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Pluripotent stem cells are classified as naïve and primed cells, based on their in vitro growth characteristics and potential to differentiate into various types of cells. Human-induced pluripotent stem cells (iPSCs, also known as epiblast stem cells [EpiSCs]) have limited capacity to differentiate and are slightly more differentiated than naïve stem cells (NSCs). Although there are several in vitro protocols that allow iPSCs to differentiate into pancreatic lineage, data concerning generation of β-cells from these iPSCs are limited. Based on the pluripotentiality of NSCs, it was hypothesized that NSCs can differentiate into pancreatic β-cells when placed under an appropriate differentiation induction condition. We examined whether NSCs can be efficiently induced to form potentially pancreatic β cells after being subjected to an in vitro protocol. Several colonies resembling in vitro-produced β-cell foci, with β-cell-specific marker expression, were observed when NSC-derived embryoid bodies (EBs) were induced to differentiate into β-cell lineage. Conversely, EpiSC-derived EBs failed to form such foci in vitro. Intrapancreatic grafting of the in vitro-formed β-cell foci into nude mice (BALB/c-nu/nu) generated a cell mass containing insulin-producing cells (IPCs), without noticeable tumorigenesis. These NSCs can be used as a promising resource for curing type 1 diabetes.

DOI： 10.3390/jcm9092838

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A major component of gastric acid is hydrochloric acid (HCl), which can activate transient receptor potential vanilloid 1 (TRPV1). In the present study, we investigated how sustained laryngeal TRPV1 activation affects the frequency of the swallowing reflex. Experiments were carried out on 85 male Sprague-Dawley rats. The effects of short and sustained application of chemicals (3 µl of 0.1 N HCl or capsaicin) on the frequency of swallowing and on time-dependent changes in the occurrence of swallowing evoked by supralaryngeal nerve stimulation were determined. To evaluate vascular permeability of the larynx, Evans blue dye was intravenously injected after 5 or 60 min of sustained TRPV1 activation. SB366791 (a TRPV1 inhibitor) and Cap/QX-314 (a TRPV1-expressed neuronal inhibitor) significantly inhibited HCl/capsaicin-induced swallowing, but air flow-induced swallowing was not affected. Although the number of air flow-induced swallows followed by capsaicin stimulation was not affected within 5 min, it was significantly reduced by 60-min capsaicin or HCl application. The swallowing threshold associated with supralaryngeal nerve stimulation did not significantly change throughout the recording period. Evans blue dye concentrations in the larynx were significantly higher at 60 min in the 10-5 M capsaicin group than in the control group. Our results suggest that sustained TPRV1 activation not only desensitizes TRPV1 but also inactivates mechanoreceptors, which may be attributed to increases in vascular permeability and edema, as part of an inflammatory process.NEW & NOTEWORTHY Although a transient receptor potential vanilloid 1 (TRPV1) inhibitor or TRPV1-expressed neuronal inhibitor significantly inhibited HCl/capsaicin-evoked swallowing, air flow-induced swallowing was not affected. The number of air flow-induced swallows was significantly reduced within 60 min of TRPV1 activation. Evans blue dye concentration in the larynx increased 60 min after capsaicin application. TPRV1 activation not only desensitizes TRPV1 but also inactivates mechanoreceptors caused by increases in vascular permeability and edema.

DOI： 10.1152/ajpgi.00082.2020

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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 GABAB receptor (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 STATEMENT Dysregulation of GABAB receptors (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.

DOI： 10.1523/JNEUROSCI.2654-19.2020

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Human tissue-specific stem cells (hTSCs), found throughout the body, can differentiate into several lineages under appropriate conditions in vitro and in vivo. By transfecting terminally differentiated cells with reprogramming factors, we previously produced induced TSCs from the pancreas and hepatocytes that exhibit additional properties than iPSCs, as exemplified by very low tumour formation after xenogenic transplantation. We hypothesised that hTSCs, being partially reprogrammed in a state just prior to iPSC transition, could be isolated from any terminally differentiated cell type through transient reprogramming factor overexpression. Cytochemical staining of human deciduous tooth-derived dental pulp cells (HDDPCs) and human skin-derived fibroblasts following transfection with Yamanaka's factors demonstrated increased ALP activity, a stem cell marker, three weeks after transfection albeit in a small percentage of clones. Repeated transfections (≤3) led to more efficient iPSC generation, with HDDPCs exhibiting greater multipotentiality at two weeks post-transfection than the parental intact HDDPCs. These results indicated the utility of iPSC technology to isolate TSCs from HDDPCs and fibroblasts. Generally, a step-wise loss of pluripotential phenotypes in ESCs/iPSCs occurs during their differentiation process. Our present findings suggest that the reverse phenomenon can also occur upon repeated introduction of reprogramming factors into differentiated cells such as HDDPCs and fibroblasts.

