BACKGROUND AND PURPOSE:Kinases phosphorylate ion channels, but their noncatalytic roles via protein-protein interactions (PPI) are less understood. Here, we identified the peptidomimetic ZL141 to characterize the PPI between GSK3β and Nav1.6, revealing a noncatalytic role for GSK3β in regulating Nav1.6 currents and neuronal excitability. EXPERIMENTAL APPROACH:To screen peptide inhibitors, the GSK3β/Nav1.6 C-terminal tail (CTD) PPI was reconstituted using split-luciferase complementation assay. Intrinsic fluorescence spectroscopy (IFS), surface plasmon resonance (SPR), kinase assays, and molecular docking were used to assess peptidomimetic binding and predict the PPI interface. ZL141's function was evaluated via patch-clamp electrophysiology in heterologous cells and nucleus accumbens (NAc) medium spiny neurons (MSNs), along with in vivo GSK3β silencing. KEY RESULTS:ZL141 disrupts GSK3β/Nav1.6 CTD complex formation without altering GSK3β's catalytic activity. AlphaFold modelling predicted the ZL141 binding site at the surface of the GSK3β/Nav1.6 CTD PPI interface. ZL141 has a micromolar affinity for GSK3β, suppresses Na+ currents, and affects Nav1.6 activation and inactivation kinetics. In vivo, silencing GSK3β in the NAc mimics the inhibitory effects of ZL141 on MSN firing and occludes any further pharmacological activity of the compound. CONCLUSIONS AND IMPLICATIONS:These findings reveal a critical PPI role of GSK3β in transducing Nav1.6 function and contributing to MSN firing. Both GSK3β and Nav1.6 activities are dysregulated in neurological and neuropsychiatric disorders. Given that Nav1.6 hyperactivity is implicated in maladaptive reward signalling, PPI-targeted modulation using compounds like ZL141 may offer a precision pharmacological strategy for treating psychiatric disorders such as bipolar disorder or schizophrenia.

Depression is a mental illness characterized by despair behavior, inability to feel pleasure, and social withdrawal. Causes are not yet clarified, but stress is a condition that induces depression. Neuronal alterations, comprising maladaptive neuronal plasticity and excitability, are present in both responses to stress and depression. Fibroblast growth factor 14 (Fgf14) controls neuronal excitability and proper action potential firing by stabilizing voltage-dependent sodium (Nav) channels into the axon. Fgf14-Nav channels complex is regulated by glycogen synthase kinase 3. Recently, Fgf14 has been genetically associated to depression. However, little is known about its role in controlling stress-induced depression. This study demonstrates that female Fgf14-/- mice are resilient to depression, as reported by reduced level of despair behavior, anhedonia, and increased sociability. Also, a reduction of anxious-like behavior was highlighted. Fgf14-/- mice showed increased expression of cannabinoid receptor without alterations of dopaminergic system in mPFC, suggesting a link between Fgf14 and endocannabinoid system in the control mechanisms underlying depression. Neuronal activity was assessed by analyzing cFOS expression during basal and following acute stress induced by tail suspension test (TST). The analysis revealed that neuronal activation in mPFC and VTA was correlated to immobility, where ratio of cFOS expression over immobility was significantly higher in Fgf14-/- mice. This suggests that higher neuronal activity might be involved in resilience to depression. In resilient Fgf14-/- mice, TST-induced acute stress caused activation only in pyramidal neurons. Our findings suggest that Fgf14 is involved in stress-coping mechanisms and could be targeted to improve resilience to depression.

