Mitochondrial metabolism is highly responsive to nutrient availability and ongoing activity in neuronal circuits. The molecular mechanisms by which brain cells respond to an increase in cellular energy expenditure are largely unknown. Mild mitochondrial uncoupling enhances cellular energy expenditure in mitochondria and can be induced with 2,4‐dinitrophenol (DNP), a proton ionophore previously used for weight loss. We found that DNP treatment reduces mitochondrial membrane potential, increases intracellular Ca2+ levels and reduces oxidative stress in cerebral cortical neurons. Gene expression profiling of the cerebral cortex of DNP‐treated mice revealed reprogramming of signaling cascades that included suppression of the mammalian target of rapamycin (mTOR) and insulin – PI3K – MAPK pathways, and up‐regulation of tuberous sclerosis complex 2, a negative regulator of mTOR. Genes encoding proteins involved in autophagy processes were up‐regulated in response to DNP. CREB (cAMP‐response element‐binding protein) signaling, Arc and brain‐derived neurotrophic factor, which play important roles in synaptic plasticity and adaptive cellular stress responses, were up‐regulated in response to DNP, and DNP‐treated mice exhibited improved performance in a test of learning and memory. Immunoblot analysis verified that key DNP‐induced changes in gene expression resulted in corresponding changes at the protein level. Our findings suggest that mild mitochondrial uncoupling triggers an integrated signaling response in brain cells characterized by reprogramming of mTOR and insulin signaling, and up‐regulation of pathways involved in adaptive stress responses, molecular waste disposal, and synaptic plasticity.
While pre‐conditioning is induced before stroke onset, ischemic post‐conditioning (IPostC) is performed after reperfusion, which typically refers to a series of mechanical interruption of blood reperfusion after stroke. IPostC is known to reduce infarction in wild‐type animals. We investigated if IPostC protects against brain injury induced by focal ischemia in Tcell–deficient nude rats and to examine its effects on Akt and the mammalian target of rapamycin (mTOR) pathway. Although IPostC reduced infarct size at 2 days post‐stroke in wild‐type rats, it did not attenuate infarction in nude rats. Despite the unaltered infarct size in nude rats, IPostC increased levels of phosphorylated Akt (p‐Akt) and Akt isoforms (Akt1, Akt2, Akt3), and p‐mTOR, p‐S6K and p‐4EBP1 in the mTOR pathway, as well as growth associated Protein 43 (GAP43), both in the peri‐infarct area and core, 24 h after stroke. IPostC improved neurological function in nude rats 1–30 days after stroke and reduced the extent of brain damage 30 days after stroke. The mTOR inhibitor rapamycin abolished the long‐term protective effects of IPostC. We determined that IPostC did not inhibit acute infarction in nude rats but did provide long‐term protection by enhancing Akt and mTOR activity during the acute post‐stroke phase.
The effect of psychoactive drugs on depression has usually been studied in cases of prolonged drug addiction and/or withdrawal, without much emphasis on the effects of subchronic or recreational drug use. To address this issue, we exposed laboratory rats to subchronic regimens of heroin or cocaine and tested long‐term effects on (i) depressive‐like behaviors, (ii) brain‐derived neurotrophic factor (BDNF) levels in reward‐related brain regions, and (iii) depressive‐like behavior following an additional chronic mild stress procedure. The long‐term effect of subchronic cocaine exposure was a general reduction in locomotor activity whereas heroin exposure induced a more specific increase in immobility during the forced swim test. Both cocaine and heroin exposure induced alterations in BDNF levels that are similar to those observed in several animal models of depression. Finally, both cocaine and heroin exposure significantly enhanced the anhedonic effect of chronic mild stress. These results suggest that subchronic drug exposure induces depressive‐like behavior which is accompanied by modifications in BDNF expression and increases the vulnerability to develop depressive‐like behavior following chronic stress. Implications for recreational and small‐scale drug users are discussed.
