RESUMO
Safinamide (Xadago) is a novel dual-mechanism drug that has been approved in the European Union and United States as add-on treatment to levodopa in Parkinson's disease therapy. In addition to its selective and reversible monoamine oxidase B inhibition, safinamide through use-dependent sodium channel blockade reduces overactive glutamatergic transmission in basal ganglia, which is believed to contribute to motor symptoms and complications including levodopa-induced dyskinesia (LID). The present study investigated the effects of safinamide on the development of LID in 6-hydroxydopamine (6-OHDA)-lesioned rats, evaluating behavioral, molecular, and neurochemical parameters associated with LID appearance. 6-OHDA-lesioned rats were treated with saline, levodopa (6 mg/kg), or levodopa plus safinamide (15 mg/kg) for 21 days. Abnormal involuntary movements, motor performance, molecular composition of the striatal glutamatergic synapse, glutamate, and GABA release were analyzed. In the striatum, safinamide prevented the rearrangement of the subunit composition of N-methyl-d-aspartate receptors and the levodopa-induced increase of glutamate release associated with dyskinesia without affecting the levodopa-stimulated motor performance and dyskinesia. Overall, these findings suggest that the striatal glutamate-modulating component of safinamide's activity may contribute to its clinical effects, where its long-term use as levodopa add-on therapy significantly improves motor function and "on" time without troublesome dyskinesia.
Assuntos
Alanina/análogos & derivados , Benzilaminas/farmacologia , Discinesia Induzida por Medicamentos/tratamento farmacológico , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Levodopa/farmacologia , Transdução de Sinais/efeitos dos fármacos , Alanina/farmacologia , Animais , Antiparkinsonianos/farmacologia , Corpo Estriado/efeitos dos fármacos , Modelos Animais de Doenças , Dopaminérgicos/farmacologia , Masculino , Oxidopamina/farmacologia , Doença de Parkinson/tratamento farmacológico , Ratos , Ratos Sprague-DawleyRESUMO
A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.
RESUMO
Frontotemporal Dementia (FTD) is a neurodegenerative disorder mainly characterised by Tau or TDP43 inclusions. A co-autoimmune aetiology has been hypothesised. In this study, we aimed at defining the pathogenetic role of anti-AMPA GluA3 antibodies in FTD. Serum and cerebrospinal fluid (CSF) anti-GluA3 antibody dosage was carried out and the effect of CSF with and without anti-GluA3 antibodies was tested in rat hippocampal neuronal primary cultures and in differentiated neurons from human induced pluripotent stem cells (hiPSCs). TDP43 and Tau expression in hiPSCs exposed to CSF was assayed. Forty-one out of 175 screened FTD sera were positive for the presence of anti-GluA3 antibodies (23.4%). FTD patients with anti-GluA3 antibodies more often presented presenile onset, behavioural variant FTD with bitemporal atrophy. Incubation of rat hippocampal neuronal primary cultures with CSF with anti-GluA3 antibodies led to a decrease of GluA3 subunit synaptic localization of the AMPA receptor (AMPAR) and loss of dendritic spines. These results were confirmed in differentiated neurons from hiPSCs, with a significant reduction of the GluA3 subunit in the postsynaptic fraction along with increased levels of neuronal Tau. In conclusion, autoimmune mechanism might represent a new potentially treatable target in FTD and might open new lights in the disease underpinnings.
