Accumulation Of Fragmented Mitochondria Research Articles

Expansion Microscopy-based imaging for visualization of mitochondria in Drosophila ovarian germline stem cells.

Recent studies have shown that mitochondrial morphology can modulate organelle function and greatly affect stem cell behavior, thus affecting tissue homeostasis. As such, we previously showed that the accumulation of fragmented mitochondria in aged Drosophila ovarian germline stem cells (GSCs) contributes to age-dependent GSC loss. However, standard immunofluorescence methods to examine mitochondrial morphology yield images with insufficient resolution for rigorous analysis, while 3-dimensional electron microscopy examination of mitochondrial morphology is labor intensive and allows only limited sampling of mitochondria. To overcome these issues, we utilized the expansion microscopy technique to expand GSC samples by 4-fold in combination with mitochondrial immunofluorescence labeling. Here, we present a simple, inexpensive method for nanoscale optical imaging of mitochondria in the germline. This protocol may be beneficial for studies that require visualization of mitochondria or other fine subcellular structures in the Drosophila ovary.

Abstract 15660: Impaired Mitochondrial Quality Control Drives the Development of Cardiomyopathy in Anderson-Fabry Disease

Introduction: Anderson-Fabry disease (AFD) is a systemic disorder caused by impaired alpha-galactosidase (GLA) activity, leading to a progressive cardiomyopathy, with hypertrophy and diastolic dysfunction. However, the molecular mechanisms underlying these features are unknown. Aim: To explore the role of mitochondria as a pathogenic mechanism of cardiac dysfunction in AFD. Methods: We tested a transgenic mouse that lacks endogenous GLA and expresses human R301Q-GLA [Tg/GLA knockout]. We performed serial ecochardiographic, histological, and biochemical analyses on cardiac tissue and single cell contractility on isolated adult cardiomyocytes (CM). Moreover, we assessed mitochondrial morphology/function in mouse embryonic fibroblasts (MEFs). Results: AFD-MEFs display an impaired mitochondrial respiration and reduced ATP synthesis. Increased mitochondrial levels of DRP1, LC3II, and MFN-ubiquitination along with accumulation of fragmented mitochondria and autophagosomes indicate the presence of mitophagy-targeted mitochondria that were not removed. Reduced PGC-1a and mitochondrial-DNA synthesis in AFD-MEFs indicate impaired mitochondrial biogenesis as well. Altogether, our data indicate impaired mitophagy and quality control of AFD-mitochondria. At echocardiography, AFD mice showed diastolic dysfunction and hypertrophy starting from 9 months. Isolated AFD-CMs exhibit reduced inotropic response to isoproterenol. Remarkably, alterations of cardiac mitochondrial function were observed starting from 3 months, including increased levels of MFN2-ubiquitination, DRP1 and LC3II, decreased levels of PGC-1a along with accumulation of disarranged mitochondria and autophagosomes. Frank impairment of mitochondrial respiration in AFD-CMs and reduced myocardial ATP-content was detected from 6 months, together with increased release of Cytochrome-C in the cytosol. Strikingly, mitochondrial dysfunction was also detected in fibroblasts isolated from AFD patients, which have a significantly reduced electron transport chain activity. Conclusions: In AFD, energetic alterations associated with impaired autophagy/mitophagy, are evident at 3 months and progressively lead to cardiac hypertrophy and dysfunction.

PINK1/TAX1BP1-directed mitophagy attenuates vascular endothelial injury induced by copper oxide nanoparticles

