Mitochondrial peptidase IMMP2L mutation causes early onset of age-associated disorders and impairs adult stem cell self-renewal

Mitochondrial reactive oxygen species (ROS) are proposed to play a central role in aging and age-associated disorders, although direct in vivo evidence is lacking. We recently generated a mouse mutant with mutated inner mitochondrial membrane peptidase 2-like (Immp2l) gene, which impairs the signal peptide sequence processing of mitochondrial proteins cytochrome c1 and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion and cause impaired fertility in both sexes. Here, we design experiments to examine the effects of excessive mitochondrial ROS generation on health span. We show that Immp2l mutation increases oxidative stress in multiple organs such as the brain and the kidney, although expression of superoxide dismutases in these tissues of the mutants is also increased. The mutants show multiple aging-associated phenotypes, including wasting, sarcopenia, loss of subcutaneous fat, kyphosis, and ataxia, with female mutants showing earlier onset and more severe age-associated disorders than male mutants. The loss of body weight and fat was unrelated to food intake. Adipose-derived stromal cells (ADSC) from mutant mice showed impaired proliferation capability, formed significantly less and smaller colonies in colony formation assays, although they retained adipogenic differentiation capability in vitro. This functional impairment was accompanied by increased levels of oxidative stress. Our data showed that mitochondrial ROS is the driving force of accelerated aging and suggested that ROS damage to adult stem cells could be one of the mechanisms for age-associated disorders.     

Oxidative stress is involved in age-dependent spermatogenic damage of Immp2l mutant mice

Mitochondrial reactive oxygen species (ROS) have been implicated in spermatogenic damage, although direct in vivo evidence is lacking. We recently generated a mouse in which the inner mitochondrial membrane peptidase 2-like (Immp2l) gene is mutated. This Immp2l mutation impairs the processing of signal peptide sequences from mitochondrial cytochrome c₁ and glycerol phosphate dehydrogenase 2. The mitochondria from mutant mice generate elevated levels of superoxide ion, which causes age-dependent spermatogenic damage. Here we confirm age-dependent spermatogenic damage in a new cohort of mutants, which started at the age of 10.5 months. Compared with age-matched controls, protein carbonyl content was normal in testes of 2- to 5-month-old mutants, but significantly elevated in testes of 13-month-old mutants, indicating elevated oxidative stress in the testes at the time of impaired spermatogenesis. Testicular expression of superoxide dismutases was not different between control and mutant mice, whereas that of catalase was increased in young and old mutants. The expression of cytosolic glutathione peroxidase 4 (phospholipid hydroperoxidase) in testes was significantly reduced in 13-month-old mutants, concomitant with impaired spermatogenesis. Apoptosis of all testicular populations was increased in mutant mice with spermatogenic damage. The mitochondrial DNA (mtDNA) mutation rate in germ cells of mutant mice with impaired spermatogenesis was unchanged, excluding a major role of mtDNA mutation in ROS-mediated spermatogenic damage. Our data show that increased mitochondrial ROS are one of the driving forces for spermatogenic impairment.     

Stress-dependent opposing roles for mitophagy in aging of the ascomycete Podospora anserina

Mitochondrial dysfunction is causatively linked to organismal aging and the development of degenerative diseases. Here we describe stress-dependent opposing roles of mitophagy, the selective autophagic degradation of mitochondria, in aging and life-span control. We report that the ablation of the mitochondrial superoxide dismutase which is involved in reactive oxygen species (ROS) balancing, does not affect life span of the fungal aging model Podospora anserina, although superoxide levels are strongly increased and complex I-dependent respiration is impaired. This unexpected phenotype depends on functional autophagy, particularly mitophagy, which is upregulated during aging of this mutant. It identifies mitophagy as a prosurvival response involved in the control of mitohormesis, the well-known beneficial effect of mild mitochondrial oxidative stress. In contrast, excessive superoxide stress turns mitophagy to a prodeath pathway and leads to accelerated aging. Overall our data uncover mitophagy as a dynamic pathway that specifically responds to different levels of mitochondrial oxidative stress and thereby affects organismal aging.     