DOI： 10.1038/s41598-018-37291-2

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Alcohol is associated with nearly half of all violent crimes committed in the United States; yet, a potential neural basis for this type of pathological aggression remains elusive. Alcohol may act on N-methyl-d-aspartate receptors (NMDARs) within cortical circuits to impede processing and to promote aggression. Here, male mice were characterized as alcohol-heightened (AHAs) or alcohol non-heightened aggressors (ANAs) during resident-intruder confrontations after self-administering 1.0 g/kg alcohol (6% w/v) or water. Alcohol produced a pathological-like pattern of aggression in AHAs; these mice shifted their bites to more vulnerable locations on the body of a submissive animal, including the anterior back and ventrum after consuming alcohol. In addition, through immunoblotting, we found that AHAs overexpressed the NMDAR GluN2D subunit in the prefrontal cortex (PFC) as compared to ANAs while the two phenotypes expressed similar levels of GluN1, GluN2A and GluN2B. After identifying several behavioral and molecular characteristics that distinguish AHAs from ANAs, we tested additional mice for their aggression following preferential antagonism of GluN2D-containing NMDARs. In these experiments, groups of AHAs and ANAs self-administered 1.0 g/kg alcohol (6% w/v) or water before receiving intraperitoneal (i.p.) doses of ketamine or memantine, or infusions of memantine directly into the prelimbic (PLmPFC) or infralimbic medial PFC (ILmPFC). Moderate doses of IP ketamine, IP memantine, or intra-PLmPFC memantine increased aggression in AHAs, but only in the absence of alcohol. Prior alcohol intake blocked the pro-aggressive effects of ketamine or memantine. In contrast, only memantine, administered systemically or intra-PLmPFC, interacted with prior alcohol intake to escalate aggression in ANAs. Intra-ILmPFC memantine had no effect on aggression in either AHAs or ANAs. In sum, this work illustrates a potential role of GluN2D-containing NMDARs in the PLmPFC in alcohol-heightened aggression. GluN2D-containing NMDARs are highly expressed on cortical parvalbumin-containing interneurons, suggesting that, in a subset of individuals, alcohol may functionally alter signal integration within cortical microcircuits to dysregulate threat reactivity and promote aggression. This work suggests that targeting GluN2D-NMDARs may be of use in reducing the impact of alcohol-related violence in the human population.

DOI： 10.1038/npp.2017.253

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

γ-aminobutyric acid type B (GABAB) receptors are broadly expressed in the nervous system and play an important role in neuronal excitability. GABAB receptors are G protein-coupled receptors that mediate slow and prolonged inhibitory action, via activation of Gαi/o-type proteins. GABAB receptors mediate their inhibitory action through activating inwardly rectifying K+ channels, inactivating voltage-gated Ca2+ channels, and inhibiting adenylate cyclase. Functional GABAB receptors are obligate heterodimers formed by the co-assembly of R1 and R2 subunits. It is well established that GABAB receptors interact not only with G proteins and effectors but also with various proteins. This review summarizes the structure, subunit isoforms, and function of GABAB receptors, and discusses the complexity of GABAB receptors, including how receptors are localized in specific subcellular compartments, the mechanism regulating cell surface expression and mobility of the receptors, and the diversity of receptor signaling through receptor crosstalk and interacting proteins.

DOI： 10.2183/pjab.94.026

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UNLABELLED: Repeated exposure to psychostimulants induces locomotor sensitization and leads to persistent changes in the circuitry of the mesocorticolimbic dopamine (DA) system. G-protein-gated inwardly rectifying potassium (GIRK; also known as Kir3) channels mediate a slow IPSC and control the excitability of DA neurons. Repeated 5 d exposure to psychostimulants decreases the size of the GABAB receptor (GABABR)-activated GIRK currents (IBaclofen) in ventral tegmental area (VTA) DA neurons of mice, but the mechanism underlying this plasticity is poorly understood. Here, we show that methamphetamine-dependent attenuation of GABABR-GIRK currents in VTA DA neurons required activation of both D1R-like and D2R-like receptors. The methamphetamine-dependent decrease in GABABR-GIRK currents in VTA DA neurons did not depend on a mechanism of dephosphorylation of the GABAB R2 subunit found previously for other neurons in the reward pathway. Rather, the presence of the GIRK3 subunit appeared critical for the methamphetamine-dependent decrease of GABABR-GIRK current in VTA DA neurons. Together, these results highlight different regulatory mechanisms in the learning-evoked changes that occur in the VTA with repeated exposure to psychostimulants. SIGNIFICANCE STATEMENT: Exposure to addictive drugs such as psychostimulants produces persistent adaptations in inhibitory circuits within the mesolimbic dopamine system, suggesting that addictive behaviors are encoded by changes in the reward neural circuitry. One form of neuroadaptation that occurs with repeated exposure to psychostimulants is a decrease in slow inhibition, mediated by a GABAB receptor and a potassium channel. Here, we examine the subcellular mechanism that links psychostimulant exposure with changes in slow inhibition and reveal that one type of potassium channel subunit is important for mediating the effect of repeated psychostimulant exposure. Dissecting out the components of drug-dependent plasticity and uncovering novel protein targets in the reward circuit may lead to the development of new therapeutics for treating addiction.

