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Fast synaptic inhibition is dependent on targeting specific GABA

GABA type A receptors (GABAARs) mediate fast synaptic inhibition and are trafficked to functionally diverse synapses. However, the precise molecular mechanisms that regulate the synaptic targeting of these receptors are unclear. Whereas it has been previously shown that phosphorylation events in a4, b,and g subunits of GABAARs govern their function and trafficking, phosphorylation of other subunits has not yet been demonstrated. Here, we show that the a2 subunit of GABAARs is phosphorylated at Ser-359 and enables dynamic regulation of GABAAR binding to the scaffolding proteins gephyrin and collybistin. We initially identified Ser-359 phosphorylation by MS analysis, and additional experiments revealed that it is regulated by the activities of cAMP-dependent protein kinase (PKA) and the protein phosphatase 1 (PP1) and/or PP2A. GST-based pulldowns and coimmunoprecipitation experiments demonstrate preferential binding of both gephyrin and collybistin to WT and an S359A phosphonull variant, but not to an S359D phosphomimetic variant. Furthermore, the decreased capacity of the a2 S359D variant to bind collybistin and gephyrin decreased the density of synaptic a2-containing GABAAR clusters and caused an absence of a2 enrichment in the axon initial segment. These results suggest that PKA-mediated phosphorylation and PP1/PP2A-dependent dephosphorylation of the a2 subunit play a role in the dynamic regulation of GABAAR accumulation at inhibitory synapses, thereby regulating the strength of synaptic inhibition. The MS data have been deposited to ProteomeXchange, with the data set identifier PXD019597.

Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the brain's reward circuit, GABAergic neurons control the excitability of dopamine (DA) projection neurons and are the site of initial psychostimulant-dependent changes in signaling. Previous work established that cocaine/methamphetamine exposure increases protein phosphatase 2A (PP2A) activity, which dephosphorylates the GABABR2 subunit, promotes internalization of the GABABreceptor (GABABR) and leads to smaller GABABR-activated G-protein-gated inwardly rectifying potassium (GIRK) currents in VTA GABA neurons. How the actions of PP2A become selective for a particular signaling pathway is poorly understood. Here, we demonstrate that PP2A can associate directly with a short peptide sequence in the C terminal domain of the GABABR1 subunit, and that GABABRs and PP2A are in close proximity in rodent neurons (mouse/rat; mixed sexes). We show that this PP2A-GABABR interaction can be regulated by intracellular Ca2+. Finally, a peptide that potentially reduces recruitment of PP2A to GABABRs and thereby limits receptor dephosphorylation increases the magnitude of baclofen-induced GIRK currents. Thus, limiting PP2A-dependent dephosphorylation of GABABRs may be a useful strategy to increase receptor signaling for treating diseases.SIGNIFICANCE STATEMENTDysregulation of GABABreceptors (GABABRs) underlies altered neurotransmission in many neurological disorders. Protein phosphatase 2A (PP2A) is involved in dephosphorylating and subsequent internalization of GABABRs in models of addiction and depression. Here, we provide new evidence that PP2A B55 regulatory subunit interacts directly with a small region of the C-terminal domain of the GABABR1 subunit, and that this interaction is sensitive to intracellular Ca2+. We demonstrate that a short peptide corresponding to the PP2A interaction site on GABABR1 competes for PP2A binding, enhances phosphorylation GABABR2 S783, and affects functional signaling through GIRK channels. Our study highlights how targeting PP2A dependent dephosphorylation of GABABRs may provide a specific strategy to modulate GABABR signaling in disease conditions.

Pharmaceutical breakthroughs for anxiety have been lackluster in the last half-century. Converging behavior and limbic molecular heterogeneity has the potential to revolutionize biomarker-driven interventions. However, current in vivo models too often deploy artificial systems including directed evolution, mutations and fear induction, which poorly mirror clinical manifestations. Here, we explore transcriptional heterogeneity of the amygdala in isogenic mice using an unbiased multi-dimensional computational approach that segregates intra-cohort reactions to moderate situational adversity and intersects it with high content molecular profiling. We show that while the computational approach stratifies known features of clinical anxiety including nitric oxide, opioid and corticotropin signaling, previously unrecognized druggable biomarkers emerge, such as calpain11 and scand1. Through ingenuity pathway analyses, we further describe a role for neurosteroid estradiol signaling, heat shock proteins, ubiquitin ligases and lipid metabolism. In addition, we report a remarkable behavioral pattern that maps to molecular features of anxiety in mice through counterphobic social attitudes, which manifest as increased, yet spatially distant socialization. These findings provide an unbiased approach for interrogating anxiolytics, and hint toward biomarkers underpinning behavioral and social patterns that merit further exploration.

