Accumulation Of Abnormal Mitochondria Research Articles

StarD7 deficiency switches on glycolysis and promotes mitophagy flux in C2C12 myoblasts.

StarD7 is a member of the START protein family required for phosphatidylcholine delivery to the mitochondria, thus key to maintain mitochondrial structure. Its deficiency has been associated with an impairment of cellular processes, such as proliferation and migration, and it has also been reported that it is needed in myogenic differentiation. Here, we show that StarD7 deficiency in C2C12 muscle cells results in the accumulation of abnormal mitochondria, a reduced number of mitochondria per cell area and increased glycolysis. In addition, StarD7-deficient cells undergo an increase in mitochondria-ER contact sites, reduced connexin 43 expression, and disturbances in lipid handling, evidenced by lipid droplet accumulation and decreased levels in phosphatidylserine synthase 1 and 2 expression. Interestingly, StarD7-deficient cells showed alterations in mitophagy markers. We observed accumulation of LC3B-II and BNIP3 proteins in mitochondria-enriched fractions and accumulation of autophagolysosomal and lysosomal vesicles in StarD7-deficient cells. Furthermore, live-cell imaging experiments of StarD7 knockdown cells expressing mitochondria-targeted mKeima indicated an enhanced mitochondria delivery into lysosomes. Importantly, StarD7 reconstitution in StarD7-deficient cells restores LC3B-II expression in mitochondria-enriched fractions at similar levels to those observed in control cells. Collectively, these findings suggest that StarD7-deficient C2C12 myoblasts are associated with altered cristae structure, disturbances in neutral lipid accumulation, glucose metabolism, and increased mitophagy flux. The alterations mentioned above allow for the maintenance of mitochondrial function.

Insights into the mechanisms of telbivudine-induced myopathy associated with mitochondrial dysfunction

As a nucleotide analogue (NA), telbivudine was widely used in the treatment for chronic hepatitis B (CHB) by interfering with reverse transcriptase of hepatitis B virus. However, the use of NAs for hepatitis B treatment has been accompanied by numerous reports highlighting the occurrence of neuromyopathy, particularly in the case of telbivudine. This study aimed to investigate the underlying mechanisms responsible for telbivudine-induced myopathy. We established animal and cell models of telbivudine-induced myopathy using C57BL/6 mice and C2C12 cells, respectively. Our findings revealed that telbivudine significantly reduced mitochondrial DNA (mtDNA) copy number and caused increase of oxidative stress. Telbivudine treatment significantly inhibited mitochondrial complex I and IV expression, impairing the oxidative phosphorylation function of the respiratory chain. Modified Gomori trichrome (MGT) staining of the muscle sections displayed an increase in ragged red fibers (RRFs), indicating abnormal mitochondrial accumulation. In conclusion, our study provides compelling evidence suggesting that telbivudine-induced myopathy is associated with mitochondrial toxicity and impaired energy metabolism. The observed muscle pathology, depletion of mtDNA, elevation of oxidative stress and altered mitochondrial function support the hypothesis that telbivudine disrupts mitochondrial homeostasis, ultimately leading to muscle damage. This may be also a common mechanism for NAs to cause neuromyopathy.

Application of left ventricular endomyocardial biopsy in the diagnosis of mitochondrial cardiomyopathy: a case report

BackgroundThe clinical features of mitochondrial cardiomyopathy (MCM) are diverse. It can present as hypertrophic cardiomyopathy or dilated cardiomyopathy. The diagnosis of MCM is challenging and usually based on biopsy.Case presentationThe 30-year-old man was admitted to hospital due to dyspnea for 1 month and edema of both lower extremities for 1 week. Echocardiography suggested a whole heart enlargement, a whole heart diminished function. Renal impairment and diabetes were observed. Coronary angiography showed single-vessel disease (90% stenosis in the ostium of a small marginal branch). Left ventricular endomyocardial biopsy was performed.ConclusionMyocardial histopathology demonstrated a large number of abnormal mitochondrial accumulation, so the diagnosis was considered as mitochondrial cardiomyopathy.

Parkin Precipitates on Mitochondria via Aggregation and Autoubiquitination.