Assuntos
Autoanticorpos/líquido cefalorraquidiano , Autoimunidade , Proteínas de Ligação a DNA/imunologia , Demência Frontotemporal/imunologia , Hipocampo/imunologia , Neurônios/imunologia , Receptores de AMPA/antagonistas & inibidores , Idoso , Animais , Autoanticorpos/farmacologia , Células COS , Estudos de Casos e Controles , Diferenciação Celular/efeitos dos fármacos , Chlorocebus aethiops , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Feminino , Demência Frontotemporal/líquido cefalorraquidiano , Demência Frontotemporal/diagnóstico , Demência Frontotemporal/genética , Expressão Gênica , Hipocampo/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Neurônios/efeitos dos fármacos , Neurônios/patologia , Cultura Primária de Células , Ratos , Receptores de AMPA/genética , Receptores de AMPA/imunologia , Proteínas tau/genética , Proteínas tau/imunologiaRESUMO
A disintegrin and metalloproteinase 10 (ADAM10) is the major α-secretase that catalyzes the amyloid precursor protein (APP) ectodomain shedding in the brain and prevents amyloid formation. Its activity depends on correct intracellular trafficking and on synaptic membrane insertion. Here, we describe that in hippocampal neurons the synapse-associated protein-97 (SAP97), an excitatory synapse scaffolding element, governs ADAM10 trafficking from dendritic Golgi outposts to synaptic membranes. This process is mediated by a previously uncharacterized protein kinase C phosphosite in SAP97 SRC homology 3 domain that modulates SAP97 association with ADAM10. Such mechanism is essential for ADAM10 trafficking from the Golgi outposts to the synapse, but does not affect ADAM10 transport from the endoplasmic reticulum. Notably, this process is altered in Alzheimer's disease brains. These results help in understanding the mechanism responsible for the modulation of ADAM10 intracellular path, and can constitute an innovative therapeutic strategy to finely tune ADAM10 shedding activity towards APP.
Assuntos
Proteínas ADAM/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismo , Proteínas ADAM/química , Proteína ADAM10 , Proteínas Adaptadoras de Transdução de Sinal/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/química , Animais , Células COS , Chlorocebus aethiops , Proteína 1 Homóloga a Discs-Large , Ativação Enzimática , Células HEK293 , Humanos , Proteínas de Membrana/química , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Fosfotreonina/metabolismo , Densidade Pós-Sináptica/metabolismo , Ligação Proteica , Ratos , Sinapses/metabolismoRESUMO
An increasing body of experimental evidence demonstrates that the glutamatergic system is involved in the genesis of l-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID). Indeed, the N-methyl-d-aspartate (NMDA) receptor antagonist amantadine is the only anti-dyskinetic compound used in patients, albeit with limited efficacy and side effects. In this study, we investigated the anti-dyskinetic properties of memantine, a non-competitive NMDA receptor antagonist in clinical use for the treatment of dementia, in the 6-hydroxy-dopamine (6-OHDA)-lesion rat model of Parkinson's disease. For comparison, parallel experiments were also performed with amantadine. First, we investigated the acute effect of different doses of memantine (5, 10, 15 and 20mg/kg), and amantadine (10, 20, 40, 60mg/kg) on established dyskinesia induced by L-DOPA (6mg/kg plus benserazide). Results showed that both memantine and amantadine produced a significant reduction of LID. Afterward, drug-naïve and L-DOPA-primed 6-OHDA-lesioned rats were sub-chronically treated with daily injections of L-DOPA (6mg/kg plus benserazide) alone, or in combination with the effective doses of memantine, while amantadine was tested in already dyskinetic rats. Results showed that memantine significantly dampened dyskinesia in both drug-naïve and L-DOPA-primed rats, but only during the first few days of administration. In fact, the anti-dyskinetic effect of memantine was completely lost already at the fifth administration, indicating a rapid induction of tolerance. Interestingly, a 3-week washout period was not sufficient to restore the anti-dyskinetic effect of the drug. Similarly, amantadine was able to dampen already established dyskinesia only during the first day of administration. Moreover, memantine partially decreased the therapeutic effect of L-DOPA, as showed by the result of the stepping test. Finally, loss of the anti-dyskinetic effect of memantine was associated to increased synaptic GluN2A/GluN2B ratio at striatal synaptic membranes. Our results are in line with clinical observations suggesting that NMDA receptor blockade may only be transiently effective against LID in PD patients.