Significance Phosphodiesterase 10A (PDE10A) is as a target of interest in Huntington’s disease (HD) as levels of the enzyme have been shown to decrease prior to the development of the hallmark motor symptoms. Clearly, a better understanding of how PDE10A protein levels change as HD develops is required. Here we show that mutations in the regulatory GAF domains of PDE10A that cause hyperkinetic syndromes in humans lead to misprocessing of the PDE10A enzyme that ultimately leads to targeted degradation by the ubiquitin proteasome system or clearance by autophagy. Both mechanisms result in a paucity of PDE10A activity that lead to a loss of movement coordination. Our research suggests that similar mechanisms may underpin PDE10A loss during HD., A robust body of evidence supports the concept that phosphodiesterase 10A (PDE10A) activity in the basal ganglia orchestrates the control of coordinated movement in human subjects. Although human mutations in the PDE10A gene manifest in hyperkinetic movement disorders that phenocopy many features of early Huntington’s disease, characterization of the maladapted molecular mechanisms and aberrant signaling processes that underpin these conditions remains scarce. Recessive mutations in the GAF-A domain have been shown to impair PDE10A function due to the loss of striatal PDE10A protein levels, but here we show that this paucity is caused by irregular intracellular trafficking and increased PDE10A degradation in the cytosolic compartment. In contrast to GAF-A mutants, dominant mutations in the GAF-B domain of PDE10A induce PDE10A misfolding, a common pathological phenotype in many neurodegenerative diseases. These data demonstrate that the function of striatal PDE10A is compromised in disorders where disease-associated mutations trigger a reduction in the fidelity of PDE compartmentalization.

We previously showed that apremilast, an FDA-approved PDE4 inhibitor, selectively alters behavioral responses to ethanol and certain GABAergic drugs in a PKA-dependent manner in C57BL6/J mice. Here, we investigated if PKA phosphorylation of β3 GABAsubA/subreceptor subunits is involved in apremilast regulation of ethanol, propofol, or diazepam responses. Apremilast prolonged rotarod ataxia and loss of the righting reflex by ethanol and propofol in wild-type mice, but not in β3-S408A/S409A knock-in mice. In contrast, apremilast hastened recovery from the ataxic and sedative effects of diazepam in both genotypes. These findings suggest that apremilast modulation of ethanol and propofol behaviors in wild-type mice is mediated by β3 subunit phosphorylation, whereas its actions on diazepam responses involve a different mechanism. The PKA inhibitor H-89 prevented apremilast modulation of ethanol-induced ataxia. Apremilast sensitized wild-type males to ethanol-induced ataxia and decreased acute functional tolerance (AFT) in females but had no effect in β3-S408A/S409A mice of either sex. These results could not be attributed to genotype differences in blood ethanol clearance. There were also no baseline genotype differences in ethanol consumption and preference in two different voluntary drinking procedures. However, the ability of apremilast to reduce ethanol consumption was diminished in β3-S408A/S409A mice. Our results provide strong evidence that PKA-dependent phosphorylation of β3 GABAsubA/subreceptor subunits is an important mechanism by which apremilast increases acute sensitivity to alcohol, decreases AFT, and decreases ethanol drinking.

Neuroactive steroids (NASs) have potent anxiolytic, anticonvulsant, sedative, and hypnotic actions, that reflect in part their efficacy as GABA

First-in-line benzodiazepine treatment fails to terminate seizures in about 30% of epilepsy patients, highlighting a need for novel antiseizure strategies. Impaired GABAergic inhibition is key to the development of such benzodiazepine-resistant seizures, as well as the pathophysiology of status epilepticus (SE). It is emerging that reduced or impaired neuronal K+/Cl- cotransporter 2 (KCC2) activity contributes to deficits in γ-aminobutyric acid (GABA)-mediated inhibition and increased seizure vulnerability. The with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich (SPAK) kinase signaling pathway inhibits neuronal KCC2 via KCC2-T1007 phosphorylation. A selective WNK kinase inhibitor, WNK463, was recently synthesized by Novartis. Exploiting WNK463, we test the hypothesis that pharmacological WNK inhibition will enhance KCC2 activity, increase the efficacy of GABAergic inhibition, and thereby limit seizure activity in animal models. Immunoprecipitation and Western blot analysis were used to examine WNK463's effects on KCC2-T1007 phosphorylation, in vitro and in vivo. A thallium (Tl+) uptake assay was used in human embryonic kidney (HEK-293) cells expressing KCC2 to test WNK463's effects on KCC2-mediated Tl+ transport. Gramicidin-perforated- and whole-cell patch-clamp recordings in cortical rat neurons were used to examine WNK463's effects on KCC2-mediated Cl- transport. In mouse brain slices (entorhinal cortex), field recordings were utilized to examine WNK463's effects on 4-aminopyridine-induced seizure activity. Last, WNK463 was directly deliver to the mouse hippocampus in vivo and tested in a kainic acid model of diazepam-resistant SE. WNK463 significantly reduces KCC2-T1007 phosphorylation in vitro and in vivo (mice). In human embryonic kidney 293 (HEK-293) cells expressing KCC2, WNK463 greatly enhanced the rates Tl+ transport. However, the drug did not enhance Tl+ transport in cells expressing a KCC2-phospho null T1007 mutant. In cultured rat neurons, WNK463 rapidly reduced intracellular Cl- and consequently hyperpolarized the Cl- reversal potential (EGABA). In mature neurons that were artificially loaded with 30 mM Cl-, WNK463 significantly enhanced KCC2-mediated Cl- export and hyperpolarized EGABA. In a 4-aminopyridine model of acute seizures, WNK463 reduced the frequency and number of seizure-like events (SLEs). Finally, in an in vivo kainic acid (KA) model of diazepam-resistant SE, WNK463 slowed the onset and reduced the severity of KA-induced status epilepticus. Last, WNK463 prevented the development of pharmaco-resistance to diazepam in drug-treated mice. Our findings demonstrate that acute WNK463 treatment potentiates KCC2 activity in neurons and limits seizure burden in two well-established models of seizures and epilepsy. Our work suggests that agents which act to increase KCC2 activity may be useful adjunct therapeutics to alleviate diazepam-resistant SE.