The mechanism by which the drug rapamycin inhibits the mechanistic target of rapamycin (mTOR) is of intense interest because of its likely relevance in cancer biology, aging, and other age‐related diseases. While rapamycin acutely and directly inhibits mTORC1, only chronic administration of rapamycin can inhibit mTORC2 in some, but not all, cell lines or tissues. The mechanism leading to cell specificity of mTORC2 inhibition by rapamycin is not understood and is especially important because many of the negative metabolic side effects of rapamycin, reported in mouse studies and human clinical trials, have been attributed recently to mTORC2 inhibition. Here, we identify the expression level of different FK506‐binding proteins (FKBPs), primarily FKBP12 and FKBP51, as the key determinants for rapamycin‐mediated inhibition of mTORC2. In support, enforced reduction of FKBP12 completely converts a cell line that is sensitive to mTORC2 inhibition to an insensitive cell line, and increased expression can enhance mTORC2 inhibition. Further reduction of FKBP12 in cell lines with already low FKBP12 levels completely blocks mTORC1 inhibition by rapamycin, indicating that relative FKBP12 levels are critical for both mTORC1 and mTORC2 inhibition, but at different levels. In contrast, reduction of FKBP51 renders cells more sensitive to mTORC2 inhibition. Our findings reveal that the expression of FKBP12 and FKBP51 is the rate limiting factor that determines the responsiveness of a cell line or tissue to rapamycin. These findings have implications for treating specific diseases, including neurodegeneration and cancer, as well as targeting aging in general. 相似文献
Brain‐derived neurotrophic factor (BDNF), corticotropin‐releasing factor (CRF), and hypothalamic neuronal histamine are anorexigenic substances within the hypothalamus. This study examined the interactions among BDNF, CRF, and histamine during the regulation of feeding behavior in rodents. Food intake was measured after treatment with BDNF, α‐fluoromethyl histidine (FMH; a specific suicide inhibitor of histidine decarboxylase that depletes hypothalamic neuronal histamine), or CRF antagonist. We measured food intake in wild‐type mice and mice with targeted disruption of the histamine H1 receptor (H1KO mice) after central BDNF infusion. Furthermore, we investigated CRF content and histamine turnover in the hypothalamus after BDNF treatment, and conversely, BDNF content in the hypothalamus after histamine treatment. We used immunohistochemical staining for histamine H1 receptors (H1‐R) in BDNF neurons. BDNF‐induced feeding suppression was partially attenuated in rats pre‐treated with FMH or a CRF antagonist, and in H1KO mice. BDNF treatment increased CRF content and histamine turnover in the hypothalamus. Histamine increased BDNF content in the hypothalamus. Immunohistochemical analysis revealed that H1‐Rs were expressed on BDNF neurons in the ventromedial nucleus of the hypothalamus. These results indicate that CRF and hypothalamic neuronal histamine mediate the suppressive effects of BDNF on feeding behavior and body weight. 相似文献
Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient‐sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome‐lysosome reformation (ALR). Here we identify a lysosomal pool of phosphatidylinositol 3‐phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3‐kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process. 相似文献
D2/D3 dopamine receptors (D2R/D3R) agonists regulate Akt, but their effects display a complex time‐course. In addition, the respective roles of D2R and D3R are not defined and downstream targets remain poorly characterized, especially in vivo. These issues were addressed here for D3R. Systemic administration of quinelorane, a D2R/D3R agonist, transiently increased phosphorylation of Akt and GSK‐3β in rat nucleus accumbens and dorsal striatum with maximal effects 10 min after injection. Akt activation was associated with phosphorylation of several effectors of the mammalian target of rapamycin complex 1 (mTORC1): p70S6 kinase, ribosomal protein‐S6 (Ser240/244), and eukaryotic initiation factor‐4E binding protein‐1. The action of quinelorane was antagonized by a D2/D3R antagonist, raclopride, and the selective D3R antagonist S33084, inactive by themselves. Furthermore, no effect of quinerolane was seen in knock‐out mice lacking D3R. In drd1a‐EGFP transgenic mice, quinelorane activated Akt/GSK‐3β in both neurons expressing and lacking D1 receptor. Thus, the stimulation of D3R transiently activates the Akt/GSK‐3β pathway in the two populations of medium‐size spiny neurons of the nucleus accumbens and dorsal striatum. This effect may contribute to the influence of D3R ligands on reward, cognition, and processes disrupted in schizophrenia, drug abuse, and Parkinson's disease. 相似文献
Doxorubicin is the mainstay of treatment for various haematological malignancies and solid tumours. However, its clinical application may be hampered by dose‐dependent cardiotoxicity. The mechanism of doxorubicin‐induced cardiotoxicity may involve various signalling pathways including free radical generation, peroxynitrite formation, calcium overloading, mitochondrial dysfunction and alteration in apoptosis and autophagy. Interestingly, the use of resveratrol in combination with doxorubicin has been reported to prevent cardiac toxicity as well as to exert a synergistic effect against tumour cells both in vivo and in vitro. Thus, the aim of this review is to summarize current knowledge and to elucidate the protective effect of resveratrol in doxorubicin‐induced cardiotoxicity. 相似文献
