Cross species review of the physiological role of D-serine in translationally relevant behaviors.

Bridging the gap between preclinical models of neurological and psychiatric disorders with their human manifestations is necessary to understand their underlying mechanisms, identify biomarkers, and develop novel therapeutics. Cognitive and social impairments underlie multiple neuropsychiatric and neurological disorders and are often comorbid with sleep disturbances, which can exacerbate poor outcomes. Importantly, many symptoms are conserved between vertebrates and invertebrates, although they may have subtle differences. Therefore, it is essential to determine the molecular mechanisms underlying these behaviors across different species and their translatability to humans. Genome-wide association studies have indicated an association between glutamatergic gene variants and both the risk and frequency of psychiatric disorders such as schizophrenia, bipolar disorder, and autism spectrum disorder. For example, changes in glutamatergic neurotransmission, such as glutamate receptor subtype N-methyl-D-aspartate receptor (NMDAR) hypofunction, have been shown to contribute to the pathophysiology of schizophrenia. Furthermore, in neurological disorders, such as traumatic brain injury and Alzheimer's disease, hyperactivation of NMDARs leads to synaptic damage. In addition to glutamate binding, NMDARs require the binding of a co-agonist D-serine or glycine to the GluN1 subunit to open. D-serine, which is racemized from L-serine by the neuronal enzyme serine racemase (SRR), and both SRR and D-serine are enriched in cortico-limbic brain regions. D-serine is critical for complex behaviors, such as cognition and social behavior, where dysregulation of its synthesis and release has been implicated in many pathological conditions. In this review, we explore the role of D-serine in behaviors that are translationally relevant to multiple psychiatric and neurological disorders in different models across species.

Inhibition of glial D-serine release rescues synaptic damage after brain injury.

Synaptic damage is one of the most prevalent pathophysiological responses to traumatic CNS injury and underlies much of the associated cognitive dysfunction; however, it is poorly understood. The D-amino acid, D-serine, serves as the primary co-agonist at synaptic NMDA receptors (NDMARs) and is a critical mediator of NMDAR-dependent transmission and synaptic plasticity. In physiological conditions, D-serine is produced and released by neurons from the enzymatic conversion of L-serine by serine racemase (SRR). However, under inflammatory conditions, glial cells become a major source of D-serine. Here, we report that D-serine synthesized by reactive glia plays a critical role in synaptic damage after traumatic brain injury (TBI) and identify the therapeutic potential of inhibiting glial D-serine release though the transporter Slc1a4 (ASCT1). Furthermore, using cell-specific genetic strategies and pharmacology, we demonstrate that TBI-induced synaptic damage and memory impairment requires D-serine synthesis and release from both reactive astrocytes and microglia. Analysis of the murine cortex and acutely resected human TBI brain also show increased SRR and Slc1a4 levels. Together, these findings support a novel role for glial D-serine in acute pathological dysfunction following brain trauma, whereby these reactive cells provide the excess co-agonist levels necessary to initiate NMDAR-mediated synaptic damage.

Postsynaptic Serine Racemase Regulates NMDA Receptor Function.

d-serine is the primary NMDAR coagonist at mature forebrain synapses and is synthesized by the enzyme serine racemase (SR). However, our understanding of the mechanisms regulating the availability of synaptic d-serine remains limited. Though early studies suggested d-serine is synthesized and released from astrocytes, more recent studies have demonstrated a predominantly neuronal localization of SR. More specifically, recent work intriguingly suggests that SR may be found at the postsynaptic density, yet the functional implications of postsynaptic SR on synaptic transmission are not yet known. Here, we show an age-dependent dendritic and postsynaptic localization of SR and d-serine by immunohistochemistry and electron microscopy in mouse CA1 pyramidal neurons. In addition, using a single-neuron genetic approach in SR conditional KO mice from both sexes, we demonstrate a cell-autonomous role for SR in regulating synaptic NMDAR function at Schaffer collateral (CA3)-CA1 synapses. Importantly, single-neuron genetic deletion of SR resulted in the elimination of LTP at 1 month of age, which could be rescued by exogenous d-serine. Interestingly, there was a restoration of LTP by 2 months of age that was associated with an upregulation of synaptic GluN2B. Our findings support a cell-autonomous role for postsynaptic neuronal SR in regulating synaptic NMDAR function and suggests a possible autocrine mode of d-serine action.SIGNIFICANCE STATEMENT NMDARs are key regulators of neurodevelopment and synaptic plasticity and are unique in their requirement for binding of a coagonist, which is d-serine at most forebrain synapses. However, our understanding of the mechanisms regulating synaptic d-serine availability remains limited. d-serine is synthesized in the brain by the neuronal enzyme serine racemase (SR). Here, we show dendritic and postsynaptic localization of SR and d-serine in CA1 pyramidal neurons. In addition, using single-neuron genetic deletion of SR, we establish a role of postsynaptic SR in regulating NMDAR function. These results support an autocrine mode of d-serine action at synapses.