Mitochondrial superoxide plays a crucial role in the development of mitochondrial dysfunction during high glucose exposure in rat renal proximal tubular cells

Diabetic nephropathy is the leading cause of end-stage renal disease in the United States. Despite several studies indicating a role for mitochondrial oxidative stress and mitochondrial dysfunction in the development of diabetic complications, the precise mechanisms underlying renal mitochondrial dysfunction and renal cell injury remain unclear. The hypothesis of the current study was that high-glucose-mediated generation of mitochondrial superoxide is a key early event that leads to mitochondrial injury in renal proximal tubular cells. To ascertain the role of mitochondrial superoxide we have tested whether overexpression of the primary mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), protects against hyperglycemia-induced renal injury using normal rat renal proximal tubular cells (NRK). NRK cells were exposed to high glucose (25 mM) and the changes in the mitochondrial membrane potential, ATP levels, and superoxide generation and the loss of cell viability were measured at 24 and 48 h after high glucose exposure. Our results indicate that high glucose first induced superoxide generation and hyperpolarization in the mitochondria, followed by a secondary event, which involved a decline in ATP levels, partial Complex III inactivation, and loss of cell viability. These high-glucose-induced changes were completely prevented by overexpression of MnSOD in NRK cells. However, MnSOD activity was not changed after high glucose exposure in vitro or during the early stages of diabetes using the streptozotocin rat model. These findings show for the first time that hyperglycemic induction of superoxide production within the mitochondria initiates specific mitochondrial injury (i.e., Complex III) via a mechanism independent of MnSOD inactivation.     

4-hydroxy-2-nonenal and uncoupling proteins: an approach for regulation of mitochondrial ROS production

One factor that has the potential to regulate reactive oxygen species (ROS) generation is the mild uncoupling of oxidative phosphorylation, i.e. proton (H(+)) leak across the mitochondrial inner membrane. Proton leak has been shown to attenuate ROS generation, whereas ROS and their derivatives (such as superoxide and hydroxynonenal) have been shown to induce H(+) leak through uncoupling proteins (UCPs). This suggests the existence of a feedback loop between ROS and H(+) leak mediated through UCPs. Although the physiological functions of the new UCPs, such as UCP2 and UCP3, are still not established, extensive data support the idea that these mitochondrial carrier proteins are involved in the control of ROS generation. The molecular basis of both ROS generation and hydroxynonenal-induced uncoupling through UCPs is reviewed and the consequences of their interaction for protection against excessive ROS production at the expense of energy production is discussed.     

The Immp2l mutation causes age‐dependent degeneration of cerebellar granule neurons prevented by antioxidant treatment

Reactive oxygen species are implicated in age‐associated neurodegeneration, although direct in vivo evidence is lacking. We recently showed that mice with a mutation in the Inner Mitochondrial Membrane Peptidase 2‐like (Immp2l) gene had elevated levels of mitochondrial superoxide, impaired fertility and age‐associated phenotypes, including kyphosis and ataxia. Here we show that ataxia and cerebellar hypoplasia occur in old mutant mice (> 16 months). Cerebellar granule neurons (CGNs) are significantly underrepresented; Purkinje cells and cells in the molecular layer are not affected. Treating mutant mice with the mitochondria‐targeted antioxidant SkQ1 from 6 weeks to 21 months protected cerebellar granule neurons. Apoptotic granule neurons were observed in mutant mice but not in age‐matched normal control mice or SkQ1‐treated mice. Old mutant mice showed increased serum protein carbonyl content, cerebellar 4‐hydroxynonenal (HNE), and nitrotyrosine modification compared to old normal control mice. SOD2 expression was increased in Purkinje cells but decreased in granule neurons of old mutant mice. Mitochondrial marker protein VDAC1 also was decreased in CGNs of old mutant mice, suggesting decreased mitochondrial number. SkQ1 treatment decreased HNE and nitrotyrosine modification, and restored SOD2 and VDAC1 expression in CGNs of old mutant mice. Neuronal expression of nitric oxide synthase was increased in cerebella of young mutant mice but decreased in old mutant mice. Our work provides evidence for a causal role of oxidative stress in neurodegeneration of Immp2l mutant mice. The Immp2l mutant mouse model could be valuable in elucidating the role of oxidative stress in age‐associated neurodegeneration.     

Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm

Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase‐type activity of sperm mitochondrial complex I (MCI) and PHB expression were also determined. We demonstrate that mROS and lipid peroxidation levels are significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression are significantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase‐type activity levels of sperm MCI in the three groups studied suggests that the catalytic subunits of MCI in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCI associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, and it may regulate sperm motility via increases in low MMP and lipid peroxidation. This is the first report on the involvement of PHB in human sperm motility loss associated with increased generation of mROS at MCI.     

A novel mitochondrial ubiquitin ligase plays a critical role in mitochondrial dynamics

In this study, we have identified a novel mitochondrial ubiquitin ligase, designated MITOL, which is localized in the mitochondrial outer membrane. MITOL possesses a Plant Homeo-Domain (PHD) motif responsible for E3 ubiquitin ligase activity and predicted four-transmembrane domains. MITOL displayed a rapid degradation by autoubiquitination activity in a PHD-dependent manner. HeLa cells stably expressing a MITOL mutant lacking ubiquitin ligase activity or MITOL-deficient cells by small interfering RNA showed an aberrant mitochondrial morphology such as fragmentation, suggesting the enhancement of mitochondrial fission by MITOL dysfunction. Indeed, a dominant-negative expression of Drp1 mutant blocked mitochondrial fragmentation induced by MITOL depletion. We found that MITOL associated with and ubiquitinated mitochondrial fission protein hFis1 and Drp1. Pulse-chase experiment showed that MITOL overexpression increased turnover of these fission proteins. In addition, overexpression phenotype of hFis1 could be reverted by MITOL co-overexpression. Our finding indicates that MITOL plays a critical role in mitochondrial dynamics through the control of mitochondrial fission proteins.     

Comparisons of coenzyme Q bound to mitochondrial membrane proteins among different mammalian species.

The objective of this study was to elucidate the mechanisms that govern the variations in the rates of mitochondrial superoxide anion radical (O2-*) generation in different species. The amounts of coenzyme Q (CoQ) associated with mitochondrial membrane proteins were compared in five different mammalian species, namely mouse, rat, rabbit, pig, and cow. Micelles of cardiac mitochondria were prepared using Triton X-100 or deoxycholate (DOC) as detergents, and the micelles containing mitochondrial proteins were sedimented by sucrose density ultracentrifugation. The amount of CoQ present in both types of micelles varied in different species, whereas alpha-tocopherol, another lipoidal molecule in mitochondrial membranes, could not be detected in the micelles of any of these species. The amounts of CoQ bound to mitochondrial proteins in DOC micelles were higher in those mammalian species where CoQ10 was the predominant CoQ homologue, and the amounts were found to be inversely correlated with the rate of mitochondrial 02-* generation among different species. Results also indicated that mitochondrial CoQ exists in at least two distinct pools, one of which is associated with the membrane proteins. The degree of association between CoQ and membrane proteins appears to be a factor determining the rate of mitochondrial O2-* generation.     

