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We used microarray analysis to identify renal cell transcripts that were upregulated with low magnesium. One transcript, identified as NIPA2 (nonimprinted in Prader-Willi/Angelman syndrome) subtype 2, was increased over twofold relative to cells cultured in normal magnesium. The deduced sequence comprises 129 amino acids with 8 predicted transmembrane regions. As the secondary structure of NIPA2 conformed to a membrane transport protein, we expressed it in Xenopus oocytes and determined that it mediated [Mg.sup.2+] uptake with two-electrode voltage-clamp and fluorescence studies. [Mg.sup.2+] transport was electrogenic, voltage dependent, and saturable, demonstrating a Michaelis affinity constant of 0.31 mM. Unlike other reported [Mg.sup.2+] transporters, NIPA2 was very selective for the [Mg.sup.2+] cation. NIPA2 mRNA is found in many tissues but particularly abundant in renal cells. With the use of immunofluorescence, it was shown that NIPA2 protein was normally localized to the early endosomes and plasma membrane and was recruited to the plasma membrane in response to low extracellular magnesium. We conclude that NIPA2 plays a role in magnesium metabolism and regulation of renal magnesium conservation. nonimprinted in Prader-Willi/Angelman syndrome

Despite long-standing concerns regarding the abuse liability of benzodiazepines, the mechanisms underlying properties of benzodiazepines that may be relevant to abuse are still poorly understood. Earlier studies showed that compounds selective for α1-containing GABAA receptors (α1GABAARs) are abused by humans and self-administered by animals, and that these receptors may underlie a preference for benzodiazepines as well as neuroplastic changes observed in the ventral tegmental area following benzodiazepine administration. There is some evidence, however, that even L-838, 417, a compound with antagonistic properties at α1GABAARs and agonistic properties at the other three benzodiazepine-sensitive GABAA receptor subtypes, is self-administered, and that the α2GABAARs may have a role in benzodiazepine-induced reward enhancement. Using a two-bottle choice drinking paradigm to evaluate midazolam preference and an intracranial self-stimulation (ICSS) paradigm to evaluate the impact of midazolam on reward enhancement, we demonstrated that mice carrying a histidine-to-arginine point mutation in the α2 subunit which renders it insensitive to benzodiazepines (α2(H101R) mice) did not prefer midazolam and did not show midazolam-induced reward enhancement in ICSS, in contrast to wild-type controls, suggesting that α2GABAARs are necessary for the reward enhancing effects and preference for oral benzodiazepines. Through a viral-mediated knockdown of α2GABAARs in the nucleus accumbens (NAc), we demonstrated that α2 in the NAc is necessary for the preference for midazolam. Findings imply that α2GABAARs in the NAc are involved in at least some reward-related properties of benzodiazepines, which might partially underlie repeated drug-taking behavior. [ABSTRACT FROM AUTHOR]

In schizophrenia, cognitive dysfunction is highly predictive of poor patient outcomes and is not responsive to current medications. Postmortem studies have suggested that cognitive deficits in schizophrenia are correlated with modifications in the number and size of inhibitory synapses. To test if these modifications lead to cognitive deficits, we have created a dominant-negative virus [adeno-associated (AAV)-DN1] that disrupts the clustering of γ-aminobutyric acid type A receptors (GABAARs) at postsynaptic inhibitory specializations. When injected into the frontal cortex of mice, AAV-DN1 impairs GABAAR α2 subunit and GABA transporter 1 (GAT-1) clustering, but increases GABAAR a1 subunit clustering on the perisomatic region, with no influence on axon-initial segment clustering. Mice expressing AAV-DN1 have prepulse inhibition deficits and impairments in working memory. Significantly, these behavioral deficits are paralleled by a reduction in electroencephalography γ-power. Collectively, our study provides functional evidence revealing that GABAergic synapses in the prefrontal cortex directly contribute to cognition and γ-power. [ABSTRACT FROM AUTHOR]

Brain-derived neurotrophic factor (BDNF) is a potent regulator of neuronal activity, neurogenesis, and depressive-like behaviors; however, downstream effectors by which BDNF exerts these varying actions remain to be determined. Here we reveal that BDNF induces long-lasting enhancements in the efficacy of synaptic inhibition by stabilizingγ2 subunit-containingGABAA receptors (GABAARs) at the cell surface, leading to persistent reductions in neuronal excitability. This effect is dependent upon enhanced phosphorylation of tyrosines 365 and 367 (Y365/7) in the GABAAR γ2 subunit as revealed using mice in which these residues have been mutated to phenyalanines (Y365/7F). Heterozygotes for this mutation exhibit an antidepressant-like phenotype, as shown using behavioral-despair models of depression. In addition, heterozygous Y365/7F mice show increased levels of hippocampal neurogenesis, which has been strongly connected with antidepressant action. Both the antidepressant phenotype and the increased neurogenesis seen in these mice are insensitive to further modulation by BDNF, which produces robust antidepressant-like activity and neurogenesis in wild-type mice. Collectively, our results suggest a critical role for GABAAR γ2 subunit Y365/7 phosphorylation and function in regulating the effects of BDNF. [ABSTRACT FROM AUTHOR]

