Miroslav N. Nenov

The Fibroblast Growth Factor 14·Voltage-gated Sodium Channel Complex Is a New Target of Glycogen Synthase Kinase 3 (GSK3)

Background: Fibroblast growth factor 14 (FGF14) binds to and regulates the voltage-gated Na+ (Nav) channel. Results: Inhibition of glycogen synthase kinase 3 (GSK3) modifies FGF14/Nav channel interaction, with effects on Na+ currents and subcellular distribution of the FGF14·Nav channel complex. Conclusion: The FGF14·Nav channel complex is a new target of GSK3. Significance: We provide evidence for modulation of Nav channels by GSK3 through FGF14. The FGF14 protein controls biophysical properties and subcellular distribution of neuronal voltage-gated Na+ (Nav) channels through direct binding to the channel C terminus. To gain insights into the dynamic regulation of this protein/protein interaction complex, we employed the split luciferase complementation assay to screen a small molecule library of kinase inhibitors against the FGF14·Nav1.6 channel complex and identified inhibitors of GSK3 as hits. Through a combination of a luminescence-based counter-screening, co-immunoprecipitation, patch clamp electrophysiology, and quantitative confocal immunofluorescence, we demonstrate that inhibition of GSK3 reduces the assembly of the FGF14·Nav channel complex, modifies FGF14-dependent regulation of Na+ currents, and induces dissociation and subcellular redistribution of the native FGF14·Nav channel complex in hippocampal neurons. These results further emphasize the role of FGF14 as a critical component of the Nav channel macromolecular complex, providing evidence for a novel GSK3-dependent signaling pathway that might control excitability through specific protein/protein interactions.

Cognitive Enhancing Treatment with a PPARγ Agonist Normalizes Dentate Granule Cell Presynaptic Function in Tg2576 APP Mice

Hippocampal network hyperexcitability is considered an early indicator of Alzheimers disease (AD) memory impairment. Some AD mouse models exhibit similar network phenotypes. In this study we focused on dentate gyrus (DG) granule cell spontaneous and evoked properties in 9-month-old Tg2576 mice that model AD amyloidosis and cognitive deficits. Using whole-cell patch-clamp recordings, we found that Tg2576 DG granule cells exhibited spontaneous EPSCs that were higher in frequency but not amplitude compared with wild-type mice, suggesting hyperactivity of DG granule cells via a presynaptic mechanism. Further support of a presynaptic mechanism was revealed by increased I–O relationships and probability of release in Tg2576 DG granule cells. Since we and others have shown that activation of the peroxisome proliferator-activated receptor gamma (PPARγ) axis improves hippocampal cognition in mouse models for AD as well as benefitting memory performance in some humans with early AD, we investigated how PPARγ agonism affected synaptic activity in Tg2576 DG. We found that PPARγ agonism normalized the I–O relationship of evoked EPSCs, frequency of spontaneous EPSCs, and probability of release that, in turn, correlated with selective expression of DG proteins essential for presynaptic SNARE function that are altered in patients with AD. These findings provide evidence that DG principal cells may contribute to early AD hippocampal network hyperexcitability via a presynaptic mechanism, and that hippocampal cognitive enhancement via PPARγ activation occurs through regulation of presynaptic vesicular proteins critical for proper glutamatergic neurotransmitter release, synaptic transmission, and short-term plasticity.

Quantitative Proteomics Reveals Protein–Protein Interactions with Fibroblast Growth Factor 12 as a Component of the Voltage-Gated Sodium Channel 1.2 (Nav1.2) Macromolecular Complex in Mammalian Brain