The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability

Mitochondria are well known as sites of electron transport and generators of cellular ATP. Mitochondria also appear to be sites of cell survival regulation. In the process of programmed cell death, mediators of apoptosis can be released from mitochondria through disruptions in the outer mitochondrial membrane; these mediators then participate in the activation of caspases and of DNA degradation. Thus the regulation of outer mitochondrial membrane integrity is an important control point for apoptosis. The Bcl-2 family is made up of outer mitochondrial membrane proteins that can regulate cell survival, but the mechanisms by which Bcl-2 family proteins act remain controversial. Most metabolites are permeant to the outer membrane through the voltage dependent anion channel (VDAC), and Bcl-2 family proteins appear to be able to regulate VDAC function. In addition, many Bcl-2 family proteins can form channels in vitro, and some pro-apoptotic members may form multimeric channels large enough to release apoptosis promoting proteins from the intermembrane space. Alternatively, Bcl-2 family proteins have been hypothesized to coordinate the permeability of both the outer and inner mitochondrial membranes through the permeability transition (PT) pore. Increasing evidence suggests that alterations in cellular metabolism can lead to pro-apoptotic changes, including changes in intracellular pH, redox potential and ion transport. By regulating mitochondrial membrane physiology, Bcl-2 proteins also affect mitochondrial energy generation, and thus influence cellular bioenergetics. Cell Death and Differentiation (2000) 7, 1182 - 1191     

Mitochondrial Ubiquitin Ligase MITOL Ubiquitinates Mutant SOD1 and Attenuates Mutant SOD1-induced Reactive Oxygen Species Generation

We have previously identified a novel mitochondrial ubiquitin ligase, MITOL, which is localized in the mitochondrial outer membrane and is involved in the control of mitochondrial dynamics. In this study, we examined whether MITOL eliminates misfolded proteins localized to mitochondria. Mutant superoxide dismutase1 (mSOD1), one of misfolded proteins, has been shown to localize in mitochondria and induce mitochondrial dysfunction, possibly involving in the onset and progression of amyotrophic lateral sclerosis. We found that in the mitochondria, MITOL interacted with and ubiquitinated mSOD1 but not wild-type SOD1. In vitro ubiquitination assay revealed that MITOL directly ubiquitinates mSOD1. Cycloheximide-chase assay in the Neuro2a cells indicated that MITOL overexpression promoted mSOD1 degradation and suppressed both the mitochondrial accumulation of mSOD1 and mSOD1-induced reactive oxygen species (ROS) generation. Conversely, the overexpression of MITOL CS mutant and MITOL knockdown by specific siRNAs resulted in increased accumulation of mSOD1 in mitochondria, which enhanced mSOD1-induced ROS generation and cell death. Thus, our findings indicate that MITOL plays a protective role against mitochondrial dysfunction caused by the mitochondrial accumulation of mSOD1 via the ubiquitin–proteasome pathway.     

Disruption of fusion results in mitochondrial heterogeneity and dysfunction

Mitochondria undergo continual cycles of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. Paradoxically, cells invest many resources to maintain tubular mitochondrial morphology, when reducing both fusion and fission simultaneously achieves the same end. This observation suggests a requirement for mitochondrial fusion, beyond maintenance of organelle morphology. Here, we show that cells with targeted null mutations in Mfn1 or Mfn2 retained low levels of mitochondrial fusion and escaped major cellular dysfunction. Analysis of these mutant cells showed that both homotypic and heterotypic interactions of Mfns are capable of fusion. In contrast, cells lacking both Mfn1 and Mfn2 completely lacked mitochondrial fusion and showed severe cellular defects, including poor cell growth, widespread heterogeneity of mitochondrial membrane potential, and decreased cellular respiration. Disruption of OPA1 by RNAi also blocked all mitochondrial fusion and resulted in similar cellular defects. These defects in Mfn-null or OPA1-RNAi mammalian cells were corrected upon restoration of mitochondrial fusion, unlike the irreversible defects found in fzodelta yeast. In contrast, fragmentation of mitochondria, without severe loss of fusion, did not result in such cellular defects. Our results showed that key cellular functions decline as mitochondrial fusion is progressively abrogated.     