The loss of the E3 ligase Parkin, in a familial form of Parkinson's disease, is thought to cause the failure of both the polyubiquitination of abnormal mitochondria and the consequent induction of mitophagy, resulting in abnormal mitochondrial accumulation. However, this has not been confirmed in patient autopsy cases or animal models. More recently, the function of Parkin as a redox molecule that directly scavenges hydrogen peroxide has attracted much attention. To determine the role of Parkin as a redox molecule in the mitochondria, we overexpressed various combinations of Parkin, along with its substrates FAF1, PINK1, and ubiquitin in cell culture systems. Here, we observed that the E3 Parkin monomer was surprisingly not recruited to abnormal mitochondria but self-aggregated with or without self-ubiquitination into the inner and outer membranes, becoming insoluble. Parkin overexpression alone generated aggregates without self-ubiquitination, but it activated autophagy. These results suggest that for damaged mitochondria, the polyubiquitination of Parkin substrates on the mitochondria is not indispensable for mitophagy.

Mitochondrial Autophagy in Ischemic Aged Livers

Mitochondrial autophagy (mitophagy) is a central catabolic event for mitochondrial quality control. Defective or insufficient mitophagy, thus, can result in mitochondrial dysfunction, and ultimately cell death. There is a strong causal relationship between ischemia/reperfusion (I/R) injury and mitochondrial dysfunction following liver resection and transplantation. Compared to young patients, elderly patients poorly tolerate I/R injury. Accumulation of abnormal mitochondria after I/R is more prominent in aged livers than in young counterparts. This review highlights how altered autophagy is mechanistically involved in age-dependent hypersensitivity to reperfusion injury.

Autophagy in asthma and chronic obstructive pulmonary disease.

Autophagy (or macroautophagy) is a key cellular process that removes damaged molecules (particularly proteins) and subcellular organelles to maintain cellular homeostasis. There is growing evidence that abnormalities in autophagy may contribute to the pathogenesis of many chronic diseases, including asthma and chronic obstructive pulmonary disease (COPD). In asthma, increased autophagy plays a role in promoting type 2 immune responses and eosinophilic inflammation, whereas decreased autophagy may be important in neutrophilic asthma. Acute exposure to cigarette smoke may activate autophagy, resulting in ciliary dysfunction and death of airway epithelial cells, whereas in stable COPD most studies have demonstrated an impairment in autophagy, with reduced autophagic flux and accumulation of abnormal mitochondria (defective mitophagy) and linked to cellular senescence. Autophagy may be increased or decreased in different cell types and depending on the cellular environment, making it difficult to target autophagy therapeutically. Several existing drugs may activate autophagy, including rapamycin, metformin, carbamazepine, cardiac glycosides and statins, whereas others, such as chloroquine, inhibit this process. However, these drugs are nonspecific and more selective drugs are now in development, which may prove useful as novel agents to treat asthma and COPD in the future.

Early-onset dietary restriction maintains mitochondrial health, autophagy and ER function in the left ventricle during aging

Dietary restriction (DR) exerts healthy benefits, including heart functions. However, the cardioprotective role of DR is till controversial among researchers due to the variation of DR conditions. The present study focuses on the protective effect of early-onset DR on cardiac injury using mitochondrial structure and expression of protein associated with mitochondrial homeostasis, autophagy and endoplasmic reticulum (ER) function as measures. 2-month-old mice were fed with a breeding diet ad libitum (AL) or DR (60% of AL) for 3 (Young) or 20 (Aged) months. Body weight increased with aging, whereas DR treatment kept body weight consistent. DR mice exhibited a higher relative heart weight than AL mice. DR mice displayed lower plasma glucose levels, compared with AL groups. Furthermore, Aged-AL, but not Aged-DR mice, had increased collagen content and morphological distortions in the left ventricle (LV). Aged-DR mice had a higher ATP and lower TBARS in the LV than Aged-AL mice. Mitochondrial morphology was detected by electron microscopy; Aged-AL mice had increased abnormal morphology of mitochondria. Treatment with DR reduced abnormal mitochondrial accumulation. Aging elevated the protein expressions of mitochondrial functions and ER-induced apoptosis. Aging downregulated autophagy related proteins and chaperones in the heart. Dietary restriction reversed those protein expressions. The present study demonstrated a beneficial effect of early onset DR on cardiac aging. The age-dependent mitochondrial dysfunction and protein quality control dysregulation was significantly reversed by long-term DR, demonstrating a concordance with the beneficial effect in the heart.

The Link of the Prion Protein with Ca2+ Metabolism and ROS Production, and the Possible Implication in Aβ Toxicity

The cellular prion protein (PrPC) is an ubiquitous cell surface protein mostly expressed in neurons, where it localizes to both pre- and post-synaptic membranes. PrPC aberrant conformers are the major components of mammalian prions, the infectious agents responsible for incurable neurodegenerative disorders. PrPC was also proposed to bind aggregated misfolded proteins/peptides, and to mediate their neurotoxic signal. In spite of long-lasting research, a general consensus on the precise pathophysiologic mechanisms of PrPC has not yet been reached. Here we review our recent data, obtained by comparing primary neurons from PrP-expressing and PrP-knockout mice, indicating a central role of PrPC in synaptic transmission and Ca2+ homeostasis. Indeed, by controlling gene expression and signaling cascades, PrPC is able to optimize glutamate secretion and regulate Ca2+ entry via store-operated channels and ionotropic glutamate receptors, thereby protecting neurons from threatening Ca2+ overloads and excitotoxicity. We will also illustrate and discuss past and unpublished results demonstrating that Aβ oligomers perturb Ca2+ homeostasis and cause abnormal mitochondrial accumulation of reactive oxygen species by possibly affecting the PrP-dependent downregulation of Fyn kinase activity.

Mitochondrial quality control in cardiac cells: Mechanisms and role in cardiac cell injury and disease.

Mitochondria play an important role in maintaining cardiac homeostasis by supplying the major energy required for cardiac excitation-contraction coupling as well as controlling the key intracellular survival and death pathways. Healthy mitochondria generate ATP molecules through an aerobic process known as oxidative phosphorylation (OXPHOS). Mitochondrial injury during myocardial infarction (MI) impairs OXPHOS and results in the excessive production of reactive oxygen species (ROS), bioenergetic insufficiency, and contributes to the development of cardiovascular diseases. Therefore, mitochondrial biogenesis along with proper mitochondrial quality control machinery, which removes unhealthy mitochondria is pivotal for mitochondrial homeostasis and cardiac health. Upon damage to the mitochondrial network, mitochondrial quality control components are recruited to segregate the unhealthy mitochondria and target aberrant mitochondrial proteins for degradation and elimination. Impairment of mitochondrial quality control and accumulation of abnormal mitochondria have been reported in the pathogenesis of various cardiac disorders and heart failure. Here, we provide an overview of the recent studies describing various mechanistic pathways underlying mitochondrial homeostasis with the main focus on cardiac cells. In addition, this review demonstrates the potential effects of mitochondrial quality control dysregulation in the development of cardiovascular disease.

MicroRNA-378 promotes autophagy and inhibits apoptosis in skeletal muscle

The metabolic regulation of cell death is sophisticated. A growing body of evidence suggests the existence of multiple metabolic checkpoints that dictate cell fate in response to metabolic fluctuations. However, whether microRNAs (miRNAs) are able to respond to metabolic stress, reset the threshold of cell death, and attempt to reestablish homeostasis is largely unknown. Here, we show that miR-378/378* KO mice cannot maintain normal muscle weight and have poor running performance, which is accompanied by impaired autophagy, accumulation of abnormal mitochondria, and excessive apoptosis in skeletal muscle, whereas miR-378 overexpression is able to enhance autophagy and repress apoptosis in skeletal muscle of mice. Our in vitro data show that metabolic stress-responsive miR-378 promotes autophagy and inhibits apoptosis in a cell-autonomous manner. Mechanistically, miR-378 promotes autophagy initiation through the mammalian target of rapamycin (mTOR)/unc-51-like autophagy activating kinase 1 (ULK1) pathway and sustains autophagy via Forkhead box class O (FoxO)-mediated transcriptional reinforcement by targeting phosphoinositide-dependent protein kinase 1 (PDK1). Meanwhile, miR-378 suppresses intrinsic apoptosis initiation directly through targeting an initiator caspase-Caspase 9. Thus, we propose that miR-378 is a critical component of metabolic checkpoints, which integrates metabolic information into an adaptive response to reduce the propensity of myocytes to undergo apoptosis by enhancing the autophagic process and blocking apoptotic initiation. Lastly, our data suggest that inflammation-induced down-regulation of miR-378 might contribute to the pathogenesis of muscle dystrophy.

