RESUMO
It has been shown that subunit composition is the main determinant of the synaptic or extrasynaptic localization of GABAA receptors (GABAARs). Synaptic and extrasynaptic GABAARs are involved in phasic and tonic inhibition, respectively. It has been proposed that synaptic GABAARs bind to the postsynaptic gephyrin/collybistin (Geph/CB) lattice, but not the typically extrasynaptic GABAARs. Nevertheless, there are no studies of the direct binding of various types of GABAARs with the submembranous Geph/CB lattice in the absence of other synaptic proteins, some of which are known to interact with GABAARs. We have reconstituted GABAARs of various subunit compositions, together with the Geph/CB scaffold, in HEK293 cells, and have investigated the recruitment of surface GABAARs by submembranous Geph/CB clusters. Results show that the typically synaptic α1ß3γ2 GABAARs were trapped by submembranous Geph/CB clusters. The α5ß3γ2 GABAARs, which are both synaptic and extrasynaptic, were also trapped by Geph/CB clusters. Extrasynaptic α4ß3δ GABAARs consistently showed little or no trapping by the Geph/CB clusters. However, the extrasynaptic α6ß3δ, α1ß3, α6ß3 (and less α4ß3) GABAARs were highly trapped by the Geph/CB clusters. AMPA and NMDA glutamate receptors were not trapped. The results suggest: (I) in the absence of other synaptic molecules, the Geph/CB lattice has the capacity to trap not only synaptic but also several typically extrasynaptic GABAARs; (II) the Geph/CB lattice is important but does not play a decisive role in the synaptic localization of GABAARs; and (III) in neurons there must be mechanisms preventing the trapping of several typically extrasynaptic GABAARs by the postsynaptic Geph/CB lattice.
Assuntos
Receptores de GABA-A , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Sinapses , Membrana Celular/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
Collybistin (CB) is a guanine nucleotide exchange factor (GEF) selectively localized at GABAergic and glycinergic postsynapses. Analysis of mRNA shows that several isoforms of collybistin are expressed in the brain. Some of the isoforms have a SH3 domain (CBSH3+) and some have no SH3 domain (CBSH3-). The CBSH3+ mRNAs are predominantly expressed over CBSH3-. However, in an immunoblot study of mouse brain homogenates, only CBSH3+ protein isoforms were detected, proposing that CBSH3- protein might not be expressed in the brain. The expression or lack of expression of CBSH3- protein is an important issue because CBSH3- has a strong effect in promoting the postsynaptic clustering of gephyrin and GABA-A receptors (GABAA Rs). Moreover CBSH3- is constitutively active; therefore lower expression of CBSH3- protein might play a relatively stronger functional role than the more abundant but self-inhibited CBSH3+ isoforms, which need to be activated. We are now showing that: (a) CBSH3- protein is expressed in the brain; (b) parvalbumin positive (PV+) interneurons show higher expression of CBSH3- protein than other neurons; (c) CBSH3- is associated with GABAergic synapses in various regions of the brain and (d) knocking down CBSH3- in hippocampal neurons decreases the synaptic clustering of gephyrin and GABAA Rs. The results show that CBSH3- protein is expressed in the brain and that it plays a significant role in the size regulation of the GABAergic postsynapse.
Assuntos
Encéfalo/metabolismo , Proteínas de Membrana/metabolismo , Receptores de GABA-A/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Sinapses/metabolismo , Animais , Masculino , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Domínios de Homologia de srcRESUMO
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 ß3 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.
