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.

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.

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.