Intravenous anesthetics exert a component of their actions via potentiating inhibitory neurotransmission mediated by y-aminobutyric type-A receptors (GABAARS). Phasic and tonic inhibition is mediated by distinct populations of GABAARS, with the majority of phasic inhibition by subtypes composed of a 1 -3βγ2 subunits, whereas tonic inhibition is dependent on subtypes assembled from α4 - 6βƍ subunits. To explore the contribution that these distinct forms of inhibition play in mediating intravenous anesthesia, we have used mice in which tyrosine residues 365/7 within the γ2 subunit are mutated to phenyalanines (Y365/7F). Here we demonstrate that this mutation leads to increased accumulation of the α4 subunit containing GABAARS in the thalamus and dentate gyrus of female Y365/7F but not male Y365/7F mice. Y365/7F mice exhibited a gender-specific enhancement of tonic inhibition in the dentate gyrus that was more sensitive to modulation by the anesthetic etomidate, together with a deficit in long-term potentiation. Consistent with this, female Y365/7F, but not male Y365/7F, mice exhibited a dramatic increase in the duration of etomidate- and propofol-mediated hypnosis. Moreover, the amnestic actions of etomidate were selectively potentiated in female Y365/7F mice. Collectively, these observations suggest that potentiation of tonic inhibition mediated by a4 subunit containing GABAARS contributes to the hypnotic and amnestic actions of the intravenous anesthetics, etomidate and propofol. [ABSTRACT FROM AUTHOR]

The Golgi-specific zinc finger protein GODZ (palmitoyl acyl- transferase/DHHC-3) mediates the palmitoylation and post- translational modification of many protein substrates that regulate membrane-protein interactions. Here, we show that GODZ also mediates Ca2+ transport in expressing Xenopus laevis oocytes. Two-electrode voltage-clamp, fluorescence, and 45Ca2+ isotopic uptake determinations demonstrated voltage- and concentration-dependent, saturable, and substrate-inhibitable Ca2+ transport in oocytes expressing GODZ cRNA but not in oocytes injected with water alone. Moreover, we show that GODZ-mediated Ca2+ transport is regulated by palmitoylation, as the palmitoyl acyltransferase inhibitor 2-bromopalmitate or alteration of the acyltransferase DHHC motif (GODZ- DHHS) diminished GODZ-mediated Ca2+ transport by ∼80%. The GODZ mutation V61R abolished Ca2+ transport but did not affect palmitoyl acyltransferase activity. Coexpression of GODZ-V61R with GODZ-DHHS restored GODZ-DHHS-mediated Ca2+ uptake to values observed with wild-type GODZ, excluding an endogenous effect of palmitoylation. Coexpression of an independent palmitoyl acyltransferase (HIP14) with the GODZ-DHHS mutant also rescued Ca2+ transport. HIP14 did not mediate Ca2+ transport when expressed alone. Immunocytochemistry studies showed that GODZ and HIP14 co-localized to the Golgi and the same post-Golgi vesicles, suggesting that heteropalmitoylation might play a physiological role in addition to a biochemical function. We conclude that GODZ encodes a Ca2+ transport protein in addition to its ability to palmitoylate protein substrates. [ABSTRACT FROM AUTHOR]