Assuntos
Proteínas de Transporte/metabolismo , Receptores de GABA-B/metabolismo , Animais , Encéfalo/embriologia , Regulação da Expressão Gênica , Cobaias , Células HEK293 , Hipocampo/embriologia , Hipocampo/metabolismo , Humanos , Lisossomos/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico , Ratos , Sinapses/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
We have found that the γ2 subunit of the GABA(A) receptor (γ2-GABA(A)R) specifically interacts with protocadherin-γC5 (Pcdh-γC5) in the rat brain. The interaction occurs between the large intracellular loop of the γ2-GABA(A)R and the cytoplasmic domain of Pcdh-γC5. In brain extracts, Pcdh-γC5 coimmunoprecipitates with GABA(A)Rs. In cotransfected HEK293 cells, Pcdh-γC5 promotes the transfer of γ2-GABA(A)R to the cell surface. We have previously shown that, in cultured hippocampal neurons, endogenous Pcdh-γC5 forms clusters, some of which associate with GABAergic synapses. Overexpression of Pcdh-γC5 in hippocampal neurons increases the density of γ2-GABA(A)R clusters but has no significant effect on the number of GABAergic contacts that these neurons receive, indicating that Pcdh-γC5 is not synaptogenic. Deletion of the cytoplasmic domain of Pcdh-γC5 enhanced its surface expression but decreased the association with both γ2-GABA(A)R clusters and presynaptic GABAergic contacts. Cultured hippocampal neurons from the Pcdh-γ triple C-type isoform knock-out (TCKO) mouse (Pcdhg(tcko/tcko)) showed plenty of GABAergic synaptic contacts, although their density was reduced compared with sister cultures from wild-type and heterozygous mice. Knocking down Pcdh-γC5 expression with shRNA decreased γ2-GABA(A)R cluster density and GABAergic innervation. The results indicate that, although Pcdh-γC5 is not essential for GABAergic synapse formation or GABA(A)R clustering, (1) Pcdh-γC5 regulates the surface expression of GABA(A)Rs via cis-cytoplasmic interaction with γ2-GABA(A)R, and (2) Pcdh-γC5 plays a role in the stabilization and maintenance of some GABAergic synapses.
Assuntos
Caderinas/metabolismo , Receptores de GABA-A/metabolismo , Animais , Biotinilação , Proteínas Relacionadas a Caderinas , Caderinas/genética , Linhagem Celular Transformada , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica/genética , Glutamato Descarboxilase/metabolismo , Proteínas de Fluorescência Verde/genética , Guanilato Quinases/metabolismo , Hipocampo/citologia , Humanos , Imunoprecipitação , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Receptores de GABA-A/genética , Transfecção , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismoRESUMO
GABA(A) receptors (GABA(A)-Rs) are localized at both synaptic and extrasynaptic sites, mediating phasic and tonic inhibition, respectively. Previous studies suggest an important role of γ2 and δ subunits in synaptic versus extrasynaptic targeting of GABA(A)-Rs. Here, we demonstrate differential function of α2 and α6 subunits in guiding the localization of GABA(A)-Rs. To study the targeting of specific subtypes of GABA(A)-Rs, we used a molecularly engineered GABAergic synapse model to precisely control the GABA(A)-R subunit composition. We found that in neuron-HEK cell heterosynapses, GABAergic events mediated by α2ß3γ2 receptors were very fast (rise time â¼2 ms), whereas events mediated by α6ß3δ receptors were very slow (rise time â¼20 ms). Such an order of magnitude difference in rise time could not be attributed to the minute differences in receptor kinetics. Interestingly, synaptic events mediated by α6ß3 or α6ß3γ2 receptors were significantly slower than those mediated by α2ß3 or α2ß3γ2 receptors, suggesting a differential role of α subunit in receptor targeting. This was confirmed by differential targeting of the same δ-γ2 chimeric subunits to synaptic or extrasynaptic sites, depending on whether it was co-assembled with the α2 or α6 subunit. In addition, insertion of a gephyrin-binding site into the intracellular domain of α6 and δ subunits brought α6ß3δ receptors closer to synaptic sites. Therefore, the α subunits, together with the γ2 and δ subunits, play a critical role in governing synaptic versus extrasynaptic targeting of GABA(A)-Rs, possibly through differential interactions with gephyrin.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Proteínas de Transporte/genética , Células Cultivadas , Células HEK293 , Humanos , Proteínas de Membrana/genética , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/genética , Sinapses/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