Abstract 406: Loss of Sigmar1 Leads to Impaired Mitochondrial Respiration, Altered Mitochondrial Dynamics and Development of Cardiac Contractile Dysfunction

Rationale: Studies involving different neuronal and cancer cell lines indicated that Sigma 1 receptor (Sigmar1) function as an inter-organelle signaling molecule and elicit cytoprotective functions. Though the presence of Sigmar1 in the heart was reported using ligand binding assay, all studies to date have been limited to pharmacological approaches using less selective ligands for Sigmar1. Despite all these studies, the physiological function of Sigmar1 in the heart remains unknown. Objective: We investigated the physiological function of Sigmar1 in the heart in regulating mitochondrial respiration, mitochondrial dynamics and cardiac contractility using the Sigmar1 knockout mouse (Sigmar1 -/- ). Methods and Results: Sigmar1 -/- hearts showed significantly increased hemodynamic parameters assessed by left ventricular (LV) catheterization both under basal conditions and after stimulation with increasing doses of the β 1 -adrenoceptor agonist. Noninvasive echocardiographic measurements showed the development of cardiac functional decline in Sigmar1 –/– hearts over aging indicated by decreased LV percent fraction shortening and percent ejection fraction. Histochemistry also showed significant cardiac fibrosis and increased expression of periostin in the Sigmar1 –/– hearts compared to wildtype (Wt) hearts. Ultrastructural analysis of Sigmar1 -/- cardiomyocytes showed irregularly shaped, highly fused mitochondrial network, and exhibited abnormal cristae. Mitochondrial size distribution in Sigmar1 -/- hearts showed an increased number of large mitochondria resulting in decreased numbers of mitochondria per microscopic field and also showed altered expression of mitochondrial dynamics regulatory proteins. Real-time oxygen consumption rates in isolated mitochondria showed reduced respiratory function in Sigmar1 -/- hearts compared with Wt hearts. Conclusions: All these data demonstrate a potential function of Sigmar1 in regulating normal mitochondrial organization and size in the heart. Knockdown of Sigmar1 evoked mitochondrial dysfunction, accumulation of abnormal mitochondria, enhanced adverse cardiac remodeling and resulting in cardiac contractile dysfunction.

Abstract 2405: P53-dependent mitophagy controls glycolytic shift in radioresistant head and neck cancer cells

Abstract P53 is an important biomarker in response to genotoxic stress, but it is also known to plays a key role in the regulation of metabolic homeostasis. The loss of p53 is a well-established contributor to the malignant transformation and glycolytic phenotype acquisition of cells during cancer development. However, the role of p53 in genotoxic therapy-induced metabolic shift in cancers remains unclear. Here, we attempted to elucidate how p53 participates in the glycolytic shift of head and neck cancer cell lines following irradiation. We established a stable radioresistant head and neck cancer cells (HN30-R; p53 wild type and UMSCC1-R; p53 null type) through cumulative irradiation and then analyzed their glucose metabolic profiles and mitochondria respiration. As a result, the metabolic analysis revealed no changes glycolysis of HN30-R cells, but UMSCC1-R cells exhibited increased glycolysis through increased glucose uptake and lactate production and glycolytic intermediates as well as related glycolytic enzymes, compared to UMSCC1 cells. Also, we confirmed that the mitochondrial respiration was reduced by the maximal respiration parameters of oxygen consumption rate (OCR) and that abnormal mitochondria were accumulated by electron microscopy in UMSCC1-R cells. Thus, UMSCC1-R cells exhibited an increased sensitivity to glycolysis-targeting drugs such a hexokinases inhibitor (2-deoxy-D-glucoes; 2-DG) and a lactate dehydrogenase-A inhibitor (AT101), but HN30-R cells did not shown any changes. Moreover, we identified that mitophagy limits glycolytic shift through the p53-dependent clearance of abnormal mitochondria. Taken together, these results suggest that p53 null type cells increased aerobic glycolysis to overcome the accumulation of abnormal mitochondria in radioresistnat cells. Conversely, p53 wild type cells inhibited the glycolytic shift by regulating a integrity through p53-dependent mitophagy. Thus, glycolysis-targeted drugs could be an alternative strategy for overcoming recurrent cancers after radiotherapy, and p53 status could be a biomarker for selecting participants for clinical trials. Citation Format: Hyo Won Chang, Ji Won Kim, Mi Ra Kim, Hae Yun Nam, Myungjin Lee, Won Hyeok Lee, Song Hee Kim, Da Seul Seong, Myung Woul Han, Jong Cheol Lee, Jung Je Park, Ji-hyun Seo, Seong Who Kim, Sang Yoon Kim. P53-dependent mitophagy controls glycolytic shift in radioresistant head and neck cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2405.

