RESUMO
GABAB receptor-mediated inhibition is indispensable for maintaining a healthy neuronal excitation/inhibition balance. Many neurological diseases are associated with a disturbed excitation/inhibition balance and downregulation of GABAB receptors due to enhanced sorting of the receptors to lysosomal degradation. A key event triggering the downregulation of the receptors is the phosphorylation of S867 in the GABAB1 subunit mediated by CaMKIIß. Interestingly, close to S867 in GABAB1 exists another phosphorylation site, T872. Therefore, the question arose as to whether phosphorylation of T872 is involved in downregulating the receptors and whether phosphorylation of this site is also mediated by CaMKIIß or by another protein kinase. Here, we show that mutational inactivation of T872 in GABAB1 prevented the degradation of the receptors in cultured neurons. We found that, in addition to CaMKIIß, also ERK1/2 is involved in the degradation pathway of GABAB receptors under physiological and ischemic conditions. In contrast to our previous view, CaMKIIß does not appear to directly phosphorylate S867. Instead, the data support a mechanism in which CaMKIIß activates ERK1/2, which then phosphorylates S867 and T872 in GABAB1. Blocking ERK activity after subjecting neurons to ischemic stress completely restored downregulated GABAB receptor expression to normal levels. Thus, preventing ERK1/2-mediated phosphorylation of S867/T872 in GABAB1 is an opportunity to inhibit the pathological downregulation of the receptors after ischemic stress and is expected to restore a healthy neuronal excitation/inhibition balance.
Assuntos
Sistema de Sinalização das MAP Quinases , Receptores de GABA-B , Fosforilação , Regulação para Baixo , Movimento Celular , Receptores de GABA-B/genética , Ácido gama-AminobutíricoRESUMO
Metabotropic GABA(B) receptors are abundantly expressed at glutamatergic synapses where they control excitability of the synapse. Here, we tested the hypothesis that glutamatergic neurotransmission may regulate GABA(B) receptors. We found that application of glutamate to cultured cortical neurons led to rapid down-regulation of GABA(B) receptors via lysosomal degradation. This effect was mimicked by selective activation of AMPA receptors and further accelerated by coactivation of group I metabotropic glutamate receptors. Inhibition of NMDA receptors, blockade of L-type Ca(2+) channels, and removal of extracellular Ca(2+) prevented glutamate-induced down-regulation of GABA(B) receptors, indicating that Ca(2+) influx plays a critical role. We further established that glutamate-induced down-regulation depends on the internalization of GABA(B) receptors. Glutamate did not affect the rate of GABA(B) receptor endocytosis but led to reduced recycling of the receptors back to the plasma membrane. Blockade of lysosomal activity rescued receptor recycling, indicating that glutamate redirects GABA(B) receptors from the recycling to the degradation pathway. In conclusion, the data indicate that sustained activation of AMPA receptors down-regulates GABA(B) receptors by sorting endocytosed GABA(B) receptors preferentially to lysosomes for degradation on the expense of recycling. This mechanism may relieve glutamatergic synapses from GABA(B) receptor-mediated inhibition resulting in increased synaptic excitability.
Assuntos
Regulação para Baixo , Lisossomos/metabolismo , Receptores de GABA-B/metabolismo , Receptores de Glutamato/metabolismo , Animais , Western Blotting , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/metabolismo , Células Cultivadas , Ácido Egtázico/farmacologia , Endocitose/efeitos dos fármacos , Glutamatos/farmacologia , Imuno-Histoquímica , Transporte de Íons/efeitos dos fármacos , Microscopia Confocal , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nifedipino/farmacologia , Ratos , Ratos Wistar , Receptores de AMPA/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
General consensus states that immunoglobulins are exclusively expressed by B lymphocytes to form the first line of defense against common pathogens. Here, we provide compelling evidence for the expression of two heavy chain immunoglobulin genes in subpopulations of neurons in the mouse brain and spinal cord. RNA isolated from excitatory and inhibitory neurons through ribosome affinity purification revealed Ighg3 and Ighm transcripts encoding for the constant (Fc), but not the variable regions of IgG3 and IgM. Because, in the absence of the variable immunoglobulin regions, these transcripts lack the canonical transcription initiation site used in lymphocytes, we screened for alternative 5' transcription start sites and identified a novel 5' exon adjacent to a proposed promoter element. Immunohistochemical, Western blot, and in silico analyses strongly support that these neuronal transcripts are translated into proteins containing four Immunoglobulin domains. Our data thus demonstrate the expression of two Fc-encoding genes Ighg3 and Ighm in spinal and supraspinal neurons of the murine CNS and suggest a hitherto unknown function of the encoded proteins.