Collybistin promotes submembrane clustering of gephyrin and is essential for the postsynaptic localization of gephyrin and γ-aminobutyric acid type A (GABA(A)) receptors at GABAergic synapses in hippocampus and amygdala. Four collybistin isoforms are expressed in brain neurons; CB2 and CB3 differ in the C terminus and occur with and without the Src homology 3 (SH3) domain. We have found that in transfected hippocampal neurons, all collybistin isoforms (CB2(SH3+), CB2(SH3-), CB3(SH3+), and CB3(SH3-)) target to and concentrate at GABAergic postsynapses. Moreover, in non-transfected neurons, collybistin concentrates at GABAergic synapses. Hippocampal neurons co-transfected with CB2(SH3-) and gephyrin developed very large postsynaptic gephyrin and GABA(A) receptor clusters (superclusters). This effect was accompanied by a significant increase in the amplitude of miniature inhibitory postsynaptic currents. Co-transfection with CB2(SH3+) and gephyrin induced the formation of many (supernumerary) non-synaptic clusters. Transfection with gephyrin alone did not affect cluster number or size, but gephyrin potentiated the clustering effect of CB2(SH3-) or CB2(SH3+). Co-transfection with CB2(SH3-) or CB2(SH3+) and gephyrin did not affect the density of presynaptic GABAergic terminals contacting the transfected cells, indicating that collybistin is not synaptogenic. Nevertheless, the synaptic superclusters induced by CB2(SH3-) and gephyrin were accompanied by enlarged presynaptic GABAergic terminals. The enhanced clustering of gephyrin and GABA(A) receptors induced by collybistin isoforms was not accompanied by enhanced clustering of neuroligin 2. Moreover, during the development of GABAergic synapses, the clustering of gephyrin and GABA(A) receptors preceded the clustering of neuroligin 2. We propose a model in which the SH3- isoforms play a major role in the postsynaptic accumulation of GABA(A) receptors and in GABAergic synaptic strength.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Animais , Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Células HEK293 , Humanos , Potenciais Pós-Sinápticos Inibidores , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Fatores de Troca de Nucleotídeo Guanina Rho , Transfecção , Ácido gama-Aminobutírico/metabolismo , Domínios de Homologia de srcRESUMO
Collybistin (CB) is a rho guanine exchange factor found at GABAergic and glycinergic postsynapses that interacts with the inhibitory scaffold protein, gephyrin, and induces accumulation of gephyrin and GABA type-A receptors (GABAARs) to the postsynapse. We have previously reported that the isoform without the src homology 3 (SH3) domain, CBSH3-, is particularly active in enhancing the GABAergic postsynapse in both cultured hippocampal neurons as well as in cortical pyramidal neurons after chronic in vivo expression in in utero electroporated (IUE) rats. Deficiency of CB in knock-out (KO) mice results in absence of gephyrin and gephyrin-dependent GABAARs at postsynaptic sites in several brain regions, including hippocampus. In the present study, we have generated an adeno-associated virus (AAV) that expresses CBSH3- in a cre-dependent manner. Using male and female VGLUT1-IRES-cre or VGAT-IRES-cre mice, we explore the effect of overexpression of CBSH3- in hippocampal pyramidal cells or hippocampal interneurons. The results show that: (1) the accumulation of gephyrin and GABAARs at inhibitory postsynapses in hippocampal pyramidal neurons or interneurons can be enhanced by CBSH3- overexpression; (2) overexpression of CBSH3- in hippocampal pyramidal cells can enhance the strength of inhibitory neurotransmission; and (3) these enhanced inhibitory synapses provide protection against pentylenetetrazole (PTZ)-induced seizures. The results indicate that this AAV vector carrying CBSH3- can be used for in vivo enhancement of GABAergic synaptic transmission in selected target neurons in the brain.