Copper oxide nanoparticles (CuONPs) are widely used metal oxide NPs owing to their excellent physical–chemical properties. Circulation translocation of CuONPs after inhalation leads to vascular endothelial injury. Mitochondria, an important regulatory hub for maintaining cell functions, are signaling organelles in responses to NPs-induced injury. However, how mitochondrial dynamics (fission and fusion) and mitophagy (an autophagy process to degrade damaged mitochondria) are elaborately orchestrated to maintain mitochondrial homeostasis in CuONPs-induced vascular endothelial injury is still unclear. In this study, we demonstrated that CuONPs exposure disturbed mitochondrial dynamics through oxidative stress-dependent manner in vascular endothelial cells, as evidenced by the increase of mitochondrial fission and the accumulation of fragmented mitochondria. Inhibition of mitochondrial fission with Mdivi-1 aggravated CuONPs-induced mtROS production and cell death. Furthermore, we found that mitochondrial fission led to the activation of PINK1-mediated mitophagy, and pharmacological inhibition with wortmannin, chloroquine or genetical inhibition with siRNA-mediated knockdown of PINK1 profoundly repressed mitophagy, suggesting that the protective role of mitochondrial fission and PINK1-mediated mitophagy in CuONPs-induced toxicity. Intriguingly, we identified that TAX1BP1 was the primary receptor to link the ubiquitinated mitochondria with autophagosomes, since TAX1BP1 knockdown elevated mtROS production, decreased mitochondrial clearance and aggravated CuONPs-induced cells death. More importantly, we verified that urolithin A, a mitophagy activator, promoted mtROS clearance and the removal of damaged mitochondria induced by CuONPs exposure both in vitro and in vivo. Overall, our findings indicated that modulating mitophagy may be a therapeutic strategy for pathological vascular endothelial injury caused by NPs exposure.Graphical

Mitochondrial fission in Huntington's disease mouse striatum disrupts ER-mitochondria contacts leading to disturbances in Ca2+ efflux and Reactive Oxygen Species (ROS) homeostasis

Mitochondria-associated membranes (MAMs) are dynamic structures that communicate endoplasmic reticulum (ER) and mitochondria allowing calcium transfer between these two organelles. Since calcium dysregulation is an important hallmark of several neurodegenerative diseases, disruption of MAMs has been speculated to contribute to pathological features associated with these neurodegenerative processes. In Huntington's disease (HD), mutant huntingtin induces the selective loss of medium spiny neurons within the striatum. The cause of this specific susceptibility remain unclear. However, defects on mitochondrial dynamics and bioenergetics have been proposed as critical contributors, causing accumulation of fragmented mitochondria and subsequent Ca2+ homeostasis alterations. In the present work, we show that aberrant Drp1-mediated mitochondrial fragmentation within the striatum of HD mutant mice, forces mitochondria to place far away from the ER disrupting the ER-mitochondria association and therefore causing drawbacks in Ca2+ efflux and an excessive production of mitochondria superoxide species. Accordingly, inhibition of Drp1 activity by Mdivi-1 treatment restored ER-mitochondria contacts, mitochondria dysfunction and Ca2+ homeostasis.In sum, our results give new insight on how defects on mitochondria dynamics may contribute to striatal vulnerability in HD and highlights MAMs dysfunction as an important factor involved in HD striatal pathology.

Altered mitochondrial quality control in autophagy-deficient vascular smooth muscle cells is linked to high-risk atherosclerotic plaque phenotype

Atherosclerosis is one of the most common causes of stroke worldwide. Intracranial or extracranial atherosclerosis may occur concomitantly with systemic atherosclerosis involving other arterial beds, such as coronary, or peripheral arteries. A major mechanism of stroke, involves unstable plaque as described in the coronary model. Vascular smooth muscle cells (VSMCs) regulate various aspects of vessel homeostasis, including contraction, dilation, and vessel remodeling. Consequently, VSMCs are one of the main cellular determinants in arterial pathology and several evidence indicates that apoptosis of VSMCs is associated with features of high-risk/vulnerable atherosclerotic lesions. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques. By using a mouse model of atherosclerosis carrying a VSMC-specific deletion of the essential autophagy gene Atg7, we observed that impaired VSMC autophagy promotes an unstable plaque phenotype as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate the apoptosis of VSMCs and plaque vulnerability. Overall, our results indicate that mitochondrial quality control could be a promising therapeutic target to stabilize atherosclerotic plaques and to decrease the risk of stroke.

Cholix toxin, an eukaryotic elongation factor 2 ADP-ribosyltransferase, interacts with Prohibitins and induces apoptosis with mitochondrial dysfunction in human hepatocytes.