Identification of peptidomimetics as novel chemical probes modulating fibroblast growth factor 14 (FGF14) and voltage-gated sodium channel 1.6 (Nav1.6) protein-protein interactions.

The voltage-gated sodium (Nav) channel is the molecular determinant of action potential in neurons. Protein-protein interactions (PPI) between the intracellular Nav1.6 C-tail and its regulatory protein fibroblast growth factor 14 (FGF14) provide an ideal and largely untapped opportunity for development of neurochemical probes. Based on a previously identified peptide FLPK, mapped to the FGF14:FGF14 PPI interface, we have designed and synthesized a series of peptidomimetics with the intent of increasing clogP values and improving cell permeability relative to the parental lead peptide. In-cell screening using the split-luciferase complementation (LCA) assay identified ZL0177 (13) as the most potent inhibitor of the FGF14:Nav1.6 channel complex assembly with an apparent IC50 of 11 µM. Whole-cell patch-clamp recordings demonstrated that ZL0177 significantly reduced Nav1.6-mediated transient current density and induced a depolarizing shift of the channel voltage-dependence of activation. Docking studies revealed strong interactions between ZL0177 and Nav1.6, mediated by hydrogen bonds, cation-π interactions and hydrophobic contacts. All together these results suggest that ZL0177 retains some key features of FGF14-dependent modulation of Nav1.6 currents. Overall, ZL0177 provides a chemical scaffold for developing Nav channel modulators as pharmacological probes with therapeutic potential of interest for a broad range of CNS and PNS disorders.

Overexpression of heat shock factor 1 maintains TAR DNA binding protein 43 solubility via induction of inducible heat shock protein 70 in cultured cells.

TAR DNA binding protein 43 (TDP-43) is a nuclear protein that has been shown to have altered homeostasis in the form of neuronal nuclear and cytoplasmic aggregates in some familial and almost all cases of sporadic amyotrophic lateral sclerosis as well as 51% of frontotemporal lobar degeneration and 57% of Alzheimer's disease cases. Heat shock proteins (HSPs), such as HSP70, recognize misfolded or aggregated proteins and refold, disaggregate, or turn them over and are upregulated by the master transcription factor heat shock factor 1 (HSF1). Here, we explore the effect of HSF1 overexpression on proteotoxic stress-related alterations in TDP-43 solubility, proteolytic processing, and cytotoxicity. HSF1 overexpression reduced TDP-43-positive puncta concomitantly with upregulating HSP70 and HSP90 protein levels. HSF1 overexpression or pharmacological activation sustained TDP-43 solubility and significantly reduced truncation of TDP-43 in response to inhibition of the proteasome with Z-Leu-Leu-Leu-al, and this was reversed by HSF1 inhibition. HSF1 activation conferred protection against toxicity associated with TDP-43 C-terminal fragments without globally increasing the activity of the ubiquitin proteasome system (UPS) while concomitantly reducing the induction of autophagy, suggesting that HSF1 protection is an early event. In support of this, inhibition of HSP70 ATPase activity further reduced TDP-43 solubility. HSF1 knockout significantly increased TDP-43 insolubility and accelerated TDP-43 fragmentation in response to proteotoxic stress. Overall, this study shows that HSF1 overexpression protects against TDP-43 pathology by upregulation of chaperones, especially HSP70, rather than enhancing autophagy or the UPS during times of proteotoxic stress. © 2016 Wiley Periodicals, Inc.