Assuntos
Proteínas de Transporte , Pentilenotetrazol , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Feminino , Hipocampo/metabolismo , Masculino , Camundongos , Pentilenotetrazol/toxicidade , Células Piramidais/metabolismo , Ratos , Receptores de GABA-A/metabolismo , Convulsões/induzido quimicamente , Sinapses/metabolismo , Transmissão SinápticaRESUMO
Mass spectrometry and immunoblot analysis of a rat brain fraction enriched in type-II postsynaptic densities and postsynaptic GABAergic markers showed enrichment in the protein septin 11. Septin 11 is expressed throughout the brain, being particularly high in the spiny branchlets of the Purkinje cells in the molecular layer of cerebellum and in the olfactory bulb. Immunofluorescence of cultured hippocampal neurons showed that 54 +/- 4% of the GABAergic synapses and 25 +/- 2% of the glutamatergic synapses had colocalizing septin 11 clusters. Similar colocalization numbers were found in the molecular layer of cerebellar sections. In cultured hippocampal neurons, septin 11 clusters were frequently present at the base of dendritic protrusions and at the bifurcation points of the dendritic branches. Electron microscopy immunocytochemistry of the rat brain cerebellum revealed the accumulation of septin 11 at the neck of dendritic spines, at the bifurcation of dendritic branches, and at some GABAergic synapses. Knocking down septin 11 in cultured hippocampal neurons with septin 11 small hairpin RNAs showed (i) reduced dendritic arborization; (ii) decreased density and increased length of dendritic protrusions; and (iii) decreased GABAergic synaptic contacts that these neurons receive. The results indicate that septin 11 plays important roles in the cytoarchitecture of neurons, including dendritic arborization and dendritic spines, and that septin 11 also plays a role in GABAergic synaptic connectivity.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Células Cultivadas , Cerebelo/metabolismo , Cerebelo/ultraestrutura , Clonagem Molecular , Dendritos/metabolismo , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , Hipocampo/citologia , Hipocampo/metabolismo , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SeptinasRESUMO
It has been proposed that the combinatorial expression of γ-protocadherins (Pcdh-γs) and other clustered protocadherins (Pcdhs) provides a code of molecular identity and individuality to neurons, which plays a major role in the establishment of specific synaptic connectivity and formation of neuronal circuits. Particular attention has been directed to the Pcdh-γ family, for which experimental evidence derived from Pcdh-γ-deficient mice shows that they are involved in dendrite self-avoidance, synapse development, dendritic arborization, spine maturation, and prevention of apoptosis of some neurons. Moreover, a triple-mutant mouse deficient in the three C-type members of the Pcdh-γ family (Pcdh-γC3, Pcdh-γC4, and Pcdh-γC5) shows a phenotype similar to the mouse deficient in whole Pcdh-γ family, indicating that the latter is largely due to the absence of C-type Pcdh-γs. The role of each individual C-type Pcdh-γ is not known. We have developed a specific antibody to Pcdh-γC4 to reveal the expression of this protein in the rat brain. The results show that although Pcdh-γC4 is expressed at higher levels in the embryo and earlier postnatal weeks, it is also expressed in the adult rat brain. Pcdh-γC4 is expressed in both neurons and astrocytes. In the adult brain, the regional distribution of Pcdh-γC4 immunoreactivity is similar to that of Pcdh-γC4 mRNA, being highest in the olfactory bulb, dentate gyrus, and cerebellum. Pcdh-γC4 forms puncta that are frequently apposed to glutamatergic and GABAergic synapses. They are also frequently associated with neuron-astrocyte contacts. The results provide new insights into the cell recognition function of Pcdh-γC4 in neurons and astrocytes.
Assuntos
Encéfalo/metabolismo , Caderinas/biossíntese , Animais , Astrócitos/metabolismo , Proteínas Relacionadas a Caderinas , Feminino , Masculino , Camundongos , Neurônios/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Local neocortical circuits play critical roles in information processing, including synaptic plasticity, circuit physiology, and learning, and GABAergic inhibitory interneurons have key roles in these circuits. Moreover, specific neurological disorders, including schizophrenia and autism, are associated with deficits in GABAergic transmission in these circuits. GABAergic synapses represent a small fraction of neocortical synapses, and are embedded in complex local circuits that contain many neuron and synapse types. Thus, it is challenging to study the physiological roles of GABAergic inhibitory interneurons and their synapses, and to develop treatments for the specific disorders caused by dysfunction at these GABAergic synapses. To these ends, we report a novel technology that can deliver different genes into pre- and post-synaptic neocortical interneurons connected by a GABAergic synapse: First, standard gene transfer into the presynaptic neurons delivers a synthetic peptide neurotransmitter, containing three domains, a dense core vesicle sorting domain, a GABAA receptor-binding domain, a single-chain variable fragment anti-GABAA ß2 or ß3, and the His tag. Second, upon release, this synthetic peptide neurotransmitter binds to GABAA receptors on the postsynaptic neurons. Third, as the synthetic peptide neurotransmitter contains the His tag, antibody-mediated, targeted gene transfer using anti-His tag antibodies is selective for these neurons. We established this technology by expressing the synthetic peptide neurotransmitter in GABAergic neurons in the middle layers of postrhinal cortex, and the delivering the postsynaptic vector into connected GABAergic neurons in the upper neocortical layers. Targeted gene transfer was 61% specific for the connected neurons, but untargeted gene transfer was only 21% specific for these neurons. This technology may support studies on the roles of GABAergic inhibitory interneurons in circuit physiology and learning, and support gene therapy treatments for specific disorders associated with deficits at GABAergic synapses.
