RESUMO
BACKGROUND: The AMP-activated protein kinase (AMPK) is a sensor of cellular energy and nutrient status, with substantial amount of cross talk with other signaling pathways, including its phosphorylation by Akt, PKA, and GSK3ß. AIMS: Various signaling pathways and energy-consuming transport of glutamate receptors subunits are required in synaptic plasticity. However, it is unknown which energy sensors integrate the signaling pathways in these processes. In this article, we elucidated the role of AMPK activation and GSK3ß phosphorylation after HFS during the inducement of early-phase long-term potentiation (E-LTP). METHODS: Synaptic LTP in vivo was induced by high-frequency stimulation (HFS at 200 Hz at a 5-s interval). In addition, phosphorylation of AMPK and glycogen synthase kinase 3ß (GSK3ß) were measured using Western blotting. The amount of hippocampal AMP, ADP and ATP was measured by HPLC. RESULTS: We showed that the phosphorylation of AMPK and GSK3ß was significantly increased by HFS in vivo. HFS-induced AMPK activation occurred via increased (AMP + ADP)/ATP ratio and activation of Ca(2+) /calmodulin-dependent kinase kinase beta (CaMKKß). Pharmacological inhibition of AMPK by compound C (CC) prevented HFS-induced inhibitory phosphorylation of GSK3ß and the induction of LTP in dentate gyrus (DG) area in vivo. CONCLUSIONS: Our findings reveal that HFS-triggered AMPK activation phosphorylates GSK3ß and induces E-LTP in vivo.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Giro Denteado/citologia , Estimulação Elétrica/métodos , Regulação da Expressão Gênica/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Potenciação de Longa Duração/fisiologia , Neurônios/fisiologia , Nucleotídeos de Adenina/metabolismo , Análise de Variância , Animais , Cromatografia Líquida de Alta Pressão , Ativação Enzimática/fisiologia , Ativação Enzimática/efeitos da radiação , Masculino , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Deficit in synaptic plasticity in the hippocampus frequently occurs during normal aging. Although the protein level and calcium permeability of AMPARs alter with aging, the alteration of AMPARs and their regulatory proteins during aging are far from understanding. Dynamics of GluR2 subunit are dependent on the function of protein interacting with Cα kinase 1 (PICK1), PKCα and calcineurin (CaN). Here, we firstly show that the expression of PICK1 and CaN B decreased significantly in the hippocampus of old rats compared to that of young and adult rats. The decrease was accompanied by a reduction of GluR2 and PKCα and an increase in CaN A. Next, we found that in young and adult rats, the distribution of PICK1 and GluR2 diffused in the cytoplasm of hippocampal neurons, but closely around perinuclear in the hippocampal neurons of old rats. These results suggest that the expression of GluR2, PICK1, PKCα and CaN B significant decreased in the hippocampus and these alterations may lead to altered distribution of GluR2 and PICK1 during aging.
Assuntos
Envelhecimento/fisiologia , Proteínas de Transporte/biossíntese , Hipocampo/metabolismo , Proteínas Nucleares/biossíntese , Receptores de AMPA/biossíntese , Animais , Western Blotting , Calcineurina/biossíntese , Proteínas do Citoesqueleto , Imunofluorescência , Expressão Gênica , Perfilação da Expressão Gênica , Masculino , Proteína Quinase C/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
The accumulation of malondialdehyde (MDA), a lipid peroxidation by-product that has been used as an indicator of cellular oxidation status, is significantly increased in many neurological diseases such as brain ischemia/reperfusion, Alzheimer's disease and Parkinson's disease in vivo. In the present study, we found that MDA treatment in vitro reduced cortical neuronal viability in a time- and dose-dependent manner and induced cellular apoptosis as well as necrosis simultaneously. Furthermore, exposure to MDA led to accumulation of intracellular reactive oxygen species, dysfunction of mitochondria (denoted by the loss of mitochondrial transmembrane potential (Δψm)) and activation of JNK and ERK. Carnosine exhibited better protection against MDA-induced cell injury than antioxidant N-acetyl-cysteine (NAC) with its multi-potency, which alleviated MDA-induced protein cross-linking, Δψm decrease, reactive oxygen species burst, JNK and ERK activation. In conclusion, our results suggest that MDA induced cell injury in vitro via protein cross-linking and successive mitochondrial dysfunction, and the activation of reactive oxygen species-dependent MAPK signaling pathway. Carnosine alleviated all these alterations induced by MDA, but NAC merely inhibited Bcl-2 family-related activation of JNK and ERK. These results prompt the possibility that carnosine, but not other conventional antioxidants, can protect neurons against MDA-induced injury through decomposition of protein cross-linking toxicity and may serve as a novel agent in the treatment of neurodegenerative diseases.