Vibrio cholerae produced-Cholix toxin (Cholix) is a cytotoxin that ADP-ribosylates eukaryotic elongation factor 2, inhibiting protein synthesis, and inducing apoptosis. Here, we identified prohibitin (PHB) 1 and 2 as novel Cholix-interacting membrane proteins in immortalised human hepatocytes and HepG2 cells by Cholix immunoprecipitation assays. The expression level of PHB1 was decreased by Cholix after a 12hr incubation. Cholix-induced poly (ADP-ribose) polymerase (PARP) cleavage was significantly enhanced in PHB (PHB1 or PHB2) knockdown cells. In contrast, transiently overexpressed PHB in hepatocytes attenuated Cholix-induced Bax/Bak conformational changes and PARP cleavage. In addition, Cholix-induced reactive oxygen species production and accumulation of fragmented mitochondria were enhanced in PHB-knockdown cells. Furthermore, Cholix induced activation of Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which was enhanced in PHB-knockdown cells, followed by actin filament depolymerisation and accumulation of tubulin in the blebbing cells. Inhibition of ROCK1 by siRNA or its inhibitor suppressed Cholix-induced PARP cleavage and reactive oxygen species generation. Our findings identify PHB as a new protein that interacts with Cholix and is involved in Cholix-induced mitochondrial dysfunction and cytoskeletal rearrangement by ROCK1 activation during apoptosis.

Altered mitochondrial quality control in Atg7-deficient VSMCs promotes enhanced apoptosis and is linked to unstable atherosclerotic plaque phenotype

Vascular smooth muscle cells (VSMCs) are one of the main cellular determinants in arterial pathology. A large body of evidence indicates that death of VSMCs is associated with features of high-risk/vulnerable atherosclerotic plaques. Mitochondrial turnover is an essential aspect of the mitochondrial quality control in which dysfunctional mitochondria are selectively eliminated through autophagy and replaced through expansion of preexisting mitochondria. Even though successful autophagy promotes VSMC survival, it is unclear whether reduced autophagic flux affects mitochondrial quality control of VSMCs in atherosclerotic plaques. By using apolipoprotein E-deficient (ApoE−/−) mice carrying a VSMC-specific deletion of the essential autophagy gene Atg7, we show in the present study that impaired VSMC autophagy promotes an unstable plaque phenotype, as well as the accumulation of fragmented mitochondria with reduced bioenergetic efficiency and more oxidative stress. Furthermore, we demonstrate that disrupted autophagic flux is linked to defective mitophagy and biogenesis of mitochondria, which exacerbate VSMC apoptosis and in turn plaque vulnerability. Overall, our data indicate that mitochondrial quality control is a promising therapeutic target to stabilize atherosclerotic plaques.

Exercise reestablishes autophagic flux and mitochondrial quality control in heart failure

ABSTRACT We previously reported that facilitating the clearance of damaged mitochondria through macroautophagy/autophagy protects against acute myocardial infarction. Here we characterize the impact of exercise, a safe strategy against cardiovascular disease, on cardiac autophagy and its contribution to mitochondrial quality control, bioenergetics and oxidative damage in a post-myocardial infarction-induced heart failure animal model. We found that failing hearts displayed reduced autophagic flux depicted by accumulation of autophagy-related markers and loss of responsiveness to chloroquine treatment at 4 and 12 wk after myocardial infarction. These changes were accompanied by accumulation of fragmented mitochondria with reduced O2 consumption, elevated H2O2 release and increased Ca2+-induced mitochondrial permeability transition pore opening. Of interest, disruption of autophagic flux was sufficient to decrease cardiac mitochondrial function in sham-treated animals and increase cardiomyocyte toxicity upon mitochondrial stress. Importantly, 8 wk of exercise training, starting 4 wk after myocardial infarction at a time when autophagy and mitochondrial oxidative capacity were already impaired, improved cardiac autophagic flux. These changes were followed by reduced mitochondrial number:size ratio, increased mitochondrial bioenergetics and better cardiac function. Moreover, exercise training increased cardiac mitochondrial number, size and oxidative capacity without affecting autophagic flux in sham-treated animals. Further supporting an autophagy mechanism for exercise-induced improvements of mitochondrial bioenergetics in heart failure, acute in vivo inhibition of autophagic flux was sufficient to mitigate the increased mitochondrial oxidative capacity triggered by exercise in failing hearts. Collectively, our findings uncover the potential contribution of exercise in restoring cardiac autophagy flux in heart failure, which is associated with better mitochondrial quality control, bioenergetics and cardiac function.