DOI： 10.1523/JNEUROSCI.1327-15.2016

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Alterations in the efficacy of neuronal inhibition mediated by GABAA receptors (GABAARs) containing β3 subunits are continually implicated in autism spectrum disorders (ASDs). In vitro, the plasma membrane stability of GABAARs is potentiated via phosphorylation of serine residues 408 and 409 (S408/9) in the β3 subunit, an effect that is mimicked by their mutation to alanines. To assess if modifications in β3 subunit expression contribute to ASDs, we have created a mouse in which S408/9 have been mutated to alanines (S408/9A). S408/9A homozygotes exhibited increased phasic, but decreased tonic, inhibition, events that correlated with alterations in the membrane stability and synaptic accumulation of the receptor subtypes that mediate these distinct forms of inhibition. S408/9A mice exhibited alterations in dendritic spine structure, increased repetitive behavior, and decreased social interaction, hallmarks of ASDs. ASDs are frequently comorbid with epilepsy, and consistent with this comorbidity, S408/9A mice exhibited a marked increase in sensitivity to seizures induced by the convulsant kainic acid. To assess the relevance of our studies using S408/9A mice for the pathophysiology of ASDs, we measured S408/9 phosphorylation in Fmr1 KO mice, a model of fragile X syndrome, the most common monogenetic cause of ASDs. Phosphorylation of S408/9 was selectively and significantly enhanced in Fmr1 KO mice. Collectively, our results suggest that alterations in phosphorylation and/or activity of β3-containing GABAARs may directly contribute to the pathophysiology of ASDs.

DOI： 10.1073/pnas.1514657112

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Gamma-aminobutyric acid B receptors (GABABRs) are heterodimeric G-protein coupled receptors, which mediate slow synaptic inhibition in the brain. Emerging evidence suggests astrocytes also express GABABRs, although their physiological significance remains unknown. To begin addressing this issue, we have used imaging and biochemical analysis to examine the role GABABRs play in regulating astrocytic Ca(2+) signalling. Using live imaging of cultured cortical astrocytes loaded with calcium indicator Fluo-4/AM, we found that astrocytic GABABRs are able to induce astrocytic calcium transients only if they are pre-activated by P2 purinoceptors (P2YRs). The GABABR-mediated calcium transients were attenuated by the removal of extracellular calcium. Furthermore, P2YRs enhance the phosphorylation of astrocytic GABABR R2 subunits on both serine 783 (S783) and serine 892 (S892), two phosphorylation sites that are well known to regulate the activity and the cell surface stability of GABABRs. Collectively these results suggest that P2YR mediated signalling is an important determinant of GABABR activity and phosphorylation in astrocytes.

DOI： 10.1016/j.neuropharm.2014.09.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Emerging evidence suggests that functional γ-aminobutyric acid B receptors (GABABRs) are expressed by astrocytes within the mammalian brain. GABABRs are heterodimeric G-protein-coupled receptors that are composed of R1/R2 subunits. To date, they have been characterized in neurons as the principal mediators of sustained inhibitory signaling; however their roles in astrocytic physiology have been ill defined. Here we reveal that the cytoplasmic tail of the GABABR2 subunit binds directly to the astrocytic protein glutamine synthetase (GS) and that this interaction determines the subcellular localization of GS. We further demonstrate that the binding of GS to GABABR2 increases the steady state expression levels of GS in heterologous cells and in mouse primary astrocyte culture. Mechanistically this increased stability of GS in the presence of GABABR2 occurs via reduced proteasomal degradation. Collectively, our results suggest a novel role for GABABRs as regulators of GS stability. Given the critical role that GS plays in the glutamine-glutamate cycle, astrocytic GABABRs may play a critical role in supporting both inhibitory and excitatory neurotransmission.

DOI： 10.1074/jbc.M114.583534

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Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABAARs). Although neurosteroids are accepted allosteric modulators of GABAARs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABAARs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABAAR subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABAAR subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABAARs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior.

DOI： 10.1073/pnas.1403285111

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Cognitive dysfunction is a common symptom in many neuropsychiatric disorders and directly correlates with poor patient outcomes. The majority of prolonged inhibitory signaling in the brain is mediated via GABAB receptors (GABABRs), but the molecular function of these receptors in cognition is ill defined. To explore the significance of GABABRs in neuronal activity and cognition, we created mice with enhanced postsynaptic GABABR signaling by mutating the serine 783 in receptor R2 subunit (S783A), which decreased GABABR degradation. Enhanced GABABR activity reduced the expression of immediate-early gene-encoded protein Arc/Arg3.1, effectors that are critical for long-lasting memory. Intriguingly, S783A mice exhibited increased numbers of excitatory synapses and surface AMPA receptors, effects that are consistent with decreased Arc/Arg3.1 expression. These deficits in Arc/Arg3.1 and neuronal morphology lead to a deficit in spatial memory consolidation. Collectively our results suggest a novel and unappreciated role for GABABR activity in determining excitatory neuronal architecture and spatial memory via their ability to regulate Arc/Arg3.1.