Assuntos
Antiparkinsonianos/toxicidade , Discinesias/tratamento farmacológico , Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Levodopa/toxicidade , Memantina/uso terapêutico , Amantadina/administração & dosagem , Amantadina/uso terapêutico , Animais , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Masculino , Memantina/administração & dosagem , Oxidopamina , Doença de Parkinson Secundária/induzido quimicamente , Ratos , Ratos Sprague-DawleyRESUMO
Cerebrospinal fluid (CSF) total Tau levels vary widely in neurodegenerative disorders, thus being not useful in their discrimination over Alzheimer disease. No CSF marker for progressive supranuclear palsy (PSP) is currently available. The aim of this study was to characterise and measure Tau forms in order to verify the differential patterns among neurodegenerative disorders. Seventy-eight patients with neurodegenerative disorders and 26 controls were included in the study. Each patient underwent a standardised clinical and neuropsychological evaluation, MRI, and CSF total-Tau and phospho-Tau dosage. In CSF and cerebral cortex, a quantitative immunoprecipitation was developed. An extended (55 kDa), and a truncated (33 kDa) forms of Tau were recognised. CSF samples were assayed, the optical density of the two Tau forms was measured, and the ratio calculated (Tau ratio, 33 kDa/55 kDa forms). Tau ratio 33 kDa/55 kDa was significantly decreased in patients with PSP (0.46+/-0.16) when compared to controls, including healthy subjects (1.16+/-0.46, P=0.002) and Alzheimer disease (1.38+/-0.68, P<0.001), and when compared to frontotemporal dementia (0.98+/-0.30, P=0.008) or corticobasal degeneration syndrome (0.98+/-0.48, P=0.02). Moreover, in PSP patients Tau form ratio was lower than in other neurodegenerative extrapyramidal disorders, such as Parkinson disease (1.16+/-0.26, P=0.002) and dementia with lewy bodies (1.44+/-0.48, P<0.001). Tau ratio 33 kDa/55 kDa did not correlate either with demographic characteristics, cognitive performances or with motor impairment severity. Truncated Tau production shows a different pattern in PSP compared to other neurodegenerative disorders, supporting the view of disease-specific pathological pathways. These findings are promising in suggesting the identification of a marker for PSP diagnosis in clinical practice.
Assuntos
Paralisia Supranuclear Progressiva/líquido cefalorraquidiano , Paralisia Supranuclear Progressiva/diagnóstico , Proteínas tau/líquido cefalorraquidiano , Proteínas tau/classificação , Idoso , Biomarcadores/líquido cefalorraquidiano , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Proteínas tau/químicaRESUMO
Membrane associated guanylate kinase proteins (MAGUKs) play a key role in the regulation of the intracellular trafficking and synaptic localization of ionotropic glutamate receptors. In particular, the postsynaptic density-95-like subfamily of MAGUKs (PSD-MAGUKs) organizes ionotropic glutamate receptors and their associated signaling proteins in the postsynaptic density of the excitatory synapse regulating the strength of synaptic activity. Several recent observations clearly put forward the idea that alterations of PSD-MAGUK protein function such as alterations of PSD-MAGUK protein interaction with N-methyl-D-aspartate (NMDA) receptors regulatory subunits are common events in several CNS disorders. With this view, a better knowledge and understanding of PSD-MAGUK function as well as of the molecular events regulating PSD-MAGUK-mediated interactions in the glutamatergic synapse could lead to the identification of new pharmaceutical targets for the therapy of CNS disorders.
