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.

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.

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 GSK3ß 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 GSK3ß 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.

ESI-MS/MS and MALDI-IMS Localization Reveal Alterations in Phosphatidic Acid, Diacylglycerol, and DHA in Glioma Stem Cell Xenografts.

Glioblastoma (GBM) is the most common adult primary brain tumor. Despite aggressive multimodal therapy, the survival of patients with GBM remains dismal. However, recent evidence has demonstrated the promise of bone marrow-derived mesenchymal stem cells (BM-hMSCs) as a therapeutic delivery vehicle for anti-glioma agents due to their ability to migrate or home to human gliomas. While several studies have demonstrated the feasibility of harnessing the homing capacity of BM-hMSCs for targeted delivery of cancer therapeutics, it is now also evident, based on clinically relevant glioma stem cell (GSC) models of GBMs, that BM-hMSCs demonstrate variable tropism toward these tumors. In this study, we compared the lipid environment of GSC xenografts that attract BM-hMSCs (N = 9) with those that do not attract (N = 9) to identify lipid modalities that are conducive to homing of BM-hMSC to GBMs. We identified lipids directly from tissue by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) of lipid extracts. Several species of signaling lipids, including phosphatidic acid (PA 36:2, PA 40:5, PA 42:5, and PA 42:7) and diacylglycerol (DAG 34:0, DAG 34:1, DAG 36:1, DAG 38:4, DAG 38:6, and DAG 40:6), were lower in attracting xenografts. Molecular lipid images showed that PA (36:2), DAG (40:6), and docosahexaenoic acid (DHA) were decreased within tumor regions of attracting xenografts. Our results provide the first evidence for lipid signaling pathways and lipid-mediated tumor inflammatory responses in the homing of BM-hMSCs to GSC xenografts. Our studies provide new fundamental knowledge on the molecular correlates of the differential homing capacity of BM-hMSCs toward GSC xenografts.

Integrated Transcriptomic and Glycomic Profiling of Glioma Stem Cell Xenografts.

Bone marrow-derived human mesenchymal stem cells (BM-hMSCs) have the innate ability to migrate or home toward and engraft in tumors such as glioblastoma (GBM). Because of this unique property of BM-hMSCs, we have explored their use for cell-mediated therapeutic delivery for the advancement of GBM treatment. Extravasation, the process by which blood-borne cells­such as BM-hMSCs­enter the tissue, is a highly complex process but is heavily dependent upon glycosylation for glycan-glycan and glycan-protein adhesion between the cell and endothelium. However, in a translationally significant preclinical glioma stem cell xenograft (GSCX) model of GBM, BM-hMSCs demonstrate unequal tropism toward these tumors. We hypothesized that there may be differences in the glycan compositions between the GSCXs that elicit homing ("attractors") and those that do not ("non-attractors") that facilitate or impede the engraftment of BM-hMSCs in the tumor. In this study, glycotranscriptomic analysis revealed significant heterogeneity within the attractor phenotype and the enrichment of high mannose type N-glycan biosynthesis in the non-attractor phenotype. Orthogonal validation with topical PNGase F deglycosylation on the tumor regions of xenograft tissue, followed by nLC-ESI-MS, confirmed the presence of increased high mannose type N-glycans in the non-attractors. Additional evidence provided by our glycomic study revealed the prevalence of terminal sialic acid-containing N-glycans in non-attractors and terminal galactose and N-acetyl-glucosamine N-glycans in attractors. Our results provide the first evidence for differential glycomic profiles in attractor and non-attractor GSCXs and extend the scope of molecular determinates in BM-hMSC homing to glioma.

The fibroblast growth factor 14·voltage-gated sodium channel complex is a new target of glycogen synthase kinase 3 (GSK3).

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.

Current Evidence for a Bidirectional Loop Between the Lysosome and Alpha-Synuclein Proteoforms.

Cumulative evidence collected in recent decades suggests that lysosomal dysfunction contributes to neurodegenerative diseases, especially if amyloid proteins are involved. Among these, alpha-synuclein (aSyn) that progressively accumulates and aggregates in Lewy bodies is undisputedly a main culprit in Parkinson disease (PD) pathogenesis. Lysosomal dysfunction is evident in brains of PD patients, and mutations in lysosomal enzymes are a major risk factor of PD. At first glance, the role of protein-degrading lysosomes in a disease with pathological protein accumulation seems obvious and should guide the development of straightforward and rational therapeutic targets. However, our review demonstrates that the story is more complicated for aSyn. The protein can possess diverse posttranslational modifications, aggregate formations, and truncations, all of which contribute to a growing known set of proteoforms. These interfere directly or indirectly with lysosome function, reducing their own degradation, and thereby accelerating the protein aggregation and disease process. Conversely, unbalanced lysosomal enzymatic processes can produce truncated aSyn proteoforms that may be more toxic and prone to aggregation. This highlights the possibility of enhancing lysosomal function as a treatment for PD, if it can be confirmed that this approach effectively reduces harmful aSyn proteoforms and does not produce novel, toxic proteoforms.