DOI： 10.1523/JNEUROSCI.3320-13.2013

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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 stabilizing γ2 subunit-containing GABA(A) receptors (GABA(A)Rs) 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 GABA(A)R 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 GABA(A)R γ2 subunit Y365/7 phosphorylation and function in regulating the effects of BDNF.

DOI： 10.1523/JNEUROSCI.1845-13.2013

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Intravenous anesthetics exert a component of their actions via potentiating inhibitory neurotransmission mediated by γ-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 α1-3βγ2 subunits, whereas tonic inhibition is dependent on subtypes assembled from α4-6βδ 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 α4 subunit containing GABAARs contributes to the hypnotic and amnestic actions of the intravenous anesthetics, etomidate and propofol.

DOI： 10.1523/JNEUROSCI.5475-12.2013

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Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.

DOI： 10.1016/j.neuron.2011.12.031

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Cyclin E is a component of the core cell cycle machinery, and it drives cell proliferation by regulating entry and progression of cells through the DNA synthesis phase. Cyclin E expression is normally restricted to proliferating cells. However, high levels of cyclin E are expressed in the adult brain. The function of cyclin E in quiescent, postmitotic nervous system remains unknown. Here we use a combination of in vivo quantitative proteomics and analyses of cyclin E knockout mice to demonstrate that in terminally differentiated neurons cyclin E forms complexes with Cdk5 and controls synapse function by restraining Cdk5 activity. Ablation of cyclin E led to a decreased number of synapses, reduced number and volume of dendritic spines, and resulted in impaired synaptic plasticity and memory formation in cyclin E-deficient animals. These results reveal a cell cycle-independent role for a core cell cycle protein, cyclin E, in synapse function and memory.

DOI： 10.1016/j.devcel.2011.08.009

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Chemical transmitters released from astrocytes, termed gliotransmitters, modulate synaptic transmission and neuronal function. Using astrocyte-specific inducible transgenicmice (dnSNARE mice), we have demonstrated that inhibiting gliotransmission leads to reduced activation of adenosine A1 receptors (A1R) and impaired sleep homeostasis (Halassa et al. (2009) Neuron 61:213-219); Pascual et al. (2005) Science 310:113-116). Additionally, synaptic N-methyl-D-aspartate receptor (NMDAR) currents are reduced in these astrocyte-specific transgenic animals (Fellin et al. (2009) Proc Natl Acad Sci USA 106:15037-15042). Because of the importance of adenosine and NMDA receptors to sleep processes we asked whether there is a causal linkage between changes in A1R activation and synaptic NMDA receptors. We show that astrocytic dnSNARE expression leads to reduced tyrosine phosphorylation of Srckinase and NR2 subunits concomitant with the decreased surface expression of the NR2 subunits. To test the role of A1R signaling in mediating these actions, we show that incubation of wildtype (WT) slices with an A1R antagonist reduces tyrosine phosphorylation of Src kinase and NR2B, decreases the surface expression of the NR2B subunits and leads to smaller NMDA component of miniature EPSCs. In dnSNARE mice we could rescue WT phenotype by incubation in an A1R agonist:activation of A1 receptor led to increased tyrosine phosphorylation of Src kinase and NR2B subunits as well as increased the surface expression of the NR2B subunit and increased NMDA component of the synaptic mEPSC. These results provide the first demonstration that astrocytes can affect neuronal excitability on a long time scale by regulating the surface expression of NMDA receptors through the activation of specific intracellular signaling pathways.

DOI： 10.1002/glia.21181

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Inhibition in the adult mammalian central nervous system (CNS) is mediated by γ-aminobutyric acid (GABA). The fast inhibitory actions of GABA are mediated by GABA type A receptors (GABA(A)Rs); they mediate both phasic and tonic inhibition in the brain and are the principle sites of action for anticonvulsant, anxiolytic, and sedative-hypnotic agents that include benzodiazepines, barbiturates, neurosteroids, and some general anesthetics. GABA(A)Rs are heteropentameric ligand-gated ion channels that are found concentrated at inhibitory postsynaptic sites where they mediate phasic inhibition and at extrasynaptic sites where they mediate tonic inhibition. The efficacy of inhibition and thus neuronal excitability is critically dependent on the accumulation of specific GABA(A)R subtypes at inhibitory synapses. Here we evaluate how neurons control the number of GABA(A)Rs on the neuronal plasma membrane together with their selective stabilization at synaptic sites. We then go on to examine the impact that these processes have on the strength of synaptic inhibition and behavior.