Intellectual disability (ID) is a common neurodevelopmental disorder that can arise from genetic mutations ranging from trisomy to single nucleotide polymorphism. Mutations in a growing number of single genes have been identified as causative in ID, including ARHGEF9. Evaluation of 41 ARHGEF9 patient reports shows ubiquitous inclusion of ID, along with other frequently reported symptoms of epilepsy, abnormal baseline EEG activity, behavioral symptoms, and sleep disturbances. ARHGEF9 codes for the Cdc42 Guanine Nucleotide Exchange Factor 9 collybistin (Cb), a known regulator of inhibitory synapse function via direct interaction with the adhesion molecule neuroligin-2 and the α2 subunit of GABA

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder typically characterized by insoluble inclusions of hyperphosphorylated TDP-43. The mechanisms underlying toxic TDP-43 accumulation are not understood. Persistent activation of p38 mitogen-activated protein kinase (MAPK) is implicated in ALS. However, it is unclear how p38 MAPK affects TDP-43 proteinopathy. Here, we demonstrate that inhibition of p38α MAPK reduces pathological TDP-43 phosphorylation, aggregation, cytoplasmic mislocalization, and neurotoxicity. We establish that p38α MAPK phosphorylates TDP-43 at pathological serine 409/410 (S409/S410) and serine 292 (S292), which reduces TDP-43 liquid-liquid phase separation (LLPS) but allows pathological TDP-43 aggregation. Moreover, we show that protein arginine methyltransferase 1 methylates TDP-43 at R293. Importantly, S292 phosphorylation reduces R293 methylation, and R293 methylation reduces S409/S410 phosphorylation. R293 methylation permits TDP-43 LLPS and reduces pathological TDP-43 aggregation. Thus, strategies to reduce p38α-mediated TDP-43 phosphorylation and promote R293 methylation could have therapeutic utility for ALS and related TDP-43 proteinopathies.

Neuroactive steroids (NASs) are synthesized within the brain and exert profound effects on behavior. These effects are primarily believed to arise from the activities of NASs as positive allosteric modulators (PAMs) of the GABA-type A receptor (GABA(A)R). NASs also activate a family of G protein–coupled receptors known as membrane progesterone receptors (mPRs). Here, using surface-biotinylation assays and electrophysiology techniques, we examined mPRs' role in mediating the effects of NAS on the efficacy of GABAergic inhibition. Selective mPR activation enhanced phosphorylation of Ser-408 and Ser-409 (Ser-408/9) within the GABA(A)R β3 subunit, which depended on the activity of cAMP-dependent protein kinase A (PKA) and protein kinase C (PKC). mPR activation did not directly modify GABA(A)R activity and had no acute effects on phasic or tonic inhibition. Instead, mPR activation induced a sustained elevation in tonic current, which was blocked by PKA and PKC inhibition. Substitution of Ser-408/9 to alanine residues also prevented the effects of mPR activation on tonic current. Furthermore, this substitution abolished the effects of sustained NAS exposure on tonic inhibition. Interestingly, the allosteric effects of NAS on GABAergic inhibition were independent of Ser-408/9 in the β3 subunit. Additionally, although allosteric effects of NAS on GABAergic inhibition were sensitive to a recently developed “NAS antagonist,” the sustained effects of NAS on tonic inhibition were not. We conclude that metabotropic effects of NAS on GABAergic inhibition are mediated by mPR-dependent modulation of GABA(A)R phosphorylation. We propose that this mechanism may contribute to the varying behavioral effects of NAS.

