The finely tuned regulation of neuronal firing relies on the integrity of ion channel macromolecular complexes. Minimal disturbances of these tightly regulated networks can lead to persistent maladaptive plasticity of brain circuitry. The intracellular fibroblast growth factor 14 (FGF14) belongs to the nexus of proteins interacting with voltage-gated Na+ (Nav) channels at the axonal initial segment. At this site, FGF14 acts as a molecular determinant of neuronal excitability. Through isoform-specific interactions with the intracellular C-terminal tail of neuronal Nav channels (Nav1.1, Nav1.2, Nav1.6), FGF14 controls channel gating, axonal targeting and phosphorylation in neurons effecting excitability, synaptic transmission, plasticity and neurogenesis in the cortico-mesolimbic circuit with cognitive and affective behavioral outcomes. In translational studies, interest in FGF14 continues to rise with a growing list of associative links to diseases of the cognitive and affective domains such as neurodegeneration, depression, anxiety, addictive behaviors and recently schizophrenia, suggesting its role as a converging node in the etiology of complex brain disorders. Yet, a full understanding of FGF14 function in neurons is far from being complete and likely to involve other functions unrelated to the direct regulation of Nav channels. The goal of this Mini Review is to provide a summary of the most recent studies on the emerging role of FGF14 in complex brain disorders.

Novel proteoforms with single amino acid variations represent proteins that often have altered biological functions but are less explored in the human proteome. We have developed an approach, searching high quality shotgun proteomic data against an extended protein database, to identify expressed mutant proteoforms in glioma stem cell (GSC) lines. The systematic search of MS/MS spectra using PEAKS 7.0 as the search engine has recognized 17 chromosome 19 proteins in GSCs with altered amino acid sequences. The results were further verified by manual spectral examination, validating 19 proteoforms. One of the novel findings, a mutant form of branched-chain aminotransferase 2 (p.Thr186Arg), was verified at the transcript level and by targeted proteomics in several glioma stem cell lines. The structure of this proteoform was examined by molecular modeling in order to estimate conformational changes due to mutation that might lead to functional modifications potentially linked to glioma. Based on our initial findings, we believe that our approach presented could contribute to construct a more complete map of the human functional proteome.

Multiple voltage-gated Na⁺ (Nav) channelopathies can be ascribed to subtle changes in the Nav macromolecular complex. Fibroblast growth factor 14 (FGF14) is a functionally relevant component of the Nav1.6 channel complex, a causative link to spinocerebellar ataxia 27 (SCA27) and an emerging risk factor for neuropsychiatric disorders. Yet, how this protein:channel complex is regulated in the cell is still poorly understood. To search for key cellular pathways upstream of the FGF14:Nav1.6 complex, we have developed, miniaturized and optimized an in-cell assay in 384-well plates by stably reconstituting the FGF14:Nav1.6 complex using the split-luciferase complementation assay. We then conducted a high-throughput screening (HTS) of 267 FDA-approved compounds targeting known mediators of cellular signaling. Of the 65 hits initially detected, 24 were excluded based on counter-screening and cellular toxicity. Based on target analysis, potency and dose-response relationships, 5 compounds were subsequently repurchased for validation and confirmed as hits. Among those, the tyrosine kinase inhibitor lestaurtinib was highest ranked, exhibiting submicromolar inhibition of FGF14:Nav1.6 assembly. While providing evidence for a robust in-cell HTS platform that can be adapted to search for any channelopathy-associated regulatory proteins, these results lay the potential groundwork for repurposing cancer drugs for neuropsychopharmacology.

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 β8–β9 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⁺ (Na_v) channels are the primary molecular determinant of the action potential. Among the nine isoforms of the Na_v channel α subunit that have been described (Na_v1.1-Na_v1.9), Na_v1.1, Na_v1.2, and Na_v1.6 are the primary isoforms expressed in the central nervous system (CNS). Crucially, these three CNS Na_v 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 Na_v channel isoforms could represent promising targets for the development of targeted neuromodulators. However, current therapeutics that target Na_v channels lack selectivity, which results in deleterious side effects due to modulation of off-target Na_v channel isoforms. Among the structural components of the Na_v channel α subunit that could be pharmacologically targeted to achieve isoform selectivity, the C-terminal domains (CTD) of Na_v channels represent promising candidates on account of displaying appreciable amino acid sequence divergence that enables functionally unique protein–protein interactions (PPIs) with Na_v 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 Na_v1.6 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 Na_v1.6 channel. In this work, we show that whereas the compound displays dose-dependent effects on the activity of Na_v1.6 channels in heterologous cells, the compound does not affect Na_v1.1 or Na_v1.2 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 Na_v1.6 channel is a strategy to achieve isoform-selective modulation, and, more broadly, that sites on the CTDs of Na_v channels interacted with by auxiliary proteins could represent candidates for the development of targeted therapeutics.