DOI： 10.1152/physrev.00015.2010

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Slow and persistent synaptic inhibition is mediated by metabotropic GABAB receptors (GABABRs). GABABRs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABABRs are predominantly found on dendritic spines, adjacent to excitatory synapses, but the control of their plasma membrane availability is still controversial. Here, we explore the role of glutamate receptor activation in regulating the function and surface availability of GABABRs in central neurons. We demonstrate that prolonged activation of NMDA receptors (NMDA-Rs) leads to endocytosis, a diversion from a recycling route, and subsequent lysosomal degradation of GABABRs. These sorting events are paralleled by a reduction in GABABR-dependent activation of inwardly rectifying K+ channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABABR2 subunit, an established substrate of AMP-dependent protein kinase (AMPK). NMDA-R activation leads to a rapid increase in phosphorylation of S783, followed by a slower dephosphorylation, which results from the activity of AMPK and protein phosphatase 2A, respectively. Agonist activation of GABABRs counters the effects of NMDA. Thus, NMDA-R activation alters the phosphorylation state of S783 and acts as a molecular switch to decrease the abundance of GABABRs at the neuronal plasma membrane. Such a mechanism may be of significance during synaptic plasticity or pathological conditions, such as ischemia or epilepsy, which lead to prolonged activation of glutamate receptors.

DOI： 10.1073/pnas.1000853107

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GABA(B) receptors (GABA(B)R) are heterodimeric G protein-coupled receptors (GPCRs) that mediate slow and prolonged inhibitory signals in the central nervous system. The signaling of GPCRs is under stringent control and is subject to regulation by multiple posttranslational mechanisms. The beta-adrenergic receptor is a prototypic GPCR. Like most GPCRs, prolonged exposure of this receptor to agonist induces phosphorylation of multiple intracellular residues that is largely dependent upon the activity of G protein-coupled receptor kinases (GRKs). Phosphorylation terminates receptor-effector coupling and promotes both interaction with beta-arrestins and removal from the plasma membrane via clathrin-dependent endocytosis. Emerging evidence for GABA(B)Rs suggests that these GPCRs do not conform to this mode of regulation. Studies using both native and recombinant receptor preparations have demonstrated that GABA(B)Rs do not undergo agonist-induced internalization and are not GRK substrates. Moreover, whilst GABA(B)Rs undergo clathrin-dependent constitutive endocytosis, it is generally accepted that their rates of internalization are not modified by prolonged agonist exposure. Biochemical studies have revealed that GABA(B)Rs are phosphorylated on multiple residues within the cytoplasmic domains of both the R1 and R2 subunits by cAMP-dependent protein kinase and 5'AMP-dependent protein kinase (AMPK). Here we discuss the role that this phosphorylation plays in determining GABA(B)R effector coupling and their trafficking within the endocytic pathway and go on to evaluate the significance of GABA(B)R phosphorylation in controlling neuronal excitability under normal and pathological conditions.

DOI： 10.1016/S1054-3589(10)58005-0

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Downregulation of G-protein-coupled receptors (GPCRs) provides an important mechanism for reducing neurotransmitter signaling during sustained stimulation. Chronic stimulation of M-2 muscarinic receptors (M(2)Rs) causes internalization of M2R and G-protein-activated inwardly rectifying potassium (GIRK) channels in neuronal PC12 cells, resulting in loss of function. Here, we show that coexpression of GABA(B) R2 receptors (GBR2s) rescues both surface expression and function of M2R, including M2R-induced activation of GIRKs and inhibition of cAMP production. GBR2 showed significant association with M2R at the plasma membrane but not other GPCRs (M1R, mu-opioid receptor), as detected by fluorescence resonance energy transfer measured with total internal reflection fluorescence microscopy. Unique regions of the proximal C-terminal domains of GBR2 and M2R mediate specific binding between M2R and GBR2. In the brain, GBR2, but not GBR1, biochemically coprecipitates with M2R and overlaps with M2R expression in cortical neurons. This novel heteromeric association between M2R and GBR2 provides a possible mechanism for altering muscarinic signaling in the brain and represents a previously unrecognized role for GBR2.

DOI： 10.1523/JNEUROSCI.4103-09.2009

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Fast synaptic inhibition in the brain is largely mediated by gamma-aminobutyric acid receptors (GABA(A)R). While the pharmacological manipulation of GABA(A)R function by therapeutic agents, such as benzodiazepines can have profound effects on neuronal excitation and behavior, the endogenous mechanisms neurons use to regulate the efficacy of synaptic inhibition and their impact on behavior remains poorly understood. To address this issue, we created a knock-in mouse in which tyrosine phosphorylation of the GABA(A)Rs gamma 2 subunit, a posttranslational modification that is critical for their functional modulation, has been ablated. These animals exhibited enhanced GABA(A)R accumulation at postsynaptic inhibitory synaptic specializations on pyramidal neurons within the CA3 subdomain of the hippocampus, primarily due to aberrant trafficking within the endocytic pathway. This enhanced inhibition correlated with a specific deficit in spatial object recognition, a behavioral paradigm dependent upon CA3. Thus, phospho-dependent regulation of GABA(A)R function involving just two tyrosine residues in the gamma 2 subunit provides an input-specific mechanism that not only regulates the efficacy of synaptic inhibition, but has behavioral consequences.