Developing cortical GABAergic interneurons rely on genetic programs, neuronal activity, and environmental cues to construct inhibitory circuits during early postnatal development. Disruption of these events can cause long-term changes in cortical inhibition and may be involved in neurological disorders associated with inhibitory circuit dysfunction. We hypothesized that tonic glutamate signaling in the neonatal cortex contributes to, and is necessary for, the maturation of cortical interneurons. To test this hypothesis, we used mice of both sexes to quantify extracellular glutamate concentrations in the cortex during development, measure ambient glutamate-mediated activation of developing cortical interneurons, and manipulate tonic glutamate signaling using subtype-specific NMDA receptor antagonistsin vitroandin vivo. We report that ambient glutamate levels are high (≈100 nm) in the neonatal cortex and decrease (to ≈50 nm) during the first weeks of life, coincident with increases in astrocytic glutamate uptake. Consistent with elevated ambient glutamate, putative parvalbumin-positive interneurons in the cortex (identified usingG42:GAD1-eGFPreporter mice) exhibit a transient, tonic NMDA current at the end of the first postnatal week. GluN2C/GluN2D-containing NMDA receptors mediate the majority of this current and contribute to the resting membrane potential and intrinsic properties of developing putative parvalbumin interneurons. Pharmacological blockade of GluN2C/GluN2D-containing NMDA receptorsin vivoduring the period of tonic interneuron activation, but not later, leads to lasting decreases in interneuron morphological complexity and causes deficits in cortical inhibition later in life. These results demonstrate that dynamic ambient glutamate signaling contributes to cortical interneuron maturation via tonic activation of GluN2C/GluN2D-containing NMDA receptors.SIGNIFICANCE STATEMENTInhibitory GABAergic interneurons make up 20% of cortical neurons and are critical to controlling cortical network activity. Dysfunction of cortical inhibition is associated with multiple neurological disorders, including epilepsy. Establishing inhibitory cortical networks requiresin uteroproliferation, differentiation, and migration of immature GABAergic interneurons, and subsequent postnatal morphological maturation and circuit integration. Here, we demonstrate that ambient glutamate provides tonic activation of immature, putative parvalbumin-positive GABAergic interneurons in the neonatal cortex via high-affinity NMDA receptors. When this activation is blocked, GABAergic interneuron maturation is disrupted, and cortical networks exhibit lasting abnormal hyperexcitability. We conclude that temporally precise activation of developing cortical interneurons by ambient glutamate is critically important for establishing normal cortical inhibition.

Hyperammonemia is known to cause various neurological dysfunctions such as seizures and cognitive impairment. Several studies have suggested that hyperammonemia may also be linked to the development of Alzheimer's disease (AD). However, the direct evidence for a role of ammonia in the pathophysiology of AD remains to be discovered. Herein, we report that hyperammonemia increases the amount of mature amyloid precursor protein (mAPP) in astrocytes, the largest and most prevalent type of glial cells in the central nervous system that are capable of metabolizing glutamate and ammonia, and promotes amyloid beta (Aβ) production. We demonstrate the accumulation of mAPP in astrocytes was primarily due to enhanced endocytosis of mAPP from the plasma membrane. A large proportion of internalized mAPP was targeted not to the lysosome, but to the endoplasmic reticulum, where processing enzymes β-secretase BACE1 (beta-site APP cleaving enzyme 1) and γ-secretase presenilin-1 are expressed, and mAPP is cleaved to produce Aβ. Finally, we show the ammonia-induced production of Aβ in astrocytic endoplasmic reticulum was specific to Aβ42, a principal component of senile plaques in AD patients. Our studies uncover a novel mechanism of Aβ42 production in astrocytes and also provide the first evidence that ammonia induces the pathogenesis of AD by regulating astrocyte function.

Intellectual disability (ID) is a common neurodevelopmental disorder that can arise from genetic mutations ranging from trisomy to single nucleotide polymorphism. Mutations in a growing number of single genes have been identified as causative in ID, including ARHGEF9. Evaluation of 41 ARHGEF9 patient reports shows ubiquitous inclusion of ID, along with other frequently reported symptoms of epilepsy, abnormal baseline EEG activity, behavioral symptoms, and sleep disturbances. ARHGEF9 codes for the Cdc42 Guanine Nucleotide Exchange Factor 9 collybistin (Cb), a known regulator of inhibitory synapse function via direct interaction with the adhesion molecule neuroligin-2 and the α2 subunit of GABAA receptors. We mutate the Cb binding motif within the large intracellular loop of α2 replacing it with the binding motif for gephyrin from the α1 subunit (Gabra2-1). The Gabra2-1 mutation causes a strong downregulation of Cb expression, particularly at cholecystokinin basket cell inhibitory synapses. Gabra2-1 mice have deficits in working and recognition memory, as well as hyperactivity, anxiety, and reduced social preference, recapitulating the frequently reported features of ARHGEF9 patients. Gabra2-1 mice also have spontaneous seizures during postnatal development which can lead to mortality, and baseline abnormalities in low-frequency wavelengths of the EEG. EEG abnormalities are vigilance state-specific and manifest as sleep disturbance including increased time in wake and a loss of free-running rhythmicity in the absence of light as zeitgeber. Gabra2-1 mice phenocopy multiple features of human ARHGEF9 mutation, and reveal α2 subunit-containing GABAA receptors as a druggable target for treatment of this complex ID syndrome.