Assuntos
Sistema Nervoso Central/metabolismo , Regiões Constantes de Imunoglobulina/genética , Neurônios/metabolismo , Animais , Linfócitos B/metabolismo , Sequência de Bases/genética , Expressão Gênica/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Regiões Constantes de Imunoglobulina/imunologia , Domínios de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Transcriptoma/genéticaRESUMO
Endocytosis is considered as an important mechanism for regulating cell surface numbers and thereby signaling strength of G protein-coupled receptors. Currently, little is known about the endocytotic pathways of GABA(B) receptors in neurons. Here we report that GABA(B) receptors are constitutively internalized presumably via clathrin-dependent endocytosis in cultured cortical neurons. Colocalization of GABA(B) receptors with endosomal marker proteins indicated sorting of GABA(B) receptors from early endosomes to recycling endosomes and to lysosomes. Cell surface biotinylation experiments revealed fast constitutive recycling of GABA(B) receptors as the predominant pathway that was accelerated by the GABA(B) receptor agonist baclofen. Finally, degradation of GABA(B) receptors in lysosomes was demonstrated by their intracellular accumulation upon inhibition of lysosomal proteases and by blocking recycling which resulted in the redirection of receptors to lysosomes for degradation. These data imply rapid constitutive - agonist-accelerated - recycling of GABA(B) receptors presumably via clathrin-coated pits and their final targeting to lysosomes for degradation.
Assuntos
Córtex Cerebral/citologia , Endocitose/fisiologia , Lisossomos/metabolismo , Neurônios/metabolismo , Receptores de GABA-B , Animais , Baclofeno/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Células Cultivadas , Córtex Cerebral/metabolismo , Clatrina/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Endossomos/metabolismo , Feminino , Agonistas GABAérgicos/metabolismo , Agonistas dos Receptores de GABA-B , Humanos , Ionóforos/metabolismo , Leupeptinas/metabolismo , Monensin/metabolismo , Neurônios/citologia , Gravidez , Ratos , Ratos Wistar , Receptores de GABA-B/metabolismoRESUMO
The G protein-coupled GABAB receptors, constituted from GABAB1 and GABAB2 subunits, are important regulators of neuronal excitability by mediating long-lasting inhibition. One factor that determines receptor availability and thereby the strength of inhibition is regulated protein degradation. GABAB receptors are constitutively internalized from the plasma membrane and are either recycled to the cell surface or degraded in lysosomes. Lys-63-linked ubiquitination mediated by the E3 ligase Mind bomb-2 (MIB2) is the signal that sorts GABAB receptors to lysosomes. However, it is unknown how Lys-63-linked ubiquitination and thereby lysosomal degradation of the receptors is regulated. Here, we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) promotes MIB2-mediated Lys-63-linked ubiquitination of GABAB receptors. We found that inhibition of CaMKII in cultured rat cortical neurons increased cell surface GABAB receptors, whereas overexpression of CaMKIIß, but not CaMKIIα, decreased receptor levels. This effect was conveyed by Lys-63-linked ubiquitination of GABAB1 at multiple sites mediated by the E3 ligase MIB2. Inactivation of the CaMKII phosphorylation site on GABAB1(Ser-867) strongly reduced Lys-63-linked ubiquitination of GABAB receptors and increased their cell surface expression, whereas the phosphomimetic mutant GABAB1(S867D) exhibited strongly increased Lys-63-linked ubiquitination and reduced cell surface expression. Finally, triggering lysosomal degradation of GABAB receptors by sustained activation of glutamate receptors, a condition occurring in brain ischemia, was accompanied with a massive increase of GABAB1(Ser-867) phosphorylation-dependent Lys-63-linked ubiquitination of GABAB receptors. These findings indicate that CaMKIIß-dependent Lys-63-linked ubiquitination of GABAB1 at multiple sites controls sorting of GABAB receptors to lysosomes for degradation under physiological and pathological condition.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , Receptores de GABA-B/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Benzilaminas/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Células HEK293 , Humanos , Neurônios/efeitos dos fármacos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ratos , Sulfonamidas/farmacologia , UbiquitinaçãoRESUMO