DOI： 10.1073/pnas.0908840106

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Gliotransmission, the release of molecules from astrocytes, regulates neuronal excitability and synaptic transmission in situ. Whether this process affects neuronal network activity in vivo is not known. Using a combination of astrocyte-specific molecular genetics, with in vivo electrophysiology and pharmacology, we determined that gliotransmission modulates cortical slow oscillations, a rhythm characterizing nonrapid eye movement sleep. Inhibition of gliotransmission by the expression of a dominant negative SNARE domain in astrocytes affected cortical slow oscillations, reducing the duration of neuronal depolarizations and causing prolonged hyperpolarizations. These network effects result from the astrocytic modulation of intracortical synaptic transmission at two sites: a hypofunction of postsynaptic NMDA receptors, and by reducing extracellular adenosine, a loss of tonic A1 receptor-mediated inhibition. These results demonstrate that rhythmic brain activity is generated by the coordinated action of the neuronal and glial networks.

DOI： 10.1073/pnas.0906419106

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The efficacy of synaptic transmission depends on the availability of ionotropic and metabotropic neurotransmitter receptors at the plasma membrane, but the contribution of the endocytic and recycling pathways in the regulation of gamma-aminobutyric acid type B (GABA(B)) receptors remains controversial. To understand the mechanisms that regulate the abundance of GABA(B) receptors, we have studied their turnover combining surface biotin labeling and a microscopic immunoendocytosis assay in hippocampal and cortical neurons. We report that internalization of GABA(B) receptors is agonist-independent. We also demonstrate that receptors endocytose in the cell body and dendrites but not in axons. Additionally, we show that GABA(B) receptors endocytose as heterodimers via clathrin- and dynamin-1-dependent mechanisms and that they recycle to the plasma membrane after endocytosis. More importantly, we show that glutamate decreases the levels of cell surface receptors in a manner dependent on an intact proteasome pathway. These observations indicate that glutamate and not GABA controls the abundance of surface GABA(B) receptors in central neurons, consistent with their enrichment at glutamatergic synapses.

DOI： 10.1074/jbc.M802419200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

Status epilepticus ( SE) is a progressive and often lethal human disorder characterized by continuous or rapidly repeating seizures. Of major significance in the pathology of SE are deficits in the functional expression of GABA(A) receptors (GABA(A)Rs), the major sites of fast synaptic inhibition in the brain. We demonstrate that SE selectively decreases the phosphorylation of GABAARs on serine residues 408/9 (S408/9) in the beta 3 subunit by intimately associated protein kinase C isoforms. Dephosphorylation of S408/9 unmasks a basic patch-binding motif for the clathrin adaptor AP2, enhancing the endocytosis of selected GABA(A)R subtypes from the plasma membrane during SE. In agreement with this, enhancing S408/9 phosphorylation or selectively blocking the binding of the beta 3 subunit to AP2 increased GABA(A)R cell surface expression levels and restored the efficacy of synaptic inhibition in SE. Thus, enhancing phosphorylation of GABA(A)Rs or selectively blocking their interaction with AP2 may provide novel therapeutic strategies to ameliorate SE.

DOI： 10.1523/JNEUROSCI.4346-07.2008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

GABA(A) receptor-mediated inhibition depends on the maintenance of intracellular Cl- concentration ([Cl-](in)) at low levels. In neurons in the developing CNS, [Cl-](in) is elevated, E-GABA is depolarizing, and GABA consequently is excitatory. Depolarizing GABAergic synaptic responses may be recapitulated in various neuropathological conditions, including epilepsy. In the present study, rat hippocampal dentate granule cells were recorded using gramicidin perforated patch techniques at varying times (1-60 d) after an epileptogenic injury, pilocarpine-induced status epilepticus (STEP). In normal, non-epileptic animals, these strongly inhibited dentate granule cells act as a gate, regulating hippocampal excitation, controlling seizure initiation and/or propagation. For 2 weeks after STEP, we found that E-GABA was positively shifted in granule cells. This shift in E-GABA altered synaptic integration, increased granule cell excitability, and resulted in compromised "gate" function of the dentate gyrus. E-GABA recovered to control values at longer latencies post-STEP (2-8 weeks), when animals had developed epilepsy. During this period of shifted E-GABA, expression of the Cl- extruding K-/Cl- cotransporter, KCC2 was decreased. Application of the KCC2 blocker, furosemide, to control neurons mimicked E-GABA shifts evident in granule cells post-STEP. Furthermore, post-STEP and furosemide effects interacted occlusively, both on E-GABA in granule cells, and on gatekeeper function of the dentate gyrus. This suggests a shared mechanism, reduced KCC2 function. These findings demonstrate that decreased expression of KCC2 persists for weeks after an epileptogenic injury, reducing inhibitory efficacy and enhancing dentate granule cell excitability. This pathophysiological process may constitute a significant mechanism linking injury to the subsequent development of epilepsy.