Assuntos
Neurônios GABAérgicos/metabolismo , Interneurônios/metabolismo , Neocórtex/metabolismo , Neurotransmissores/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-B/genética , Sinapses/metabolismo , Animais , Técnicas de Transferência de Genes , Vetores Genéticos , Camundongos , Neurotransmissores/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Receptores de GABA-A/imunologia , Receptores de GABA-A/metabolismo , Receptores de GABA-B/imunologia , Receptores de GABA-B/metabolismo , Anticorpos de Cadeia Única/imunologiaRESUMO
The recruitment of inhibitory GABAA receptors to neuronal synapses requires a complex interplay between receptors, neuroligins, the scaffolding protein gephyrin and the GDP-GTP exchange factor collybistin (CB). Collybistin is regulated by protein-protein interactions at the N-terminal SH3 domain, which can bind neuroligins 2/4 and the GABAAR α2 subunit. Collybistin also harbors a RhoGEF domain which mediates interactions with gephyrin and catalyzes GDP-GTP exchange on Cdc42. Lastly, collybistin has a pleckstrin homology (PH) domain, which binds phosphoinositides, such as phosphatidylinositol 3-phosphate (PI3P/PtdIns3P) and phosphatidylinositol 4-monophosphate (PI4P/PtdIns4P). PI3P located in early/sorting endosomes has recently been shown to regulate the postsynaptic clustering of gephyrin and GABAA receptors and consequently the strength of inhibitory synapses in cultured hippocampal neurons. This process is disrupted by mutations in the collybistin gene (ARHGEF9), which cause X-linked intellectual disability (XLID) by a variety of mechanisms converging on disrupted gephyrin and GABAA receptor clustering at central synapses. Here we report a novel missense mutation (chrX:62875607C>T, p.R356Q) in ARHGEF9 that affects one of the two paired arginine residues in the PH domain that were predicted to be vital for binding phosphoinositides. Functional assays revealed that recombinant collybistin CB3SH3- R356Q was deficient in PI3P binding and was not able to translocate EGFP-gephyrin to submembrane microaggregates in an in vitro clustering assay. Expression of the PI3P-binding mutants CB3SH3- R356Q and CB3SH3- R356N/R357N in cultured hippocampal neurones revealed that the mutant proteins did not accumulate at inhibitory synapses, but instead resulted in a clear decrease in the overall number of synaptic gephyrin clusters compared to controls. Molecular dynamics simulations suggest that the p.R356Q substitution influences PI3P binding by altering the range of structural conformations adopted by collybistin. Taken together, these results suggest that the p.R356Q mutation in ARHGEF9 is the underlying cause of XLID in the probands, disrupting gephyrin clustering at inhibitory GABAergic synapses via loss of collybistin PH domain phosphoinositide binding.
RESUMO
We have recently shown that disrupting the expression and post-synaptic clustering of gephyrin in cultured hippocampal pyramidal cells, by either gephyrin RNAi (RNA interference) or over-expression of a dominant negative gephyrin-enhanced green fluorescent protein (EGFP) fusion protein, leads to decreased number of post-synaptic gephyrin and GABA(A) receptor clusters and to reduced GABAergic innervation of these cells. On the other hand, increasing gephyrin expression led to a small increase in the number of gephyrin and GABA(A) receptor clusters and to little or no effect on GABAergic innervation. We are now reporting that altering gephyrin expression and clustering affects the size but not the density of glutamatergic synaptic contacts. Knocking down gephyrin with gephyrin RNAi, or preventing gephyrin clustering by over-expression of the dominant negative gephyrin-enhanced green fluorescent protein fusion protein, leads to larger post-synaptic PSD-95 clusters and larger pre-synaptic glutamatergic terminals. On the other hand, over-expression of gephyrin leads to slightly smaller PSD-95 clusters and pre-synaptic glutamatergic terminals. The change in size of PSD-95 clusters were accompanied by a parallel change in the size of NR2-NMDA receptor clusters. It is concluded that the levels of expression and clustering of gephyrin, a protein that concentrates at the post-synaptic complex of the inhibitory synapses, not only has homotypic effects on GABAergic synaptic contacts, but also has heterotypic effects on glutamatergic synaptic contacts. We are proposing that gephyrin is a counterpart of the post-synaptic glutamatergic scaffold protein PSD-95 in regulating the number and/or size of the excitatory and inhibitory synaptic contacts.