The NMDA receptor is thought to play a central role in some forms of neuronal plasticity, including the induction of long-term potentiation. NMDA receptor hypofunction can result in mnemonic impairment and has been implicated in the cognitive symptoms of schizophrenia. The activity of NMDA receptors is controlled by its endogenous coagonist glycine, and a local elevation of glycine levels is expected to enhance NMDA receptor function. Here, we achieved this by the generation of a novel mouse line (CamKIIalphaCre;Glyt1tm1.2fl/fl) with a neuron and forebrain selective disruption of glycine transporter 1 (GlyT1). The mutation led to a significant reduction of GlyT1 and a corresponding reduction of glycine reuptake in forebrain samples, without affecting NMDA receptor expression. NMDA (but not AMPA) receptor-evoked EPSCs recorded in hippocampal slices of mutant mice were 2.5 times of those recorded in littermate controls, suggesting that neuronal GlyT1 normally assumes a specific role in the regulation of NMDA receptor responses. Concomitantly, the mutants were less responsive to phencyclidine than controls. The mutation enhanced aversive Pavlovian conditioning without affecting spontaneous anxiety-like behavior in the elevated plus maze and augmented a form of attentional learning called latent inhibition in three different experimental paradigms: conditioned freezing, conditioned active avoidance, conditioned taste aversion. The CamKIIalphaCre;Glyt1tm1.2fl/fl mouse model thus suggests that augmentation of forebrain neuronal glycine transmission is promnesic and may also offer an effective therapeutic intervention against the cognitive and attentional impairments characteristic of schizophrenia.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Glicina/metabolismo , Neurônios/metabolismo , Prosencéfalo/crescimento & desenvolvimento , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/prevenção & controle , Animais , Transtornos de Ansiedade/genética , Atenção/fisiologia , Aprendizagem da Esquiva/fisiologia , Cognição/fisiologia , Modelos Animais de Doenças , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Aprendizagem/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes Neurológicos , Mutação/genética , Técnicas de Cultura de Órgãos , Fenótipo , Prosencéfalo/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Transmissão Sináptica/genética , Regulação para Cima/genéticaRESUMO
Receptor internalization is recognized as an important mechanism for rapidly regulating cell surface numbers of receptors. However, there are conflicting results on the existence of rapid endocytosis of gamma-aminobutyric acid, type B (GABAB) receptors. Therefore, we analyzed internalization of GABAB receptors expressed in HEK 293 cells qualitatively and quantitatively using immunocytochemical, cell surface enzyme-linked immunosorbent assay, and biotinylation methods. The data indicate the existence of rapid constitutive receptor internalization, with the first endocytosed receptors being observed in proximity of the plasma membrane after 10 min. After 120 min, a loss of about 40-50% of cell surface receptors was detected. Stimulation of GABAB receptors with GABA or baclofen did not enhance endocytosis of receptors, indicating the lack of agonist-induced internalization. The data suggest that GABAB receptors were endocytosed via the classical dynamin- and clathrin-dependent pathway and accumulated in an endosomal sorting compartment before being targeted to lysosomes for degradation. No evidence for recycling of receptors back to the cell surface was found. In conclusion, the results indicate the presence of constitutive internalization of GABAB receptors via clathrin-coated pits, which resulted in lysosomal degradation of the receptors.
Assuntos
Clatrina/fisiologia , Endocitose , Lisossomos/fisiologia , Receptores de GABA/metabolismo , Linhagem Celular , Vesículas Revestidas por Clatrina , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Transporte ProteicoRESUMO
The metabotropic gamma-aminobutyric acid, type B (GABA(B)) receptors mediate the slow component of GABAergic transmission in the brain. Functional GABA(B) receptors are heterodimers of the two subunits GABA(B1) and GABA(B2), of which GABA(B1) exists in two main isoforms, GABA(B1a) and GABA(B1b). The significance of the structural heterogeneity of GABA(B) receptors, the mechanism leading to their differential targeting in neurons as well as the regulation of cell surface numbers of GABA(B) receptors, is poorly understood. To gain insights into these processes, we searched for proteins interacting with the C-terminal domain of GABA(B2). Here, we showed that the transcription factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) directly interacts with GABA(B) receptors in a subtype-selective manner to regulate cell surface expression of GABA(B1a)/GABA(B2) receptors upon co-expression in HEK 293 cells. The interaction of CHOP with GABA(B1a)/GABA(B2) receptors resulted in their intracellular accumulation and in a reduced number of cell surface receptors. This regulation required the interaction of CHOP via two distinct domains with the heterodimeric receptor; its C-terminal leucine zipper associates with the leucine zipper present in the C-terminal domain of GABA(B2), and its N-terminal domain associates with an as yet unidentified site on GABA(B1a). In conclusion, the data indicated a subtype-selective regulation of cell surface receptors by interaction with the transcription factor CHOP.