Voltage-gated Na+ (Nav) channels are the primary determinants of the action potential in excitable cells. Nav channels rely on a wide and diverse array of intracellular protein-protein interactions (PPIs) to achieve their full function. Glycogen synthase kinase 3β (GSK3β) has been previously identified as a modulator of Nav1.6-encoded currents and neuronal excitability through PPI formation with Nav1.6 and phosphorylation of its C-terminal domain (CTD). Here, we hypothesized that GSK3β functions as a scaffold in a regulatory PPI complex with the Nav1.6 CTD. Mutagenesis screening using the split-luciferase complementation assay indicated that the axin-binding domain (ABD) of GSK3β (262-299) is necessary for complex formation between the Nav1.6 CTD and GSK3β, and that residues within this domain are drivers of GSK3β-mediated regulation of the channel. Overexpression of an ABD-GFP fusion construct in human embryonic kidney 293 cells stably expressing Nav1.6 significantly reduced Nav1.6 nanocluster density compared with GFP alone. In addition, overexpression of the ABD-GFP fusion construct ablates GSK3β-mediated potentiation of Nav1.6-encoded currents and alters channel kinetics. Finally, in vivo AAV-mediated overexpression of the ABD-GFP construct in the CA1 hippocampal region induced a reduction in maximal action potential firing and an increase in action potential current threshold in a manner resembling previously reported effects of GSK3β silencing in neurons. Taken together, these results not only suggest that GSK3β-mediated regulation of Nav1.6 extends beyond transient phosphorylation but also implicates the ABD as a critical regulatory domain that facilitates GSK3β's functional effects on Nav1.6 and neuronal excitability.

Protein/protein interactions (PPI) play crucial roles in neuronal functions. Yet, their potential as drug targets for brain disorders remains underexplored. The fibroblast growth factor 14 (FGF14)/voltage-gated Na+ channel 1.6 (Nav1.6) complex regulates excitability of medium spiny neurons (MSN) of the nucleus accumbens (NAc), a central hub of reward circuitry that controls motivated behaviors. Here, we identified compound 1028 (IUPAC: ethyl 3-(2-(3-(hydroxymethyl)-1H-indol-1-yl)acetamido)benzoate), a brain-permeable small molecule that targets FGF14R117, a critical residue located within a druggable pocket at the FGF14/Nav1.6 PPI interface. We found that 1028 modulates FGF14/Nav1.6 complex assembly and depolarizes the voltage-dependence of Nav1.6 channel inactivation with nanomolar potency by modulating the intramolecular interaction between the III-IV linker and C-terminal domain of the Nav1.6 channel. Consistent with the compound's effects on Nav1.6 channel inactivation, 1028 enhances MSN excitability ex vivo and accumbal neuron firing rate in vivo in murine models. Systemic administration of 1028 maintains behavioral motivation preferentially during motivationally deficient conditions in murine models. These behavioral effects were abrogated by in vivo gene silencing of Fgf14 in the NAc and were accompanied by a selective reduction in accumbal dopamine levels during reward consumption in murine models. These findings underscore the potential to selectively regulate complex behaviors associated with neuropsychiatric disorders through targeting of PPIs in neurons.

Voltage-gated Na+ channels (Nav) are the molecular determinants of action potential initiation and propagation. Among the nine voltage-gated Na+ channel isoforms (Nav1.1–Nav1.9), Nav1.2 and Nav1.6 are of particular interest because of their developmental expression profile throughout the central nervous system (CNS) and their association with channelopathies. Although the α-subunit coded by each of the nine isoforms can sufficiently confer transient Na+ currents (INa), in vivo these channels are modulated by auxiliary proteins like intracellular fibroblast growth factor (iFGFs) through protein–protein interaction (PPI), and probes developed from iFGF/Nav PPI complexes have been shown to precisely modulate Nav channels. Previous studies identified ZL0177, a peptidomimetic derived from a short peptide sequence at the FGF14/Nav1.6 PPI interface, as a functional modulator of Nav1.6-mediated INa+. However, the isoform specificity, binding sites, and putative physiological impact of ZL0177 on neuronal excitability remain unexplored. Here, we used automated planar patch-clamp electrophysiology to assess ZL0177’s functional activity in cells stably expressing Nav1.2 or Nav1.6. While ZL0177 was found to suppress INa in both Nav1.2- and Nav1.6-expressing cells, ZL0177 elicited functionally divergent effects on channel kinetics that were isoform-specific and supported by differential docking of the compound to AlphaFold structures of the two channel isoforms. Computational modeling predicts that ZL0177 modulates Nav1.2 and Nav1.6 in an isoform-specific manner, eliciting phenotypically divergent effects on action potential discharge. Taken together, these results highlight the potential of PPI derivatives for isoform-specific regulation of Nav channels and the development of therapeutics for channelopathies.