The K+/Cl– co-transporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl−levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors GABAARs. In accordance with this, compromised KCC2 activity results in seizures but whether such deficits directly contribute to the subsequent changes in neuronal viability that lead to epileptogenesis, remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression resulted in the rapid activation of the extrinsic apoptotic pathway, in mature hippocampal neurons. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization or dendritic structure. However ablating KCC2 expression in immature neurons was sufficient to prevent the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to loss of KCC2 function. In contrast KCC2 appears to play a minimal role in mediating neuronal development or architecture.

KCC2 plays a critical role in determining the efficacy of synaptic inhibition and deficits in its activity lead to epilepsy and neurodevelopmental delay. Here we use unbiased proteomic analyses to demonstrate that KCC2 forms stable protein complexes in the neuronal plasma membrane with 96 autism and/or epilepsy risk gene (ASD/Epi) products including ANKB, ANKG, CNTN1, ITPR1, NCKAP1, SCN2A, SHANK3, SPTAN1, and SPTBN1. Many of these proteins are also targets of Fragile-X mental retardation protein (FMRP), the inactivation of which is the leading monogenic cause of autism. Accordingly, the expression of a subset of these KCC2-binding partners was decreased in Fmr1 knockout mice. Fmr1 knockout compromised KCC2 phosphorylation, a key regulatory mechanism for transporter activity and the postnatal development of GABAergic inhibition. Thus, KCC2 is a point of convergence for multiple ASD/Epi risk genes and therapies targeting this transporter may have broad utility in alleviating these heterogeneous disorders and their associated epilepsies.

Loss of glutamatergic synapses is thought to be a key cellular pathology associated with neuropsychiatric disorders including schizophrenia (SCZ) and major depressive disorder (MDD). Genetic and cellular studies of SCZ and MDD using in vivo and in vitro systems have supported a key role for dysfunction of excitatory synapses in the pathophysiology of these disorders. Recent clinical studies have demonstrated that the estrogen, 17β-estradiol can ameliorate many of the symptoms experienced by patients. Yet, to date, our understanding of how 17β-estradiol exerted these beneficial effects is limited. In this study, we have tested the hypothesis that 17β-estradiol can restore dendritic spine number in a cellular model that recapitulates the loss of synapses associated with SCZ and MDD. Ectopic expression of wildtype, mutant or shRNA-mediated knockdown of Disrupted in Schizophrenia (DISC1) reduced dendritic spine density in primary cortical neurons. Acute or chronic treatment with 17β-estradiol increased spine density to control levels in neurons with altered DISC1 levels. In addition, 17β-estradiol reduced the extent to which ectopic wildtype and mutant DISC1 aggregated. Furthermore, 17β-estradiol also caused the enrichment of synaptic proteins at synapses and increased the number of dendritic spines containing PSD-95 or that overlapped with the pre-synaptic marker bassoon. Taken together, our data indicates that estrogens can restore lost excitatory synapses caused by altered DISC1 expression, potentially through the trafficking of DISC1 and its interacting partners. These data highlight the possibility that estrogens exert their beneficial effects in SCZ and MDD in part by modulating dendritic spine number.

Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABAA receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro, zuranolone enhanced GABAA receptor current at nine unique human recombinant receptor subtypes, including representative receptors for both synaptic (γ subunit-containing) and extrasynaptic (δ subunit-containing) configurations. At a representative synaptic subunit configuration, α1β2γ2, zuranolone potentiated GABA currents synergistically with the benzodiazepine diazepam, consistent with the non-competitive activity and distinct binding sites of the two classes of compounds at synaptic receptors. In a brain slice preparation, zuranolone produced a sustained increase in GABA currents consistent with metabotropic trafficking of GABAA receptors to the cell surface. In vivo, zuranolone exhibited potent activity, indicating its ability to modulate GABAA receptors in the central nervous system after oral dosing by protecting against chemo-convulsant seizures in a mouse model and enhancing electroencephalogram β-frequency power in rats. Together, these data establish zuranolone as a potent and efficacious neuroactive steroid GABAA receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABAA receptor positive modulators for treating mood disorders; brexanolone is the first therapeutic approved specifically for the treatment of postpartum depression. Zuranolone is currently under clinical investigation for the treatment of major depressive episodes in major depressive disorder, postpartum depression, and bipolar depression.