Dynamin-related Protein 1 Inhibition Mitigates Bisphenol A-mediated Alterations in Mitochondrial Dynamics and Neural Stem Cell Proliferation and Differentiation

The regulatory dynamics of mitochondria comprises well orchestrated distribution and mitochondrial turnover to maintain the mitochondrial circuitry and homeostasis inside the cells. Several pieces of evidence suggested impaired mitochondrial dynamics and its association with the pathogenesis of neurodegenerative disorders. We found that chronic exposure of synthetic xenoestrogen bisphenol A (BPA), a component of consumer plastic products, impaired autophagy-mediated mitochondrial turnover, leading to increased oxidative stress, mitochondrial fragmentation, and apoptosis in hippocampal neural stem cells (NSCs). It also inhibited hippocampal derived NSC proliferation and differentiation, as evident by the decreased number of BrdU- and β-III tubulin-positive cells. All these effects were reversed by the inhibition of oxidative stress using N-acetyl cysteine. BPA up-regulated the levels of Drp-1 (dynamin-related protein 1) and enhanced its mitochondrial translocation, with no effect on Fis-1, Mfn-1, Mfn-2, and Opa-1 in vitro and in the hippocampus. Moreover, transmission electron microscopy studies suggested increased mitochondrial fission and accumulation of fragmented mitochondria and decreased elongated mitochondria in the hippocampus of the rat brain. Impaired mitochondrial dynamics by BPA resulted in increased reactive oxygen species and malondialdehyde levels, disruption of mitochondrial membrane potential, and ATP decline. Pharmacological (Mdivi-1) and genetic (Drp-1siRNA) inhibition of Drp-1 reversed BPA-induced mitochondrial dysfunctions, fragmentation, and apoptosis. Interestingly, BPA-mediated inhibitory effects on NSC proliferation and neuronal differentiations were also mitigated by Drp-1 inhibition. On the other hand, Drp-1 inhibition blocked BPA-mediated Drp-1 translocation, leading to decreased apoptosis of NSC. Overall, our studies implicate Drp-1 as a potential therapeutic target against BPA-mediated impaired mitochondrial dynamics and neurodegeneration in the hippocampus.

Mitochondrial dysfunction and mitophagy: the beginning and end to diabetic nephropathy?

Diabetic nephropathy (DN) is a progressive microvascular complication arising from diabetes. Within the kidney, the glomeruli, tubules, vessels and interstitium are disrupted, ultimately impairing renal function and leading to end-stage renal disease (ESRD). Current pharmacological therapies used in individuals with DN do not prevent the inevitable progression to ESRD; therefore, new targets of therapy are urgently required. Studies from animal models indicate that disturbances in mitochondrial homeostasis are central to the pathogenesis of DN. Since renal proximal tubule cells rely on oxidative phosphorylation to provide adequate ATP for tubular reabsorption, an impairment of mitochondrial bioenergetics can result in renal functional decline. Defects at the level of the electron transport chain have long been established in DN, promoting electron leakage and formation of superoxide radicals, mediating microinflammation and contributing to the renal lesion. More recent studies suggest that mitochondrial-associated proteins may be directly involved in the pathogenesis of tubulointerstitial fibrosis and glomerulosclerosis. An accumulation of fragmented mitochondria are found in the renal cortex in both humans and animals with DN, suggesting that in tandem with a shift in dynamics, mitochondrial clearance mechanisms may be impaired. The process of mitophagy is the selective targeting of damaged or dysfunctional mitochondria to autophagosomes for degradation through the autophagy pathway. The current review explores the concept that an impairment in the mitophagy system leads to the accelerated progression of renal pathology. A better understanding of the cellular and molecular events that govern mitophagy and dynamics in DN may lead to improved therapeutic strategies.