Assuntos
Proteínas de Transporte/fisiologia , Expressão Gênica/fisiologia , Ácido Glutâmico/metabolismo , Proteínas de Membrana/fisiologia , Terminações Pré-Sinápticas/metabolismo , Sinapses/fisiologia , Animais , Proteínas de Transporte/genética , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Embrião de Mamíferos , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Terminações Pré-Sinápticas/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Receptores de GABA-A/genética , Sinapses/efeitos dos fármacos , Transfecção/métodos , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
We have previously shown that the glutamate receptor interacting protein 1 (GRIP1) splice forms GRIP1a/b and GRIP1c4-7 are present at the GABAergic post-synaptic complex. Nevertheless, the role that these GRIP1 protein isoforms play at the GABAergic post-synaptic complex is not known. We are now showing that GRIP1c4-7 and GRIP1a/b interact with gephyrin, the main post-synaptic scaffold protein of GABAergic and glycinergic synapses. Gephyrin coprecipitates with GRIP1c4-7 or GRIP1a/b from rat brain extracts and from extracts of human embryonic kidney 293 cells that have been cotransfected with gephyrin and one of the GRIP1 protein isoforms. Moreover, purified gephyrin binds to purified GRIP1c4-7 or GRIP1a/b, indicating that gephyrin directly interacts with the common region of these GRIP1 proteins, which includes PDZ domains 4-7. An engineered deletion construct of GRIP1a/b (GRIP1a4-7), which both contains the aforementioned common region and binds to gephyrin, targets to the post-synaptic GABAergic complex of transfected cultured hippocampal neurons. In these hippocampal cultures, endogenous gephyrin colocalizes with endogenous GRIP1c4-7 and GRIP1a/b in over 90% of the GABAergic synapses. Double-labeling electron microscopy immunogold reveals that in the rat brain GRIP1c4-7 and GRIP1a/b colocalize with gephyrin at the post-synaptic complex of individual synapses. These results indicate that GRIP1c4-7 and GRIP1a/b colocalize and interact with gephyrin at the GABAergic post-synaptic complex and suggest that this interaction plays a role in GABAergic synaptic function.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transmissão Sináptica/genética , Ácido gama-Aminobutírico/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Linhagem Celular , Células Cultivadas , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Ligação Proteica/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Transmissão Sináptica/fisiologia , TransfecçãoRESUMO
In this article we present a comprehensive review of relevant research and reports on the GABA(A) receptor in the aged and Alzheimer's disease (AD) brain. In comparison to glutamatergic and cholinergic systems, the GABAergic system is relatively spared in AD, but the precise mechanisms underlying differential vulnerability are not well understood. Using several methods, investigations demonstrate that despite resistance of the GABAergic system to neurodegeneration, particular subunits of the GABA(A) receptor are altered with age and AD, which can induce compensatory increases in GABA(A) receptor subunits within surrounding cells. We conclude that although aging- and disease-related changes in GABA(A) receptor subunits may be modest, the mechanisms that compensate for these changes may alter the pharmacokinetic and physiological properties of the receptor. It is therefore crucial to understand the subunit composition of individual GABA(A) receptors in the diseased brain when developing therapeutics that act at these receptors.