Mesial temporal lobe epilepsy (mTLE) is the most common form of epilepsy, believed to arise in part from compromised GABAergic inhibition. The neuronal specific K+/Cl− co-transporter 2 (KCC2) is a critical determinant of the efficacy of GABAergic inhibition and deficits in its activity are observed in mTLE patients and animal models of epilepsy. To test if reductions of KCC2 activity directly contribute to the pathophysiology of mTLE, we locally ablated KCC2 expression in a subset of principal neurons within the adult hippocampus. Deletion of KCC2 resulted in compromised GABAergic inhibition and the development of spontaneous, recurrent generalized seizures. Moreover, local ablation of KCC2 activity resulted in hippocampal sclerosis, a key pathological change seen in mTLE. Collectively, our results demonstrate that local deficits in KCC2 activity within the hippocampus are sufficient to precipitate mTLE. Keywords: KCC2, GABA, Epilepsy, Hippocampal sclerosis

The stress-induced activation of the hypothalamic-pituitary-adrenal (HPA) axis is normally suppressed during pregnancy. Dysregulation of the HPA axis has been proposed to play a role in postpartum depression. However, direct investigation into the relationship between the HPA axis and postpartum depression has been hindered by the lack of useful animal models. Building on our discovery of a role for the K+/Cl− co-transporter, KCC2, in the GABAergic regulation of CRH neurons in the paraventricular nucleus of the hypothalamus (PVN), critical for mounting the body’s physiological response to stress, we assessed the role of KCC2 in the regulation of the HPA axis during pregnancy and the postpartum period. Here we demonstrate that the normal suppression of the stress-induced activation of the HPA axis during the peripartum period involves maintenance of KCC2 in the PVN. Mice lacking KCC2 specifically in corticosterone-releasing hormone (CRH) neurons, which govern the activity of the HPA axis (KCC2/Crh mice), exhibit dysregulation of the HPA axis and abnormal postpartum behaviors. Loss of KCC2 specifically in CRH neurons in the PVN is sufficient to reproduce the depression-like phenotype and deficits in maternal behaviors during the postpartum period. Similarly, chemogenetic activation of CRH neurons in the PVN is sufficient to induce abnormal postpartum behaviors and chemogenetic silencing of CRH neurons in the PVN can ameliorate abnormal postpartum behaviors observed in KCC2/Crh mice. This study demonstrates that dysregulation of the HPA axis is sufficient to induce abnormal postpartum behaviors and deficits in maternal behaviors in mice, providing empirical support for a role of HPA axis dysfunction in the pathophysiology of postpartum depression

apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.

K+/Cl− cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl− levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABAA) receptors, which are Cl−-permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.

KCC2 is a neuron specific K+-Cl− co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into “head” and “core” domains connected by a flexible “linker”. Dimers are asymmetrical and display a bent “S-shape” architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2.

Estrogen plays a critical role in many physiological processes and exerts profound effects on behavior by regulating neuronal excitability. While estrogen has been established to exert effects on dendritic morphology and excitatory neurotransmission its role in regulating neuronal inhibition is poorly understood. Fast synaptic inhibition in the adult brain is mediated by specialized populations of γ-c aA receptors (GABAARs) that are selectively enriched at synapses, a process dependent upon their interaction with the inhibitory scaffold protein gephyrin. Here we have assessed the role that estradiol (E2) plays in regulating the dynamics of GABAARs and stability of inhibitory synapses. Treatment of cultured cortical neurons with E2 reduced the accumulation of GABAARs and gephyrin at inhibitory synapses. However, E2 exposure did not modify the expression of either the total or the plasma membrane GABAARs or gephyrin. Mechanistically, single-particle tracking revealed that E2 treatment selectively reduced the dwell time and thereby decreased the confinement of GABAARs at inhibitory synapses. Consistent with our cell biology measurements, we observed a significant reduction in amplitude of inhibitory synaptic currents in both cultured neurons and hippocampal slices exposed to E2, while their frequency was unaffected. Collectively, our results suggest that acute exposure of neurons to E2 leads to destabilization of GABAARs and gephyrin at inhibitory synapses, leading to reductions in the efficacy of GABAergic inhibition via a postsynaptic mechanism.

Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the brain's reward circuit, GABAergic neurons control the excitability of dopamine (DA) projection neurons and are the site of initial psychostimulant-dependent changes in signaling. Previous work established that cocaine/methamphetamine exposure increases protein phosphatase 2A (PP2A) activity, which dephosphorylates the GABA(B)R2 subunit, promotes internalization of the GABA(B) receptor (GABA(B)R) and leads to smaller GABA(B)R-activated G-protein-gated inwardly rectifying potassium (GIRK) currents in VTA GABA neurons. How the actions of PP2A become selective for a particular signaling pathway is poorly understood. Here, we demonstrate that PP2A can associate directly with a short peptide sequence in the C terminal domain of the GABA(B)R1 subunit, and that GABA(B)Rs and PP2A are in close proximity in rodent neurons (mouse/rat; mixed sexes). We show that this PP2A-GABA(B)R interaction can be regulated by intracellular Ca(2+). Finally, a peptide that potentially reduces recruitment of PP2A to GABA(B)Rs and thereby limits receptor dephosphorylation increases the magnitude of baclofen-induced GIRK currents. Thus, limiting PP2A-dependent dephosphorylation of GABA(B)Rs may be a useful strategy to increase receptor signaling for treating diseases. SIGNIFICANCE STATEMENT Dysregulation of GABA(B) receptors (GABA(B)Rs) underlies altered neurotransmission in many neurological disorders. Protein phosphatase 2A (PP2A) is involved in dephosphorylating and subsequent internalization of GABA(B)Rs in models of addiction and depression. Here, we provide new evidence that PP2A B55 regulatory subunit interacts directly with a small region of the C-terminal domain of the GABA(B)R1 subunit, and that this interaction is sensitive to intracellular Ca(2+). We demonstrate that a short peptide corresponding to the PP2A interaction site on GABA(B)R1 competes for PP2A binding, enhances phosphorylation GABA(B)R2 S783, and affects functional signaling through GIRK channels. Our study highlights how targeting PP2A dependent dephosphorylation of GABA(B)Rs may provide a specific strategy to modulate GABA(B)R signaling in disease conditions.

USP14 is a cysteine protease deubiquitinase associated with the proteasome and plays important catalytic and allosteric roles in proteasomal degradation. USP14 inhibition has been considered a therapeutic strategy for accelerating degradation of aggregation-prone proteins in neurodegenerative diseases and for inhibiting proteasome function to induce apoptotic cell death in cancers. Here we studied the effects of USP14 inhibition in mammalian cells using small molecule inhibitors and an inactive USP14 mutant C114A. Neither the inhibitors nor USP14 C114A showed consistent or significant effects on the level of TDP-43, tau or α-synuclein in HEK293T cells. However, USP14 C114A led to a robust accumulation of ubiquitinated proteins, which were isolated by ubiquitin immunoprecipitation and identified by mass spectrometry. Among these proteins we confirmed that ubiquitinated β-catenin accumulated in the cells expressing USP14 C114A with immunoblotting and immunoprecipitation experiments. The proteasome binding domain of USP14 C114A is required for its effect on ubiquitinated proteins. UCHL5 is the other cysteine protease deubiquitinase associated with the proteasome. Interestingly, the inactive mutant of UCHL5 C88A also caused an accumulation of ubiquitinated proteins in HEK293T cells but did not affect β-catenin, demonstrating USP14 but not UCHL5 has a specific effect on β-catenin. We used ubiquitin immunoprecipitation and mass spectrometry to identify the accumulated ubiquitinated proteins in UCHL5 C88A expressing cells which are mostly distinct from those identified in USP14 C114A expressing cells. Among the identified proteins are well established proteasome substrates and proteasome subunits. Besides β-catenin, we also verified with immunoblotting that UCHL5 C88A inhibits its own deubiquitination and USP14 C114A inhibits deubiquitination of two proteasomal subunits PSMC1 and PSMD4. Together our data suggest that USP14 and UCHL5 can deubiquitinate distinct substrates at the proteasome and regulate the ubiquitination of the proteasome itself which is tightly linked to its function.

The axon initial segment (AIS) is the site of action potential (AP) initiation in most neurons and is thus a critical site in the regulation of neuronal excitability. Normal function within the discrete AIS compartment requires intricate molecular machinery to ensure the proper concentration and organization of voltage-gated and ligand-gated ion channels; in humans, dysfunction at the AIS due to channel mutations is commonly associated with epileptic disorders. In this review, we will examine the molecular mechanisms underlying the formation of the only synapses found at the AIS: synapses containing γ-aminobutyric type A receptors (GABAARs). GABAARs are heteropentamers assembled from 19 possible subunits and are the primary mediators of fast synaptic inhibition in the brain. Although the total GABAAR population is incredibly heterogeneous, only one specific GABAAR subtype-the α2-containing receptor-is enriched at the AIS. These AIS synapses are innervated by GABAergic chandelier cells, and this inhibitory signaling is thought to contribute to the tight control of AP firing. Here, we will summarize the progress made in understanding the regulation of GABAAR synapse formation, concentrating on post-translational modifications of subunits and on interactions with intracellular proteins. We will then discuss subtype-specific synapse formation, with a focus on synapses found at the AIS, and how these synapses influence neuronal excitation.