Effect of mutations in Tom40 on stability of the translocase of the outer mitochondrial membrane (TOM) complex, assembly of Tom40, and import of mitochondrial preproteins

Mitochondrial preproteins synthesized in the cytosol are imported through the mitochondrial outer membrane by the translocase of the outer mitochondrial membrane (TOM) complex. Tom40 is the major component of the complex and is essential for cell viability. We generated 21 different mutations in conserved regions of the Neurospora crassa Tom40 protein. The mutant genes were transformed into a tom40 null nucleus maintained in a sheltered heterokaryon, and 17 of the mutant genes gave rise to viable strains. All mutations reduced the efficiency of the altered Tom40 molecules to assemble into the TOM complex. Mitochondria isolated from seven of the mutant strains had defects for importing mitochondrial preproteins. Only one strain had a general import defect for all preproteins examined. Another mutation resulted in defects in the import of a matrix-destined preprotein and an outer membrane beta-barrel protein, but import of the ADP/ATP carrier to the inner membrane was unaffected. Five strains showed deficiencies in the import of beta-barrel proteins. The latter results suggest that the TOM complex distinguishes beta-barrel proteins from other classes of preprotein during import. This supports the idea that the TOM complex plays an active role in the transfer of preproteins to subsequent translocases for insertion into the correct mitochondrial subcompartment.     

MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline

MITOPLD is a member of the phospholipase D superfamily proteins conserved among diverse species. Zucchini (Zuc), the Drosophila homolog of MITOPLD, has been implicated in primary biogenesis of Piwi-interacting RNAs (piRNAs). By contrast, MITOPLD has been shown to hydrolyze cardiolipin in the outer membrane of mitochondria to generate phosphatidic acid, which is a signaling molecule. To assess whether the mammalian MITOPLD is involved in piRNA biogenesis, we generated Mitopld mutant mice. The mice display meiotic arrest during spermatogenesis, demethylation and derepression of retrotransposons, and defects in primary piRNA biogenesis. Furthermore, in mutant germ cells, mitochondria and the components of the nuage, a perinuclear structure involved in piRNA biogenesis/function, are mislocalized to regions around the centrosome, suggesting that MITOPLD may be involved in microtubule-dependent localization of mitochondria and these proteins. Our results indicate a conserved role for MITOPLD/Zuc in the piRNA pathway and link mitochondrial membrane metabolism/signaling to small RNA biogenesis.     

Mutant huntingtin expression induces mitochondrial calcium handling defects in clonal striatal cells: functional consequences

Huntington disease (HD) is caused by a pathological elongation of CAG repeats in the huntingtin protein gene and is characterized by atrophy and neuronal loss primarily in the striatum. Mitochondrial dysfunction and impaired Ca2+ homeostasis in HD have been suggested previously. Here, we elucidate the effects of Ca2+ on mitochondria from the wild type (STHdhQ7/Q7) and mutant (STHdhQ111/Q111) huntingtin-expressing cells of striatal origin. When treated with increasing Ca2+ concentrations, mitochondria from mutant huntingtin-expressing cells showed enhanced sensitivity to Ca2+, as they were more sensitive to Ca2+-induced decreases in state 3 respiration and DeltaPsim, than mitochondria from wild type cells. Further, mutant huntingtin-expressing cells had a reduced mitochondrial Ca2+ uptake capacity in comparison with wild type cells. Decreases in state 3 respiration were associated with increased mitochondrial membrane permeability. The DeltaPsim defect was attenuated in the presence of ADP and the decreases in Ca2+ uptake capacity were abolished in the presence of Permeability Transition Pore (PTP) inhibitors. These findings clearly indicate that mutant huntingtin-expressing cells have mitochondrial Ca2+ handling defects that result in respiratory deficits and that the increased sensitivity to Ca2+ induced mitochondrial permeabilization maybe a contributing mechanism to the mitochondrial dysfunction in HD.     

Protein carbonylation and adipocyte mitochondrial function

Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1α subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1α subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte.