Assembly rules for GABAA receptor complexes in the brain.
ABSTRACT: GABAA receptor (GABAAR) pentamers are assembled from a pool of 19 subunits, and variety in subunit combinations diversifies GABAAR functions to tune brain activity. Pentamers with distinct subunit compositions localize differentially at synaptic and non-synaptic sites to mediate phasic and tonic inhibition, respectively. Despite multitudes of theoretical permutations, limited subunit combinations have been identified in the brain. Currently, no molecular model exists for combinatorial GABAAR assembly in vivo. Here, we reveal assembly rules of native GABAAR complexes that explain GABAAR subunit subcellular distributions using mice and Xenopus laevis oocytes. First, ? subunits possess intrinsic signals to segregate into distinct pentamers. Second, ?2 is essential for GABAAR assembly with Neuroligin-2 (NL2) and GARLHs, which localize GABAARs at synapses. Third, ? suppresses ?6 synaptic localization by preventing assembly with GARLHs/NL2. These findings establish the first molecular model for combinatorial GABAAR assembly in vivo and reveal an assembly pathway regulating GABAAR synaptic localization.
Project description:GABA type A receptors (GABAARs) mediate the majority of fast inhibitory neurotransmission in the central nervous system (CNS). Most prevalent as heteropentamers composed of two ?, two ?, and a ?2 subunit, these ligand-gated ionotropic chloride channels are capable of extensive genetic diversity (?1-6, ?1-3, ?1-3, ?, ?, ?, ?, ?1-3). Part of this selective GABAAR assembly arises from the critical role for ?2 in maintaining synaptic receptor localization and function. Accordingly, mutations in this subunit account for over half of the known epilepsy-associated genetic anomalies identified in GABAARs. Fundamental structure-function studies and cellular pathology investigations have revealed dynamic GABAAR trafficking and synaptic scaffolding as critical regulators of GABAergic inhibition. Here, we introduce in vitro and in vivo findings regarding the specific role of the ?2 subunit in receptor trafficking. We then examine ?2 subunit human genetic variation and assess disease related phenotypes and the potential role of altered GABAAR trafficking. Finally, we discuss new-age imaging techniques and their potential to provide novel insight into critical regulatory mechanisms of GABAAR function.
Project description:In contrast with numerous studies of glutamate receptor-associated proteins and their involvement in the modulation of excitatory synapses, much less is known about mechanisms controlling postsynaptic GABAA receptor (GABAAR) numbers. Using tandem affinity purification from tagged GABAAR ?2 subunit transgenic mice and proteomic analysis, we isolated several GABAAR-associated proteins, including Cleft lip and palate transmembrane protein 1 (Clptm1). Clptm1 interacted with all GABAAR subunits tested and promoted GABAAR trapping in the endoplasmic reticulum. Overexpression of Clptm1 reduced GABAAR-mediated currents in a recombinant system, in cultured hippocampal neurons, and in brain, with no effect on glycine or AMPA receptor-mediated currents. Conversely, knockdown of Clptm1 increased phasic and tonic inhibitory transmission with no effect on excitatory synaptic transmission. Furthermore, altering the expression level of Clptm1 mimicked activity-induced inhibitory synaptic scaling. Thus, in complement to other GABAAR-associated proteins that promote receptor surface expression, Clptm1 limits GABAAR forward trafficking and regulates inhibitory homeostatic plasticity.
Project description:The cell adhesion molecule Neuroligin2 (NL2) is localized selectively at GABAergic synapses, where it interacts with the scaffolding protein gephyrin in the post-synaptic density. However, the role of this interaction for formation and plasticity of GABAergic synapses is unclear. Here, we demonstrate that endogenous NL2 undergoes proline-directed phosphorylation at its unique S714-P consensus site, leading to the recruitment of the peptidyl-prolyl cis-trans isomerase Pin1. This signalling cascade negatively regulates NL2's ability to interact with gephyrin at GABAergic post-synaptic sites. As a consequence, enhanced accumulation of NL2, gephyrin and GABAA receptors was detected at GABAergic synapses in the hippocampus of Pin1-knockout mice (Pin1-/-) associated with an increase in amplitude of spontaneous GABAA-mediated post-synaptic currents. Our results suggest that Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating NL2/gephyrin interaction.