Trans-synaptic cell-adhesion molecules have been implicated in regulating CNS synaptogenesis. Among these, the Neuroligin (NL) family (NLs 1-4) of postsynaptic adhesion proteins has been shown to promote the development and specification of excitatory versus inhibitory synapses. NLs form a heterophilic complex with the presynaptic transmembrane protein Neurexin (NRX). A differential association of NLs with postsynaptic scaffolding proteins and NRX isoforms has been suggested to regulate the ratio of excitatory to inhibitory synapses ( E/I ratio). Using transgenic mice, we have tested this hypothesis by overexpressing NL1 in vivo to determine whether the relative levels of these cell adhesion molecules may influence synapse maturation, long-term potentiation (LTP), and/or learning. We found that NL1-overexpressing mice show significant deficits in memory acquisition, but not in memory retrieval. Golgi and electron microscopy analysis revealed changes in synapse morphology indicative of increased maturation of excitatory synapses. In parallel, electrophysiological examination indicated a shift in the synaptic activity toward increased excitation as well as impairment in LTP induction. Our results demonstrate that altered balance in the expression of molecules necessary for synapse specification and development (such as NL1) can lead to defects in memory formation and synaptic plasticity and outline the importance of rigidly controlled synaptic maturation processes. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Polyglutamine expansions of huntingtin protein are responsible for the Huntington neurological disorder. HIP14 protein has been shown to interact with huntingtin. HIP14 and a HIP14- like protein, HIP14L, with a 69% similarity reside in the Golgi and possess palmitoyl acyltransferase activity through innate cysteine-rich domains, DHHC. Here, we used microarray analysis to show that reduced extracellular magnesium concentration increases HIP14L mRNA suggesting a role in cellular magnesium metabolism. Because HIP14 was not on the microarray platform, we used real-time reverse transcriptase-PCR to show that HIP14 and HIP14L transcripts were up-regulated 3-fold with low magnesium. Western analysis with a specific HIP14 antibody also showed that endogenous HIP14 protein increased with diminished magnesium. Furthermore, we demonstrate that when expressed in Xenopus oocytes, HIP14 and HIP14L mediate Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with Michaelis constants of 0.87 ± 0.02 and 0.74 ± 0.07 mM, respectively. Diminished magnesium leads to an apparent increase in HIP14-green fluorescent protein and HIP14L-green fluorescent fusion proteins in the Golgi complex and subplasma membrane post-Golgi vesicles of transfected epithelial cells. We also show that inhibition of palmitoylation with 2-bromopalmitate, or deletion of the DHHC motif HIP14 Δ DHHC,diminishes HIP14-mediatedMg2+ transport by about 50%. Coexpression of an independent protein acyltransferase, GODZ, with the deleted HIP14 Δ DHHCmutant restored Mg2+ transport to values observed with wild-type HIP14. Although we did not directly measure palmitoylation of HIP14 in these studies, the data are consistent with a regulatory role of autopalmitoylation in HIP14-mediated Mg2+ transport. We conclude that the huntingtin interacting protein genes, HIP14 and HIP14L, encode Mg2+ transport proteins that are regulated by their innate palmitoyl acyltransferases thus fulfilling the characteristics of "chanzymes." [ABSTRACT FROM AUTHOR]

The level of excitation in the brain is kept under control through inhibitory signals mainly exerted by GABA neurons. However, the molecular machinery that regulates the balance between excitation and inhibition (E/I) remains unclear. Candidate molecules implicated in this process are neuroligin (NL) adhesion molecules, which are differentially enriched at either excitatory or inhibitory contacts. In this study, we use transgenic mouse models expressing NL1 or NL2 to examine whether enhanced expression of specific NLs results in synaptic imbalance and altered neuronal excitability and animal behavior. Our analysis reveals several abnormalities selectively manifested in transgenic mice with enhanced expression of NL2 but not NL1. A small change in NL2 expression results in enlarged synaptic contact size and vesicle reserve pool in frontal cortex synapses and an overall reduction in the E/I ratio. The frequency of miniature inhibitory synaptic currents was also found to be increased in the frontal cortex of transgenic NL2 mice. These animals also manifested stereotyped jumping behavior, anxiety, impaired social interactions, and enhanced incidence of spike-wave discharges, as depicted by EEG analysis in freely moving animals. These findings may provide the neural basis for E/I imbalance and altered behavior associated with neurodevelopmental disorders. [ABSTRACT FROM AUTHOR]

Mutations in the NIPA1(SPG6) gene, named for ‘nonim-printed in Prader-Willi/Angelman’ has been implicated in one form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and weakness. However, the function of NIPA1 is unknown. Here, we show that reduced magnesium concentration enhances expression of NIPA1 suggesting a role in cellular magnesium metabolism. Indeed NIPA1 mediates Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with a Michaelis constant of 0.69 ± 0.21 mM when expressed in Xenopus oocytes. Subcellular localization with immunofluorescence showed that endogenous NIPA1 protein associates with early endosomes and the cell surface in a variety of neuronal and epithelial cells. As expected of a magnesium-responsive gene, we find that altered magnesium concentration leads to a redistribution between the endosomal compartment and the plasma membrane; high magnesium results in diminished cell surface NIPA1 whereas low magnesium leads to accumulation in early endosomes and recruitment to the plasma membrane. The mouse NIPA1 mutants, T39R and G100R, corresponding to the respective human mutants showed a loss-of-function when expressed in oocytes and altered trafficking in transfected COS7 cells. We conclude that NIPA1 normally encodes a Mg2+ transporter and the loss-of function of NIPA1(SPG6) due to abnormal trafficking of the mutated protein provides the basis of the HSP phenotype. [ABSTRACT FROM AUTHOR]