Assuntos
Carnosina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Malondialdeído/toxicidade , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/citologia , Reagentes de Ligações Cruzadas/toxicidade , Relação Dose-Resposta a Droga , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , Necrose/induzido quimicamente , Neurônios/citologia , Neurônios/metabolismo , Proteínas/química , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Astrocytes are vital structures that support and/or protect neighboring neurons from pathology. Although it is generally accepted that glutamate receptors mediate most astrocyte effects, acid-evoked currents have recently attracted attention for their role in this regard. Here, we identified the existence and characteristics of acid-sensing ion channels (ASICs) and the transient receptor potential vanilloid type 1 (TRPV1) in astrocytes. There were two types of currents recorded under the application of acidic solution (pH 6.0) in cultured rat astrocytes. Transient currents were exhibited by 10% of the astrocytes, and sustained currents were exhibited by the other 90%, consistent with the features of ASIC and TRPV1 currents, respectively. Western blotting and immunofluorescence confirmed the expression of ASIC1, ASIC2a, ASIC3, and TRPV1 in cultured and in situ astrocytes. Unlike the ASICs expressed in neurons, which were mainly distributed in the cell membrane/cytoplasm, most of the ASICs in astrocytes were expressed in the nucleus. TRPV1 was more permeable to Na(+) in cultured astrocytes, which differed from the typical neuronal TRPV1 that was mainly permeable to Ca(2+). This study demonstrates that there are two kinds of acid-evoked currents in rat astrocytes, which may provide a new understanding about the functions of ligand-gated ion channels in astrocytes.
Assuntos
Ácidos/farmacologia , Astrócitos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Canais Iônicos Sensíveis a Ácido , Amilorida/farmacologia , Animais , Animais Recém-Nascidos , Astrócitos/fisiologia , Cálcio/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Masculino , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/classificação , Canais de Sódio/metabolismo , Canais de Cátion TRPV/metabolismoRESUMO
Deficits in learning and memory accompanied by age-related neurodegenerative diseases are closely related to the impairment of synaptic plasticity. In this study, we investigated the role of thiol redox status in the modulation of the N-methyl-d-aspartate receptor (NMDAR)-dependent long-term potentiation (LTP) in CA1 areas of hippocampal slices. Our results demonstrated that the impaired LTP induced by aging could be reversed by acute administration of reductants that can regulate thiol redox status directly, such as dithiothreitol or ß-mercaptoethanol, but not by classical anti-oxidants such as vitamin C or trolox. This repair was mediated by the recruitment of aging-related deficits in NMDAR function induced by these reductants and was mimicked by glutathione, which can restore the age-associated alterations in endogenous thiol redox status. Moreover, antioxidant prevented but failed to reverse H(2)O(2) -induced impairment of NMDAR-mediated synaptic plasticity. These results indicate that the restoring of thiol redox status may be a more effective strategy than the scavenging of oxidants in the treatment of pre-existing oxidative injury in learning and memory.
Assuntos
Envelhecimento , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Plasticidade Neuronal/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Compostos de Sulfidrila/metabolismo , Animais , Ditiotreitol/farmacologia , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Mercaptoetanol/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Oxirredução , Ratos , Ratos Sprague-DawleyRESUMO
The adipocyte-derived hormone leptin and the pancreatic beta-cell-derived hormone insulin function as afferent signals to the hypothalamus in an endocrine feedback loop that regulates body adiposity. They act in hypothalamic centers to modulate the function of specific neuronal subtypes, such as neuropeptide Y (NPY) neurons, by modifying neuronal electrical activity. To investigate the intrinsic activity of these neurons and their responses to insulin and leptin, we used a combination of morphological features and immunocytochemical technique to identify the NPY neurons of hypothalamic arcuate nucleus (ARC) and record whole cell large-conductance Ca(2+)-activated potassium (BK) currents on them. We found that both of the hormones increase the peak amplitude of BK currents, shifting the steady-state activation curve to the left. The effect of both insulin and leptin can be prevented by pretreatment with inhibitors of tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) but not MAPK. These data indicate that PI3K-mediated signals are the common regulators of BK channels by insulin and leptin and mediated the two hormones' identical activatory effects on ARC NPY neurons. The effect of insulin and leptin together was similar to that of insulin or leptin alone, and leptin or insulin pretreatment did not lead to insulin- or leptin-sensitizing effects, respectively. These intracellular signaling mechanisms may play key roles in regulating ARC NPY neuron activity and physiological processes such as the control of food intake and body weight, which are under the combined control of insulin and leptin.