SILK studies - capturing the turnover of proteins linked to neurodegenerative diseases.

Alzheimer disease (AD) is one of several neurodegenerative diseases characterized by dysregulation, misfolding and accumulation of specific proteins in the CNS. The stable isotope labelling kinetics (SILK) technique is based on generating amino acids labelled with naturally occurring stable (that is, nonradioactive) isotopes of carbon and/or nitrogen. These labelled amino acids can then be incorporated into proteins, enabling rates of protein production and clearance to be determined in vivo and in vitro without the use of radioactive or chemical labels. Over the past decade, SILK studies have been used to determine the turnover of key pathogenic proteins amyloid-ß (Aß), tau and superoxide dismutase 1 (SOD1) in the cerebrospinal fluid of healthy individuals, patients with AD and those with other neurodegenerative diseases. These studies led to the identification of several factors that alter the production and/or clearance of these proteins, including age, sleep and disease-causing genetic mutations. SILK studies have also been used to measure Aß turnover in blood and within brain tissue. SILK studies offer the potential to elucidate the mechanisms underlying various neurodegenerative disease mechanisms, including neuroinflammation and synaptic dysfunction, and to demonstrate target engagement of novel disease-modifying therapies.

Amyloid-ß Plaques in Clinical Alzheimer's Disease Brain Incorporate Stable Isotope Tracer In Vivo and Exhibit Nanoscale Heterogeneity.

Alzheimer's disease (AD) is a neurodegenerative disorder with clinical manifestations of progressive memory decline and loss of executive function and language. AD affects an estimated 5.3 million Americans alone and is the most common form of age-related dementia with a rapidly growing prevalence among the aging population-those 65 years of age or older. AD is characterized by accumulation of aggregated amyloid-beta (Aß) in the brain, which leads to one of the pathological hallmarks of AD-Aß plaques. As a result, Aß plaques have been extensively studied after being first described over a century ago. Advances in brain imaging and quantitative measures of Aß in biological fluids have yielded insight into the time course of plaque development decades before and after AD symptom onset. However, despite the fundamental role of Aß plaques in AD, in vivo measures of individual plaque growth, growth distribution, and dynamics are still lacking. To address this question, we combined stable isotope labeling kinetics (SILK) and nanoscale secondary ion mass spectrometry (NanoSIMS) imaging in an approach termed SILK-SIMS to resolve plaque dynamics in three human AD brains. In human AD brain, plaques exhibit incorporation of a stable isotope tracer. Tracer enrichment was highly variable between plaques and the spatial distribution asymmetric with both quiescent and active nanometer sub-regions of tracer incorporation. These data reveal that Aß plaques are dynamic structures with deposition rates over days indicating a highly active process. Here, we report the first, direct quantitative measures of in vivo deposition into plaques in human AD brain. Our SILK-SIMS studies will provide invaluable information on plaque dynamics in the normal and diseased brain and offer many new avenues for investigation into pathological mechanisms of the disease, with implications for therapeutic development.

Environmental Enrichment and Social Isolation Mediate Neuroplasticity of Medium Spiny Neurons through the GSK3 Pathway.

Resilience and vulnerability to neuropsychiatric disorders are linked to molecular changes underlying excitability that are still poorly understood. Here, we identify glycogen-synthase kinase 3ß (GSK3ß) and voltage-gated Na+ channel Nav1.6 as regulators of neuroplasticity induced by environmentally enriched (EC) or isolated (IC) conditions-models for resilience and vulnerability. Transcriptomic studies in the nucleus accumbens from EC and IC rats predicted low levels of GSK3ß and SCN8A mRNA as a protective phenotype associated with reduced excitability in medium spiny neurons (MSNs). In vivo genetic manipulations demonstrate that GSK3ß and Nav1.6 are molecular determinants of MSN excitability and that silencing of GSK3ß prevents maladaptive plasticity of IC MSNs. In vitro studies reveal direct interaction of GSK3ß with Nav1.6 and phosphorylation at Nav1.6T1936 by GSK3ß. A GSK3ß-Nav1.6T1936 competing peptide reduces MSNs excitability in IC, but not EC rats. These results identify GSK3ß regulation of Nav1.6 as a biosignature of MSNs maladaptive plasticity.

Diversity of Amyloid-beta Proteoforms in the Alzheimer's Disease Brain.

Amyloid-beta (Aß) plays a key role in the pathogenesis of Alzheimer's disease (AD), but little is known about the proteoforms present in AD brain. We used high-resolution mass spectrometry to analyze intact Aß from soluble aggregates and insoluble material in brains of six cases with severe dementia and pathologically confirmed AD. The soluble aggregates are especially relevant because they are believed to be the most toxic form of Aß. We found a diversity of Aß peptides, with 26 unique proteoforms including various N- and C-terminal truncations. N- and C-terminal truncations comprised 73% and 30%, respectively, of the total Aß proteoforms detected. The Aß proteoforms segregated between the soluble and more insoluble aggregates with N-terminal truncations predominating in the insoluble material and C- terminal truncations segregating into the soluble aggregates. In contrast, canonical Aß comprised the minority of the identified proteoforms (15.3%) and did not distinguish between the soluble and more insoluble aggregates. The relative abundance of many truncated Aß proteoforms did not correlate with post-mortem interval, suggesting they are not artefacts. This heterogeneity of Aß proteoforms deepens our understanding of AD and offers many new avenues for investigation into pathological mechanisms of the disease, with implications for therapeutic development.