The use of the mammal target of rapamycin (mTOR) inhibitors has been consolidated as the therapy of election for preventing graft rejection in kidney transplant patients, despite their immunosuppressive activity is less strong than anti‐calcineurin agents like tacrolimus and cyclosporine A. Furthermore, as mTOR is widely expressed, rapamycin (a macrolide antibiotic produced by Streptomyces hygroscopicus) is recommended in patients presenting neoplasia due to its antiproliferative actions. Hence, we have investigated whether rapamycin presents side effects in the physiology of other cell types different from leucocytes, such as platelets. Blood samples were drawn from healthy volunteers and kidney transplant patients long‐term medicated with rapamycin: sirolimus and everolimus. Platelets were either loaded with fura‐2 or directly stimulated, and immunoassayed or fixed with Laemmli's buffer to perform the subsequent analysis of platelet physiology. Our results indicate that rapamycin evokes a biphasic time‐dependent alteration in calcium homeostasis and function in platelets from kidney transplant patients under rapamycin regime, as demonstrated by the reduction in granule secretion observed and subsequent impairment of platelet aggregation in these patients compared with healthy volunteers. Platelet count was also reduced in these patients, thus 41% of patients presented thrombocytopenia. All together our results show that long‐term administration of rapamycin to kidney transplant patients evokes alteration in platelet function. 相似文献
Aging is a risk factor for Alzheimer's disease (AD) and is associated with cognitive decline. However, underlying molecular mechanisms of brain aging are not clear. Recent studies suggest epigenetic influences on gene expression in AD, as DNA methylation levels influence protein and mRNA expression in postmortem AD brain. We hypothesized that some of these changes occur with normal aging. To test this hypothesis, we measured markers of the arachidonic acid (AA) cascade, neuroinflammation, pro‐ and anti‐apoptosis factors, and gene specific epigenetic modifications in postmortem frontal cortex from nine middle‐aged [41 ± 1 (SEM) years] and 10 aged subjects (70 ± 3 years). The aged compared with middle‐aged brain showed elevated levels of neuroinflammatory and AA cascade markers, altered pro and anti‐apoptosis factors and loss of synaptophysin. Some of these changes correlated with promoter hypermethylation of brain derived neurotrophic factor (BDNF), cyclic AMP responsive element binding protein (CREB), and synaptophysin and hypomethylation of BCL‐2 associated X protein (BAX). These molecular alterations in aging are different from or more subtle than changes associated with AD pathology. The degree to which they are related to changes in cognition or behavior during normal aging remains to be evaluated. 相似文献
Autophagy is the main cellular catabolic process responsible for degrading organelles and large protein aggregates. It is initiated by the formation of a unique membrane structure, the phagophore, which engulfs part of the cytoplasm and forms a double‐membrane vesicle termed the autophagosome. Fusion of the outer autophagosomal membrane with the lysosome and degradation of the inner membrane contents complete the process. The extent of autophagy must be tightly regulated to avoid destruction of proteins and organelles essential for cell survival. Autophagic activity is thus regulated by external and internal cues, which initiate the formation of well‐defined autophagy‐related protein complexes that mediate autophagosome formation and selective cargo recruitment into these organelles. Autophagosome formation and the signaling pathways that regulate it have recently attracted substantial attention. In this review, we analyze the different signaling pathways that regulate autophagy and discuss recent progress in our understanding of autophagosome biogenesis. 相似文献
Amino acids regulate TOR complex 1 (TORC1) via two counteracting mechanisms, one activating and one inactivating. The presence of amino acids causes TORC1 recruitment to lysosomes where TORC1 is activated by binding Rheb. How the absence of amino acids inactivates TORC1 is less well understood. Amino acid starvation recruits the TSC1/TSC2 complex to the vicinity of TORC1 to inhibit Rheb; however, the upstream mechanisms regulating TSC2 are not known. We identify here the eIF4A‐containing eIF4F translation initiation complex as an upstream regulator of TSC2 in response to amino acid withdrawal in Drosophila. We find that TORC1 and translation preinitiation complexes bind each other. Cells lacking eIF4F components retain elevated TORC1 activity upon amino acid removal. This effect is specific for eIF4F and not a general consequence of blocked translation. This study identifies specific components of the translation machinery as important mediators of TORC1 inactivation upon amino acid removal. 相似文献
Stroke is a multi‐factorial polygenic disease and is a major cause of death and adult disability. Administration of bone marrow stem cells protects ischemic rat brain by facilitating recovery of neurological functions. But the molecular mechanism of stem cells action and their effect on gene expression is not well explored. In this study, we have transplanted 1 × 106 human bone marrow mesenchymal stem cells (hBMMSCs) in middle cerebral artery occluded (MCAo) adult male Wistar rats through intracarotid artery route at 24 h after surgery. Motor behavioral tests (rotarod and open field) were performed to assess the changes in motor functions at day 0 and day1, 4, 8 and 14. The expression of studied genes at mRNA and protein level was quantified by using Q‐PCR and western blotting, respectively. Further, we have assessed the methylation pattern of promoter of these genes by using methylation‐specific PCR. Data were analyzed statistically and correlated. A significant improvement in behavioral deficits was observed in stem cells treated group after 14th day post stroke. Significantly (p < 0.05) increased mRNA and protein levels of brain derived neurotrophic factor and ANP genes in hBMMSCs treated group along with decrease in methylation level at their promoter was observed. On the other hand, significantly decreased mRNA and protein level of TSP1 and WNK1 in hBMMSCs treated group was observed. In conclusion, hBMMSCs administration significantly improves the behavioral deficits by improving motor and locomotor coordination. The promoter of TSP1 and WNK1 genes was found to be hyper‐methylated in hBMMSCs group resulting in their decreased expression while the promoter of ANP and brain derived neurotrophic factor was found to be hypo‐methylated. This study might shed a light on how hBMMSCs affect the gene expression by modulating methylation status.
Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates cell growth, metabolism, and autophagy to maintain cellular homeostasis. Its activity is inhibited by adverse conditions, including nutrient limitation, hypoxia, and DNA damage. In this study, we demonstrate that Che‐1, a RNA polymerase II‐binding protein activated by the DNA damage response, inhibits mTOR activity in response to stress conditions. We found that, under stress, Che‐1 induces the expression of two important mTOR inhibitors, Redd1 and Deptor, and that this activity is required for sustaining stress‐induced autophagy. Strikingly, Che‐1 expression correlates with the progression of multiple myeloma and is required for cell growth and survival, a malignancy characterized by high autophagy response. 相似文献
Apoptosis plays a critical role in the development of myocardial infarction. Cardiomyocytes are enriched with mitochondria and excessive mitochondrial fission can trigger cellular apoptosis. Recently, the mitochondrial ubiquitin ligase (MITOL), localized in the mitochondrial outer membrane, was reported to play an important role in the regulation of mitochondrial dynamics and apoptosis. However, the underlying mechanism of its action remains uncertain. The present study was aimed at uncovering the role of MITOL in the regulation of cardiomyocyte apoptosis. Our results showed that MITOL expression was up‐regulated in cardiomyocytes in response to apoptotic stimulation. Mitochondrial ubiquitin ligase overexpression blocked dynamin‐related protein 1 accumulation in the mitochondria, and attenuated the mitochondrial fission induced by hydrogen peroxide. Conversely, MITOL knockdown sensitized cardiomyocytes to undergo mitochondrial fission, resulting in subsequent apoptosis. These findings suggest that MITOL plays a protective role against apoptosis in cardiomyocytes, and may serve as a potential therapeutic target for apoptosis‐related cardiac diseases. 相似文献
Long non‐coding RNAs (lncRNAs) have emerged as new and important regulators of pathological processes including tumour development. In this study, we demonstrated that differentiation antagonizing non‐protein coding RNA (DANCR) was up‐regulated in lung adenocarcinoma (ADC) and that the knockdown of DANCR inhibited tumour cell proliferation, migration and invasion and restored cell apoptosis rescued; cotransfection with a miR‐496 inhibitor reversed these effects. Luciferase reporter assays showed that miR‐496 directly modulated DANCR; additionally, we used RNA‐binding protein immunoprecipitation (RIP) and RNA pull‐down assays to further confirm that the suppression of DANCR by miR‐496 was RISC‐dependent. Our study also indicated that mTOR was a target of miR‐496 and that DANCR could modulate the expression levels of mTOR by working as a competing endogenous RNA (ceRNA). Furthermore, the knockdown of DANCR reduced tumour volumes in vivo compared with those of the control group. In conclusion, this study showed that DANCR might be an oncogenic lncRNA that regulates mTOR expression through directly binding to miR‐496. DANCR may be regarded as a biomarker or therapeutic target for ADC. 相似文献
The traditional Chinese medicine Danshensu (DSS) has a protective effect on cardiac ischaemia/reperfusion (I/R) injury. However, the molecular mechanisms underlying the DSS action remain undefined. We investigated the potential role of DSS in autophagy and apoptosis using cardiac I/R injury models of cardiomyocytes and isolated rat hearts. Cultured neonatal rat cardiomyocytes were subjected to 6 hrs of hypoxia followed by 18 hrs of reoxygenation to induce cell damage. The isolated rat hearts were used to perform global ischaemia for 30 min., followed by 60 min. reperfusion. Ischaemia/reperfusion injury decreased the haemodynamic parameters on cardiac function, damaged cardiomyocytes or even caused cell death. Pre‐treatment of DSS significantly improved cell survival and protected against I/R‐induced deterioration of cardiac function. The improved cell survival upon DSS treatment was associated with activation of mammalian target of rapamycin (mTOR) (as manifested by increased phosphorylation of S6K and S6), which was accompanied with attenuated autophagy flux and decreased expression of autophagy‐ and apoptosis‐related proteins (including p62, LC3‐II, Beclin‐1, Bax, and Caspase‐3) at both protein and mRNA levels. These results suggest that alleviation of cardiac I/R injury by pre‐treatment with DSS may be attributable to inhibiting excessive autophagy and apoptosis through mTOR activation. 相似文献
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