Assuntos
Encéfalo/metabolismo , Proteínas de Membrana/metabolismo , Sinapses/metabolismo , Membranas Sinápticas/metabolismo , Animais , Encéfalo/fisiopatologia , Proteína 4 Homóloga a Disks-Large , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Receptores de Glutamato/metabolismo , Sinapses/ultraestrutura , Membranas Sinápticas/ultraestrutura , Transmissão Sináptica/fisiologiaRESUMO
OBJECTIVE: In CSF, extended (55 kDa) and truncated (33 kDa) tau forms have been previously recognized, and the tau 33 kDa/55 kDa ratio has been found significantly reduced in progressive supranuclear palsy (PSP) vs in other neurodegenerative disorders. The aim of this study was to evaluate the diagnostic value of the CSF tau form ratio as a biomarker of PSP and to correlate the structural anatomic changes as measured by means of voxel-based morphometry (VBM) to CSF tau form ratio decrease. METHODS: A total of 166 subjects were included in the study (21 PSP, 20 corticobasal degeneration syndrome, 44 frontotemporal dementia, 29 Alzheimer disease, 10 Parkinson disease, 15 dementia with Lewy bodies, and 27 individuals without any neurodegenerative disorder). Each patient underwent a standardized clinical and neuropsychological evaluation. In CSF, a semiquantitative immunoprecipitation was developed to evaluate CSF tau 33 kDa/55 kDa ratio. MRI assessment and VBM analysis was carried out. RESULTS: Tau form ratio was significantly reduced in patients with PSP (0.504 +/- 0.284) when compared to age-matched controls (0.989 +/- 0.343), and to patients with other neurodegenerative conditions (range = 0.899-1.215). The area under the curve (AUC) of the receiver operating characteristic analysis in PSP vs other subgroups ranged from 0.863 to 0.937 (PSP vs others, AUC = 0.897, p < 0.0001). VBM study showed that CSF tau form ratio decrease correlated significantly with brainstem atrophy. CONCLUSIONS: Truncated tau production, which selectively affects brainstem neuron susceptibility, can be considered a specific and reliable marker for PSP. Tau form ratio was the lowest in progressive supranuclear palsy with no overlap with any other neurodegenerative illness.
Assuntos
Encéfalo/patologia , Paralisia Supranuclear Progressiva/líquido cefalorraquidiano , Paralisia Supranuclear Progressiva/diagnóstico , Proteínas tau/líquido cefalorraquidiano , Adulto , Idoso , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/diagnóstico , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Demência/líquido cefalorraquidiano , Demência/diagnóstico , Feminino , Humanos , Imunoprecipitação/métodos , Doença por Corpos de Lewy/líquido cefalorraquidiano , Doença por Corpos de Lewy/diagnóstico , Masculino , Pessoa de Meia-Idade , Transtornos Neurocognitivos/líquido cefalorraquidiano , Transtornos Neurocognitivos/diagnóstico , Doença de Parkinson/líquido cefalorraquidiano , Doença de Parkinson/diagnóstico , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Paralisia Supranuclear Progressiva/patologiaRESUMO
Synapse Associated Protein 97 (SAP97), a member of membrane-associated guanylate kinase (MAGUK) protein family, has been involved in the correct targeting and clustering of ionotropic glutamate receptors (iGluRs) at postsynaptic sites. Calcium/calmodulin kinase II (CaMKII) phosphorylates SAP97 on two major sites in vivo; one located in the N-terminal domain (Ser39) and the other in the first postsynaptic density disc large ZO1 (PDZ) domain (Ser232). CaMKII-mediated phosphorylation of SAP97-Ser39 is necessary and sufficient to drive SAP97 to the postsynaptic compartment in cultured hippocampal neurons. CaMKII-dependent phosphorylation of Ser232 disrupts SAP97 interaction with NR2A subunit, thereby regulating synaptic targeting of this NMDA receptor subunit. Here we show by means of phospho-specific antibodies that SAP97-Ser39 phosphorylation represents the driving force to release SAP97/NR2A complex from the endoplasmic reticulum. Ser39 phosphorylation does not interfere with SAP97 capability to bind NR2A. On the contrary, SAP97-Ser232 phosphorylation occurs within the postsynaptic compartment and is responsible for both the disruption of NR2A/SAP97 complex and, consequently, for NR2A insertion in the postsynaptic membrane. Thus, CaMKII-dependent phosphorylation of SAP97 in different time frames and locations within the neurons controls both NR2A trafficking and insertion.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Proteínas de Membrana/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cafeína/farmacologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Células Cultivadas , Cricetinae , Cricetulus , Interações Medicamentosas , Embrião de Mamíferos , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/citologia , Imunoprecipitação/métodos , Técnicas In Vitro , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/ultraestrutura , Inibidores de Fosfodiesterase/farmacologia , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Serina/metabolismo , Transfecção/métodosRESUMO