RationaleIn previous work, a convergent transcriptomic approach strongly suggested a role for retinoic acid (RA) in controlling the emotion- and reward-related functions of the nucleus accumbens shell (NAcSh).ObjectiveHere, we causally assess the role of NAcSh RA in controlling anxiety-, emotion-, and reward-related behavior in rats and explore cellular mechanisms that may underlie this phenotype.MethodsRats underwent bilateral knockdown of the retinoic acid synthesis enzyme Aldh1a1 in the NAcSh. Anxiety-related behavior was assessed using open-field exploration, elevated plus maze, and sucrose neophobia tests. Emotion-related behavior was assessed via sucrose preference, post-isolation social contact, and forced swim tests. Animals were subsequently allowed to self-administer fentanyl to assess reward- and frustration-related behavior. In parallel, electrophysiological testing of medium spiny neurons (MSNs) in the NAcSh was used to explore the role of RA in NAcSh cellular function.ResultsWe observed an anxiety-vulnerable, depression-resilient phenotype in knockdown animals compared to controls. During operant tasks, knockdown animals took fewer fentanyl infusions during FR5 maintenance and showed decreased demand intensity in behavioral economics sessions. Finally, electrophysiological assessment of NAcSh MSNs revealed attenuated excitability following Aldh1a1 knockdown. Altogether, our findings reveal a key role for NAcSh RA signaling in determining emotional resilience and drug-taking, likely via decreased MSN excitability.ConclusionsOur results posit the RA synthesis enzyme Aldh1a1 as a promising therapeutic target for depression-, frustration-, and addiction-associated disorders. This is the first report linking RA to frustrative nonreward, the NAcSh to operant frustration, and RA to fentanyl drug-taking behavior.

Pyrethroid pesticides have been associated with neurodevelopmental disorders including attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). While behavioral effects of pyrethroid exposure have been previously reported, the underlying mechanisms remain unclear. Here, we hypothesized that exposure to deltamethrin (DM), a widely used pyrethroid pesticide known for its neurotoxicity during early developmental stages, induces brain dysfunction through alterations in brain-derived extracellular vesicle (BDEV) signaling. Using a well-established rodent model of early life DM exposure within the recommended no observable effect level, we isolated BDEVs from postnatal 30-day-old vehicle-exposed (control) and DM-exposed mice using a differential sucrose density gradient. Following ZetaView nanoparticle tracking and electron microscopy characterization, quantitative mass spectrometry-based proteomics revealed 89 differentially expressed proteins (DEPs) in BDEVs from DM exposed animals compared to control BDEVs. Bioinformatic analysis identified convergence of DEPs on pathways associated with mitochondrial function and synaptic plasticity. PKH67-green conjugated BDEVs derived from either control or DM-exposed mice were bilaterally injected intracerebroventricularly into naive adult mice, and the brain distribution of labeled BDEVs was verified prior to extracellular field recording experiments. Strikingly, long-term potentiation (LTP) at CA3-CA1 hippocampal synapses, a functional correlate of learning and memory, was intact in control BDEVs but absent in naive mice receiving BDEVs from DM exposed mice. Notably, exogenously delivering LRRTM1, one of the DEPs found in DM BDEVs, disrupts synaptic transmission in CA1 neurons consistent with impaired LTP. Thus, differentially regulated signaling in BDEVs represents a novel mechanism of DM neurotoxicity.