Assuntos
Membrana Celular/metabolismo , Regulação da Expressão Gênica , Receptores de GABA-B/metabolismo , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Deleção de Genes , Hipocampo/citologia , Humanos , Zíper de Leucina , Microscopia de Fluorescência , Mutagênese Insercional , Neurônios/metabolismo , Testes de Precipitina , Ratos , Receptores de GABA-B/genéticaRESUMO
BACKGROUND: Accumulating evidence pinpoints to the pivotal role of mitogen-activated protein kinases (MAPKs) in the signal transduction underlying cardiac preconditioning. METHODS: PD98059, an inhibitor of extracellular signal-regulated protein kinase (MEK-ERK1/2), and SB203580, an inhibitor of p38 MAPK, were used to evaluate the role of MAPKs with respect to postischemic functional recovery in isolated perfused rat hearts subjected to ischemic preconditioning (IPC) and anesthetic preconditioning (APC). Western blot analyses were used to determine the degree of ERK1/2 and p38 MAPK activation after the application of the preconditioning stimulus and after ischemia-reperfusion. Immunohistochemical staining served to visualize subcellular localization of activated MAPKs. RESULTS: PD98059 and SB203580 abolished postischemic functional recovery in IPC but not in APC. IPC but not APC markedly activated ERK1/2 and p38 MAPK, which were abrogated by coadministration of the specific blockers. Conversely, IPC and APC enhanced ERK1/2 activity after ischemia-reperfusion as compared to nonpreconditioned hearts, and IPC in addition enhanced p38 MAPK activity. Coadministration of PD98059 and SB203580 during IPC but not during APC inhibited postischemically enhanced MAPK activities. Moreover, chelerythrine and 5-hydroxydecanoate, effective blockers of IPC and APC, annihilated IPC- and APC-induced enhanced postischemic responses of MAPKs. Finally, administration of PD98059 during ischemia-reperfusion diminished the protective effects of IPC and APC. Immunohistochemistry revealed increased ERK1/2 activity primarily in intercalated discs and nuclei and increased p38 MAPK activity in the sarcolemma and nuclei of IPC-treated hearts. CONCLUSIONS: Although MAPKs may orchestrate cardioprotection as triggers and mediators in IPC, they are devoid of triggering, but they may have mediator effects in APC.
Assuntos
Anestésicos/farmacologia , Coração/fisiologia , Precondicionamento Isquêmico Miocárdico , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Western Blotting , Ativação Enzimática/fisiologia , Coração/efeitos dos fármacos , Proteínas de Choque Térmico/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Miocárdio/enzimologia , Fenótipo , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
BACKGROUND: Translocation of protein kinase C (PKC) to subcellular targets is a pivotal signaling step in ischemic preconditioning (IPC). However, to date, it is unknown whether PKC isoforms translocate in anesthetic preconditioning (APC). METHODS: The PKC blockers chelerythrine and rottlerin and the adenosine triphosphate-dependent potassium (K(ATP)) channel blockers HMR-1098 and 5-hydroxydecanoate were used to assess the role of PKC and K(ATP) channels in isolated perfused rat hearts subjected to IPC or APC (1.5 minimum alveolar concentration isoflurane) followed by 40 min of ischemia and 30 min of reperfusion. Immunohistochemical techniques were used to visualize PKC translocation after preconditioning. In addition, the phosphorylation status of PKC isoforms was assessed. RESULTS: Chelerythrine, rottlerin, and 5-hydroxydecanoate blocked IPC and APC with respect to functional recovery, albeit IPC at higher concentrations. HMR-1098 did not affect IPC or APC. PKCdelta and PKCepsilon translocated to nuclei in both IPC and APC, which was inhibited by chelerythrine and rottlerin. PKCdelta translocated to mitochondria but not to the sarcolemma, and PKCepsilon translocated to the sarcolemma and intercalated disks but not to mitochondria. Interestingly, PKCepsilon was accumulated at the intercalated disks in control and preconditioned hearts. Phosphorylation of PKCdelta on serine643 was increased in IPC and APC and blocked by chelerythrine and rottlerin, whereas phosphorylation of PKCdelta on threonine505 was increased only in IPC and not blocked by chelerythrine or rottlerin. PKCepsilon on serine729 did not change its phosphorylation status. CONCLUSIONS: This study indicates that translocation of PKCdelta plays a pivotal role in IPC and APC and suggests that phosphorylation of PKCdelta on serine643 may be of particular relevance in transferring the APC stimulus to mitochondrial K(ATP) channels.