Disruption and therapeutic rescue of autophagy in a human neuronal model of Niemann Pick type C1

An unresolved issue about many neurodegenerative diseases is why neurons are particularly sensitive to defects in ubiquitous cellular processes. One example is Niemann Pick type C1, caused by defects in cholesterol trafficking in all cells, but where neurons are preferentially damaged. Understanding this selective failure is limited by the difficulty in obtaining live human neurons from affected patients. To solve this problem, we generated neurons with decreased function of NPC1 from human embryonic stem cells and used them to test the hypothesis that defective cholesterol handling leads to enhanced pathological phenotypes in neurons. We found that human NPC1 neurons have strong spontaneous activation of autophagy, and, contrary to previous reports in patient fibroblasts, a block of autophagic progression leading to defective mitochondrial clearance. Mitochondrial fragmentation is an exceptionally severe phenotype in NPC1 neurons compared with fibroblasts, causing abnormal accumulation of mitochondrial proteins. Contrary to expectation, these abnormal phenotypes were rescued by treatment with the autophagy inhibitor 3-methyladenine and by treatment with the potential therapeutic cyclodextrin, which mobilizes cholesterol from the lysosomal compartment. Our findings suggest that neurons are especially sensitive to lysosomal cholesterol accumulation because of autophagy disruption and accumulation of fragmented mitochondria, thus defining a new route to effective drug development for NPC1 disease.

Mitochondrial Aberrations in Mucolipidosis Type IV

Mucolipidosis type IV is a genetic lysosomal storage disease associated with degenerative processes in the brain, eye, and other tissues. Mucolipidosis type IV results from mutations in the gene MCOLN1, which codes for the TRP family ion channel, mucolipin 1. The connection between lysosomal dysfunction and degenerative processes in mucolipidosis type IV is unclear. Here we report that mucolipidosis type IV and several unrelated lysosomal storage diseases are associated with significant mitochondrial fragmentation and decreased mitochondrial Ca2+ buffering efficiency. The mitochondrial alterations observed in these lysosomal storage diseases are reproduced in control cells by treatment with lysosomal inhibitors and with the autophagy inhibitor 3-methyladenine. This suggests that inefficient autophagolysosomal recycling of mitochondria generates fragmented, effete mitochondria in mucolipidosis. Mitochondria accumulate that cannot properly buffer calcium fluxes in the cell. A decrease in mitochondrial Ca2+ buffering capacity in cells affected by these lysosomal storage diseases is associated with increased sensitivity to apoptosis induced by Ca2+-mobilizing agonists and executed via a caspase-8-dependent pathway. Deficient Ca2+ homeostasis may represent a common mechanism of degenerative cell death in several lysosomal storage diseases.

The mitochondrial protein hFis1 regulates mitochondrial fission in mammalian cells through an interaction with the dynamin-like protein DLP1.

The yeast protein Fis1p has been shown to participate in mitochondrial fission mediated by the dynamin-related protein Dnm1p. In mammalian cells, the dynamin-like protein DLP1/Drp1 functions as a mitochondrial fission protein, but the mechanisms by which DLP1/Drp1 and the mitochondrial membrane interact during the fission process are undefined. In this study, we have tested the role of a mammalian homologue of Fis1p, hFis1, and provided new and mechanistic information about the control of mitochondrial fission in mammalian cells. Through differential tagging and deletion experiments, we demonstrate that the intact C-terminal structure of hFis1 is essential for mitochondrial localization, whereas the N-terminal region of hFis1 is necessary for mitochondrial fission. Remarkably, an increased level of cellular hFis1 strongly promotes mitochondrial fission, resulting in an accumulation of fragmented mitochondria. Conversely, cell microinjection of hFis1 antibodies or treatment with hFis1 antisense oligonucleotides induces an elongated and collapsed mitochondrial morphology. Further, fluorescence resonance energy transfer and coimmunoprecipitation studies demonstrate that hFis1 interacts with DLP1. These results suggest that hFis1 participates in mitochondrial fission through an interaction that recruits DLP1 from the cytosol. We propose that hFis1 is a limiting factor in mitochondrial fission and that the number of hFis1 molecules on the mitochondrial surface determines fission frequency.