DOI： 10.1523/JNEUROSCI.4390-07.2007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Although metabotropic glutamate receptor 5 (mGluR5) is essential for cocaine self-administration and drug-seeking behavior, there is limited knowledge of the cellular actions of this receptor in the nucleus accumbens (NAc). Although mGIuR5 has the potential to regulate neurons directly, recent studies have shown the importance of mGIuR5 in regulating Ca2+ signaling in astrocytes and, as a consequence, the Ca2+-dependent release of excitatory transmitters from these glia. In this study, we demonstrate that activation of mGIuR5 induces Ca2+ oscillations in NAc astrocytes with the correlated appearance of NMDA receptor-dependent slow inward currents detected in medium spiny neurons (MSNs). Photolysis of caged Ca2+ loaded specifically into astrocytes evoked slow inward currents demonstrating that Ca2+ elevations in astrocytes are responsible for these excitatory events. Pharmacological evaluation of these glial-evoked NMDA currents shows that they are mediated by NR2B-containing NMDA receptors, whereas synaptic NMDA receptors rely on NR2A-containing receptors. Stimulation of glutamatergic afferents activates mGluRS-dependent astrocytic Ca2+ oscillations and gliotransmission that is sustained for minutes beyond the initial stimulus. Because gliotransmission is mediated by NMDA receptors, depolarized membrane potentials exhibited during up-states augment excitation provided by gliotransmission, which drives bursts of MSN action potentials. Because the predominant mGluR5-dependent action of glutamatergic afferents is to cause the sustained activation of astrocytes, which in turn excite MSNs through extrasynaptic NMDA receptors, our results raise the potential for gliotransmission being involved in prolonged mGluR5-dependent adaptation in the NAc.

DOI： 10.1073/pnas.0609408104

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELL PRESS  

GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K+ channels (GIRKs) and inhibiting Ca2+, channels. We demonstrate here that GABAB receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABAB receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABAB receptor function to reduce excitotoxicity and thereby promotes neuronal survival.

DOI： 10.1016/j.neuron.2006.12.015

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The investigation of chemically synthesized inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogs has led to the isolation of a novel protein with a molecular size of 130 kDa, characterized as a molecule with a domain organization similar to phospholipase C (PLC)-δ1 but lacking enzymatic activity. Two isoforms of the molecule were subsequently identified, the molecule has been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and-2. Regarding its ability to bind Ins (1,4,5)-P3 via the pleckstrin homology domain, the involvement of PRIP-1 in Ins(1,4,5)P3-mediated Ca2+ signaling was first examined. Yeast two-hybrid screening of a brain cDNA library identified GABARAP (GABAA receptor-associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible neurological involvement of PRIP, particularly in GABAA receptor signaling. PRIP-1 and-2 double knock-out (DKO) mice were analyzed for GABAA receptor function with special reference to the action of benzodiazepines whose target is the γ subunit of the receptors
sensitivity to benzodiazepine was reduced, as assessed by biochemical, electrophysiological, and behavioral analyses of DKO mice, suggesting the dysfunction of γ2 subunit-containing GABAA receptors. The mesencephalic trigeminal nucleus, which mediates perceptions from periodontal mechanoreceptors and jaw-closer muscle spindles, receives many synaptic inputs, including those from GABAA receptors, indicating that PRIP might indirectly be involved in rhythmical jaw movement. In the present article, we summarize our current research and the functional significance of PRIP. © 2007, Japanese Association for Oral Biology. All rights reserved.

DOI： 10.2330/joralbiosci.49.244

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

PRIP, phospholipase C related, but catalytically inactive protein was first identified as a novel inositol 1,4,5-trisphosphate binding protein. It has a number of binding partners including protein phosphatase (PP1 and 2A), GABA(A) receptor associated protein, and the beta subunits of GABAA receptors, in addition to inositol 1,4,5-trisphosphate. The identification of these molecules led us to examine the possible involvement of PRIP in the phospho-regulation of the beta subunits of GABAA receptors using hippocampal neurons prepared from PRIP-1 and 2 double knock-out (DKO) mice. Experiments were performed with special reference to the dephosphorylation processes of the beta subunits. The phosphorylation of beta 3 subunits by the activation of protein kinase A in cortical neurons of the control mice continued for up to 5min, even after washing out of the stimulus, followed by a gradual dephosphorylation. That of DKO mice gradually increased in spite of the lower phosphorylation levels induced by the stimulation. There was little difference in the amount of cellular cyclic AMP and protein kinase A activity between the control and mutant mice, indicating that phosphatases such as PP1 and MA are primarily involved in the difference. The time course of PP1 activity changes in the vicinity of the receptors in control mice corresponded to the phosphorylation of PRIP, while that of the mutant mice decreased with the period of the incubation. This is a good agreement with the suggestion that PRIP binds to and inactivates PP1, which is regulated by the phosphorylation of PRIP at threonine 94. These results suggest that PRIP plays an important role in controlling the dynamics of GABA(A) receptor phosphorylation by through PP1 binding and, therefore, the efficacy of synaptic inhibition mediated by these receptors. (c) 2006 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.advenzreg.2006.01.006

