Glutamate transporters (also called excitatory amino acid transporters, EAATs) bind extracellular glutamate and transport it to intracellular space to regulate glutamate neurotransmission and to maintain extracellular glutamate concentrations below neurotoxic levels. We previously showed that isoflurane, a commonly used anesthetic, enhanced the activity of EAAT3, a major neuronal EAAT. This effect required a protein kinase C (PKC) ␣-dependent EAAT3 redistribution to the plasma membrane. In this study, we prepared COS7 cells stably expressing EAAT3 with or without mutations of potential PKC phosphorylation sites in the putative intracellular domains. Here we report that mutation of threonine 5 or threonine 498 to alanine did not affect the isoflurane effects on EAAT3. However, the mutation of serine 465 to alanine abolished isoflurane-induced increase of EAAT3 activity and redistribution to the plasma membrane. The mutation of serine 465 to aspartic acid increased the expression of EAAT3 in the plasma membrane and also abolished the isoflurane effects on EAAT3. These results suggest an essential role of serine 465 in the isofluraneincreased EAAT3 activity and redistribution and a direct effect of PKC on EAAT3. Consistent with these results, isoflurane induced an increase in phosphorylation of wild type, T5A, and T498A EAAT3, and this increase was absent in S465A and S465D. Our current results, together with our previous data that showed the involvement of PKC␣ in the isoflurane effects on EAAT3, suggest that the phosphorylation of serine 465 in EAAT3 by PKC␣ mediates the increased EAAT3 activity and redistribution to plasma membrane after isoflurane exposure.Glutamate is the major excitatory neurotransmitter in the central nervous system. Similar to the case with many other neurotransmitters, there is no extracellular enzyme known to metabolize glutamate. Glutamate transporters, also called excitatory amino acid transporters (EAATs), 2 transport glutamate from extracellular space into cells (1, 2). Thus, EAATs play a critical role in securing a high signal-to-noise ratio in glutamate neurotransmission and in preventing harmful over-stimulation of glutamate receptors under physiological conditions (2). Inhibition of EAATs has been shown to prolong the time course of glutamate neurotransmission (3), and decreased expression of EAATs is associated with neurodegeneration and increased infarct volume after brain ischemia (4, 5). Five EAATs have been identified so far: EAAT1-5. They have about 520 -580 amino acids. In rats, EAAT1 and EAAT2 are expressed in glial cells, EAAT3 and EAAT4 are found in neurons and EAAT5 is located in the neurons and glial cells of retina (2). EAAT1, EAAT2, and EAAT3 are distributed in many brain regions including cerebral cortex, hippocampus, and cerebellum, whereas EAAT4 is predominantly expressed in the cerebellum (6). Thus, EAAT3 is the major neuronal EAAT in the central nervous system. All five EAATs are sodium co-transporters and require potassium coupling to complete the transporting ...
Mitochondrial dynamics, a complicated cellular process consisting of mitochondrial fusion and fission, has been suggested to be involved in regulating the stemness of bone marrow mesenchymal stem cells (BMSCs). This study was undertaken to explore the relationship between mitochondrial dynamics and the maintenance of BMSCs’ stemness. Rat BMSCs were treated with fibroblast growth factor 2 (FGF2) and epithelial growth factor (EGF) to induce differentiation. Mitochondrial dynamics was determined by mitochondrial length observed by confocal microscope and DLP1 (a protein promoting mitochondrial fission), OPA1 (a protein promoting mitochondrial fusion) expression revealed by Western blotting analysis. BMSCs’ stemness was determined by flow cytometry and osteogenic/adipogenic differentiation ability. We found that in the process of BMSCs differentiation, mitochondrial length was increased, along with a decreased protein level of DLP1 and an increased protein level of OPA1 in the mitochondria, indicating a shift toward mitochondrial fusion in BMSCs during differentiation. Notably, when the mitochondrial fission was inhibited by Mdivi-1, the stemness marker, CD90, was deceased along with the reduction of DLP1 expression. Under the same condition, the potential of BMSCs to be induced into adipocytes or osteocytes was decreased. Correspondingly, when BMSCs were treated with tyrphostin A9, a reagent promoting mitochondrial fission by increasing DLP1, the stemness marker, CD54, was increased with an increased potential of BMSCs to be induced into adipocytes or osteocytes. Hence, our results demonstrated that mitochondrial fission contributed to the maintenance of BMSCs’ stemness. Impact statement How to maintain the stemness of bone marrow mesenchymal stem cells (BMSCs) in cultures is a long-standing question. The present study found that mitochondrial dynamics affects the stemness of BMSCs in cultures and the retaining of mitochondrial fission enhances the stemness of BMSCs. This work thus provides a novel insight into strategic approaches to maintain the stemness of BMSCs in cultures in relation to the clinical application of bone-marrow stem cells.
Previous studies demonstrated that mitochondrial fission arguments the stemness of bone marrow-derived mesenchymal stem cells (BMSCs). Because mitophagy is critical in removing damaged or surplus mitochondrial fragments and maintaining mitochondrial integrity, the present study was undertaken to test the hypothesis that mitophagy is involved in mitochondrial fission-enhanced stemness of BMSCs. Primary cultures of rat BMSCs were treated with tyrphostin A9 (TA9, a potent inducer of mitochondrial fission) to increase mitochondrial fission, which was accompanied by enhanced mitophagy as defined by increased co-staining of MitoTracker Green for mitochondria and LysoTracker Deep Red for lysosomes, as well as the increased co-localization of autophagy markers (LC3B, P62) and mitochondrial marker (Tom20). A mitochondrial uncoupler, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was used to promote mitophagy, which was confirmed by an increased co-localization of mitochondrial and lysosome biomarkers. The argumentation of mitophagy was associated with enhanced stemness of BMSCs as defined by increased expression of stemness markers Oct4 and Sox2, and enhanced induction of BMSCs to adipocytes or osteocytes. Conversely, transfection of BMSCs with siRNA targeting mitophagy-essential genes Pink1/ Prkn led to diminished stemness of the stem cells, as defined by depressed stemness markers. Importantly, concomitant promotion of mitochondrial fission and inhibition of mitophagy suppressed the stemness of BMSCs. These results thus demonstrate that mitophagy is critically involved in mitochondrial fission promotion of the stemness of BMSCs.
Cytochrome c oxidase (CCO) is a copper-dependent enzyme of mitochondrial respiratory chain. In pressure overload-induced cardiac hypertrophy, copper level and CCO activity are both depressed, along with disturbance in mitochondrial fusion and fission dynamics. Copper repletion leads to recovery of CCO activity and normalized mitochondrial dynamics. The present study was undertaken to define the link between CCO activity and mitochondrial dynamic changes. Primary cultures of neonatal rat cardiomyocytes were treated with phenylephrine to induce cell hypertrophy. Hypertrophic cardiomyocytes were then treated with copper to reverse hypertrophy. In the hypertrophic cardiomyocytes, CCO activity was depressed and mitochondrial fusion was suppressed. Upon copper repletion, CCO activity was recovered and mitochondrial fusion was reestablished. Depression of CCO activity by siRNA targeting CCO assembly homolog 17 (COX17), a copper chaperone for CCO, led to fragmentation of mitochondria, which was not recoverable by copper supplementation. This study thus demonstrates that copper-dependent CCO is critical for mitochondrial fusion in the regression of cardiomyocyte hypertrophy.
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