Intraflagellar transport protein IFT20 is essential for male fertility and spermiogenesis in mice.

Intraflagellar transport (IFT) is a conserved mechanism thought to be essential for the assembly and maintenance of cilia and flagella. However, little is known about its role in mammalian sperm flagella formation. To fill this gap, we disrupted the Ift20 gene in male germ cells. Homozygous mutant mice were infertile with significantly reduced sperm counts and motility. In addition, abnormally shaped elongating spermatid heads and bulbous round spermatids were found in the lumen of the seminiferous tubules. Electron microscopy revealed increased cytoplasmic vesicles, fiber-like structures, abnormal accumulation of mitochondria and a decrease in mature lysosomes. The few developed sperm had disrupted axonemes and some retained cytoplasmic lobe components on the flagella. ODF2 and SPAG16L, two sperm flagella proteins failed to be incorporated into sperm tails of the mutant mice, and in the germ cells, both were assembled into complexes with lighter density in the absence of IFT20. Disrupting IFT20 did not significantly change expression levels of IFT88, a component of IFT-B complex, and IFT140, a component of IFT-A complex. Even though the expression level of an autophagy core protein that associates with IFT20, ATG16, was reduced in the testis of the Ift20 mutant mice, expression levels of other major autophagy markers, including LC3 and ubiquitin were not changed. Our studies suggest that IFT20 is essential for male fertility and spermiogenesis in mice, and its major function is to transport cargo proteins for sperm flagella formation. It also appears to be involved in removing excess cytoplasmic components.

ATG3-dependent autophagy mediates mitochondrial homeostasis in pluripotency acquirement and maintenance

ABSTRACTPluripotent stem cells, including induced pluripotent and embryonic stem cells (ESCs), have less developed mitochondria than somatic cells and, therefore, rely more heavily on glycolysis for energy production.1-3 However, how mitochondrial homeostasis matches the demands of nuclear reprogramming and regulates pluripotency in ESCs is largely unknown. Here, we identified ATG3-dependent autophagy as an executor for both mitochondrial remodeling during somatic cell reprogramming and mitochondrial homeostasis regulation in ESCs. Dysfunctional autophagy by Atg3 deletion inhibited mitochondrial removal during pluripotency induction, resulting in decreased reprogramming efficiency and accumulation of abnormal mitochondria in established iPSCs. In Atg3 null mouse ESCs, accumulation of aberrant mitochondria was accompanied by enhanced ROS generation, defective ATP production and attenuated pluripotency gene expression, leading to abnormal self-renewal and differentiation. These defects were rescued by reacquisition of wild-type but not lipidation-deficient Atg3 expression. Taken together, our findings highlight a critical role of ATG3-dependent autophagy for mitochondrial homeostasis regulation in both pluripotency acquirement and maintenance.

Abstract 222: Sigmar1 Mediates Mitochondrial Autophagy and Protects the Heart Against Ischemia/Reperfusion Injury

Rationale: Sigma 1 receptor (Sigmar1) is a highly expressed mitochondrion-associated ER membrane resident protein in different cell lines. We recently reported that Sigmar1 is highly expressed in cardiomyocytes but it’s molecular functions and role in the stress response still remains unknown. Objective: We investigated the functional role of Sigmar1 in mediating mitochondrial autophagy, mitochondrial fission and effects on stress resistance in the heart. Methods and Results: Subcellular fractionation and biochemical experiments confirmed Sigmar1 expression in the mitochondria, where it resides as an integral mitochondrial outer membrane protein. Sigmar1 overexpression induced mitochondrial fission, increased autophagosome formation and autophagic flux in cardiomyocytes. Similarly, cardiac specific Sigmar1 transgenic (Tg) mice showed increased levels of mitochondrial fission and mitochondrial autophagy without adverse effects. Conversely, Sigmar1 knockdown induced both mitochondrial elongation and accumulation of damaged mitochondria, whereas autophagosome formation and autophagic flux were reduced at baseline and in response to glucose deprivation in cardiomyocytes. Parallel studies using Sigmar1 knockout mice showed increased accumulation of abnormal mitochondria and significantly altered cardiac contractility. To define the functional significance of Sigmar1 in the cardiac stress response, we subjected the mice to ischemia/reperfusion (I/R) injury. Sigmar1 Tg mouse showed reduced infarct size, protected from I/R-injury induced adverse cardiac remodeling, and improved cardiac function associated with enhanced mitochondrial autophagy even 12 weeks after reperfusion injury. In contrary, knockdown of Sigmar1 evoked mitochondrial dysfunction, accumulation of abnormal mitochondria, enhanced adverse cardiac remodeling, aggravated cardiac dysfunction and increased susceptibility to I/R-injury. Conclusions: Our findings suggested that Sigmar1 is an integral mitochondrial outer membrane protein dispensable for constitutive mitochondrial quality control in normal hearts. Sigmar1 regulates mitochondrial autophagy to protect the heart against I/R injury-induced cardiac remodeling and dysfunction.

Human wild-type full-length tau accumulation disrupts mitochondrial dynamics and the functions via increasing mitofusins.

Intracellular accumulation of tau protein is hallmark of sporadic Alzheimer’s disease (AD), however, the cellular mechanism whereby tau accumulation causes neurodegeneration is poorly understood. Here we report that overexpression of human wild-type full-length tau (termed htau) disrupted mitochondrial dynamics by enhancing fusion and induced their perinuclear accumulation in HEK293 cells and rat primary hippocampal neurons. The htau accumulation at later stage inhibited mitochondrial functions shown by the decreased ATP level, the ratio of ATP/ADP and complex I activity. Simultaneously, the cell viability was decreased with retraction of the cellular/neuronal processes. Further studies demonstrated that htau accumulation increased fusion proteins, including OPA1 and mitofusins (Mfn1, Mfn2) and reduced the ubiquitination of Mfn2. Downregulation of the mitofusins by shRNA to ~45% or ~52% of the control levels attenuated the htau-enhanced mitochondrial fusion and restored the functions, while downregulation of OPA1 to ~50% of the control level did not show rescue effects. Finally, abnormal mitochondrial accumulation and dysfunction were also observed in the brains of htau transgenic mice. Taken together, our data demonstrate that htau accumulation decreases cell viability and causes degeneration via enhancing mitofusin-associated mitochondrial fusion, which provides new insights into the molecular mechanisms underlying tauopathies.

Mitochondrial Protein PGAM5 Regulates Mitophagic Protection against Cell Necroptosis.

Necroptosis as a molecular program, rather than simply incidental cell death, was established by elucidating the roles of receptor interacting protein (RIP) kinases 1 and 3, along with their downstream partner, mixed lineage kinase-like domain protein (MLKL). Previous studies suggested that phosphoglycerate mutase family member 5 (PGAM5), a mitochondrial protein that associates with RIP1/RIP3/MLKL complex, promotes necroptosis. We have generated mice deficient in the pgam5 gene and surprisingly found PGAM5-deficiency exacerbated rather than reduced necroptosis in response to multiple in vitro and in vivo necroptotic stimuli, including ischemic reperfusion injury (I/R) in the heart and brain. Electron microscopy, biochemical, and confocal analysis revealed that PGAM5 is indispensable for the process of PINK1 dependent mitophagy which antagonizes necroptosis. The loss of PGAM5/PINK1 mediated mitophagy causes the accumulation of abnormal mitochondria, leading to the overproduction of reactive oxygen species (ROS) that worsen necroptosis. Our results revise the former proposal that PGAM5 acts downstream of RIP1/RIP3 to mediate necroptosis. Instead, PGAM5 protects cells from necroptosis by independently promoting mitophagy. PGAM5 promotion of mitophagy may represent a therapeutic target for stroke, myocardial infarction and other diseases caused by oxidative damage and necroptosis.

Transient assembly of F-actin on the outer mitochondrial membrane contributes to mitochondrial fission.

In addition to established membrane remodeling roles in various cellular locations, actin has recently emerged as a participant in mitochondrial fission. However, the underlying mechanisms of its participation remain largely unknown. We report that transient de novo F-actin assembly on the mitochondria occurs upon induction of mitochondrial fission and F-actin accumulates on the mitochondria without forming detectable submitochondrial foci. Impairing mitochondrial division through Drp1 knockout or inhibition prolonged the time of mitochondrial accumulation of F-actin and also led to abnormal mitochondrial accumulation of the actin regulatory factors cortactin, cofilin, and Arp2/3 complexes, suggesting that disassembly of mitochondrial F-actin depends on Drp1 activity. Furthermore, down-regulation of actin regulatory proteins led to elongation of mitochondria, associated with mitochondrial accumulation of Drp1. In addition, depletion of cortactin inhibited Mfn2 down-regulation- or FCCP-induced mitochondrial fragmentation. These data indicate that the dynamic assembly and disassembly of F-actin on the mitochondria participates in Drp1-mediated mitochondrial fission.

Inhibition of autophagy induces retinal pigment epithelial cell damage by the lipofuscin fluorophore A2E

In this study, we show augmented autophagy in the retinal pigment epithelial cell line ARPE-19 when cultured in the presence of the lipofuscin pigment A2E. A2E alone does not induce RPE cell death, but cell death was induced in the presence of A2E with the autophagy inhibitor 3-methyladenine (3MA), with a concomitant increase in the generation of mitochondrial reactive oxygen species. On the other hand, the ATP production capacity of mitochondria was decreased in the presence of A2E, and pharmacological inhibition of autophagy had no additional effects. The altered mRNA expression level of mitochondrial function markers was confirmed by real-time polymerase chain reaction, which showed that the antioxidant enzymes SOD1 and SOD2 were not reduced in the presence of A2E alone, but significantly suppressed with the addition of 3MA. Furthermore, transmission electron micrography revealed autophagic vacuole formation in the presence of A2E, and inhibition of autophagy resulted in the accumulation of abnormal mitochondria with loss of cristae. Spheroid culture of human RPE cells demonstrated debris accumulation in the presence of A2E, and this accumulation was accelerated in the presence of 3MA. These results indicate that autophagy in RPE cells is a vital cytoprotective process that prevents the accumulation of damaged cellular molecules.

Clinical, pathological and molecular biological characteristics of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episode in children

To delineate the characteristics of the clinical manifestation, pathology of skeletal muscle and gene mutations of mitochondrial encephalomyopathy with lactic acidosis and stroke-like episode (MELAS) in children. The clinical manifestation, laboratorial data, brain images, muscle pathology and mitochondrial gene mutations were analyzed in 24 patients with MELAS who were diagnosed in Department of Pediatrics, Peking University First Hospital. Their prognosis was evaluated by following up. Symptoms of central nervous system such as stroke-like episodes, vomiting, convulsion and headache were present in all the 24 cases. Nine cases had the symptoms of myopathy. Twenty cases had developmental delay. Short stature, being thin and hairy was very common in these cases. Serum lactate level increased in all the cases, pyruvate increased in 17 cases. Elevated CSF lactate was found in 2 cases. Magnetic resonance imaging (MRI) was performed on 24 cases, out of them 23 were abnormal. The lesions mainly involved cerebral lobes. Occipital lobe was the most common site of lesions. Computed tomography (CT) was performed on 13 cases, low density lesions were present in 10 cases, basal ganglia calcifications in 5 cases. Muscle biopsy was performed on 8 cases, ragged-red fibers (RRF) were found in 4/8 cases, and abnormal accumulation of mitochondria were found in 3/8 cases. The mtDNA gene mutational analysis showed A3243G mutation in these patients. The mutation rates varied from 11.6% to 75.0%. The same mutation were found in 4/5 mothers who had the genetic tests, and the mutation rates of the mothers varied from 15.0% to 23.6%. The clinical information of 11 cases was available through recent following up. Three cases died, the others had some degrees of mental retardation. Children with MELAS had various clinical manifestations. Central nervous system and skeletal muscle were usually involved. Short stature and hypertrichosis were common signs. The prognosis of this disease was disappointing. mtDNA A3243G was the most common mutation in MELAS. Fully understanding the characteristics of its clinical manifestation, laboratory tests, brain image, muscle pathology and molecular features can be helpful to the early diagnosis and treatment.