The function and pharmacology of γ-aminobutyric acid type A receptors (GABA(A)Rs) are of great physiological and clinical importance and have long been thought to be determined by the channel pore–forming subunits. We discovered that Shisa7, a single-passing transmembrane protein, localizes at GABAergic inhibitory synapses and interacts with GABA(A)Rs. Shisa7 controls receptor abundance at synapses and speeds up the channel deactivation kinetics. Shisa7 also potently enhances the action of diazepam, a classic benzodiazepine, on GABA(A)Rs. Genetic deletion of Shisa7 selectively impairs GABAergic transmission and diminishes the effects of diazepam in mice. Our data indicate that Shisa7 regulates GABA(A)R trafficking, function, and pharmacology and reveal a previously unknown molecular interaction that modulates benzodiazepine action in the brain.

The TRAF2 and NCK interacting kinase (TNIK) has been proposed to play a role in cytoskeletal organization and synaptic plasticity and has been linked, among others, to neurological disorders. However, target validation efforts for TNIK have been hampered by the limited kinase selectivity of small molecule probes and possible functional compensation in mouse models. Both issues are at least in part due to its close homology to the kinases MINK1 (or MAP4K6) and MAP4K4 (or HGK). As part of our interest in validating TNIK as a therapeutic target for neurological diseases, we set up a panel of biochemical and cellular assays, which are described herein. We then examined the activity of known amino-pyridine-based TNIK inhibitors (1, 3) and prepared structurally very close analogs that lack the ability to inhibit the target. We also developed a structurally orthogonal, naphthyridine-based TNIK inhibitor (9) and an inactive control molecule of the same chemical series. These validated small-molecule probes will enable dissection of the function of TNIK family in the context of human disease biology.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability. A reduction in neuronal inhibition mediated by γ-aminobutyric acid type A receptors (GABAARs) has been implicated in the pathophysiology of FXS. Neuroactive steroids (NASs) are known allosteric modulators of GABAAR channel function, but recent studies from our laboratory have revealed that NASs also exert persistent metabotropic effects on the efficacy of tonic inhibition by increasing the protein kinase C (PKC)-mediated phosphorylation of the α4 and β3 subunits which increase the membrane expression and boosts tonic inhibition. We have assessed the GABAergic signaling in the hippocampus of fragile X mental retardation protein (FMRP) knock-out (Fmr1 KO) mouse. The GABAergic tonic current in dentate gyrus granule cells (DGGCs) from 3- to 5-week-old (p21–35) Fmr1 KO mice was significantly reduced compared to WT mice. Additionally, spontaneous inhibitory post synaptic inhibitory current (sIPSC) amplitudes were increased in DGGCs from Fmr1 KO mice. While sIPSCs decay in both genotypes was prolonged by the prototypic benzodiazepine diazepam, those in Frm1-KO mice were selectively potentiated by RO15-4513. Consistent with this altered pharmacology, modifications in the expression levels and phosphorylation of receptor GABAAR subtypes that mediate tonic inhibition were seen in Fmr1 KO mice. Significantly, exposure to NASs induced a sustained elevation in tonic current in Fmr1 KO mice which was prevented with PKC inhibition. Likewise, exposure reduced elevated membrane excitability seen in the mutant mice. Collectively, our results suggest that NAS act to reverse the deficits of tonic inhibition seen in FXS, and thereby reduce aberrant neuronal hyperexcitability seen in this disorder.

The K+/Cl− cotransporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl− levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABAARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture, we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization, or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture.

Zuranolone (SAGE-217) is a novel, synthetic, clinical stage neuroactive steroid GABAA receptor positive allosteric modulator designed with the pharmacokinetic properties to support oral daily dosing. In vitro, zuranolone enhanced GABAA receptor current at nine unique human recombinant receptor subtypes, including representative receptors for both synaptic (γ subunit-containing) and extrasynaptic (δ subunit-containing) configurations. At a representative synaptic subunit configuration, α1β2γ2, zuranolone potentiated GABA currents synergistically with the benzodiazepine diazepam, consistent with the non-competitive activity and distinct binding sites of the two classes of compounds at synaptic receptors. In a brain slice preparation, zuranolone produced a sustained increase in GABA currents consistent with metabotropic trafficking of GABAA receptors to the cell surface. In vivo, zuranolone exhibited potent activity, indicating its ability to modulate GABAA receptors in the central nervous system after oral dosing by protecting against chemo-convulsant seizures in a mouse model and enhancing electroencephalogram β-frequency power in rats. Together, these data establish zuranolone as a potent and efficacious neuroactive steroid GABAA receptor positive allosteric modulator with drug-like properties and CNS exposure in preclinical models. Recent clinical data support the therapeutic promise of neuroactive steroid GABAA receptor positive modulators for treating mood disorders; brexanolone is the first therapeutic approved specifically for the treatment of postpartum depression. Zuranolone is currently under clinical investigation for the treatment of major depressive episodes in major depressive disorder, postpartum depression, and bipolar depression.