Thapsigargin induces biphasic fragmentation of mitochondria through calcium-mediated mitochondrial fission and apoptosis

Mitochondrial fission and fusion are the main components mediating the dynamic change of mitochondrial morphology observed in living cells. While many protein factors directly participating in mitochondrial dynamics have been identified, upstream signals that regulate mitochondrial morphology are not well understood. In this study, we tested the role of intracellular Ca(2+) in regulating mitochondrial morphology. We found that treating cells with the ER Ca(2+)-ATPase inhibitor thapsigargin (TG) induced two phases of mitochondrial fragmentation. The initial fragmentation of mitochondria occurs rapidly within minutes dependent on an increase in intracellular Ca(2+) levels, and Ca(2+) influx into mitochondria is necessary for inducing mitochondrial fragmentation. The initial mitochondrial fragmentation is a transient event, as tubular mitochondrial morphology was restored as the Ca(2+) level decreased. We were able to block the TG-induced mitochondrial fragmentation by inhibiting mitochondrial fission proteins DLP1/Drp1 or hFis1, suggesting that increased mitochondrial Ca(2+) acts upstream to activate the cellular mitochondrial fission machinery. We also found that prolonged incubation with TG induced the second phase of mitochondrial fragmentation, which was non-reversible and led to cell death as reported previously. These results suggest that Ca(2+) is involved in controlling mitochondrial morphology via intra-mitochondrial Ca(2+) signaling as well as the apoptotic process.     

Mst1 deletion reduces hyperglycemia-mediated vascular dysfunction via attenuating mitochondrial fission and modulating the JNK signaling pathway

Diabetes is a leading cause of microvascular complications, such as nephropathy and retinopathy. Recent studies have proposed that hyperglycemia-induced endothelial cell dysfunction is modulated by mitochondrial stress. Therefore, our experiment was to detect the upstream mediator of mitochondrial stress in hyperglycemia-treated endothelial cells with a focus on macrophage-stimulating 1 (Mst1) and mitochondrial fission. Our data illuminated that hyperglycemia incubation reduced cell viability, as well as increased apoptosis ratio in endothelial cell, and this alteration seemed to be associated with Mst1 upregulation. Inhibition of Mst1 via transfection of Mst1 siRNA into an endothelial cell could sustain cell viability and maintain mitochondrial function. At the molecular levels, endothelial cell death was accompanied with the activation of mitochondrial oxidative stress, mitochondrial apoptosis, and mitochondrial fission. Genetic ablation of Mst1 could reduce mitochondrial oxidative injury, block mitochondrial apoptosis, and repress mitochondrial fission. Besides, we also found Mst1 triggered mitochondrial dysfunction as well as endothelial cell damage through augmenting JNK pathway. Suppression of JNK largely ameliorated the protective actions of Mst1 silencing on hyperglycemia-treated endothelial cells and sustain mitochondrial function. The present study identifies Mst1 as a primary key mediator for hyperglycemia-induced mitochondrial damage and endothelial cell dysfunction. Increased Mst1 impairs mitochondrial function and activates endothelial cell death via opening mitochondrial death pathway through JNK.     

MKP1 overexpression reduces TNF-α-induced cardiac injury via suppressing mitochondrial fragmentation and inhibiting the JNK–MIEF1 pathways

Mitochondrial stress has been acknowledged as the pathogenesis for tumor necrosis factor-α (TNF-α)-induced septic cardiomyopathy. Recently, MAP kinase phosphatase 1 (MKP1) downregulation and mitochondrial fragmentation modulate the mitochondrial stress via multiple molecular mechanisms. Thereby, the goal of our current work is to figure out the functional role of mitochondrial fragmentation in TNF-α-induced septic cardiomyopathy. Our results exhibited that MKP1 expression was significantly repressed in hearts treated by TNF-α. Overexpression of MKP1 sustained cardiac function and attenuated cardiomyocytes death in TNF-α-treated hearts. At the molecular levels, decreased MKP1 induced mitochondrial stress, as indicated by mitochondrial calcium overloading, mitochondrial oxidative stress, mitochondrial antioxidant downregulation, mitochondrial membrane potential reduction, mitochondrial bioenergetics suppression, mitochondrial proapoptotic factors liberation, and caspase-9 apoptotic pathway activation. To the end, we illustrated that MKP1-modulated mitochondrial stress via mitochondrial fragmentation; reactivation of mitochondrial fragmentation abolished the protective effect of MKP1 overexpression on mitochondrial function. Further, MKP1 affected mitochondrial division in a mechanism through the JNK–MIEF1 axis. Blockade of JNK pathway abolished the regulatory actions of MKP1 on mitochondrial division. Altogether, our results identify MKP1 as a novel cardioprotective factor in TNF-α-related septic cardiomyopathy via affecting mitochondrial division by the way of JNK–MIEF1 signaling pathway. Therefore, MKP1 expression, mitochondrial fragmentation modification, and JNK–MIEF1 pathway modulation may be considered as potential therapeutic targets for the treatment of cardiac injury induced by sepsis.     

Mitochondrial regulation of cardiac aging

Aging is associated with progressive decline in cardiac structure and function. Accumulating evidence in model organisms and humans links cardiac aging to mitochondrial regulation, encompassing a complex interplay of mitochondrial morphology, mitochondrial ROS, mitochondrial DNA mutations, mitochondrial unfolded protein response, nicotinamide adenine dinucleotide levels and sirtuins, as well as mitophagy. This review summarizes the recent discoveries on the mitochondrial regulation of cardiac aging and the possible molecular mechanisms underlying the anti-aging effects, as well as the potential interventions that alleviate aging-related cardiac diseases and attenuate cardiac aging via the regulation of mitochondria.     

Mitochondrial medicine for neurodegenerative diseases

Mitochondrial dysfunction has been reported in a wide array of neurological disorders ranging from neuromuscular to neurodegenerative diseases. Recent studies on neurodegenerative diseases have revealed that mitochondrial pathology is generally found in inherited or sporadic neurodegenerative diseases and is believed to be involved in the pathophysiological process of these diseases. Commonly seen types of mitochondrial dysfunction in neurodegenerative diseases include excessive free radical generation, lowered ATP production, mitochondrial permeability transition, mitochondrial DNA lesions, perturbed mitochondrial dynamics and apoptosis. Mitochondrial medicine as an emerging therapeutic strategy targeted to mitochondrial dysfunction in neurodegenerative diseases has been proven to be of value, though this area of research is still at in its early stage. In this article, we report on recent progress in the development of several mitochondrial therapies including antioxidants, blockade of mitochondrial permeability transition, and mitochondrial gene therapy as evidence that mitochondrial medicine has promise in the treatment of neurodegenerative diseases.     

Down-regulation of Mortalin Exacerbates Aβ-mediated Mitochondrial Fragmentation and Dysfunction

Mitochondrial dynamics greatly influence the biogenesis and morphology of mitochondria. Mitochondria are particularly important in neurons, which have a high demand for energy. Therefore, mitochondrial dysfunction is strongly associated with neurodegenerative diseases. Until now various post-translational modifications for mitochondrial dynamic proteins and several regulatory proteins have explained complex mitochondrial dynamics. However, the precise mechanism that coordinates these complex processes remains unclear. To further understand the regulatory machinery of mitochondrial dynamics, we screened a mitochondrial siRNA library and identified mortalin as a potential regulatory protein. Both genetic and chemical inhibition of mortalin strongly induced mitochondrial fragmentation and synergistically increased Aβ-mediated cytotoxicity as well as mitochondrial dysfunction. Importantly we determined that the expression of mortalin in Alzheimer disease (AD) patients and in the triple transgenic-AD mouse model was considerably decreased. In contrast, overexpression of mortalin significantly suppressed Aβ-mediated mitochondrial fragmentation and cell death. Taken together, our results suggest that down-regulation of mortalin may potentiate Aβ-mediated mitochondrial fragmentation and dysfunction in AD.     

Elevated intracellular calcium causes distinct mitochondrial remodelling and calcineurin-dependent fission in astrocytes

Disruptions of mitochondrial dynamics have been implicated in the pathogenesis of neurodegenerative diseases. The regulation mechanisms of mitochondrial dynamics have not been fully elucidated; however, calcium has been suggested to play a role. In the present study, we examined the role of intracellular calcium in regulating mitochondrial morphology and motility in cortical astrocytes employing different concentrations of a calcium ionophore. High levels of calcium caused a dramatic reduction in mitochondrial length, the result of two distinct phenomena: mitochondrial remodelling (or "rounding") and fission. Quantitative analysis revealed that mitochondrial remodelling/rounding was the predominant process. In addition, mitochondrial motility was reduced, as reported previously in neurons. By contrast, prolonged, more modest levels of intracellular calcium resulted in a reduction in mitochondrial length without significant effects upon mitochondrial motility. This calcium-induced reduction in mitochondrial length was not affected by the presence of calcineurin inhibitors; however, when mitochondrial fission events were specifically examined, calcineurin inhibitors had a significant inhibitory effect. This suggests that changes in mitochondrial length were primarily due to mitochondrial remodelling as opposed to fission. In the present study, we have therefore dissected the effects of calcium on mitochondrial motility, remodelling and fission. Our results suggest independent mechanisms for regulating these processes.     

Mitochondrial fusion proteins: Dual regulators of morphology and metabolism

Mitochondria in mammalian cells are visualized as a network or as filaments that undergo continuous changes in shape and in localization within the cells. These changes are a consequence of the activity of different processes such as mitochondrial fusion and fission, and mitochondrial remodelling. In all, these processes are referred to as mitochondrial dynamics, and relevant questions, still unexplained, are why cells require such an active dynamics, or why mitochondria move to specific cellular regions. In this review we will summarize some of the biological functions assigned to the proteins identified as participating in mitochondrial fusion, namely mitofusin 1, mitofusin 2 and OPA1. In addition to the capacity of these proteins to promote fusion, mitofusin 2 or OPA1 regulate mitochondrial metabolism and loss-of-function reduces oxygen consumption and the capacity to oxidize substrates. We propose that mitochondrial fusion proteins operate as integrators of signals so they regulate both mitochondrial fusion and metabolism.     

The mitochondrial tRNAs of Trypanosoma brucei are nuclear encoded

The mitochondrial DNA of Trypanosoma brucei is organized as a catenated network of maxicircles and minicircles. The maxicircles are equivalent to the typical mitochondrial genome except that the genes for the mitochondrial tRNAs have not been identified by sequence analysis of the maxicircle DNA. The apparent absence of tRNA genes in the maxicircle DNA suggests that the mitochondrial tRNAs are encoded by either the minicircle or the nuclear DNA. In order to determine their genomic origin, we isolated and identified the mitochondrial tRNAs of T. brucei. We show that these mitochondrial tRNAs are truly mitochondrially located in vivo and that they are free from detectable contamination by cytosolic RNAs. By hybridization analysis, using mitochondrial tRNAs as the probe, we determined that the mitochondrial tRNAs are encoded by nuclear DNA. This implies that RNAs, like proteins, are imported into the mitochondria. We investigated the relationship between the cytosolic and the mitochondrial tRNA genes and show that there are unique cytosolic tRNA genes, unique mitochondrial tRNA genes, and tRNA genes which appear to be shared and whose products are therefore targeted to both the cytosol and the mitochondrion.     

Mitochondrial fatty acid synthesis is required for normal mitochondrial morphology and function in Trypanosoma brucei

Trypanosoma brucei use microsomal elongases for de novo synthesis of most of its fatty acids. In addition, this parasite utilizes an essential mitochondrial type II synthase for production of octanoate (a lipoic acid precursor) as well as longer fatty acids such as palmitate. Evidence from other organisms suggests that mitochondrially synthesized fatty acids are required for efficient respiration but the exact relationship remains unclear. In procyclic form trypanosomes, we also found that RNAi depletion of the mitochondrial acyl carrier protein, an important component of the fatty acid synthesis machinery, significantly reduces cytochrome-mediated respiration. This reduction was explained by RNAi-mediated inhibition of respiratory complexes II, III and IV, but not complex I. Other effects of RNAi, such as changes in mitochondrial morphology and alterations in membrane potential, raised the possibility of a change in mitochondrial membrane composition. Using mass spectrometry, we observed a decrease in total and mitochondrial phosphatidylinositol and mitochondrial phosphatidylethanolamine. Thus, we conclude that the mitochondrial synthase produces fatty acids needed for maintaining local phospholipid levels that are required for activity of respiratory complexes and preservation of mitochondrial morphology and function.     

Oxidative stress-mediated activation of extracellular signal-regulated kinase contributes to mild cognitive impairment-related mitochondrial dysfunction

Mild cognitive impairment (MCI) occurs during the predementia stage of Alzheimer disease (AD) and is characterized by a decline in cognitive abilities that frequently represents a transition between normal cognition and AD dementia. Its pathogenesis is not well understood. Here, we demonstrate the direct consequences and potential mechanisms of oxidative stress and mitochondrial dynamic and functional defects in MCI-derived mitochondria. Using a cytoplasmic hybrid (cybrid) cell model in which mitochondria from MCI or age-matched non-MCI subjects were incorporated into a human neuronal cell line depleted of endogenous mitochondrial DNA, we evaluated the mitochondrial dynamics and functions, as well as the role of oxidative stress in the resultant cybrid lines. We demonstrated that increased expression levels of mitofusin 2 (Mfn2) are markedly induced by oxidative stress in MCI-derived mitochondria along with aberrant mitochondrial functions. Inhibition of oxidative stress rescues MCI-impaired mitochondrial fusion/fission balance as shown by the suppression of Mfn2 expression, attenuation of abnormal mitochondrial morphology and distribution, and improvement in mitochondrial function. Furthermore, blockade of MCI-related stress-mediated activation of extracellular signal-regulated kinase (ERK) signaling not only attenuates aberrant mitochondrial morphology and function but also restores mitochondrial fission and fusion balance, in particular inhibition of overexpressed Mfn2. Our results provide new insights into the role of the oxidative stress–ERK–Mfn2 signal axis in MCI-related mitochondrial abnormalities, indicating that the MCI phase may be targetable for the development of new therapeutic approaches that improve mitochondrial function in age-related neurodegeneration.     