GABA system as the cause and effect in early development.

GABA is the primary inhibitory neurotransmitter in the adult brain and through its actions on GABAARs, it protects against excitotoxicity and seizure activity, ensures temporal fidelity of neurotransmission, and regulates concerted rhythmic activity of neuronal populations. In the developing brain, the development of GABAergic neurons precedes that of glutamatergic neurons and the GABA system serves as a guide and framework for the development of other brain systems. Despite this early start, the maturation of the GABA system also continues well into the early postnatal period. In this review, we organize evidence around two scenarios based on the essential and protracted nature of GABA system development: 1) disruptions in the development of the GABA system can lead to large scale disruptions in other developmental processes (i.e., GABA as the cause), 2) protracted maturation of this system makes it vulnerable to the effects of developmental insults (i.e., GABA as the effect). While ample evidence supports the importance of GABA/GABAAR system in both scenarios, large gaps in existing knowledge prevent strong mechanistic conclusions.

Serine racemase deletion alters adolescent social behavior and whole-brain cFos activation.

Background: Neurodevelopmental disorders (NDDs) can cause debilitating impairments in social cognition and aberrant functional connectivity in large-scale brain networks, leading to social isolation and diminished everyday functioning. To facilitate the treatment of social impairments, animal models of NDDs that link N- methyl-D-aspartate receptor (NMDAR) hypofunction to social deficits in adulthood have been used. However, understanding the etiology of social impairments in NDDs requires investigating social changes during sensitive windows during development. Methods: We examine social behavior during adolescence using a translational mouse model of NMDAR hypofunction (SR-/-) caused by knocking out serine racemase (SR), the enzyme needed to make D-serine, a key NMDAR coagonist. Species-typical social interactions are maintained through brain-wide neural activation patterns; therefore, we employed whole-brain cFos activity mapping to examine network-level connectivity changes caused by SR deletion. Results: In adolescent SR-/- mice, we observed disinhibited social behavior toward a novel conspecific and rapid social habituation toward familiar social partners. SR-/- mice also spent more time in the open arm of the elevated plus maze which classically points to an anxiolytic behavioral phenotype. These behavioral findings point to a generalized reduction in anxiety-like behavior in both social and non-social contexts in SR-/- mice; importantly, these findings were not associated with diminished working memory. Inter-regional patterns of cFos activation revealed greater connectivity and network density in SR-/- mice compared to controls. Discussion: These results suggest that NMDAR hypofunction - a potential biomarker for NDDs - can lead to generalized behavioral disinhibition in adolescence, potentially arising from disrupted communication between and within salience and default mode networks.

Cognitive dysfunction following brain trauma results from sex-specific reactivation of the developmental pruning processes.

Cognitive losses resulting from severe brain trauma have long been associated with the focal region of tissue damage, leading to devastating functional impairment. For decades, researchers have focused on the sequelae of cellular alterations that exist within the perilesional tissues; however, few clinical trials have been successful. Here, we employed a mouse brain injury model that resulted in expansive synaptic damage to regions outside the focal injury. Our findings demonstrate that synaptic damage results from the prolonged increase in D-serine release from activated microglia and astrocytes, which leads to hyperactivation of perisynaptic NMDARs, tagging of damaged synapses by complement components, and the reactivation of developmental pruning processes. We show that this mechanistic pathway is reversible at several stages within a prolonged and progressive period of synaptic loss. Importantly, these key factors are present in acutely injured brain tissue acquired from patients with brain injury, which supports a therapeutic neuroprotective strategy.

