Thus, peptides derived from FGF14 might exert modulatory actions on the FGF14:Nav1.6 complex that are functionally relevant. The tetrapeptide Glu-Tyr-Tyr-Val (EYYV) mimics surface residues of FGF14 at the ß8-ß9 loop, a structural region previously implicated in its binding to Nav1.6. Here, peptidomimetics derived from EYYV (6) were designed, synthesized, and pharmacologically evaluated to develop probes with improved potency. Addition of hydrophobic protective groups to 6 and truncation to a tripeptide (12) produced a potent inhibitor of FGF14:Nav1.6 complex assembly. Conversely, addition of hydrophobic protective groups to 6 followed by addition of an N-terminal benzoyl substituent (19) produced a potentiator of FGF14:Nav1.6 complex assembly. Subsequent functional evaluation using whole-cell patch-clamp electrophysiology confirmed their inverse activities, with 12 and 19 reducing and increasing Nav1.6-mediated transient current densities, respectively. Overall, we have identified a negative and positive allosteric modulator of Nav1.6, both of which could serve as scaffolds for the development of target-selective neurotherapeutics.

Voltage-Gated Na+ Channels in Alzheimer's Disease: Physiological Roles and Therapeutic Potential.

Alzheimer's disease (AD) is the most common cause of dementia and is classically characterized by two major histopathological abnormalities: extracellular plaques composed of amyloid beta (Aß) and intracellular hyperphosphorylated tau. Due to the progressive nature of the disease, it is of the utmost importance to develop disease-modifying therapeutics that tackle AD pathology in its early stages. Attenuation of hippocampal hyperactivity, one of the earliest neuronal abnormalities observed in AD brains, has emerged as a promising strategy to ameliorate cognitive deficits and abate the spread of neurotoxic species. This aberrant hyperactivity has been attributed in part to the dysfunction of voltage-gated Na+ (Nav) channels, which are central mediators of neuronal excitability. Therefore, targeting Nav channels is a promising strategy for developing disease-modifying therapeutics that can correct aberrant neuronal phenotypes in early-stage AD. This review will explore the role of Nav channels in neuronal function, their connections to AD pathology, and their potential as therapeutic targets.

Fibroblast growth factor 13-mediated regulation of medium spiny neuron excitability and cocaine self-administration.

Cocaine use disorder (CUD) is a prevalent neuropsychiatric disorder with few existing treatments. Thus, there is an unmet need for the identification of new pharmacological targets for CUD. Previous studies using environmental enrichment versus isolation paradigms have found that the latter induces increased cocaine self-administration with correlative increases in the excitability of medium spiny neurons (MSN) of the nucleus accumbens shell (NAcSh). Expanding upon these findings, we sought in the present investigation to elucidate molecular determinants of these phenomena. To that end, we first employed a secondary transcriptomic analysis and found that cocaine self-administration differentially regulates mRNA for fibroblast growth factor 13 (FGF13), which codes for a prominent auxiliary protein of the voltage-gated Na+ (Nav) channel, in the NAcSh of environmentally enriched rats (i.e., resilient behavioral phenotype) compared to environmentally isolated rats (susceptible phenotype). Based upon this finding, we used in vivo genetic silencing to study the causal functional and behavioral consequences of knocking down FGF13 in the NAcSh. Functional studies revealed that knockdown of FGF13 in the NAcSh augmented excitability of MSNs by increasing the activity of Nav channels. These electrophysiological changes were concomitant with a decrease in cocaine demand elasticity (i.e., susceptible phenotype). Taken together, these data support FGF13 as being protective against cocaine self-administration, which positions it well as a pharmacological target for CUD.

Development of Allosteric Modulators of Voltage-Gated Na+ Channels: A Novel Approach for an Old Target.

Given their primacy in governing the action potential (AP) of excitable cells, voltage-gated Na+ (Nav) channels are important pharmacological targets of therapeutics for a diverse array of clinical indications. Despite historically being a traditional drug target, therapeutics targeting Nav channels lack isoform selectivity, giving rise to off-target side effects. To develop isoform-selective modulators of Nav channels with improved target-specificity, the identification and pharmacological targeting of allosteric sites that display structural divergence among Nav channel isoforms represents an attractive approach. Despite the high homology among Nav channel α subunit isoforms (Nav1.1-Nav1.9), there is considerable amino acid sequence divergence among their constituent C-terminal domains (CTD), which enables structurally and functionally specific protein: protein interactions (PPI) with auxiliary proteins. Although pharmacological targeting of such PPI interfaces between the CTDs of Nav channels and auxiliary proteins represents an innovate approach for developing isoform-selective modulators of Nav channels, appreciable modulation of PPIs using small molecules has conventionally been difficult to achieve. After briefly discussing the challenges of modulating PPIs using small molecules, this current frontier review that follows subsequently expounds on approaches for circumventing such difficulties in the context of developing small molecule modulators of PPIs between transmembrane ion channels and their auxiliary proteins. In addition to broadly discussing such approaches, the implementation of such approaches is specifically discussed in the context of developing small molecule modulators between the CTD of Nav channels and auxiliary proteins. Developing allosteric modulators of ion channels by targeting their PPI interfaces with auxiliary proteins represents an innovative and promising strategy in ion channel drug discovery that could expand the "druggable genome" and usher in first-in-class PPI-targeting therapeutics for a multitude of channelopathies.

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.

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.

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.

Design, Synthesis, and Pharmacological Evaluation of Analogues Derived from the PLEV Tetrapeptide as Protein-Protein Interaction Modulators of Voltage-Gated Sodium Channel 1.6.

The voltage-gated Na+ (Nav) channel is the molecular determinant of excitability. Disruption of protein-protein interactions (PPIs) between Nav1.6 and fibroblast growth factor 14 (FGF14) leads to impaired excitability of neurons in clinically relevant brain areas associated with channelopathies. Here, we designed, synthesized, and pharmacologically characterized new peptidomimetics based on a PLEV tetrapeptide scaffold derived from the FGF14:Nav1.6 PPI interface. Addition of an N-terminal 1-adamantanecarbonyl pharmacophore significantly improved peptidomimetic inhibitory potency. Surface plasmon resonance studies revealed that while this moiety was sufficient to confer binding to FGF14, altering the C-terminal moiety from methoxy (21a) to π bond-containing (23a and 23b) or cycloalkane substituents (23e) abrogated the binding to Nav1.6. Whole-cell patch-clamp electrophysiology subsequently revealed that 21a had functionally relevant interactions with both the C-terminal tail of Nav1.6 and FGF14. Collectively, these findings support that 21a (PW0564) may serve as a promising lead to develop target-selective neurotherapeutics by modulating protein-channel interactions.