Interleukin (IL)-1beta is a proinflammatory cytokine implicated in various pathophysiological conditions of the CNS involving NMDA receptor activation. Circumstantial evidence suggests that IL-1beta and NMDA receptors can functionally interact. Using primary cultures of rat hippocampal neurons, we investigated whether IL-1beta affects NMDA receptor function(s) by studying (1) NMDA receptor-induced [Ca2+]i increase and (2) NMDA-mediated neurotoxicity. IL1beta (0.01-0.1 ng/ml) dose-dependently enhances NMDA-induced [Ca2+]i increases with a maximal effect of approximately 45%. This effect occurred only when neurons were pretreated with IL-1beta, whereas it was absent if IL-1beta and NMDA were applied simultaneously, and it was abolished by IL-1 receptor antagonist (50 ng/ml). Facilitation of NMDA-induced [Ca2+]i increase by IL-1beta was prevented by both lavendustin (LAV) A (500 nm) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) (1 microm), suggesting an involvement of tyrosine kinases. Increased tyrosine phosphorylation of NMDA receptor subunits 2A and 2B and coimmunoprecipitation of activated Src tyrosine kinase with these subunits was observed after exposure of hippocampal neurons to 0.05 ng/ml IL-1beta. Finally, 0.05 ng/ml IL-1beta increased by approximately 30% neuronal cell death induced by NMDA, and this effect was blocked by both lavendustin A and PP2. These data suggest that IL-1beta increases NMDA receptor function through activation of tyrosine kinases and subsequent NR2A/B subunit phosphorylation. These effects may contribute to glutamate-mediated neurodegeneration.
Assuntos
Cálcio/metabolismo , Interleucina-1/farmacologia , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Quinases da Família src/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteína Antagonista do Receptor de Interleucina 1 , Líquido Intracelular/metabolismo , N-Metilaspartato/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos , Sialoglicoproteínas/farmacologia , Quinases da Família src/efeitos dos fármacosAssuntos
Envelhecimento/metabolismo , Transtornos Cognitivos/etiologia , Complicações do Diabetes , Hipocampo/metabolismo , Plasticidade Neuronal/fisiologia , Sinapses/metabolismo , Envelhecimento/patologia , Animais , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/fisiopatologia , Diabetes Mellitus/fisiopatologia , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Sinapses/patologia , Transmissão Sináptica/fisiologiaRESUMO
In animal models of diabetes mellitus, such as the streptozotocin-diabetic rat (STZ-rat), spatial learning impairments develop in parallel with a reduced expression of long-term potentiation (LTP) and enhanced expression of long-term depression (LTD) in the hippocampus. This study examined the time course of the effects of STZ-diabetes and insulin treatment on the hippocampal post-synaptic glutamate N-methyl-D-aspartate (NMDA) receptor complex and other key proteins regulating hippocampal synaptic transmission in the post-synaptic density (PSD) fraction. In addition, the functional properties of the NMDA-receptor complex were examined. One month of STZ-diabetes did not affect the NMDA receptor complex. In contrast, 4 months after induction of diabetes NR2B subunit immunoreactivity, CaMKII and Tyr-dependent phosphorylation of the NR2A/B subunits of the NMDA receptor were reduced and alphaCaMKII autophosphorylation and its association to the NMDA receptor complex were impaired in STZ-rats compared with age-matched controls. Likewise, NMDA currents in hippocampal pyramidal neurones measured by intracellular recording were reduced in STZ-rats. Insulin treatment prevented the reduction in kinase activities, NR2B expression levels, CaMKII-NMDA receptor association and NMDA currents. These findings strengthen the hypothesis that altered post-synaptic glutamatergic transmission is related to deficits in learning and plasticity in this animal model.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Hipocampo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Doenças do Sistema Nervoso Central/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/química , Hipocampo/citologia , Masculino , Plasticidade Neuronal/fisiologia , Técnicas de Cultura de Órgãos , Fosforilação , Células Piramidais/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/análiseRESUMO
We have investigated the distribution of NMDA and neurotrophin receptor systems and their reciprocal interactions in post-synaptic densities (PSD) purified from spinal cord. NMDA receptor subunits, trkA and trkB, but not trkC, were present in spinal cord PSD. The incubation of PSD with BDNF and NGF induced the phosphorylation of NR2A and B subunits. This phosphorylation was counteracted by antibodies directed against the catalytic domain of trkA and trkB receptors and by genistein. These results suggest the existence of a previously unexplored cross-talk between neurotrophins and NMDA receptors in rat spinal cord neurons.