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Language：English   Publisher：SPRINGER SINGAPORE PTE LTD  

Investigation of chemically synthesized inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3] analogs has led to the isolation of a novel binding protein with a molecular size of 130 kDa, characterized as a molecule with similar domain organization to phospholipase C-delta 1 (PLC-delta 1) but lacking the enzymatic activity. An isoform of the molecule was subsequently identified, and these molecules have been named PRIP (PLC-related, but catalytically inactive protein), with the two isoforms named PRIP-1 and -2. Regarding its ability to bind Ins(1,4,5)P-3 via the pleckstrin homology domain, the involvement of PRIP-1 in Ins(1,4,5)P-3-mediated Ca2+ signaling was examined using COS-1 cells overexpressing PRIP-1 and cultured neurons prepared from PRIP-1 knockout mice. Yeast two hybrid screening of a brain cDNA library using a unique N-terminus as bait identified GABARAP (GABA(A) receptor associated protein) and PP1 (protein phosphatase 1), which led us to examine the possible involvement of PRIP in GABA(A) receptor signaling. For this purpose PRIP knock-out mice were analyzed for GABA(A) receptor function in relation to the action of benzodiazepines from the electrophysiological and behavioral aspects. During the course of these experiments we found that PRIP also binds to the beta-subunit of GABA(A) receptors and PP2A (protein phosphtase 2A). Here, we summarize how PRIP is involved in Ins(1,4,5)P-3-mediated Ca2+ signaling and GABA(A) receptor signaling based on the characteristics of binding molecules.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

GABA, receptors mediate most of the fast inhibitory neurotransmission in the brain, and are believed to be composed mainly of alpha, beta, and gamma subunits. It has been shown that GABA(A) receptors interact with a number of binding partners that act to regulate both receptor function and cell surface stability. Here, we reveal that GABA(A) receptors interact directly with N-ethylmaleimide-sensitive factor (NSF), a critical regulator of vesicular dependent protein trafficking, as measured by in vitro protein binding and co-immunoprecipitation assays. In addition, we established that NSF interacts with residues 395-415 of the receptor beta subunits and co-localizes with GABA(A) receptors in hippocampal neurons. We also established that NSF can regulate GABA(A) receptor cell surface expression depending upon residues 395-415 in the beta 3 subunit. Together, our results suggest an important role for NSF activity in regulating the cell surface stability of GABA(A) receptors. (c) 2005 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.mcn.2005.07.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC NEUROSCIENCE  

GABA(A) receptors are critical in controlling neuronal activity. Here, we examined the role for phospholipase C-related inactive protein type 1 (PRIP-1), which binds and inactivates protein phosphatase 1alpha (PP1alpha) in facilitating GABA(A) receptor phospho-dependent regulation using PRIP-1(-/-) mice. In wild-type animals, robust phosphorylation and functional modulation of GABA(A) receptors containing beta3 subunits by cAMP-dependent protein kinase was evident, which was diminished in PRIP-1(-/-) mice. PRIP-1(-/-) mice exhibited enhanced PP1alpha activity compared with controls. Furthermore, PRIP-1 was able to interact directly with GABA(A) receptor beta subunits, and moreover, these proteins were found to be PP1alpha substrates. Finally, phosphorylation of PRIP-1 on threonine 94 facilitated the dissociation of PP1alpha-PRIP-1 complexes, providing a local mechanism for the activation of PP1alpha. Together, these results suggest an essential role for PRIP-1 in controlling GABA(A) receptor activity via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors.

DOI： 10.1523/JNEUROSCI.1323-04.2004

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Language：Japanese  

γ-Aminobutyric acidA (GABAA) receptors mediate most of the fast inhibitory neurotransmission in the central nervous system. These ligand-gated ion channels are crucial in the control of cell and network activity. Therefore, modulating their function or cell surface stability will have major consequences for neuronal excitation. This review highlights recent findings on the regulation of GABAA-receptor expression and function, focusing on the mechanisms of sorting, targeting, synaptic clustering, and endocytic events of GABAA receptors, all which are regulated by their associated proteins. Now these topics are an area of active interest in studies on inhibitory neurotransmission.

DOI： 10.1254/fpj.123.105

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The protein p130 was originally isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but which lacks phospholipase C activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with p130 has now led to the identification of the catalytic subunit of protein phosphatase 1 alpha (PP1c alpha) as a p130-binding protein. The association between p130 and PP1c alpha was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of p130 with PP1c alpha resulted in inhibition of the catalytic activity of the latter in a p130 concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express p130 and of mouse brain extract with antibodies to p130 and to PP1c alpha also detected the presence of a complex of p130 and PP1c alpha, The activity of glycogen phosphorylase, which is negatively regulated by dephosphorylation by PP1c alpha, was higher in COS-1 cells that stably express p130 than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca2+ signaling, p130 might also contribute to regulation of protein dephosphorylation through its interaction with PP1c alpha.

DOI： 10.1074/jbc.M009677200

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Language：Japanese  

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