The D-serine biosynthetic enzyme serine racemase is expressed by reactive astrocytes in the amygdala of human and a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized behaviorally by cognitive deterioration and emotional disruption, and neuropathologically by amyloid-ß (A ß) plaques, neurofibrillary tangles, and complement C3 (C3)-expressing neurotoxic, reactive astrocytes. We previously demonstrated that C3 + reactive astrocytes in the hippocampus and entorhinal cortex of AD patients express serine racemase (SR), which produces the N-methyl-D-aspartate receptor (NMDAR) co-agonist D-serine. We show here that C3 + reactive astrocytes express SR in the amygdala of AD patients and in an amyloid mouse model of familial AD (5xFAD). 5xFAD mice also have deficits in cue fear memory recall that is dependent on intact amygdala function. Our results suggest that D-serine produced by reactive astrocytes in the amygdala could contribute to glutamate excitotoxicity and neurodegeneration observed with AD progression.

D-serine availability modulates prefrontal cortex inhibitory interneuron development and circuit maturation.

The proper development and function of telencephalic GABAergic interneurons is critical for maintaining the excitation and inhibition (E/I) balance in cortical circuits. Glutamate contributes to cortical interneuron (CIN) development via N-methyl-D-aspartate receptors (NMDARs). NMDAR activation requires the binding of a co-agonist, either glycine or D-serine. D-serine (co-agonist at many mature forebrain synapses) is racemized by the neuronal enzyme serine racemase (SR) from L-serine. We utilized constitutive SR knockout (SR-/-) mice to investigate the effect of D-serine availability on the development of CINs and inhibitory synapses in the prelimbic cortex (PrL). We found that most immature Lhx6 + CINs expressed SR and the obligatory NMDAR subunit NR1. At embryonic day 15, SR-/- mice had an accumulation of GABA and increased mitotic proliferation in the ganglionic eminence and fewer Gad1 + (glutamic acid decarboxylase 67 kDa; GAD67) cells in the E18 neocortex. Lhx6 + cells develop into parvalbumin (PV+) and somatostatin (Sst+) CINs. In the PrL of postnatal day (PND) 16 SR-/- mice, there was a significant decrease in GAD67+ and PV+, but not SST + CIN density, which was associated with reduced inhibitory postsynaptic potentials in layer 2/3 pyramidal neurons. These results demonstrate that D-serine availability is essential for prenatal CIN development and postnatal cortical circuit maturation.

Forebrain expression of serine racemase during postnatal development.

N-methyl-D-aspartate receptors (NMDARs) are important for synaptogenesis, synaptic maturation and refinement during the early postnatal weeks after birth. Defective synapse formation or refinement underlie cognitive and emotional abnormalities in various neurodevelopmental disorders (NDDs), including schizophrenia (Sz) and autism spectrum disorder (ASD). Serine racemase (SR) is a neuronal enzyme that produces D-serine, a co-agonist required for full NMDAR activation. NMDAR hypofunction as a result of genetic SR elimination and reduced synaptic availability of D-serine reduces neuronal dendritic arborization and spine density. In adult mouse brain, the expression of SR parallels that of NMDARs across forebrain regions including the striatum, amygdala, hippocampus, and medial prefrontal cortex (mPFC). However, there have yet to be studies providing a detailed characterization of the spatial and temporal expression of SR during early periods of synaptogenesis. Here, we examined the postnatal expression of SR in cortical and subcortical brain regions important for learning, memory and emotional regulation, during the first four weeks after birth. Using dual-antigen immunofluorescence, we demonstrate that the number of SR+ neurons steadily increases with postnatal age across the mPFC, amygdala, hippocampus and striatum. We also identified differences in the rate of SR protein induction both across and within brain regions. Analyzing existing human post-mortem brain in situ data, there was a similar developmental mRNA expression profile of SRR and GRIN1 (GluN1 subunit) from infancy through the first decade of life. Our findings further support a developmental role for D-serine mediated NMDAR activation regulating synaptogenesis and neural circuit refinement, which has important implications for the pathophysiology of Sz and other NDDs.

Altered neural oscillations and behavior in a genetic mouse model of NMDA receptor hypofunction.