Assuntos
Células do Corno Anterior/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Fator de Crescimento Neural/farmacologia , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Células do Corno Anterior/metabolismo , Fosforilação/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Receptores de Fator de Crescimento Neural/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismoRESUMO
NMDA receptor, Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII), and postsynaptic density 95 (PSD-95) are three major components of the PSD fraction. Both alphaCaMKII and PSD-95 have been shown previously to bind NR2 subunits of the NMDA receptor complex. The nature and mechanisms of targeting to the NMDA receptor subunits are, however, not completely understood. Here we report that the C-terminal NR2A(S1389-V1464) sequence was sufficient to guarantee the association of both native and recombinant alphaCaMKII and PSD-95. PSD-95(54-256) was able to compete with the binding of both native and recombinant alphaCaMKII to the NR2A C-tail. Accordingly, alphaCaMKII(1-325) competes with both the native PSD-95 and the native kinase itself for the binding to NR2A. In addition, Ser/Ala1289 and Ser/Asp1289 point mutations on the unique CaMKII phosphosite of NR2A did not significantly influence the binding of native alphaCaMKII and PSD-95 to the NR2A C-tail. Finally, the association-dissociation of alphaCaMKII and PSD-95 to and from the NR2A C-tail was significantly modulated by activation of NMDA receptor achieved by either pharmacological tools or long-term potentiation induction, underlining the importance of dynamic and reciprocal interactions of NMDA receptor, alphaCaMKII, and PSD-95 in hippocampal synaptic plasticity.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Ligação Competitiva/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteína 4 Homóloga a Disks-Large , Glutationa Transferase/genética , Hipocampo/química , Hipocampo/citologia , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Potenciação de Longa Duração/fisiologia , Masculino , Proteínas de Membrana , Proteínas do Tecido Nervoso/genética , Fosforilação , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sinapses/química , Sinapses/metabolismoRESUMO
The N-methyl-d-aspartate (NMDA) receptor subunits NR2 possess extended intracellular C-terminal domains by which they can directly interact with a large number of postsynaptic density (PSD) proteins involved in synaptic clustering and signaling. We have previously shown that PSD-associated alpha-calmodulin kinase II (alphaCaMKII) binds with high affinity to the C-terminal domain of the NR2A subunit. Here, we show that residues 1412-1419 of the cytosolic tail of NR2A are critical for alphaCaMKII binding, and we identify, by site directed mutagenesis, PKC-dependent phosphorylation of NR2A(Ser(1416)) as a key mechanism in inhibiting alphaCaMKII-binding and promoting dissociation of alphaCaMKII.NR2A complex. In addition, we show that stimulation of PKC activity in hippocampal slices either with phorbol esters or with the mGluRs specific agonist trans-1-amino-1,3- cyclopentanedicarboxylic acid (t-ACPD) decreases alphaCaMKII binding to NMDA receptor complex. Thus, our data provide clues on understanding the molecular basis of a direct cross-talk between alphaCaMKII and PKC pathways in the postsynaptic compartment.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteína Quinase C/metabolismo , Receptores de N-Metil-D-Aspartato/química , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Clonagem Molecular , Cicloleucina/análogos & derivados , Cicloleucina/farmacologia , Citosol/metabolismo , Ativação Enzimática , Glutationa Transferase/metabolismo , Hipocampo/metabolismo , Mutagênese Sítio-Dirigida , Fármacos Neuroprotetores/farmacologia , Fosforilação , Plasmídeos/metabolismo , Mutação Puntual , Testes de Precipitina , Ligação Proteica , Proteína Quinase C/química , Estrutura Terciária de Proteína , Ratos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de SinaisRESUMO