The social deficits following chronic stress conditions are linked to synaptic dysfunction in the brain. Complement system plays a critical role in synapse regulation. Although complement has been implicated in chronic stress-induced behavior deficits the cellular substrates and mechanisms underlying complement-mediated behavior changes under chronic stress conditions are not known. In the present study, we investigated the role of complement component 3a receptor (C3ar1) in microglia and monocytes/macrophages (Mo/MΦ) in chronic unpredictable stress (CUS)-induced synapse loss and behavior deficits in mice. We found that deletion of microglial C3ar1 attenuated stress-induced social behavior deficits and changes in neuroinflammatory as well as synaptic markers in the prefrontal cortex (PFC). RNA sequencing data revealed that microglial C3ar1 deletion attenuates CUS-mediated changes in the expression of immediate-early genes such as Fos and Nuclear Receptor Subfamily 4 Group A Member 1 (Nr4a1) in the PFC. In contrast, lack of C3ar1 in Mo/MΦ induced social behavior deficits. Together, these findings indicate opposite functions of C3ar1 signaling in microglia and Mo/MΦ under chronic stress conditions.

INTRODUCTION:Network hyperexcitability (NH) is observed in patients with early-stage Alzheimer's disease (AD), emerging decades before cognitive decline. A key molecular determinant of NH is voltage-gated Na+ channel 1.6 (Nav1.6), which mediates action potential firing in CA1 hippocampal neurons. Ameliorating NH through inhibition of the glycogen-synthase kinase 3β (GSK3β/Nav1.6 complex may provide immediate benefits to cognition and memory and slow AD progression. METHODS:Hight-throughput virtual screening and multiple in vitro biological assays were utilized to identify compound 1063. Patch-clamp electrophysiology and electroencephalogram recordings were utilized to functionally assess 1063 in models of AD neuropathology. RESULTS:Building on previous studies identifying GSK3β as a modulatory protein binding to the Nav1.6 C-terminal domain (CTD), we identified 1063, a brain-penetrant small molecule that inhibits GSK3β/Nav1.6 complex assembly and reduces NH in AD rodent models. DISCUSSION:These results demonstrate the potential of the GSK3β/Nav1.6 complex as a therapeutic target for NH in early-stage AD. HIGHLIGHTS:The glycogen synthase kinase 3-β (GSK3β)/Nav1.6 complex is a potential target for hyperexcitability in early Alzheimer's disease (AD). Compound 1063 dose-dependently decreases GSK3β/Nav1.6 complex assembly. Compound 1063 is functionally specific for Nav1.6 over other central nervous system (CNS) Nav isoforms. Ex vivo functional studies provide evidence for target engagement. 1063 dose-dependently reduces epileptiform activity in AD rodent model.

Identifying molecular mechanisms that regulate neuronal excitability is essential for developing targeted therapies for neuropsychiatric disorders. The protein-protein interaction (PPI) between fibroblast growth factor 14 (FGF14) and the voltage-gated Na+ channel Nav1.6 is critical in regulating neuronal excitability and has emerged as a promising drug target. However, the physicochemical features that drive small-molecule modulation of this interface remain elusive. Here, we apply a descriptor-based chemoinformatics approach to analyze 15 hit compounds identified via high-throughput screening, aiming to elucidate structure-activity relationships influencing their potency and binding affinity. The analysis revealed distinct subsets of physicochemical features strongly associated with either potency or binding affinity values, suggesting that these parameters are governed by largely independent molecular determinants. This independence implies that optimizing a compound for improved affinity need not compromise potency, and vice versa. Together, these findings may guide the rational optimization of first-in-class compounds aimed at controlling neuronal excitability through targeted PPI interface modulation.

Schizophrenia (SCZ) is a complex psychiatric disorder with unclear biological mechanisms. Spectrins, cytoskeletal proteins linked to neurodevelopmental disorders, are regulated by the AKT/GSK3 pathway, which is implicated in SCZ. However, the impact of SCZ-related dysregulation of this pathway on spectrin expression and distribution remains unexplored. Here, we show that βIV spectrin protein levels were reduced in neurons of the dorsolateral prefrontal cortex in SCZ postmortem samples compared to healthy control (HC) from the Human Brain Collection Core (HBCC). To investigate potential links between βIV spectrin and the AKT/GSK3 pathway, we analyzed the PsychEncode dataset, revealing elevated SPTBN4 and AKT2 mRNA levels with correlated gene transcription in both HCs and individuals with SCZ. Next, computational tools were employed to identify potential AKT and GSK3 phosphorylation sites on βIV spectrin, and two GSK3 sites were validated through in vitro assays. To assess whether βIV spectrin distribution and sensitivity to AKT/GSK3 are altered in SCZ, we used iPSC-derived neurons from two independent cohorts of patients with significantly increased familial genetic risk for the disorder. Alteration in βIV spectrin levels and sensitivity to AKT/GSK3 inhibitors were consistently observed across both cohorts. Importantly, a Random Forest classifier applied to βIV spectrin imaging achieved up to 98% accuracy in classifying cells by diagnosis in postmortem samples, and by diagnosis or diagnosis × perturbation in iPSC samples. These findings reveal altered βIV spectrin levels and AKT/GSK3 sensitivity in SCZ, identifying βIV spectrin image-based endophenotypes as robust, generalizable predictive biomarkers of SCZ, with the potential for scalable clinical applications.

Environmental exposure to pesticides at levels deemed safe by regulatory agencies has been linked to increased risk for neurodevelopmental disorders. Yet, the mechanisms linking exposure to these disorders remain unclear. Here, we show that maternal exposure to the pesticide deltamethrin (DM) at the no observed adverse effect level (NOAEL) disrupts long-term potentiation (LTP) in the hippocampus of adult male offspring three months after exposure, a phenotype absent in female offspring. Clonazepam, a GABAa receptor agonist, rescued this deficit, indicating impaired hippocampal GABAergic signaling. Recordings from CA1 pyramidal neurons, complemented by MALDI mass spectrometry imaging, showed an imbalance in excitatory/inhibitory tone. Using a combination of parvalbumin (PV)-Cre transgenic mice and hippocampal injection of designer receptors exclusively activated by designer drugs (DREADDs), we show that developmental DM exposure reduces hippocampal PV interneuron intrinsic firing. DREADD activation rescued both PV interneuron firing and LTP deficits. Complementary behavioral experiments revealed a deficit in social memory, a behavior relevant to autism spectrum disorder (ASD) symptomatology, which was restored by DREADD activation. Overall, these results establish a novel mechanistic link between maternal exposure to DM at the NOAEL and known cellular, circuital, and behavioral vulnerabilities, indicating it is a potential driver in the exposome of ASD.

Nociception involves complex signaling, yet intrinsic mechanisms bidirectionally regulating this process remain unexplored. Here, we show that the fibroblast growth factor 13 (FGF13)/Nav1.7 protein-protein interaction (PPI) complex bidirectionally modulates nociception, and that the FGF13/Nav1.7 ratio is upregulated in type 2 diabetic neuropathy (T2DN). PW164, an FGF13/Nav1.7 channel C-terminal tail domain (CTD) PPI interface inhibitor, which reduces complex assembly, selectively suppressed Na+ currents sensitized by capsaicin-induced activation of TRPV1 channels in human induced pluripotent stem cell-derived (hIPSC-derived) sensory neurons and inhibited mechanical and thermal hyperalgesia in mice. FGF13 silencing mimics PW164 activity in culture and in vivo. Conversely, ZL192, an FGF13 ligand that stabilizes FGF13/Nav1.7 CTD assembly, sensitized Na+ currents in hIPSC-derived sensory neurons and exerted pronociceptive behavioral responses in mice. ZL192's effects were abrogated by FGF13 silencing in culture and in vivo and recapitulated by FGF13 overexpression. In a model of T2DN, PW164 injection reduced mechanical hyperalgesia locally and contralaterally without systemic side effects. In donor-derived dorsal root ganglia neurons, FGF13 and Nav1.7 proteins colocalized, and the FGF13/Nav1.7 protein ratio was upregulated in patients with T2DN. Lastly, we found that SCN9A variant V1831F, associated with painless diabetic neuropathy, abolished PW164-directed modulation of the FGF13/Nav1.7 PPI interface. Thus, FGF13 is a rheostat of nociception and promising therapeutic target for diabetic neuropathy pain.

The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1—critical to the cell cycle—selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (INa) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak INa density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor’s opposite effects on INa. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase—in concert with the AKT/GSK3 pathway—in regulating the Nav1.2 channelosome.