Voltage-gated sodium channels (Nav1.1–Nav1.9) are responsible for the initiation and propagation of action potentials in neurons, controlling firing patterns, synaptic transmission and plasticity of the brain circuit. Yet, it is the protein–protein interactions of the macromolecular complex that exert diverse modulatory actions on the channel, dictating its ultimate functional outcome. Despite the fundamental role of Nav channels in the brain, information on its proteome is still lacking. Here we used affinity purification from crude membrane extracts of whole brain followed by quantitative high-resolution mass spectrometry to resolve the identity of Nav1.2 protein interactors. Of the identified putative protein interactors, fibroblast growth factor 12 (FGF12), a member of the nonsecreted intracellular FGF family, exhibited 30-fold enrichment in Nav1.2 purifications compared with other identified proteins. Using confocal microscopy, we visualized native FGF12 in the brain tissue and confirmed that FGF12 forms a complex with Nav1.2 channels at the axonal initial segment, the subcellular specialized domain of neurons required for action potential initiation. Co-immunoprecipitation studies in a heterologous expression system validate Nav1.2 and FGF12 as interactors, whereas patch-clamp electrophysiology reveals that FGF12 acts synergistically with CaMKII, a known kinase regulator of Nav channels, to modulate Nav1.2-encoded currents. In the presence of CaMKII inhibitors we found that FGF12 produces a bidirectional shift in the voltage-dependence of activation (more depolarized) and the steady-state inactivation (more hyperpolarized) of Nav1.2, increasing the channel availability. Although providing the first characterization of the Nav1.2 CNS proteome, we identify FGF12 as a new functionally relevant interactor. Our studies will provide invaluable information to parse out the molecular determinant underlying neuronal excitability and plasticity, and extending the relevance of iFGFs signaling in the normal and diseased brain.

Identifying a Kinase Network Regulating FGF14:Nav1.6 Complex Assembly Using Split-Luciferase Complementation

Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA) is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14). Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC) as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3) and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the normal and diseased brain.

The Nav1.2 channel is regulated by GSK3

BACKGROUND Phosphorylation plays an essential role in regulating voltage-gated sodium (Na(v)) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Na(v) channels. We posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Na(v) channels. METHODS We used patch-clamp electrophysiology to record sodium currents from Na(v)1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues. RESULTS We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Na(v)1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3β suppressed Na(v)1.2-encoded currents. Neither mRNA nor total protein expression was changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Na(v)1.2 channel indicates that cell surface expression of CD4-Na(v)1.2 C-tail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3β phosphorylates T(1966) at the C-terminal tail of Na(v)1.2. CONCLUSION These findings provide evidence for a new mechanism by which GSK3 modulates Na(v) channel function via its C-terminal tail. GENERAL SIGNIFICANCE These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders.

Impaired firing properties of dentate granule neurons in an Alzheimer's disease animal model are rescued by PPARγ agonism

Early cognitive impairment in Alzheimers disease (AD) correlates with medial temporal lobe dysfunction, including two areas essential for memory formation: the entorhinal cortex and dentate gyrus (DG). In the Tg2576 animal model for AD amyloidosis, activation of the peroxisome proliferator-activated receptor-gamma (PPARγ) with rosiglitazone (RSG) ameliorates hippocampus-dependent cognitive impairment and restores aberrant synaptic activity at the entorhinal cortex to DG granule neuron inputs. It is unknown, however, whether intrinsic firing properties of DG granule neurons in these animals are affected by amyloid-β pathology and if they are sensitive to RSG treatment. Here, we report that granule neurons from 9-mo-old wild-type and Tg2576 animals can be segregated into two cell types with distinct firing properties and input resistance that correlate with less mature type I and more mature type II neurons. The DG type I cell population was greater than type II in wild-type littermates. In the Tg2576 animals, the type I and type II cell populations were nearly equal but could be restored to wild-type levels through cognitive enhancement with RSG. Furthermore, Tg2576 cell firing frequency and spike after depolarization were decreased in type I and increased in type II cells, both of which could also be restored to wild-type levels upon RSG treatment. That these parameters were restored by PPARγ activation emphasizes the therapeutic value of RSG against early AD cognitive impairment.

Genetic deletion of fibroblast growth factor 14 recapitulates phenotypic alterations underlying cognitive impairment associated with schizophrenia.

