Parkinson's disease-associated DJ-1 mutations impair mitochondrial dynamics and cause mitochondrial dysfunction

Mitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson's disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H(2) O(2)) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.     

Fzo1, a protein involved in mitochondrial fusion, inhibits apoptosis

Mitochondrial morphology and physiology are regulated by the processes of fusion and fission. Some forms of apoptosis are reported to be associated with mitochondrial fragmentation. We showed that overexpression of Fzo1A/B (rat) proteins involved in mitochondrial fusion, or silencing of Dnm1 (rat)/Drp1 (human) (a mitochondrial fission protein), increased elongated mitochondria in healthy cells. After apoptotic stimulation, these interventions inhibited mitochondrial fragmentation and cell death, suggesting that a process involved in mitochondrial fusion/fission might play a role in the regulation of apoptosis. Consistently, silencing of Fzo1A/B or Mfn1/2 (a human homolog of Fzo1A/B) led to an increase of shorter mitochondria and enhanced apoptotic death. Overexpression of Fzo1 inhibited cytochrome c release and activation of Bax/Bak, as assessed from conformational changes and oligomerization. Silencing of Mfn or Drp1 caused an increase or decrease of mitochondrial sensitivity to apoptotic stimulation, respectively. These results indicate that some of the proteins involved in mitochondrial fusion/fission modulate apoptotic cell death at the mitochondrial level.     

Bcl-2 prevents abnormal mitochondrial proliferation during etoposide-induced apoptosis

At the late stage of etoposide-induced apoptosis in HL-60 cells, marked by condensation of chromatin, mitochondria increase in numbers. There is also a drastic increase in mitochondrial DNA content. This increase in mitochondrial numbers and DNA content is an indicator of mitochondrial proliferation during apoptosis. These proliferating mitochondria exhibit abnormal morphology and are impaired, which is demonstrated by decrease in mitochondrial membrane potential and ATP content. The described apoptosis-induced abnormal mitochondrial proliferation was inhibited by overexpression of Bcl-2 protein, which also diminishes mitochondrial impairment. The increase in mitochondrial DNA levels correlated with elevated expression of one of the regulators of mitochondrial DNA replication, mtSSB. Our data suggest that proliferation of mitochondria may be an integral part of a cascade of apoptotic events.     

Human Misato regulates mitochondrial distribution and morphology

Misato of Drosophila melanogaster and Saccharomyces cerevisiae DML1 are conserved proteins having a homologous region with a part of the GTPase family that includes eukaryotic tubulin and prokaryotic FtsZ. We characterized human Misato sharing homology with Misato of D. melanogaster and S. cerevisiae DML1. Tissue distribution of Misato exhibited ubiquitous distribution. Subcellular localization of the protein studied using anti-Misato antibody suggested that it is localized to the mitochondria. Further experiments of fractionating mitochondria revealed that Misato was localized to the outer membrane. The transfection of Misato siRNA led to growth deficiencies compared with control siRNA transfected HeLa cells, and the Misato-depleted HeLa cells showed apoptotic nuclear fragmentation resulting in cell death. After silencing of Misato, the filamentous mitochondrial network disappeared and fragmented mitochondria were observed, indicating human Misato has a role in mitochondrial fusion. To examine the effects of overexpression, COS-7 cells were transfected with cDNA encoding EGFP-Misato. Its overexpression resulted in the formation of perinuclear aggregations of mitochondria in these cells. The Misato-overexpressing cells showed low viability and had no nuclei or a small and structurally unusual ones. These results indicated that human Misato has a role(s) in mitochondrial distribution and morphology and that its unregulated expression leads to cell death.     

Hypermethylation-mediated silencing of NDRG4 promotes pancreatic ductal adenocarcinoma by regulating mitochondrial function

The N-myc downstream regulated gene (NDRG) family members are dysregulated in several tumors. Functionally, NDRGs play an important role in the malignant progression of cancer cells. However, little is known about the potential implications of NDRG4 in pancreatic ductal adenocarcinoma (PDAC). The aim of the current study was to elucidate the expression pattern of NDRG4 in PDAC and evaluate its potential cellular biological effects. Here, we firstly report that epigenetic-mediated silencing of NDRG4 promotes PDAC by regulating mitochondrial function. Data mining demonstrated that NDRG4 was significantly down-regulated in PDAC tissues and cells. PDAC patients with low NDRG4 expression showed poor prognosis. Epigenetic regulation by DNA methylation was closely associated with NDRG4 down-regulation. NDRG4 overexpression dramatically suppressed PDAC cell growth and metastasis. Further functional analysis demonstrated that up-regulated NDRG4 in SW1990 and Canpan1 cells resulted in attenuated mitochondrial function, including reduced ATP production, decreased mitochondrial membrane potential, and increased fragmented mitochondria. However, opposite results were obtained for HPNE cells with NDRG4 knockdown. These results indicate that hypermethylation-driven silencing of NDRG4 can promote PDAC by regulating mitochondrial function and that NDRG4 could be as a potential biomarker for PDAC patients.     