Assuntos
Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/metabolismo , Receptores de GABA-A/fisiologia , Ácido gama-Aminobutírico/metabolismo , Doença de Alzheimer/patologia , Animais , Humanos , Subunidades Proteicas/fisiologia , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/fisiologiaRESUMO
Collybistin (CB) is a guanine nucleotide exchange factor selectively localized to γ-aminobutyric acid (GABA)ergic and glycinergic postsynapses. Active CB interacts with gephyrin, inducing the submembranous clustering and the postsynaptic accumulation of gephyrin, which is a scaffold protein that recruits GABAA receptors (GABAA Rs) at the postsynapse. CB is expressed with or without a src homology 3 (SH3) domain. We have previously reported the effects on GABAergic synapses of the acute overexpression of CBSH3- or CBSH3+ in cultured hippocampal (HP) neurons. In the present communication, we are studying the effects on GABAergic synapses after chronic in vivo transgenic expression of CB2SH3- or CB2SH3+ in neurons of the adult rat cerebral cortex. The embryonic precursors of these cortical neurons were in utero electroporated with CBSH3- or CBSH3+ DNAs, migrated to the appropriate cortical layer, and became integrated in cortical circuits. The results show that: 1) the strength of inhibitory synapses in vivo can be enhanced by increasing the expression of CB in neurons; and 2) there are significant differences in the results between in vivo and in culture studies. J. Comp. Neurol. 525:1291-1311, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Córtex Cerebral/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Animais , Córtex Cerebral/crescimento & desenvolvimento , Embrião de Mamíferos , Feminino , Imunofluorescência , Processamento de Imagem Assistida por Computador , Masculino , Microscopia Confocal , Técnicas de Patch-Clamp , Ratos , Ratos Transgênicos , Ratos Wistar , Sinapses/metabolismoRESUMO
The alpha5 subunit of the GABA(A) receptors (GABA(A)Rs) has a restricted expression in the brain. Maximum expression of this subunit occurs in the hippocampus, cerebral cortex, and olfactory bulb. Hippocampal pyramidal cells show high expression of alpha5 subunit-containing GABA(A)Rs (alpha5-GABA(A)Rs) both in culture and in the intact brain. A large pool of alpha5-GABA(A)Rs is extrasynaptic and it has been proposed to be involved in the tonic GABAergic inhibition of the hippocampus. Nevertheless, there are no studies on the localization of the alpha5-GABA(A)Rs at the electron microscope (EM) level. By using both immunofluorescence of cultured hippocampal pyramidal cells and EM postembedding immunogold of the intact hippocampus we show that, in addition to the extrasynaptic pool, there is a pool of alpha5-GABA(A)Rs that concentrates at the GABAergic synapses in dendrites of hippocampal pyramidal cells. The results suggest that the synaptic alpha5-GABA(A)Rs might play a role in the phasic GABAergic inhibition of pyramidal neurons in hippocampus and cerebral cortex.
Assuntos
Hipocampo/metabolismo , Inibição Neural/fisiologia , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/metabolismo , Córtex Cerebral/ultraestrutura , Dendritos/metabolismo , Dendritos/ultraestrutura , Imunofluorescência , Hipocampo/ultraestrutura , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Transmissão , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , Ratos , Ratos Sprague-Dawley , Sinapses/ultraestrutura , Membranas Sinápticas/metabolismo , Membranas Sinápticas/ultraestruturaRESUMO
We have studied the effects of GABAergic innervation on the clustering of GABA(A) receptors (GABA(A)Rs) in cultured hippocampal neurons. In the absence of GABAergic innervation, pyramidal cells form small (0.36 +/- 0.01 micrometer diameter) GABA(A)R clusters at their surface in the dendrites and soma. When receiving GABAergic innervation from glutamic acid decarboxylase-containing interneurons, pyramidal cells form large (1.62 +/- 0.08 micrometer breadth) GABA(A)R clusters at GABAergic synapses. This is accompanied by a disappearance of the small GABA(A)R clusters in the local area surrounding each GABAergic synapse. Although the large synaptic GABA(A)R clusters of any neuron contained all GABA(A)R subunits and isoforms expressed by that neuron, the small clusters not localized at GABAergic synapses showed significant heterogeneity in subunit and isoform composition. Another difference between large GABAergic and small non-GABAergic GABA(A)R clusters was that a significant proportion of the latter was juxtaposed to postsynaptic markers of glutamatergic synapses such as PSD-95 and AMPA receptor GluR1 subunit. The densities of both the glutamate receptor-associated and non-associated small GABA(A)R clusters were decreased in areas surrounding GABAergic synapses. However, no effect on the density or distribution of glutamate receptor clusters was observed. The results suggest that there are local signals generated at GABAergic synapses that induce both assembly of large synaptic GABA(A)R clusters at the synapse and disappearance of the small GABA(A)R clusters in the surrounding area. In the absence of GABAergic innervation, weaker GABA(A)R-clustering signals, generated at glutamatergic synapses, induce the formation of small postsynaptic GABA(A)R clusters that remain juxtaposed to glutamate receptors at glutamatergic synapses.