We have tested whether different agonists of metabotropic glutamate receptors could induce translocation of selective protein kinase C isozymes in nerve terminals. In rat cortical synaptosomes 1S, 3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD; 100 microM) induced an increase in translocation to 124.6 +/- 5.7% of basal unstimulated conditions of the Ca++-independent protein kinase Cepsilon, but not of the Ca++-dependent isozyme beta. This effect was counteracted by 1-aminoindan-1,5-dicarboxylic acid (100 microM), an antagonist of metabotropic glutamate receptor 1. On the other hand, (+)-alpha-methyl-4-carboxyphenylglycine [(+)-MCPG], an antagonist of metabotropic glutamate receptors group I and II, did not antagonize the effect of 1S,3R-ACPD, and per se induced a translocation of protein kinase Cepsilon of 164 +/- 17.7% of basal unstimulated conditions. Because the (+)-MCPG induction of protein kinase Cepsilon translocation was not antagonized by 1-aminoindan-1, 5-dicarboxylic acid, it is suggested that 1S,3R-ACPD and (+)-MCPG activate this signal transduction pathway through distinct membrane receptors. Indeed (2-[2"-carboxy-3'-phenylcyclopropyl]glycine)-13 (300 nM), a new compound known to antagonize metabotropic glutamate receptors coupled to phospholipase D, was able to antagonize protein kinase Cepsilon translocation induced by (+)-MCPG. Moreover (+)-MCPG directly induced phospholipase D activity, measured as [3H]phosphoethanol production in cortical synaptosomes. These data suggest that in cortical nerve terminals (i) distinct metabotropic glutamate receptors, coupled to different signal transduction pathways, are present, (ii) (+)-MCPG is able to induce protein kinase Cepsilon translocation, and that (iii) a metabotropic glutamate receptor associated to phospholipase D might influence translocation of protein kinase C in a calcium-independent manner.
Assuntos
Benzoatos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glicina/análogos & derivados , Isoenzimas/metabolismo , Fosfolipase D/metabolismo , Proteína Quinase C/metabolismo , Receptores de Glutamato Metabotrópico/fisiologia , Sinaptossomos/enzimologia , Animais , Córtex Cerebral/citologia , Quelantes/farmacologia , Cicloleucina/análogos & derivados , Cicloleucina/farmacologia , Ciclopropanos/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Glicina/farmacologia , Masculino , Neurônios/química , Neurônios/enzimologia , Fármacos Neuroprotetores/farmacologia , Terminações Pré-Sinápticas/química , Terminações Pré-Sinápticas/enzimologia , Proteína Quinase C beta , Proteína Quinase C-épsilon , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Ca2+/calmodulin-dependent protein kinase II (CaMKII), a multifunctional, widely distributed enzyme, is enriched in post-synaptic densities (PSDs). Here, we demonstrate that CaMKII binds to a discrete C-terminal region of the NR2A subunit of NMDA receptors and promotes the phosphorylation of a Ser residue of this NMDA receptor subunit. Glutathione S-transferase (GST)-NR2A(1349-1464) binds native CaMKII from solubilised hippocampal PSDs in 'pull-out' and overlay experiments and this binding is competed by recombinant alphaCaMKII(1-315). The longer GST-NR2A(1244-1464), although containing the CaMKII phosphosite Ser-1289, binds the kinase with a lower efficacy. CaMKII association to NR2A(1349-1464) is positively modulated by kinase autophosphorylation in the presence of Ca2+/calmodulin. These data provide direct evidence for a mechanism modulating the synaptic strength.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Hipocampo/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Sítios de Ligação , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Citoplasma/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sinapses/metabolismoRESUMO
