Methylene blue (MB), a tricyclic phenothiazine drug, has been reported to enhance mitochondrial functions including mitochondrial respiration. By comparison, stress associated with abrupt ethanol withdrawal (EW) imped...Methylene blue (MB), a tricyclic phenothiazine drug, has been reported to enhance mitochondrial functions including mitochondrial respiration. By comparison, stress associated with abrupt ethanol withdrawal (EW) impedes mitochondrial functions. We investigated whether MB protects mitochondrial respiration and cell survival from EW stress through a key mitochondrial enzyme, cytochrome c oxidase (COX). We also investigated whether the MB’s protection involves the inhibition of an excitatory neurotransmitter, glutamate. Male rats were exposed to and withdrawn from ethanol-diet (7.5%, 5 weeks). MB (0.5 mg/kg, intraperitoneal) was injected for the last 5 days of ethanol-diet and on the first day of EW. Cerebellum was then harvested to measure mitochondrial respiration and COX expression using real-time XF respirometer and immunohistochemistry, respectively. Separately, HT22 cells (a murine hippocampal cell line) were exposed to and abruptly withdrawn for 4 hours from chronic ethanol (100 mM, 3 days). MB was administered during EW with or without a COX inhibitor (NaN3) or glutamate. Mitochondrial respiration, COX content, and cell viability were then assessed using real-time XF respirometer, an immunoblot method, and Calcein assay, respectively. MB attenuated the suppressing effects of EW on mitochondrial respiration, COX content, and cell survival. This protection was reduced after NaN3 or glutamate cotreatment. These results suggest that MB treatment help maintain mitochondrial respiratory and cellular integrity through COX-upregulation and glutamateinhibition upon EW stress. MB treatment may help identify mitochondrial mechanisms underlying hyperexcitatory CNS disorders.展开更多
Benzodiazepine (BZD) is the most prescribed CNS depressant in America to treat hyper-excitatory disorders such as anxiety and insomnia. However, the chronic use of BZD often creates adverse effects including psychomot...Benzodiazepine (BZD) is the most prescribed CNS depressant in America to treat hyper-excitatory disorders such as anxiety and insomnia. However, the chronic use of BZD often creates adverse effects including psychomotor deficit. In this study, we investigated a novel mechanism by which chronic BZD impedes motoric function in female mice. We used female mice because BZD use is much more prevalent in female than male populations. We tested the hypothesis that the accumulation of p38 (stress-activated protein) in cerebellar Purkinje neurons mediates motoric deficit induced by chronic BZD. To test this hypothesis, we generated transgenic mice that lack p38 incerebellar Purkinje neurons by crossing Pcp2 (Purkinje cell protein 2)-Cre mice with p38loxP/loxP mice. p38-knockdown mice and wild-type mice received BZD (lorazepam, 0.5 mg/kg) for 14 days. During this period, they were tested for motoric performance using Rotarod assay in which a quicker fall from rotating rod indicates poorer motoric performance. Cerebellum was then collected to detect p38 inPurkinje neurons and to measure mitochondrial respiration using immunohistochemistry and real-time XF respirometry, respectively. Compared to vehicletreated mice, BZD-treated mice showed poorer motoric performance, a higher number of Purkinje neurons containing p38, and lower mitochondrial respiration. These effects of BZD were much smaller in p38-knockdown mice. These results suggest that the excessive accumulation of p38 incerebellar Purkinje neurons contributes to motoric deficit associated with chronic BZD. They also suggest that Purkinje neuronal p38 mediates BZD-induced mitochondrial respiratory inhibition in cerebellum. Our findings may provide a new mechanistic insight into chronic BZD-induced motoric deficit.展开更多
文摘Methylene blue (MB), a tricyclic phenothiazine drug, has been reported to enhance mitochondrial functions including mitochondrial respiration. By comparison, stress associated with abrupt ethanol withdrawal (EW) impedes mitochondrial functions. We investigated whether MB protects mitochondrial respiration and cell survival from EW stress through a key mitochondrial enzyme, cytochrome c oxidase (COX). We also investigated whether the MB’s protection involves the inhibition of an excitatory neurotransmitter, glutamate. Male rats were exposed to and withdrawn from ethanol-diet (7.5%, 5 weeks). MB (0.5 mg/kg, intraperitoneal) was injected for the last 5 days of ethanol-diet and on the first day of EW. Cerebellum was then harvested to measure mitochondrial respiration and COX expression using real-time XF respirometer and immunohistochemistry, respectively. Separately, HT22 cells (a murine hippocampal cell line) were exposed to and abruptly withdrawn for 4 hours from chronic ethanol (100 mM, 3 days). MB was administered during EW with or without a COX inhibitor (NaN3) or glutamate. Mitochondrial respiration, COX content, and cell viability were then assessed using real-time XF respirometer, an immunoblot method, and Calcein assay, respectively. MB attenuated the suppressing effects of EW on mitochondrial respiration, COX content, and cell survival. This protection was reduced after NaN3 or glutamate cotreatment. These results suggest that MB treatment help maintain mitochondrial respiratory and cellular integrity through COX-upregulation and glutamateinhibition upon EW stress. MB treatment may help identify mitochondrial mechanisms underlying hyperexcitatory CNS disorders.
文摘Benzodiazepine (BZD) is the most prescribed CNS depressant in America to treat hyper-excitatory disorders such as anxiety and insomnia. However, the chronic use of BZD often creates adverse effects including psychomotor deficit. In this study, we investigated a novel mechanism by which chronic BZD impedes motoric function in female mice. We used female mice because BZD use is much more prevalent in female than male populations. We tested the hypothesis that the accumulation of p38 (stress-activated protein) in cerebellar Purkinje neurons mediates motoric deficit induced by chronic BZD. To test this hypothesis, we generated transgenic mice that lack p38 incerebellar Purkinje neurons by crossing Pcp2 (Purkinje cell protein 2)-Cre mice with p38loxP/loxP mice. p38-knockdown mice and wild-type mice received BZD (lorazepam, 0.5 mg/kg) for 14 days. During this period, they were tested for motoric performance using Rotarod assay in which a quicker fall from rotating rod indicates poorer motoric performance. Cerebellum was then collected to detect p38 inPurkinje neurons and to measure mitochondrial respiration using immunohistochemistry and real-time XF respirometry, respectively. Compared to vehicletreated mice, BZD-treated mice showed poorer motoric performance, a higher number of Purkinje neurons containing p38, and lower mitochondrial respiration. These effects of BZD were much smaller in p38-knockdown mice. These results suggest that the excessive accumulation of p38 incerebellar Purkinje neurons contributes to motoric deficit associated with chronic BZD. They also suggest that Purkinje neuronal p38 mediates BZD-induced mitochondrial respiratory inhibition in cerebellum. Our findings may provide a new mechanistic insight into chronic BZD-induced motoric deficit.