Assuntos
Hipocampo/metabolismo , Neurônios/metabolismo , Agregação de Receptores/fisiologia , Receptores de GABA-A/metabolismo , Sinapses/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Proteínas de Transporte/metabolismo , Contagem de Células , Células Cultivadas , Dendritos/metabolismo , Imunofluorescência , Glutamato Descarboxilase/metabolismo , Hipocampo/citologia , Interneurônios/metabolismo , Proteínas de Membrana/metabolismo , Neurônios/citologia , Isoformas de Proteínas/metabolismo , Subunidades Proteicas , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos , Ratos Wistar , Receptores de GABA-A/classificação , Receptores de Glutamato/metabolismoRESUMO
We studied the cellular and subcellular distribution of GABA(A) receptors in the Bergmann glia and Purkinje cells in the molecular layer of the cerebellum by using electron microscopy postembedding immunogold techniques. Gold particles corresponding to alpha2 and gamma1 immunoreactivity were localized in Bergmann glia processes that wrapped Purkinje cell somata, dendritic shafts, and some dendritic spines. The gold particles were mainly located on the glial plasma membrane or intracellularly but near the plasma membrane. The density of gold particles corresponding to alpha2 and gamma1 GABA(A) receptor subunits was 4.3-fold higher in the glial processes wrapping Purkinje cell somata than in the glial processes wrapping Purkinje cell dendritic spines. Moreover, the Bergmann glia GABA(A) receptors were often located in close proximity to the type II GABAergic synapses made by the basket cell axons on Purkinje cell somata. These GABAergic synapses were enriched in neuronal GABA(A) receptors containing alpha1 and beta2/3 subunits. Unexpectedly, 2.8% of the Purkinje cell dendritic spines also showed immunoreactivity for the neuronal alpha1 or beta2/3 subunits, which were located on the spine in type I synapses or extrasynaptically. Double-labeling immunogold experiments showed that approximately 50% of the dendritic spines that were immunolabeled with the neuronal GABA(A) receptors were wrapped by Bergmann glia processes containing glial GABA(A) receptors. These results are consistent with a role of the Bergmann glial GABA(A) receptors in sensing GABAergic synaptic function.
Assuntos
Astrócitos/química , Cerebelo/citologia , Células de Purkinje , Receptores de GABA-A/análise , Sinapses , Animais , Astrócitos/metabolismo , Astrócitos/ultraestrutura , Membrana Celular/química , Extensões da Superfície Celular/química , Extensões da Superfície Celular/ultraestrutura , Cerebelo/química , Cerebelo/ultraestrutura , Dendritos/química , Dendritos/ultraestrutura , Imuno-Histoquímica , Inibição Neural , Subunidades Proteicas , Células de Purkinje/química , Células de Purkinje/ultraestrutura , Ratos , Receptores de GABA-A/imunologia , Receptores de GABA-A/metabolismo , Sinapses/química , Sinapses/ultraestruturaRESUMO
The glutamate receptor-interacting protein GRIP1 is present in glutamatergic synapses and interacts with the GluR2/3/4c subunits of the AMPA receptors. This interaction plays important roles in trafficking, synaptic targeting, and recycling of AMPA receptors as well as in the plasticity of glutamatergic synapses. Although GRIP1 has been shown to be present at GABAergic synapses in cultured neurons, the use of EM (electron microscopy) immunocytochemistry in the intact brain has failed to convincingly reveal the presence of GRIP1 in GABAergic synapses. Therefore, most studies on GRIP1 have focused on glutamatergic synapses. By using mild tissue fixation and embedding in EM, we show that in the intact brain the 7-PDZ domain GRIP1a/b is present not only in glutamatergic synapses but also in GABAergic synapses. In GABAergic synapses GRIP1a/b localizes both at the presynaptic terminals and postsynaptically, being frequently localized on the synaptic membranes or the synaptic junctional complex. Considerably higher density of GRIP1a/b is found in the presynaptic GABAergic terminals than in the glutamatergic terminals, while the density of GRIP1a/b in the postsynaptic complex is similar in both types of synapses. The results also show that the 7-PDZ and the shorter 4-PDZ domain splice forms of GRIP1 (GRIP1c 4-7) frequently colocalize with each other in individual GABAergic and glutamatergic synapses. The results suggest that GRIP1 splice forms might play important roles in brain GABAergic synapses.