ROS-induced mitochondrial depolarization initiates PARK2/PARKIN-dependent mitochondrial degradation by autophagy

Reactive oxygen species (ROS) have been implicated as a signal for general autophagy. Both mitochondrial-produced and exogenous ROS induce autophagosome formation. However, it is unclear whether ROS are required for the selective autophagic degradation of mitochondria, a process called mitophagy. Recent work using carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial-uncoupling reagent, has been shown to induce mitophagy. However, CCCP treatment may not be biologically relevant since it causes the depolarization of the entire mitochondrial network. Since mitochondria are the main ROS production sites in mammalian cells, we propose that short bursts of ROS produced within mitochondria may be involved in the signaling for mitophagy. To test this hypothesis, we induced an acute burst of ROS within mitochondria using a mitochondrial-targeted photosensitizer, mitochondrial KillerRed (mtKR). Using mtKR, we increased ROS levels in the mitochondrial matrix, which resulted in the loss of membrane potential and the subsequent activation of PARK2-dependent mitophagy. Importantly, we showed that overexpression of the mitochondrial antioxidant protein, superoxide dismutase-2, can squelch mtKR-induced mitophagy, demonstrating that mitochondrial ROS are responsible for mitophagy activation. Using this assay, we examined the impact of mitochondrial morphology on mitophagy. It was shown recently that elongated mitochondria are more resistant to mitophagy through unknown mechanisms. Here, we show that elongated mitochondria are more resistant to ROS-induced damage and mitophagy compared with fragmented mitochondria, suggesting that mitochondrial morphology has an important role in regulating ROS and mitophagy. Together, our results suggest that ROS-induced mitochondrial damage may be an important upstream activator of mitophagy.     

虱目裂化线粒体基因组研究进展

虱目是哺乳类和鸟类体表的专性寄生虫。在虱科、阴虱科、长角鸟虱科和兽羽虱科的某些寄生虱种中发现了线粒体基因组裂化现象, 其线粒体基因组裂化成了多个环状的线粒体染色体, 如体虱(Pediculus humanus)、头虱(pediculus capitis)和阴虱(Pthirus pubis)的线粒体基因组分别裂化形成20个、20个和14个微环染色体。微环染色体可能是基因删除和同源重组的结果, 关于线粒体基因组裂化的具体原因和机制, 目前并不清楚, 推测可能是进化选择或随机遗传漂变的结果或与线粒体单链DNA结合蛋白的缺失有关。鉴于线粒体基因组裂化研究对于深入理解线粒体的起源和进化方面具有重要意义, 文章以虱目裂化线粒体基因组为主线, 列举了动物裂化线粒体基因组和裂化特征, 阐述了虱目裂化线粒体基因组的研究现状, 分析了虱目线粒体基因组裂化的类型、原因和机制, 并对该领域未来的研究方向进行了展望。     

Mitochondrial dysfunction in cancer cells due to aberrant mitochondrial replication

Warburg effect is a hallmark of cancer manifested by continuous prevalence of glycolysis and dysregulation of oxidative metabolism. Glycolysis provides survival advantage to cancer cells. To investigate molecular mechanisms underlying the Warburg effect, we first compared oxygen consumption among hFOB osteoblasts, benign osteosarcoma cells, Saos2, and aggressive osteosarcoma cells, 143B. We demonstrate that, as both proliferation and invasiveness increase in osteosarcoma, cells utilize significantly less oxygen. We proceeded to evaluate mitochondrial morphology and function. Electron microscopy showed that in 143B cells, mitochondria are enlarged and increase in number. Quantitative PCR revealed an increase in mtDNA in 143B cells when compared with hFOB and Saos2 cells. Gene expression studies showed that mitochondrial single-strand DNA-binding protein (mtSSB), a key catalyst of mitochondrial replication, was significantly up-regulated in 143B cells. In addition, increased levels of the mitochondrial respiratory complexes were accompanied by significant reduction of their activities. These changes indicate hyperactive mitochondrial replication in 143B cells. Forced overexpression of mtSSB in Saos2 cells caused an increase in mtDNA and a decrease in oxygen consumption. In contrast, knockdown of mtSSB in 143B cells was accompanied by a decrease in mtDNA, increase in oxygen consumption, and retardation of cell growth in vitro and in vivo. In summary, we have found that mitochondrial dysfunction in cancer cells correlates with abnormally increased mitochondrial replication, which according to our gain- and loss-of-function experiments, may be due to overexpression of mtSSB. Our study provides insight into mechanisms of mitochondrial dysfunction in cancer and may offer potential therapeutic targets.     