Cognitive processing is highly dependent on the functional integrity of gamma-amino-butyric acid (GABA) interneurons in the brain. These cells regulate excitability and synaptic plasticity of principal neurons balancing the excitatory/inhibitory tone of cortical networks. Reduced function of parvalbumin (PV) interneurons and disruption of GABAergic synapses in the cortical circuitry result in desynchronized network activity associated with cognitive impairment across many psychiatric disorders, including schizophrenia. However, the mechanisms underlying these complex phenotypes are still poorly understood. Here we show that in animal models, genetic deletion of fibroblast growth factor 14 (Fgf14), a regulator of neuronal excitability and synaptic transmission, leads to loss of PV interneurons in the CA1 hippocampal region, a critical area for cognitive function. Strikingly, this cellular phenotype associates with decreased expression of glutamic acid decarboxylase 67 (GAD67) and vesicular GABA transporter (VGAT) and also coincides with disrupted CA1 inhibitory circuitry, reduced in vivo gamma frequency oscillations and impaired working memory. Bioinformatics analysis of schizophrenia transcriptomics revealed functional co-clustering of FGF14 and genes enriched within the GABAergic pathway along with correlatively decreased expression of FGF14, PVALB, GAD67 and VGAT in the disease context. These results indicate that Fgf14−/− mice recapitulate salient molecular, cellular, functional and behavioral features associated with human cognitive impairment, and FGF14 loss of function might be associated with the biology of complex brain disorders such as schizophrenia.

Glycogen synthase kinase 3 beta alters anxiety-, depression-, and addiction-related behaviors and neuronal activity in the nucleus accumbens shell

&NA; Psychiatric disorders such as anxiety, depression and addiction are often comorbid brain pathologies thought to share common mechanistic biology. As part of the cortico‐limbic circuit, the nucleus accumbens shell (NAcSh) plays a fundamental role in integrating information in the circuit, such that modulation of NAcSh circuitry alters anxiety, depression, and addiction‐related behaviors. Intracellular kinase cascades in the NAcSh have proven important mediators of behavior. To investigate glycogen‐synthase kinase 3 (GSK3) beta signaling in the NAcSh in vivo we knocked down GSK3beta expression with a novel adeno‐associated viral vector (AAV2) and assessed changes in anxiety‐ and depression‐like behavior and cocaine self‐administration in GSK3beta knockdown rats. GSK3beta knockdown reduced anxiety‐like behavior while increasing depression‐like behavior and cocaine self‐administration. Correlative electrophysiological recordings in acute brain slices were used to assess the effect of AAV‐shGSK3beta on spontaneous firing and intrinsic excitability of tonically active interneurons (TANs), cells required for input and output signal integration in the NAcSh and for processing reward‐related behaviors. Loose‐patch recordings showed that TANs transduced by AAV‐shGSK3beta exhibited reduction in tonic firing and increased spike half width. When assessed by whole‐cell patch clamp recordings these changes were mirrored by reduction in action potential firing and accompanied by decreased hyperpolarization‐induced depolarizing sag potentials, increased action potential current threshold, and decreased maximum rise time. These results suggest that silencing of GSK3beta in the NAcSh increases depression‐ and addiction‐related behavior, possibly by decreasing intrinsic excitability of TANs. However, this study does not rule out contributions from other neuronal sub‐types. HighlightsSpecific knockdown of GSK3 beta in the NAc shell induces an anxiolytic‐like effect.Knockdown of GSK3 beta in the NAcSh induces depression‐like behavior.Viral‐mediated knockdown of GSK3 beta increases cocaine self‐administration.Knockdown also reduces spontaneous firing and alters intrinsic excitability of TANs.

PPARgamma agonists rescue increased phosphorylation of FGF14 at S226 in the Tg2576 mouse model of Alzheimer's disease

Background Cognitive impairment in humans with Alzheimers disease (AD) and in animal models of A&bgr;‐pathology can be ameliorated by treatments with the nuclear receptor peroxisome proliferator‐activated receptor‐gamma (PPAR&ggr;) agonists, such as rosiglitazone (RSG). Previously, we demonstrated that in the Tg2576 animal model of AD, RSG treatment rescued cognitive deficits and reduced aberrant activity of granule neurons in the dentate gyrus (DG), an area critical for memory formation. Methods We used a combination of mass spectrometry, confocal imaging, electrophysiology and split‐luciferase assay and in vitro phosphorylation and Ingenuity Pathway Analysis. Results Using an unbiased, quantitative nano‐LC‐MS/MS screening, we searched for potential molecular targets of the RSG‐dependent rescue of DG granule neurons. We found that S226 phosphorylation of fibroblast growth factor 14 (FGF14), an accessory protein of the voltage‐gated Na+ (Nav) channels required for neuronal firing, was reduced in Tg2576 mice upon treatment with RSG. Using confocal microscopy, we confirmed that the Tg2576 condition decreased PanNav channels at the AIS of the DG, and that RSG treatment of Tg2576 mice reversed the reduction in PanNav channels. Analysis from previously published data sets identified correlative changes in action potential kinetics in RSG‐treated T2576 compared to untreated and wildtype controls. In vitro phosphorylation and mass spectrometry confirmed that the multifunctional kinase GSK–3&bgr;, a downstream target of insulin signaling highly implicated in AD, phosphorylated FGF14 at S226. Assembly of the FGF14:Nav1.6 channel complex and functional regulation of Nav1.6‐mediated currents by FGF14 was impaired by a phosphosilent S226A mutation. Bioinformatics pathway analysis of mass spectrometry and biochemistry data revealed a highly interconnected network encompassing PPAR&ggr;, FGF14, SCN8A (Nav 1.6), and the kinases GSK–3 &bgr;, casein kinase 2&bgr;, and ERK1/2. Conclusions These results identify FGF14 as a potential PPAR&ggr;‐sensitive target controlling A&bgr;‐induced dysfunctions of neuronal activity in the DG underlying memory loss in early AD. HighlightsPhosphorylation of FGF14 at S226 in Tg2576 animals is reduced by rosiglitazone (RSG).Tg2576 condition decreases PanNav channels at AIS, which is reversed by RSG.GSK‐3&bgr; phosphorylates FGF14 at S226.Assembly of FGF14:Nav1.6 channel complex is reduced by a S226A mutationFunctional regulation of Nav1.6‐mediated currents was impaired by a S226A mutation.

CK2 activity is required for the interaction of FGF14 with voltage-gated sodium channels and neuronal excitability

Recent data shows that fibroblast growth factor 14 (FGF14) binds to and controls the function of the voltage‐gated sodium (Nav) channel with phenotypic outcomes on neuronal excitability. Mutations in the FGF14 gene in humans have been associated with brain disorders that are partially recapitulated in Fgf14–/– mice. Thus, signaling pathways that modulate the FGF14:Nav channel interaction may be important therapeutic targets. Bioluminescence‐based screening of small molecule modulators of the FGF14:Nav1.6 complex identified 4,5,6,7‐tetrabromobenzotriazole (TBB), a potent casein kinase 2 (CK2) inhibitor, as a strong suppressor of FGF14:Nav1.6 interaction. Inhibition of CK2 through TBB reduces the interaction of FGF14 with Nav1.6 and Nav1.2 channels. Mass spectrometry confirmed direct phosphorylation of FGF14 by CK2 at S228 and S230, and mutation to alanine at these sites modified FGF14 modulation of Nav1.6‐mediated currents. In 1 d in vitro hippocampal neurons, TBB induced a reduction in FGF14 expression, a decrease in transient Na+ current amplitude, and a hyperpolarizing shift in the voltage dependence of Nav channel steady‐state inactivation. In mature neurons, TBB reduces the axodendritic polarity of FGF14. In cornu ammonis area 1 hippocampal slices from wild‐type mice, TBB impairs neuronal excitability by increasing action potential threshold and lowering firing frequency. Importantly, these changes in excitability are recapitulated in Fgf14–/– mice, and deletion of Fgf14 occludes TBB‐dependent phenotypes observed in wild‐type mice. These results suggest that a CK2‐FGF14 axis may regulate Nav channels and neuronal excitability.—Hsu, W.‐C. J., Scala, F., Nenov, M. N., Wildburger, N. C., Elferink, H., Singh, A. K., Chesson, C. B., Buzhdygan, T., Sohail, M., Shavkunov, A. S., Panova, N. I., Nilsson, C. L., Rudra, J. S., Lichti, C. F., Laezza, F. CK2 activity is required for the interaction of FGF14 with voltage‐gated sodium channels and neuronal excitability. FASEB J. 30, 2171–2186 (2016). www.fasebj.org