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

BACKGROUND: Cognitive impairment in humans with Alzheimer's disease (AD) and in animal models of Aß-pathology can be ameliorated by treatments with the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ) 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-3ß, 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 PPARγ, FGF14, SCN8A (Nav 1.6), and the kinases GSK-3 ß, casein kinase 2ß, and ERK1/2. CONCLUSIONS: These results identify FGF14 as a potential PPARγ-sensitive target controlling Aß-induced dysfunctions of neuronal activity in the DG underlying memory loss in early AD.

Soluble Amyloid-beta Aggregates from Human Alzheimer's Disease Brains.

Soluble amyloid-beta (Aß) aggregates likely contribute substantially to the dementia that characterizes Alzheimer's disease. However, despite intensive study of in vitro preparations and animal models, little is known about the characteristics of soluble Aß aggregates in the human Alzheimer's disease brain. Here we present a new method for extracting soluble Aß aggregates from human brains, separating them from insoluble aggregates and Aß monomers using differential ultracentrifugation, and purifying them >6000 fold by dual antibody immunoprecipitation. The method resulted in <40% loss of starting material, no detectible ex vivo aggregation of monomeric Aß, and no apparent ex vivo alterations in soluble aggregate sizes. By immunoelectron microscopy, soluble Aß aggregates typically appear as clusters of 10-20 nanometer diameter ovoid structures with 2-3 amino-terminal Aß antibody binding sites, distinct from previously characterized structures. This approach may facilitate investigation into the characteristics of native soluble Aß aggregates, and deepen our understanding of Alzheimer's dementia.

Control of neuronal ion channel function by glycogen synthase kinase-3: new prospective for an old kinase.

Glycogen synthase kinase 3 (GSK-3) is an evolutionarily conserved multifaceted ubiquitous enzyme. In the central nervous system (CNS), GSK-3 acts through an intricate network of intracellular signaling pathways culminating in a highly divergent cascade of phosphorylations that control neuronal function during development and adulthood. Accumulated evidence indicates that altered levels of GSK-3 correlate with maladaptive plasticity of neuronal circuitries in psychiatric disorders, addictive behaviors, and neurodegenerative diseases, and pharmacological interventions known to limit GSK-3 can counteract some of these deficits. Thus, targeting the GSK-3 cascade for therapeutic interventions against this broad spectrum of brain diseases has raised a tremendous interest. Yet, the multitude of GSK-3 downstream effectors poses a substantial challenge in the development of selective and potent medications that could efficiently block or modulate the activity of this enzyme. Although the full range of GSK-3 molecular targets are far from resolved, exciting new evidence indicates that ion channels regulating excitability, neurotransmitter release, and synaptic transmission, which ultimately contribute to the mechanisms underling brain plasticity and higher level cognitive and emotional processing, are new promising targets of this enzyme. Here, we will revise this new emerging role of GSK-3 in controling the activity of voltage-gated Na(+), K(+), Ca(2+) channels and ligand-gated glutamate receptors with the goal of highlighting new relevant endpoints of the neuronal GSK-3 cascade that could provide a platform for a better understanding of the mechanisms underlying the dysfunction of this kinase in the CNS and serve as a guidance for medication development against the broad range of GSK-3-linked human diseases.

Neuroprotective effects of blockers for T-type calcium channels.

Cognitive and functional decline with age is correlated with deregulation of intracellular calcium, which can lead to neuronal death in the brain. Previous studies have found protective effects of various calcium channel blockers in pathological conditions. However, little has been done to explore possible protective effects of blockers for T-type calcium channels, which forms a family of FDA approved anti-epileptic drugs. In this study, we found that neurons showed an increase in viability after treatment with either L-type or T-type calcium channel antagonists. The family of low-voltage activated, or T-type calcium channels, comprise of three members (Cav3.1, Cav3.2, and Cav3.3) based on their respective main pore-forming alpha subunits: alpha1G, alpha1H, and alpha1I. Among these three subunits, alpha1H is highly expressed in hippocampus and certain cortical regions. However, T-type calcium channel blockers can protect neurons derived from alpha1H-/- mice, suggesting that neuroprotection demonstrated by these drugs is not through the alpha1H subunit. In addition, blockers for T-type calcium channels were not able to confer any protection to neurons in long-term cultures, while blockers of L-type calcium channels could protect neurons. These data indicate a new function of blockers for T-type calcium channels, and also suggest different mechanisms to regulate neuronal survival by calcium signaling pathways. Thus, our findings have important implications in the development of new treatment for age-related neurodegenerative disorders.