Mitophagy promotes the stemness of bone marrow-derived mesenchymal 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.     

Monitoring mitochondrial translation in living cells

Mitochondria possess a small genome that codes for core subunits of the oxidative phosphorylation system and whose expression is essential for energy production. Information on the regulation and spatial organization of mitochondrial gene expression in the cellular context has been difficult to obtain. Here we devise an imaging approach to analyze mitochondrial translation within the context of single cells, by following the incorporation of clickable non‐canonical amino acids. We apply this method to multiple cell types, including specialized cells such as cardiomyocytes and neurons, and monitor with spatial resolution mitochondrial translation in axons and dendrites. We also show that translation imaging allows to monitor mitochondrial protein expression in patient fibroblasts. Approaching mitochondrial translation with click chemistry opens new avenues to understand how mitochondrial biogenesis is integrated into the cellular context and can be used to assess mitochondrial gene expression in mitochondrial diseases.     

p21WAF1/CIP1 deficiency induces mitochondrial dysfunction in HCT116 colon cancer cells

p21^WAF1/CIP1 is a critical regulator of cell cycle progression. However, the role of p21 in mitochondrial function remains poorly understood. In this study, we examined the effect of p21 deficiency on mitochondrial function in HCT116 human colon cancer cells. We found that there was a significant increase in the mitochondrial mass of p21^?/? HCT116 cells, as measured by 10-N-nonyl-acridine orange staining, as well as an increase in the mitochondrial DNA content. In contrast, p53^?/? cells had a mitochondrial mass comparable to that of wild-type HCT116 cells. In addition, the expression levels of the mitochondrial biogenesis regulators PGC-1α and TFAM and AMPK activity were also elevated in p21^?/? cells, indicating that p21 deficiency induces the rate of mitochondrial biogenesis through the AMPK-PGC-1α axis. However, the increase in mitochondrial biogenesis in p21^?/? cells did not accompany an increase in the cellular steady-state level of ATP. Furthermore, p21^?/? cells exhibited significant proliferation impairment in galactose medium, suggesting that p21 deficiency induces a defect in the mitochondrial respiratory chain in HCT116 cells. Taken together, our results suggest that the loss of p21 results in an aberrant increase in the mitochondrial mass and in mitochondrial dysfunction in HCT116 cells, indicating that p21 is required to maintain proper mitochondrial mass and respiratory function.     

Comparison of the levels of the 21S mitochondrial rRNA in derepressed and glucose-repressed Saccharomyces cerevisiae.

A cDNA preparation, synthesized by using Saccharomyces cerevisiae mitochondrial RNA as template and oligodeoxythymidylic acid as primer, was found to specifically hybridize to the mitochondrial 21S rRNA by the following criteria: (i) it hybridizes only to the 21S RNA species in mitochondrial RNA and not to RNA from a [rho0] mutant, and (ii) it hybridizes to fragments in restriction digests of mitochondrial DNA that contain the 21S rRNA gene but not to nuclear DNA. This cDNA was used as a probe to demonstrate that a 2.6-fold decrease in the cellular level of the mitochondrial large rRNA is associated with glucose repression of mitochondrial function in S. cerevisiae. A corresponding decrease in the level of mitochondrial DNA was not observed.     

Control of mitochondrial volume by mitochondrial metabolic water

It is well-known that mitochondrial volume largely controls mitochondrial functioning. We investigate whether metabolic water produced by oxidative phosphorylation could be involved in mitochondrial volume regulation. We modulated the generation of this water in liver mitochondria and assess their volume by two independent techniques.In liver mitochondria, the mitochondrial volume was specifically decreased when no water was produced independently of energetic parameters and uncoupling activity. In all other conditions associated with water generation, there was no significant change in mitochondrial metabolic volume.Altogether these data demonstrate that mitochondrial volume is regulated, independently of energetic status, by the mitochondrial metabolic water that acts as a signal.