The modulation of neuronal cell firing is mediated by the release of the neurotransmitter GABA (γ-aminobuytric acid), which binds to two major families of receptors. The ionotropic GABAA receptors (GABAARs) are composed of five distinct subunits that vary in expression by brain region and cell type. The action of GABA on GABAARs is modulated by a variety of clinically and pharmacologically important drugs such as benzodiazepines and alcohol. Exposure to and abuse of these substances disrupts homeostasis and induces plasticity in GABAergic neurotransmission, often via the regulation of receptor expression. Here, we review the regulation of GABAAR subunit expression in adaptive and pathological plasticity, with a focus on substance use. We examine the factors influencing the expression of GABAAR subunit genes including the regulation of the 5′ and 3′ untranslated regions, variations in DNA methylation, immediate early genes and transcription factors that regulate subunit expression, translational and post-translational modifications, and other forms of receptor regulation beyond expression. Advancing our understanding of the factors regulating GABAAR subunit expression during adaptive plasticity, as well as during substance use and withdrawal will provide insight into the role of GABAergic signaling in substance use disorders, and contribute to the development of novel targeted therapies. [ABSTRACT FROM AUTHOR]

Fast inhibitory synaptic transmission is mediated by γ-aminobutyric acid type A receptors (GABAARs) that are enriched at functionally diverse synapses via mechanisms that remain unclear. Using isothermal titration calorimetry and complementary methods we demonstrate an exclusive low micromolar binding of collybistin to the α2-subunit of GABAARs. To explore the biological relevance of collybistin-α2-subunit selectivity, we generate mice with a mutation in the α2-subunit-collybistin binding region (Gabra2-1). The mutation results in loss of a distinct subset of inhibitory synapses and decreased amplitude of inhibitory synaptic currents. Gabra2-1 mice have a striking phenotype characterized by increased susceptibility to seizures and early mortality. Surviving Gabra2-1 mice show anxiety and elevations in electroencephalogram δ power, which are ameliorated by treatment with the α2/α3-selective positive modulator, AZD7325. Taken together, our results demonstrate an α2-subunit selective binding of collybistin, which plays a key role in patterned brain activity, particularly during development. The inhibitory synaptic protein collybistin (CB) and GABAAR-α subunits are thought to interact, but strength and specificity are unclear. Here the authors study the CB-α2 interaction and show that a mouse mutated in the CB-binding region of α2 displays a loss of specific synapses and seizure. [ABSTRACT FROM AUTHOR]

The fidelity of inhibitory neurotransmission is dependent on the accumulation of γ-aminobutyric acid type A receptors (GABA A Rs) at the appropriate synaptic sites. Synaptic GABA A Rs are constructed from α(1-3), β(1-3), and γ2 subunits, and neurons can target these subtypes to specific synapses. Here, we identify a 15-amino acid inhibitory synapse targeting motif (ISTM) within the α2 subunit that promotes the association between GABA A Rs and the inhibitory scaffold proteins collybistin and gephyrin. Using mice in which the ISTM has been introduced into the α1 subunit (Gabra1-2 mice), we show that the ISTM is critical for axo-axonic synapse formation, the efficacy of GABAergic neurotransmission, and seizure sensitivity. The Gabra1-2 mutation rescues seizure-induced lethality in Gabra2-1 mice, which lack axo-axonic synapses due to the deletion of the ISTM from the α2 subunit. Taken together, our data demonstrate that the ISTM plays a critical role in promoting inhibitory synapse formation, both in the axonic and somatodendritic compartments., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

In schizophrenia, cognitive dysfunction is highly predictive of poor patient outcomes and is not responsive to current medications. Postmortem studies have suggested that cognitive deficits in schizophrenia are correlated with modifications in the number and size of inhibitory synapses. To test if these modifications lead to cognitive deficits, we have created a dominant-negative virus [adeno-associated (AAV)-DN1] that disrupts the clustering of γ-aminobutyric acid type A receptors (GABA(A)Rs) at postsynaptic inhibitory specializations. When injected into the frontal cortex of mice, AAV-DN1 impairs GABA(A)R α2 subunit and GABA transporter 1 (GAT-1) clustering, but increases GABA(A)R α1 subunit clustering on the perisomatic region, with no influence on axon-initial segment clustering. Mice expressing AAV-DN1 have prepulse inhibition deficits and impairments in working memory. Significantly, these behavioral deficits are paralleled by a reduction in electroencephalography γ-power. Collectively, our study provides functional evidence revealing that GABAergic synapses in the prefrontal cortex directly contribute to cognition and γ-power.