Acrolein is a mitochondrial toxin: effects on respiratory function and enzyme activities in isolated rat liver mitochondria

Acrolein is an air pollutant from cigarette smoking and other pollutions and also a by-product of lipid peroxidation. Studies have demonstrated that acrolein causes cytotoxicity and genotoxicity, including liver damage and death of hepatocytes. However, the toxic effects and the underlying mechanisms of acrolein on mitochondria, especially, on liver mitochondria, have not been well studied. In the present study, we investigated the toxic effects and mechanisms of acrolein on mitochondria isolated from rat liver by examining mitochondrial respiration, dehydrogenases, complex I, II, III, IV and V, permeability transition, and protein oxidation. Acrolein incubation (10-1000 microM, or 0.02-2 micromol/mg protein) with mitochondria caused dose-dependent inhibition of NADH- and succinate-linked mitochondrial respiration chain, change of mitochondrial permeability transition, increase in protein carbonyls, and selective enzyme inhibition of mitochondrial complex I, II, pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, but no effects on mitochondrial complex III, IV, V and malate dehydrogenase. These results suggest that acrolein is a mitochondrial toxin and that mitochondrial dysfunction caused by acrolein may play an important role in acrolein toxicity such as hepatotoxicity and also smoking-related diseases.     

Alkyladenine DNA glycosylase (AAG) localizes to mitochondria and interacts with mitochondrial single-stranded binding protein (mtSSB)

Due to a harsh environment mitochondrial genomes accumulate high levels of DNA damage, in particular oxidation, hydrolytic deamination, and alkylation adducts. While repair of alkylated bases in nuclear DNA has been explored in detail, much less is known about the repair of DNA alkylation damage in mitochondria. Alkyladenine DNA glycosylase (AAG) recognizes and removes numerous alkylated bases, but to date AAG has only been detected in the nucleus, even though mammalian mitochondria are known to repair DNA lesions that are specific substrates of AAG. Here we use immunofluorescence to show that AAG localizes to mitochondria, and we find that native AAG is present in purified human mitochondrial extracts, as well as that exposure to alkylating agent promotes AAG accumulation in the mitochondria. We identify mitochondrial single-stranded binding protein (mtSSB) as a novel interacting partner of AAG; interaction between mtSSB and AAG is direct and increases upon methyl methanesulfonate (MMS) treatment. The consequence of this interaction is specific inhibition of AAG glycosylase activity in the context of a single-stranded DNA (ssDNA), but not a double-stranded DNA (dsDNA) substrate. By inhibiting AAG-initiated processing of damaged bases, mtSSB potentially prevents formation of DNA breaks in ssDNA, ensuring that base removal primarily occurs in dsDNA. In summary, our findings suggest the existence of AAG-initiated BER in mitochondria and further support a role for mtSSB in DNA repair.     

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.     

ChChd3, an inner mitochondrial membrane protein, is essential for maintaining crista integrity and mitochondrial function

The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952-14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function.     

Molecular characterization of mitocalcin, a novel mitochondrial Ca2+-binding protein with EF-hand and coiled-coil domains

Here we have identified and characterized a novel mitochondrial Ca2+-binding protein, mitocalcin. Western blot analysis demonstrated that mitocalcin was widely expressed in mouse tissues. The expression in brain was increased during post-natal to adult development. Further analyses were carried out in newly established neural cell lines. The protein was expressed specifically in neurons but not in glial cells. Double-labeling studies revealed that mitocalcin was colocalized with mitochondria in neurons differentiated from 2Y-3t cells. In addition, mitocalcin was enriched in the mitochondrial fraction purified from the cells. Immunohistochemical studies on mouse cerebellum revealed that the expression pattern of mitocalcin in glomeruli of the internal granular and molecular layers was well overlapped by the distribution pattern of mitochondria. Immunogold electron microscopy showed that mitocalcin was associated with mitochondrial inner membrane. Overexpression of mitocalcin in 2Y-3t cells resulted in neurite extension. Inhibition of the expression in 2Y-3t cells caused suppression of neurite outgrowth and then cell death. These findings suggest that mitocalcin may play roles in neuronal differentiation and function through the control of mitochondrial function.     

Involvement of p32 and microtubules in alteration of mitochondrial functions by rubella virus

The interaction of the rubella virus (RV) capsid (C) protein and the mitochondrial p32 protein is believed to participate in virus replication. In this study, the physiological significance of the association of RV with mitochondria was investigated by silencing p32 through RNA interference. It was demonstrated that downregulation of p32 interferes with microtubule-directed redistribution of mitochondria in RV-infected cells. However, the association of the viral C protein with mitochondria was not affected. When cell lines either pretreated with respiratory chain inhibitors or cultivated under (mild) hypoxic conditions were infected with RV, viral replication was reduced in a time-dependent fashion. Additionally, RV infection induces increased activity of mitochondrial electron transport chain complex III, which was associated with an increase in the mitochondrial membrane potential. These effects are outstanding among the examples of mitochondrial alterations caused by viruses. In contrast to the preferential localization of p32 to the mitochondrial matrix in most cell lines, RV-permissive cell lines were characterized by an almost exclusive membrane association of p32. Conceivably, this contributes to p32 function(s) during RV replication. The data presented suggest that p32 fulfills an essential function for RV replication in directing trafficking of mitochondria near sites of viral replication to meet the energy demands of the virus.