Project description:Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive motor symptoms that are preceded by cognitive deficits and is considered as a disorder that primarily affects forebrain striatal neurons. To gain a better understanding of the molecular and cellular mechanisms associated with disease progression, we analyzed the expression of proteins involved in GABAergic neurotransmission in the striatum of the R6/1 transgenic mouse model. Western blot, quantitative PCR and immunohistochemical analyses were conducted on male R6/1 mice and age-matched wild type littermates. Analyses were performed on 2 and 6 month-old animals, respectively, before and after the onset of motor symptoms. Expression of GAD 67, GAD 65, NL2, or gephyrin proteins, involved in GABA synthesis or synapse formation did not display major changes. In contrast, expression of ?1, ?3 and ?5 GABAAR subunits was increased while the expression of ? was decreased, suggesting a change in tonic- and phasic inhibitory transmission. Western blot analysis of the striatum from 8 month-old Hdh Q111, a knock-in mouse model of HD with mild deficits, confirmed the ?1 subunit increased expression. From immunohistochemical analyses, we also found that ?1 subunit expression is increased in medium-sized spiny projection neurons (MSN) and decreased in parvalbumin (PV)-expressing interneurons at 2 and 6 months in R6/1 mice. Moreover, ?2 subunit labeling on the PV and MSN cell membranes was increased at 2 months and decreased at 6 months. Alteration of gene expression in the striatum and modification of GABAA receptor subtypes in both interneurons and projection neurons suggested that HD mutation has a profound effect on synaptic plasticity at an early stage, before the onset of motor symptoms. These results also indicate that cognitive and other behavioral deficits may be associated with changes in GABAergic neurotransmission that consequently could be a relevant target for early therapeutic treatment.
Project description:Despite 50+ years of clinical use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of ?2 subunit containing GABA type A receptors (GABAARs). Here we define key molecular events impacting ?2 GABAAR and the inhibitory synapse gephyrin scaffold following initial sustained BZD exposure in vitro and in vivo. Using immunofluorescence and biochemical experiments, we found that cultured cortical neurons treated with the classical BZD, diazepam (DZP), presented no substantial change in surface or synaptic levels of ?2-GABAARs. In contrast, both ?2 and the postsynaptic scaffolding protein gephyrin showed diminished total protein levels following a single DZP treatment in vitro and in mouse cortical tissue. We further identified DZP treatment enhanced phosphorylation of gephyrin Ser270 and increased generation of gephyrin cleavage products. Selective immunoprecipitation of ?2 from cultured neurons revealed enhanced ubiquitination of this subunit following DZP exposure. To assess novel trafficking responses induced by DZP, we employed a ?2 subunit containing an N terminal fluorogen-activating peptide (FAP) and pH-sensitive green fluorescent protein (?2pHFAP). Live-imaging experiments using ?2pHFAP GABAAR expressing neurons identified enhanced lysosomal targeting of surface GABAARs and increased overall accumulation in vesicular compartments in response to DZP. Using fluorescence resonance energy transfer (FRET) measurements between ?2 and ?2 subunits within a GABAAR in neurons, we identified reductions in synaptic clusters of this subpopulation of surface BZD sensitive receptor. Additional time-series experiments revealed the gephyrin regulating kinase ERK was inactivated by DZP at multiple time points. Moreover, we found DZP simultaneously enhanced synaptic exchange of both ?2-GABAARs and gephyrin using fluorescence recovery after photobleaching (FRAP) techniques. Finally we provide the first proteomic analysis of the BZD sensitive GABAAR interactome in DZP vs. vehicle treated mice. Collectively, our results indicate DZP exposure elicits down-regulation of gephyrin scaffolding and BZD sensitive GABAAR synaptic availability via multiple dynamic trafficking processes.
Project description:We have found that the large intracellular loop of the γ2 GABAA receptor (R) subunit (γ2IL) interacts with RNF34 (an E3 ubiquitin ligase), as shown by yeast two-hybrid and in vitro pulldown assays. In brain extracts, RNF34 co-immunoprecipitates with assembled GABAARs. In co-transfected HEK293 cells, RNF34 reduces the expression of the γ2 GABAAR subunit by increasing the ratio of ubiquitinated/nonubiquitinated γ2. Mutating several lysines of the γ2IL into arginines makes the γ2 subunit resistant to RNF34-induced degradation. RNF34 also reduces the expression of the γ2 subunit when α1 and β3 subunits are co-assembled with γ2. This effect is partially reversed by leupeptin or MG132, indicating that both the lysosomal and proteasomal degradation pathways are involved. Immunofluorescence of cultured hippocampal neurons shows that RNF34 forms clusters and that a subset of these clusters is associated with GABAergic synapses. This association is also observed in the intact rat brain by electron microscopy immunocytochemistry. RNF34 is not expressed until the 2nd postnatal week of rat brain development, being highly expressed in some interneurons. Overexpression of RNF34 in hippocampal neurons decreases the density of γ2 GABAAR clusters and the number of GABAergic contacts that these neurons receive. Knocking down endogenous RNF34 with shRNA leads to increased γ2 GABAAR cluster density and GABAergic innervation. The results indicate that RNF34 regulates postsynaptic γ2-GABAAR clustering and GABAergic synaptic innervation by interacting with and ubiquitinating the γ2-GABAAR subunit promoting GABAAR degradation.
Project description:Neuroligins are cell adhesion molecules involved in synapse formation and/or function. Neurons express four neuroligins (NL1-NL4), of which NL1 is specific to excitatory and NL2 to inhibitory synapses. Excitatory and inhibitory synapses include numerous subtypes. However, it is unknown whether NL1 performs similar functions in all excitatory and NL2 in all inhibitory synapses, or whether they regulate the formation and/or function of specific subsets of synapses. To address this central question, we performed paired recordings in primary somatosensory cortex of mice lacking NL1 or NL2. Using this system, we examined neocortical microcircuits formed by reciprocal synapses between excitatory neurons and two subtypes of inhibitory interneurons, namely, fast-spiking and somatostatin-positive interneurons. We find that the NL1 deletion had little effect on inhibitory synapses, whereas the NL2 deletion decreased (40-50%) the unitary (cell-to-cell) IPSC amplitude evoked from single fast-spiking interneurons. Strikingly, the NL2 deletion had no effect on IPSC amplitude evoked from single somatostatin-positive inhibitory interneurons. Moreover, the frequency of unitary synaptic connections between individual fast-spiking and somatostatin-positive interneurons and excitatory neurons was unchanged. The decrease in unitary IPSC amplitude originating from fast-spiking interneurons in NL2-deficient mice was due to a multiplicative and uniform downscaling of the amplitude distribution, which in turn was mediated by a decrease in both synaptic quantal amplitude and quantal content, the latter inferred from an increase in the coefficient of variation. Thus, NL2 is not necessary for establishing unitary inhibitory synaptic connections but is selectively required for "scaling up" unitary connections originating from a subset of interneurons.
Project description:Synaptic transmission depends on the matching and alignment of presynaptically released transmitters and postsynaptic neurotransmitter receptors. Neuroligin (NL) and Neurexin (Nrxn) proteins are trans-synaptic adhesion molecules that are important in validation and maturation of specific synapses. NL isoforms NL1 and NL2 have specific functional roles in excitatory and inhibitory synapses, respectively, but the molecular basis behind this distinction is still unclear. We show here that the extracellular domain of NL2 confers its unique ability to enhance inhibitory synaptic function when overexpressed in rat hippocampal pyramidal neurons, whereas NL1 normally only promotes excitatory synapses. This specificity is conferred by presynaptic Nrxn isoforms, as NL1 can also induce functional inhibitory synapse connections when the presynaptic interneurons ectopically express an Nrxn isoform that binds to NL1. Our results indicate that trans-synaptic interaction with differentially expressed presynaptic Nrxns underlies the distinct functions of NL1 and NL2, and is sufficient to induce functional inhibitory synapse formation.
Project description:GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. Here, we show that postnatal Purkinje cells (PCs) of GABA(A)alpha1 knockout (KO) mice express transiently the alpha3 subunit, leading to the assembly of functional GABA(A) receptors and initial normal formation of inhibitory synapses, that are retained until adulthood. Subsequently, down-regulation of the alpha3 subunit causes a complete loss of GABAergic postsynaptic currents, resulting in a decreased rate of inhibitory synaptogenesis and formation of mismatched synapses between GABAergic axons and PC spines. Notably, the postsynaptic adhesion molecule neuroligin-2 (NL2) is correctly targeted to inhibitory synapses lacking GABA(A) receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. Our data indicate that GABA(A) receptors are dispensable for synapse formation and maintenance and for targeting NL2 to inhibitory synapses. However, GABAergic signaling appears to be crucial for activity-dependent regulation of synapse density during neuronal maturation.
Project description:GABA type A receptors (GABAARs) mediate fast synaptic inhibition and are trafficked to functionally diverse synapses. However, the precise molecular mechanisms that regulate the synaptic targeting of these receptors are unclear. Whereas it has been previously shown that phosphorylation events in ?4, ?, and ? subunits of GABAARs govern their function and trafficking, phosphorylation of other subunits has not yet been demonstrated. Here, we show that the ?2 subunit of GABAARs is phosphorylated at Ser-359 and enables dynamic regulation of GABAAR binding to the scaffolding proteins gephyrin and collybistin. We initially identified Ser-359 phosphorylation by MS analysis, and additional experiments revealed that it is regulated by the activities of cAMP-dependent protein kinase (PKA) and the protein phosphatase 1 (PP1) and/or PP2A. GST-based pulldowns and coimmunoprecipitation experiments demonstrate preferential binding of both gephyrin and collybistin to WT and an S359A phosphonull variant, but not to an S359D phosphomimetic variant. Furthermore, the decreased capacity of the ?2 S359D variant to bind collybistin and gephyrin decreased the density of synaptic ?2-containing GABAAR clusters and caused an absence of ?2 enrichment in the axon initial segment. These results suggest that PKA-mediated phosphorylation and PP1/PP2A-dependent dephosphorylation of the ?2 subunit play a role in the dynamic regulation of GABAAR accumulation at inhibitory synapses, thereby regulating the strength of synaptic inhibition. The MS data have been deposited to ProteomeXchange, with the data set identifier PXD019597.