RESUMO
The impact of spontaneous neurotransmission on neuronal plasticity remains poorly understood. Here, we show that acute suppression of spontaneous NMDA receptor-mediated (NMDAR-mediated) neurotransmission potentiates synaptic responses in the CA1 regions of rat and mouse hippocampus. This potentiation requires protein synthesis, brain-derived neurotrophic factor expression, eukaryotic elongation factor-2 kinase function, and increased surface expression of AMPA receptors. Our behavioral studies link this same synaptic signaling pathway to the fast-acting antidepressant responses elicited by ketamine. We also show that selective neurotransmitter depletion from spontaneously recycling vesicles triggers synaptic potentiation via the same pathway as NMDAR blockade, demonstrating that presynaptic impairment of spontaneous release, without manipulation of evoked neurotransmission, is sufficient to elicit postsynaptic plasticity. These findings uncover an unexpectedly dynamic impact of spontaneous glutamate release on synaptic efficacy and provide new insight into a key synaptic substrate for rapid antidepressant action.
Assuntos
Hipocampo/citologia , Inibição Psicológica , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Análise de Variância , Animais , Animais Recém-Nascidos , Biofísica , Fator Neurotrófico Derivado do Encéfalo/deficiência , Estimulação Elétrica , Quinase do Fator 2 de Elongação/deficiência , Inibidores Enzimáticos/farmacologia , Potenciais Evocados/genética , Potenciais Evocados/fisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Comportamento Alimentar/efeitos dos fármacos , Comportamento Alimentar/fisiologia , Antagonistas GABAérgicos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Ácido Glutâmico/metabolismo , Hipocampo/fisiologia , Técnicas In Vitro , Ketamina/farmacologia , Locomoção/efeitos dos fármacos , Locomoção/genética , Camundongos , Camundongos Knockout , Inibição Neural/efeitos dos fármacos , Técnicas de Patch-Clamp , Picrotoxina/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Natação/fisiologia , Tetrodotoxina/farmacologia , Fatores de TempoRESUMO
PURPOSE: Hsp90 is important in the folding, maturation and stabilization of pro-tumorigenic client proteins and represents a viable drug target for the design of chemotherapies. Previously, we reported the development of novobiocin analogues designed to inhibit the C-terminal portion of Hsp90, which demonstrated the ability to decrease client protein expression. We now report the characterization of the novel novobiocin analogue, F-4, which demonstrates improved cytotoxicity in prostate cancer cell lines compared to the N-terminal inhibitor, 17-AAG. MATERIALS AND METHODS: LNCaP and PC-3 cells were treated with 17-AAG or F-4 in anti-proliferative, apoptosis, cell cycle and cytotoxicity assays. Western blot and prostate specific antigen (PSA) ELISAs were used to determine client protein degradation, induction of Hsp90 and to assess the functional status of the androgen receptor (AR) in response to F-4 treatment. Surface plasmon resonance (SPR) was also used to determine the binding properties of F-4 to Hsp90. RESULTS: F-4 demonstrated improved potency and efficacy compared to novobiocin in anti-proliferative assays and decreased expression of client proteins. PSA secretion was inhibited in a dose-dependent manner that paralleled a decrease in AR expression. The binding of F-4 to Hsp90 was determined to be saturable with a binding affinity (K(d)) of 100 microM. In addition, superior efficacy was demonstrated by F-4 compared to 17-AAG in experiments measuring cytotoxicity and apoptosis. CONCLUSIONS: These data reveal distinct modes of action for N-terminal and C-terminal Hsp90 inhibitors, which may offer unique therapeutic benefits for the treatment of prostate cancer.
Assuntos
Sistemas de Liberação de Medicamentos , Inibidores do Crescimento/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Novobiocina/análogos & derivados , Novobiocina/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Masculino , Neoplasias da Próstata/patologia , SpodopteraRESUMO
The 90-kDa heat shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Consequently, there is considerable interest in developing chemotherapeutic drugs that specifically disrupt the function of Hsp90. Here, we investigated the extent to which a novel novobiocin-derived C-terminal Hsp90 inhibitor, designated KU135, induced antiproliferative effects in Jurkat T-lymphocytes. The results indicated that KU135 bound directly to Hsp90, caused the degradation of known Hsp90 client proteins, and induced more potent antiproliferative effects than the established N-terminal Hsp90 inhibitor 17-allylamino-demethoxygeldanamycin (17-AAG). Closer examination of the cellular response to KU135 and 17-AAG revealed that only 17-AAG induced a strong up-regulation of Hsp70 and Hsp90. In addition, KU135 caused wild-type cells to undergo G(2)/M arrest, whereas cells treated with 17-AAG accumulated in G(1). Furthermore, KU135 but not 17-AAG was found to be a potent inducer of mitochondria-mediated apoptosis as evidenced, in part, by the fact that cell death was inhibited to a similar extent by Bcl-2/Bcl-x(L) overexpression or the depletion of apoptotic protease-activating factor-1 (Apaf-1). Together, these data suggest that KU135 inhibits cell proliferation by regulating signaling pathways that are mechanistically different from those targeted by 17-AAG and as such represents a novel opportunity for Hsp90 inhibition.