The glutamatergic synapse is the key structure in the development of activity-dependent synaptic plasticity in the central nervous system. The analysis of the complex biochemical mechanisms at the basis of the long-term changes in synaptic efficacy have received a tremendous impulse by the observation that the post-synaptic constituents of the synapse can be separated and purified through a simple procedure involving detergent treatment of synaptosomes and differential centrifugation. In this fraction, called post-synaptic density (PSD), the functional interactions of its constituents are preserved. The various subunits of ionotropic glutamate receptors are held in register with the presynaptic active zone through their interaction with linker proteins. N-methyl-D-aspartate (NMDA) subunits NR2A and NR2B, bind to the PSD protein called PSD-95, which in turn binds neuroligins, providing a handle for interacting with neurexin, located in the plasma membrane at the presynaptic active zone. Additional clustering of NMDA receptors is provided through the binding of NRI subunits to the cytoskeletal protein alpha-actinin-2. AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and kainate receptors are other important constituents of PSDs and bind to different anchoring proteins. Phosphorylation processes have long been known to modulate NMDA receptor functional activity: the finding that several protein kinases, particularly Ca2+/Calmodulin-dependent protein kinase II and protein tyrosine kinases of the src family, are major constituents of PSDs has allowed to demonstrate that these enzymes are localized in a strategic position of the glutamatergic synapse, so that their activation provides a means for NMDA receptor function regulation upon its activation. The relevance of these mechanisms has been demonstrated in experimental models of pathologies involving deficits in synaptic plasticity, such as in streptozotocin-induced diabetes and in an animal model of prenatal induced ablation of hippocampal neurons. Both animal models display disturbances in long-term potentiation and cognitive deficits, thus providing in vivo models to study pathology related changes in both the structure and the function of the excitatory synapse.
Assuntos
Sistema Nervoso Central/fisiologia , Proteínas Quinases/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Receptores de Neurotransmissores/fisiologia , Sinapses/química , Alquilantes/farmacologia , Animais , Diabetes Mellitus/patologia , Acetato de Metilazoximetanol/análogos & derivados , Acetato de Metilazoximetanol/farmacologiaRESUMO
AIMS/HYPOTHESIS: Moderate disturbances of learning and memory were recognized as a complication of diabetes mellitus in patients. The streptozotocin-diabetic rat, an animal model of insulin-dependent diabetes, shows impairments in spatial memory and in long-term potentiation expression. We have studied the effect of experimental diabetes on expression of post-synaptic glutamate N-Methyl-D-Aspartate ionotropic receptors and of other key proteins regulating synaptic transmission at the post-synaptic compartment. METHODS: In situ hybridization and Western blot analysis were used to assess expression and protein concentration of N-Methyl-D-Aspartate receptors and alpha-calcium-calmodulin-dependent kinase II. Receptor subunits alphaCaMKII-dependent phosphorylation was studied in post-synaptic densities obtained from the hippocampus and cortex of control, streptozotocin-diabetic and insulin-treated rats. RESULTS: The transcript levels of NR1 and NR2A subunits of N-Methyl-D-Aspartate were unchanged in rats with a diabetic duration of 3 months when compared with age-matched control rats. Accordingly, NR1 and NR2A as well as GluR1, GluR2/3, PSD-95 and alphaCaMKII protein concentrations in post-synaptic densities were the same in both control and diabetic rats, whereas the immunoreactivity for NR2B was reduced by about 40%. In addition, the activity of alphaCaMKII on exogenous substrates, such as syntide-2, and the phosphorylation of NR2A/B subunits of N-Methyl-D-Aspartate receptor was reduced in hippocampal post-synaptic densities of streptozotocin-diabetic rats as compared with control rats. Furthermore, we show that insulin intervention for 3 months after diabetic duration partially restored both alphaCaMKII activity and NR2B levels. CONCLUSION/INTERPRETATION: N-Methyl-D-Aspartate receptor expression and phosphorylation is possibly involved in behavioural and electrophysiological abnormalities observed in streptozotocin-diabetic rats.