Abnormalities in electroencephalographic (EEG) biomarkers occur in patients with schizophrenia and those clinically at high risk for transition to psychosis and are associated with cognitive impairment. Converging evidence suggests N-methyl-D-aspartate receptor (NMDAR) hypofunction plays a central role in the pathophysiology of schizophrenia and likely contributes to biomarker impairments. Thus, characterizing these biomarkers is of significant interest for early diagnosis of schizophrenia and development of novel treatments. We utilized in vivo EEG recordings and behavioral analyses to perform a battery of electrophysiological biomarkers in an established model of chronic NMDAR hypofunction, serine racemase knockout (SRKO) mice, and their wild-type littermates. SRKO mice displayed impairments in investigation-elicited gamma power that corresponded with reduced short-term social recognition and enhanced background (pre-investigation) gamma activity. Additionally, SRKO mice exhibited sensory gating impairments in both evoked-gamma power and event-related potential amplitude. However, other biomarkers including the auditory steady-state response, sleep spindles, and state-specific power spectral density were generally neurotypical. In conclusion, SRKO mice demonstrate how chronic NMDAR hypofunction contributes to deficits in certain translationally-relevant EEG biomarkers altered in schizophrenia. Importantly, our gamma band findings suggest an aberrant signal-to-noise ratio impairing cognition that occurs with NMDAR hypofunction, potentially tied to impaired task-dependent alteration in functional connectivity.

Effects of Deltamethrin Acute Exposure on Nav1.6 Channels and Medium Spiny Neurons of the Nucleus Accumbens.

Exposure to pyrethroids, a popular insecticide class that targets voltage-gated Na+ (Nav) channels, has been correlated to an increase in diagnosis of neurodevelopmental disorders, such as attention deficit hyperactive disorder (ADHD), in children. Dysregulation of medium spiny neurons (MSNs) firing in the nucleus accumbens (NAc) is thought to play a critical role in the pathophysiology of ADHD and other neurodevelopmental disorders. The Nav1.6 channel is the primary molecular determinant of MSN firing and is sensitive to modification by pyrethroids. Building on previous studies demonstrating that deltamethrin (DM), a commonly used pyrethroid, leads to use-dependent enhancement of sodium currents, we characterized the effect of the toxin on long-term inactivation (LTI) of the Nav1.6 channel, a parameter known to affect neuronal firing, and characterized changes in MSN intrinsic excitability. We employed whole-cell patch-clamp electrophysiology to measure sodium currents in HEK-293 cells stably expressing Nav1.6 channels and intrinsic excitability of MSNs in the brain slice preparation. We found that in response to repetitive stimulation acute exposure to 10 µM DM potentiated a build-up of residual sodium currents and modified availability of Nav1.6 by inducing LTI. In the NAc, DM modified MSN intrinsic excitability increasing evoked action potential firing frequency and inducing aberrant action potentials with low amplitude and depolarized voltage threshold, phenotypes that could be explained by DM induced changes on the Nav1.6 channel. These results provide a potential initial mechanism of toxicity of DM that could lead to disruption of the NAc circuitry overtime, increasing the risk of ADHD and other neurodevelopmental disorders.

Neuroprotective Effects of HSF1 in Retinal Ischemia-Reperfusion Injury.

Purpose: Retinal ischemia, a common cause of several vision-threatening diseases, contributes to the death of retinal neurons, particularly retinal ganglion cells (RGCs). Heat shock transcription factor 1 (HSF1), a stress-responsive protein, has been shown to be important in response to cellular stress stimuli, including ischemia. This study is to investigate whether HSF1 has a role in retinal neuronal injury in a mouse model of retinal ischemia-reperfusion (IR). Methods: IR was induced by inserting an infusion needle into the anterior chamber of the right eye and elevating a saline reservoir connected to the needle to raise the intraocular pressure to 110 mm Hg for 45 minutes. HSF1, Hsp70, molecules in the endoplasmic reticulum (ER) stress branches, tau phosphorylation, inflammatory molecules, and RGC injury were determined by immunohistochemistry, Western blot, or quantitative PCR. Results: HSF1 expression was significantly increased in the retina 6 hours after IR. Using our novel transgenic mice carrying full-length human HSF gene, we demonstrated that IR-induced retinal neuronal apoptosis and necroptosis were abrogated 12 hours after IR. RGCs and their function were preserved in the HSF1 transgenic mice 7 days after IR. Mechanistically, the beneficial effects of HSF1 may be mediated by its induction of chaperone protein Hsp70 and alleviation of ER stress, leading to decreased tau phosphorylation and attenuated inflammatory response 12 to 24 hours after IR. Conclusions: These data provide compelling evidence that HSF1 is neuroprotective against retinal IR injury, and boosting HSF1 expression may be a beneficial strategy to limit neuronal degeneration in retinal diseases.

High-throughput screening against protein:protein interaction interfaces reveals anti-cancer therapeutics as potent modulators of the voltage-gated Na+ channel complex.

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.

Functional Modulation of Voltage-Gated Sodium Channels by a FGF14-Based Peptidomimetic.

Protein-protein interactions (PPI) offer unexploited opportunities for CNS drug discovery and neurochemical probe development. Here, we present ZL181, a novel peptidomimetic targeting the PPI interface of the voltage-gated Na+ channel Nav1.6 and its regulatory protein fibroblast growth factor 14 (FGF14). ZL181 binds to FGF14 and inhibits its interaction with the Nav1.6 channel C-tail. In HEK-Nav1.6 expressing cells, ZL181 acts synergistically with FGF14 to suppress Nav1.6 current density and to slow kinetics of fast inactivation, but antagonizes FGF14 modulation of steady-state inactivation that is regulated by the N-terminal tail of the protein. In medium spiny neurons in the nucleus accumbens, ZL181 suppresses excitability by a mechanism that is dependent upon expression of FGF14 and is consistent with a state-dependent inhibition of FGF14. Overall, ZL181 and derivatives could lay the ground for developing allosteric modulators of Nav channels that are of interest for a broad range of CNS disorders.

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.

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.

Heat shock factor 1 over-expression protects against exposure of hydrophobic residues on mutant SOD1 and early mortality in a mouse model of amyotrophic lateral sclerosis.

BACKGROUND: Mutations in the Cu/Zn superoxide dismutase gene (SOD1) are responsible for 20% of familial forms of amyotrophic lateral sclerosis (ALS), and mutant SOD1 has been shown to have increased surface hydrophobicity in vitro. Mutant SOD1 may adopt a complex array of conformations with varying toxicity in vivo. We have used a novel fluorescence-based proteomic assay using 4,4'-bis-1-anilinonaphthalene-8-sulfonate (bisANS) to assess the surface hydrophobicity, and thereby distinguish between different conformations, of SOD1 and other proteins in situ. RESULTS: Covalent bisANS labeling of spinal cord extracts revealed that alterations in surface hydrophobicity of H46R/H48Q mutations in SOD1 provoke formation of high molecular weight SOD1 species with lowered solubility, likely due to increased exposure of hydrophobic surfaces. BisANS was docked on the H46R/H48Q SOD1 structure at the disordered copper binding and electrostatic loops of mutant SOD1, but not non-mutant WT SOD1. 16 non-SOD1 proteins were also identified that exhibited altered surface hydrophobicity in the H46R/H48Q mutant mouse model of ALS, including proteins involved in energy metabolism, cytoskeleton, signaling, and protein quality control. Heat shock proteins (HSPs) were also enriched in the detergent-insoluble fractions with SOD1. Given that chaperones recognize proteins with exposed hydrophobic surfaces as substrates and the importance of protein homeostasis in ALS, we crossed SOD1 H46R/H48Q mutant mice with mice over-expressing the heat shock factor 1 (HSF1) transcription factor. Here we showed that HSF1 over-expression in H46R/H48Q ALS mice enhanced proteostasis as evidenced by increased expression of HSPs in motor neurons and astrocytes and increased solubility of mutant SOD1. HSF1 over-expression significantly reduced body weight loss, delayed ALS disease onset, decreases cases of early disease, and increased survival for the 25th percentile in an H46R/H48Q SOD1 background. HSF1 overexpression did not affect macroautophagy in the ALS background, but was associated with maintenance of carboxyl terminus of Hsp70 interacting protein (CHIP) expression which declined in H46R/H48Q mice. CONCLUSION: Our results uncover the potential importance of changes in protein surface hydrophobicity of SOD1 and other non-SOD1 proteins in ALS, and how strategies that activate HSF1 are valid therapies for ALS and other age-associated proteinopathies.