Biomedical subjects

Stephen J Moss

Publications and source records attributed to Stephen J Moss.

At least 19 recordsLinked to original sources

PubMed2007

Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase.

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 Ca(2+) channels. We demonstrate here that GABA(B) 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 GABA(B) 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 GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.

Adenosine Monophosphate↗

PubMed2006

Synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts.

GABAA receptors mediate the majority of fast synaptic inhibition in the brain. The accumulation of these ligand-gated ion channels at synaptic sites is a prerequisite for neuronal inhibition, but the molecular mechanisms underlying this phenomenon remain obscure. To further understand these processes, we have examined the cellular origins of synaptic GABAA receptors. To do so, we have created fluorescent GABAA receptors that are capable of binding -bungarotoxin (Bgt), facilitating the visualization of receptor endocytosis, exocytosis and delivery to synaptic sites. Imaging with Bgt in hippocampal neurons revealed that GABAA receptor endocytosis occurred exclusively at extrasynaptic sites, consistent with the preferential colocalization of extrasynaptic receptors with the AP2 adaptin. Receptor insertion into the plasma membrane was also predominantly extrasynaptic, and pulse-chase analysis revealed that these newly inserted receptors were then able to access directly synaptic sites. Therefore, our results demonstrate that synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts. Moreover, they illustrate a dynamic mechanism for neurons to modulate GABAA receptor number at inhibitory synapses by controlling the stability of extrasynaptic receptors.

Animals↗

PubMed2006

Modulation of GABA(A) receptor phosphorylation and membrane trafficking by phospholipase C-related inactive protein/protein phosphatase 1 and 2A signaling complex underlying brain-derived neurotrophic factor-dependent regulation of GABAergic inhibition.

Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABA(A) receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABA(A) receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABA(A) receptor beta3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABA(A) receptors. We demonstrate that PRIP associates with phosphatases as well as with beta subunits. PRIP was found to colocalize with GABA(A) receptor clusters in cultured neurons and with recombinant GABA(A) receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to beta subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABA(A) receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABA(A) receptors by mediating the specific association between beta subunits of these receptors with protein phosphatases.

Animals↗

PubMed2006

Dopamine D3 receptors regulate GABAA receptor function through a phospho-dependent endocytosis mechanism in nucleus accumbens.

The dopamine D3 receptor, which is highly enriched in nucleus accumbens (NAc), has been suggested to play an important role in reinforcement and reward. To understand the potential cellular mechanism underlying D3 receptor functions, we examined the effect of D3 receptor activation on GABAA receptor (GABAAR)-mediated current and inhibitory synaptic transmission in medium spiny neurons of NAc. Application of PD128907 [(4aR,10bR)-3,4a,4,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin-9-ol hydrochloride], a specific D3 receptor agonist, caused a significant reduction of GABAAR current in acutely dissociated NAc neurons and miniature IPSC amplitude in NAc slices. This effect was blocked by dialysis with a dynamin inhibitory peptide, which prevents the clathrin/activator protein 2 (AP2)-mediated GABAA receptor endocytosis. In addition, the D3 effect on GABAAR current was prevented by agents that manipulate protein kinase A (PKA) activity. Infusion of a peptide derived from GABAAR beta subunits, which contains an atypical binding motif for the clathrin AP2 adaptor complex and the major PKA phosphorylation sites and binds with high affinity to AP2 only when dephosphorylated, diminished the D3 regulation of IPSC amplitude. The phosphorylated equivalent of the peptide was without effect. Moreover, PD128907 increased GABAAR internalization and reduced the surface expression of GABAA receptor beta subunits in NAc slices, which was prevented by dynamin inhibitory peptide or cAMP treatment. Together, our results suggest that D3 receptor activation suppresses the efficacy of inhibitory synaptic transmission in NAc by increasing the phospho-dependent endocytosis of GABAA receptors.

Analysis of Variance↗

PubMed2006

Molecular organization and assembly of the central inhibitory postsynapse.

gamma-Amino butyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. GABAA receptors play an important role in regulating neuronal excitability and in addition have been implicated in numerous neurological disorders. In order to understand synaptic inhibition it is important to comprehend the cellular mechanisms, that neurons utilize to regulate the accumulation and regulation of GABAA receptors at postsynaptic inhibitory specializations. Over the past decade a number of GABAA receptor interacting proteins have been identified allowing us to further understand the trafficking, targeting and clustering of these receptors as well as the regulation of receptor stability. In the following review we examine the proteins identified as GABAA receptor binding partners and other components of the inhibitory postsynaptic scaffold, and how they contribute to the construction of inhibitory synapses and the dynamic modulation of synaptic inhibition.

Animals↗

PubMed2005

Gephyrin regulates the cell surface dynamics of synaptic GABAA receptors.

The efficacy of fast synaptic inhibition is critically dependent on the accumulation of GABAA receptors at inhibitory synapses, a process that remains poorly understood. Here, we examined the dynamics of cell surface GABAA receptors using receptor subunits modified with N-terminal extracellular ecliptic pHluorin reporters. In hippocampal neurons, GABAA receptors incorporating pHluorin-tagged subunits were found to be clustered at synaptic sites and also expressed as diffuse extrasynaptic staining. By combining FRAP (fluorescence recovery after photobleaching) measurements with live imaging of FM4-64-labeled active presynaptic terminals, it was evident that clustered synaptic receptors exhibit significantly lower rates of mobility at the cell surface compared with their extrasynaptic counterparts. To examine the basis of this confinement, we used RNAi to inhibit the expression of gephyrin, a protein shown to regulate the accumulation of GABAA receptors at synaptic sites. However, whether gephyrin acts to control the actual formation of receptor clusters, their stability, or is simply a global regulator of receptor cell surface number remains unknown. Inhibiting gephyrin expression did not modify the total number of GABAA receptors expressed on the neuronal cell surface but significantly decreased the number of receptor clusters. Live imaging revealed that clusters that formed in the absence of gephyrin were significantly more mobile compared with those in control neurons. Together, our results demonstrate that synaptic GABAA receptors have lower levels of lateral mobility compared with their extrasynaptic counterparts, and suggest a specific role for gephyrin in reducing the diffusion of GABAA receptors, facilitating their accumulation at inhibitory synapses.

Animals↗

PubMed2005

Astrocytic purinergic signaling coordinates synaptic networks.

To investigate the role of astrocytes in regulating synaptic transmission, we generated inducible transgenic mice that express a dominant-negative SNARE domain selectively in astrocytes to block the release of transmitters from these glial cells. By releasing adenosine triphosphate, which accumulates as adenosine, astrocytes tonically suppressed synaptic transmission, thereby enhancing the dynamic range for long-term potentiation and mediated activity-dependent, heterosynaptic depression. These results indicate that astrocytes are intricately linked in the regulation of synaptic strength and plasticity and provide a pathway for synaptic cross-talk.

Adenosine↗

PubMed2005

Phospho-dependent binding of the clathrin AP2 adaptor complex to GABAA receptors regulates the efficacy of inhibitory synaptic transmission.

The efficacy of synaptic inhibition depends on the number of gamma-aminobutyric acid type A receptors (GABA(A)Rs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABA(A)R endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABA(A)R beta subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the beta3 subunit) incorporates the major sites of serine phosphorylation within receptor beta subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pepbeta3) corresponding to the AP2 binding motif in the GABA(A)R beta3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pepbeta3 peptide, but not its phosphorylated equivalent (pepbeta3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABA(A)R current. These effects of pepbeta3 on GABA(A)R current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pepbeta3 on GABA(A)R endocytosis. Therefore phospho-dependent regulation of AP2 binding to GABA(A)Rs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission.

Adaptor Protein Complex 2↗

PubMed2005

Multiple motifs regulate the trafficking of GABA(B) receptors at distinct checkpoints within the secretory pathway.

gamma-Aminobutyric acid type B receptors (GABA(B)) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABA(B)R1 and GABA(B)R2 subunits, a process that occurs within the endoplasmic reticulum (ER). However, the mechanisms that regulate receptor surface expression remain largely unknown. Here, we demonstrate that access to the cell surface for GABA(B)R1 is sequentially controlled by an RSR(R) motif and a LL motif within its cytoplasmic domain. In addition, we reveal that msec7-1, a guanine-nucleotide-exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of GTPases, critical regulators of vesicular membrane trafficking, interacts with GABA(B)R1 via the LL motif in this subunit. Finally, we establish that msec7-1 modulates the cell surface expression of GABA(B) receptors, a process that is dependent upon the integrity of the LL motif in GABA(B)R1. Together, our results demonstrate that the cell surface expression of the GABA(B)R1 subunit is regulated by multiple motifs, which act at distinct checkpoints in the secretory pathway, and also suggest a novel role for msec7-1 in regulating the membrane trafficking of GABA(B)R1 subunits.

Amino Acid Motifs↗

PubMed2005

Direct interaction of N-ethylmaleimide-sensitive factor with GABA(A) receptor beta subunits.

GABA(A) 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 beta3 subunit. Together, our results suggest an important role for NSF activity in regulating the cell surface stability of GABA(A) receptors.

Amino Acid Sequence↗

PubMed2004

Unravelling the unusual signalling properties of the GABA(B) receptor.

GABA(B) receptors are the cornerstone receptors in the modulation of inhibitory signalling in the central nervous system and continue to be targets for the amelioration of a number of neuropsychiatric and neurological disorders. Unravelling the molecular identity of this receptor has spurred much research over the past five or so years and generated a renewed interest and excitement in the field. Many questions are being answered and lessons learnt, not only about GABA(B) receptor function but also about general mechanisms of G-protein-coupled receptor signalling. However, as questions are being answered as many new questions are being raised and many GABA(B)-related conundrums continue to remain unanswered. In this report, we review some of the most recent work in the area of GABA(B) receptor research. In particular, we focus our attentions on the emerging mechanisms thought to be important in GABA(B) receptor signalling and the growing complex of associated proteins that we consider to be part of the GABA(B) receptor "signalosome."

Animals↗

PubMed2004

Association of GRIP1 with a GABA(A) receptor associated protein suggests a role for GRIP1 at inhibitory synapses.

GABA(A) receptors mediate the majority of fast synaptic inhibition in the mammalian central nervous system. GABA(A) receptors associate with a number of cytosolic proteins important for regulating their function including the GABA(A) receptor gamma2 subunit associated protein GABARAP. Here we show GABARAP associates with the synaptic PDZ domain containing protein GRIP1. GRIP1 has been localized to inhibitory synapses however the role of this protein with respect to neuronal inhibition remains unclear. Using in vitro protein interaction assays we show that GABARAP interacts directly with PDZ domains 4-6 of GRIP1. Furthermore, using coimmunoprecipitation assays we show that GABARAP interacts with GRIP1 in vivo. Finally, we show that GRIP1 colocalizes with gamma2 subunit containing GABA(A) receptors in cultured hippocampal neurons. Our findings provide evidence that GRIP1 can associate with proteins important for regulating GABA(A) receptor function and suggest that GRIP1 may play a role at inhibitory synapses.

Adaptor Proteins, Signal Transducing↗

PubMed2004

The GABA(B2) subunit is critical for the trafficking and function of native GABA(B) receptors.

Studies in heterologous systems have demonstrated that heterodimerisation of the two GABA(B) receptor subunits appears to be crucial for the trafficking and signalling of the receptor. Gene targeting of the GABA(B1) gene has demonstrated that the expression of GABA(B1) is essential for GABA(B) receptor function in the central nervous system (CNS). However, the contribution of the GABA(B2) subunit in the formation of native GABA(B) receptors is still unclear, in particular whether other proteins can substitute for this subunit. We have created a transgenic mouse in which the endogenous GABA(B2) gene has been mutated in order to express a C-terminally truncated version of the protein. As a result, the GABA(B1) subunit does not reach the cell surface and concomitantly both pre- and post-synaptic GABA(B) receptor functions are abolished. Taken together with previous gene deletion studies for the GABA(B1) subunit, this suggests that classical GABA(B) function in the brain is exclusively mediated by GABA(B1/2) heteromers.

Animals↗

PubMed2004

Huntingtin-associated protein 1 regulates inhibitory synaptic transmission by modulating gamma-aminobutyric acid type A receptor membrane trafficking.

Gamma-aminobutyric acid type A receptors (GABA(A)Rs) are the major sites of fast synaptic inhibition in the brain. An essential determinant for the efficacy of synaptic inhibition is the regulation of GABA(A)R cell surface stability. Here, we have examined the regulation of GABA(A)R endocytic sorting, a critical regulator of cell surface receptor number. In neurons, rapid constitutive endocytosis of GABA(A)Rs was evident. Internalized receptors were then either rapidly recycled back to the cell surface, or on a slower time scale, targeted for lysosomal degradation. This sorting decision was regulated by a direct interaction of GABA(A)Rs with Huntingtin-associated protein 1 (HAP1). HAP1 modulated synaptic GABA(A)R number by inhibiting receptor degradation and facilitating receptor recycling. Together these observations have identified a role for HAP1 in regulating GABA(A)R sorting, suggesting an important role for this protein in the construction and maintenance of inhibitory synapses.

Animals↗

PubMed2004

GABAA receptor phospho-dependent modulation is regulated by phospholipase C-related inactive protein type 1, a novel protein phosphatase 1 anchoring protein.

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.

Animals↗

PubMed2004

Amyloid precursor protein mRNA levels in Alzheimer's disease brain.

Insoluble beta-amyloid deposits in Alzheimer's disease (AD) brain are proteolytically derived from the membrane bound amyloid precursor protein (APP). The APP gene is differentially spliced to produce isoforms that can be classified into those containing a Kunitz-type serine protease inhibitor domain (K(+), APP(751), APP(770), APRP(365) and APRP(563)), and those without (K(-), APP(695) and APP(714)). Given the hypothesis that Abeta is a result of aberrant catabolism of APP, differential expression of mRNA isoforms containing protease inhibitors might play an active role in the pathology of AD. We took 513 cerebral cortex samples from 90 AD and 81 control brains and quantified the mRNA isoforms of APP with TaqMan real-time RT-PCR. After adjustment for age at death, brain pH and gender we found a change in the ratio of KPI(+) to KPI(-) mRNA isoforms of APP. Three separate probes, designed to recognise only KPI(+) mRNA species, gave increases of between 28% and 50% in AD brains relative to controls (p=0.002). There was no change in the mRNA levels of KPI-(APP 695) (p=0.898). Therefore, whilst KPI-mRNA levels remained stable the KPI(+) species increased specifically in the AD brains.

Adult↗

PubMed2004

Brain-derived neurotrophic factor modulates fast synaptic inhibition by regulating GABA(A) receptor phosphorylation, activity, and cell-surface stability.

The efficacy of GABAergic synaptic inhibition is a principal factor in controlling neuronal activity. We demonstrate here that brain-derived neurotrophic factor modulates the activity of GABA(A) receptors, the main sites of fast synaptic inhibition in the brain, within minutes of application. Temporally, this comprised an early enhancement in the miniature IPSC amplitude, followed by a prolonged depression. This modulation was concurrent with enhanced PKC-mediated phosphorylation, followed by protein phosphatase 2A (PP2A)-mediated dephosphorylation of the GABA(A) receptor. Mechanistically, these events were facilitated by differential recruitment of PKC, receptor for activated C-kinase, and PP2A to GABA(A) receptors, depending on the phosphorylation state of the receptor beta3-subunit. Thus, transient formation of GABA(A) receptor signaling complexes has the potential to provide a basis for acute changes in receptor function underlying GABAergic synaptic plasticity.

Animals↗

PubMed2004

Marlin-1, a novel RNA-binding protein associates with GABA receptors.

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission.

Adaptor Proteins, Signal Transducing↗