Preventing mitochondrial fission impairs mitochondrial function and leads to loss of mitochondrial DNA

Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS). At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.     

Hyperglycemia induces oxidative stress and impairs axonal transport rates in mice

Background

While hyperglycemia-induced oxidative stress damages peripheral neurons, technical limitations have, in part, prevented in vivo studies to determine the effect of hyperglycemia on the neurons in the central nervous system (CNS). While olfactory dysfunction is indicated in diabetes, the effect of hyperglycemia on olfactory receptor neurons (ORNs) remains unknown. In this study, we utilized manganese enhanced MRI (MEMRI) to assess the impact of hyperglycemia on axonal transport rates in ORNs. We hypothesize that (i) hyperglycemia induces oxidative stress and is associated with reduced axonal transport rates in the ORNs and (ii) hyperglycemia-induced oxidative stress activates the p38 MAPK pathway in association with phosphorylation of tau protein leading to the axonal transport deficits.

Research Design and Methods

T₁-weighted MEMRI imaging was used to determine axonal transport rates post-streptozotocin injection in wildtype (WT) and superoxide dismutase 2 (SOD2) overexpressing C57Bl/6 mice. SOD2 overexpression reduces mitochondrial superoxide load. Dihydroethidium staining was used to quantify the reactive oxygen species (ROS), specifically, superoxide (SO). Protein and gene expression levels were determined using western blotting and Q-PCR analysis, respectively.

Results

STZ-treated WT mice exhibited significantly reduced axonal transport rates and significantly higher levels of ROS, phosphorylated p38 MAPK and tau protein as compared to the WT vehicle treated controls and STZ-treated SOD2 mice. The gene expression levels of p38 MAPK and tau remained unchanged.

Conclusion

Increased oxidative stress in STZ-treated WT hyperglycemic mice activates the p38 MAPK pathway in association with phosphorylation of tau and attenuates axonal transport rates in the olfactory system. In STZ-treated SOD-overexpressing hyperglycemic mice in which superoxide levels are reduced, these deficits are reversed.     

Hyperglycemia: cell death in a cave

Mitochondria play a central role in mediating high glucose-induced apoptosis. A recent study has shown that increases in glucose levels induce significant alterations in caveolae components, suggesting that high glucose may affect apoptotic signaling initiated in caveolae.     

Efficient chaperone-mediated tubulin biogenesis is essential for cell division and cell migration in C. elegans

The efficient folding of actin and tubulin in vitro and in Saccharomyces cerevisiae is known to require the molecular chaperones prefoldin and CCT, yet little is known about the functions of these chaperones in multicellular organisms. Whereas none of the six prefoldin genes are essential in yeast, where prefoldin-independent folding of actin and tubulin is sufficient for viability, we demonstrate that reducing prefoldin function by RNAi in Caenorhabditis elegans causes defects in cell division that result in embryonic lethality. Our analyses suggest that these defects result mainly from a decrease in α-tubulin levels and a subsequent reduction in the microtubule growth rate. Prefoldin subunit 1 (pfd-1) mutant animals with maternally contributed PFD-1 develop to the L4 larval stage with gonadogenesis defects that include aberrant distal tip cell migration. Importantly, RNAi knockdown of prefoldin, CCT or tubulin in developing animals phenocopy the pfd-1 cell migration phenotype. Furthermore, reducing CCT function causes more severe phenotypes (compared with prefoldin knockdown) in the embryo and developing gonad, consistent with a broader role for CCT in protein folding. Overall, our results suggest that efficient chaperone-mediated tubulin biogenesis is essential in C. elegans, owing to the critical role of the microtubule cytoskeleton in metazoan development.     

Decreased mitochondrial biogenesis in temperature-sensitive cell division cycle mutants of Saccharomyces cerevisiae

The temperature-sensitive cell division cycle (cdc) G1 mutants cdc28 and cdc35 show decreased mitochondrial volumes with respect to the wild type strain A364A (WT) at the restrictive temperature. Of the three criteria of mitochondrial biogenesis studied, that is, number of mitochondria per cell, relative area of the cell occupied by mitochondria, or relative area of mitochondria occupied by inner membranes, only the second indicator was significantly lower in cdc mutants than in the WT. The mitochondrial inner membranes development did not compensate for the decrease in the organelles volume. Apparently, the reduced mitochondrial biogenesis was not due to the temperature shift because the relative area of the cell occupied by mitochondria was already significantly lower at 25°C in cdc mutants. The specific fluxes of oxygen consumption confirmed that the respiratory capacity of cdc mutants is largely impaired in respect to the WT. Cdc28 and cdc35 mutants of Saccharomyces cerevisiae had been previously shown to exhibit high respiratory quotients (from 3 to 7) in respect to the WT (RQ 1.0), which correlated with carbon and energy uncoupling probably the result of glucose-induced catabolite repression [Aon MA, Mónaco ME, Cortassa S (1995) Exp Cell Res 217, 42–51; Mónaco ME, Valdecantos PA, Aon MA (1995) Exp Cell Res 217, 52–56].     

Hyperglycemia increases mitochondrial superoxide in retina and retinal cells

Oxidative stress is believed to play a significant role in the development of diabetic retinopathy. In this study, we have investigated the effects of elevated glucose concentration on the production of superoxide anion by retina and retinal cells, the cellular source of the superoxide, the effect of therapies that are known to inhibit diabetic retinopathy on the superoxide production, and the role of the superoxide in cell death in elevated glucose concentration. Superoxide release was measured from retinas collected from streptozotocin-diabetic rats (2 months) treated with or without aminoguanidine, aspirin, or vitamin E, and from transformed retinal Müller cells (rMC-1) and bovine retinal endothelial cells (BREC) incubated in normal (5 mM) and high (25 mM) glucose. Diabetes (retina) or incubation in elevated glucose concentration (rMC-1 and BREC cells) significantly increased superoxide production, primarily from mitochondria, because an inhibitor of mitochondrial electron transport chain complex II normalized superoxide production. Inhibition of reduced nicotinamine adenine dinucleotide phosphate (NADPH) oxidase or nitric oxide synthase had little or no effect on the glucose-induced increase in superoxide. Treatment of diabetic animals with aminoguanidine, aspirin, or vitamin E for 2 months significantly inhibited the diabetes-induced increase in production of superoxide in the retinas. Despite the increased production of superoxide, no increase in protein carbonyls was detected in retinal proteins from animals diabetic for 2-6 months or rMC-1 cells incubated in 25 mM glucose for 5 d unless the activities of calpain or the proteosome were inhibited. Addition of copper/zinc-containing superoxide dismutase to the media of rMC-1 and BREC cells inhibited the apoptotic death caused by elevated glucose. Diabetes-like glucose concentration increases superoxide production in retinal cells, and the superoxide contributes to impaired viability and increased cell death under those circumstances. Three therapies that inhibit the development of diabetic retinopathy all inhibit superoxide production, raising a possibility that these therapies inhibit retinopathy in part by inhibiting a hyperglycemia-induced increase in superoxide production.     

Antibiotic anisomycin induces cell cycle arrest and apoptosis through inhibiting mitochondrial biogenesis in osteosarcoma

The anti-cancer activities of antibiotic anisomycin have been demonstrated in kidney, colon and ovarian cancers whereas its underlying mechanisms are not well elucidated. In this work, we investigated whether anisomycin is effective in sensitizes osteosarcoma cell response to chemotherapy. We show that anisomycin inhibits proliferation via inducing osteosarcoma cell arrest at G2/M phase, accompanied by the increased levels of mitotic marker cyclin B and the decreased levels of Rb and E2F-1. Anisomycin also induces apoptosis in a caspase-dependent manner in osteosarcoma cells. Importantly, anisomycin is less effective in normal control NIH3T3 cells compared to osteosarcoma cells. In addition, anisomycin inhibits osteosarcoma growth in xenograft mouse model and enhances the inhibitory effects of doxorubicin in osteosarcoma in vitro and in vivo. Mechanistically, anisomycin targets mitochondrial biogenesis in osteosarcoma as shown by the decreased mitochondrial membrane potential, suppressed mitochondrial respiration via decreasing complex I activity, reduced ATP production. Furthermore, mitochondrial biogenesis stimulator acetyl-L-Carnitine (ALCAR) significantly rescues the inhibitory effects of anisomycin in osteosarcoma cells. Our work demonstrates that anisomycin is active against osteosarcoma cells and the molecular mechanism of its action is the inhibition of mitochondrial biogenesis.     

Gamma-ray irradiation impairs dendritic cell migration to CCL19 by down-regulation of CCR7 and induction of cell apoptosis

Dendritic cells (DCs) are the most potent antigen-presenting cells and play a crucial role in the regulation of immune response and migration of DCs into secondary lymphoid tissues also play an important role in the initiation of innate and adaptive immunity. Radiation therapy is now a routine treatment for certain types of cancer and over 20 percent of cancer patients will require radiation therapy during the treatment of their disease. However, the influence of ionizing irradiation on the migratory ability of DCs is largely unknown. In this article, we report that γ ray irradiation can significantly inhibit LPS-triggered up regulation of CCR7 expression and PGE2 production by DC, thus impairing DC migration towards CCL19 in vitro and in vivo. Moreover, γ ray exposed DC also displayed an increased apoptosis rate and decreased cell viability. Furthermore, we demonstrate that exogenous PGE2 can partly reduce the gamma-ray induced migratory impairment and restored CCR7 expression of DC. Our work suggests that γ irradiation affects DC function at multiple steps during the immune response including DC migration, and that PGE2, via control of CCR7 expression, is an important regulator of DC migration.     

The mitochondrial protein C1qbp promotes cell proliferation,migration and resistance to cell death

Complement 1q-Binding Protein (C1qbp) is a mitochondrial protein reported to be upregulated in cancer. However, whether C1qbp plays a tumor suppressive or tumorigenic role in the progression of cancer is controversial. Moreover, the exact effects of C1qbp on cell proliferation, migration, and death/survival have not been definitely proven. To this end, we comprehensively examined the effects of C1qbp on mitochondrial-dependent cell death, proliferation, and migration in both normal and breast cancer cells using genetic gain- and loss-of-function approaches. In normal fibroblasts, overexpression of C1qbp protected the cells against staurosporine-induce apoptosis, increased proliferation, decreased cellular ATP, and increased cell migration in a wound-healing assay. In contrast, the opposite effects were observed in fibroblasts depleted of C1qbp by RNA interference. C1qbp expression was found to be markedly elevated in 4 different human breast cancer cell lines as well as in ductal and adenocarcinoma tumors from breast cancer patients. Stable knockdown of C1qbp by shRNA in the aggressive MDA-MB-231 breast cancer cell line greatly reduced cell proliferation, increased ATP levels, and decreased cell migration compared to control shRNA-transfected cells. Moreover, C1qbp knockdown elicited a significant increase in doxorubicin-induced apoptosis in the MDA-MB-231 cells. Finally, C1qbp upregulation was not restricted to breast cancer cells and tumors, as levels of C1qbp were also found to be significantly elevated in both human lung and colon cancer cell lines and carcinomas. Together, these results establish a pro-tumor, rather than anti-tumor, role for C1qbp, and indicate that C1qbp could serve as a molecular target for cancer therapeutics.