Mitochondrial Membrane Complex Research Articles

De novo mutations in TOMM70, a receptor of the mitochondrial import translocase, cause neurological impairment.

The translocase of outer mitochondrial membrane (TOMM) complex is the entry gate for virtually all mitochondrial proteins and is essential to build the mitochondrial proteome. TOMM70 is a receptor that assists mainly in mitochondrial protein import. Here, we report two individuals with de novo variants in the C-terminal region of TOMM70. While both individuals exhibited shared symptoms including hypotonia, hyper-reflexia, ataxia, dystonia and significant white matter abnormalities, there were differences between the two individuals, most prominently the age of symptom onset. Both individuals were undiagnosed despite extensive genetics workups. Individual 1 was found to have a p.Thr607Ile variant while Individual 2 was found to have a p.Ile554Phe variant in TOMM70. To functionally assess both TOMM70 variants, we replaced the Drosophila Tom70 coding region with a Kozak-mini-GAL4 transgene using CRISPR-Cas9. Homozygous mutant animals die as pupae, but lethality is rescued by the mini-GAL4-driven expression of human UAS-TOMM70 cDNA. Both modeled variants lead to significantly less rescue indicating that they are loss-of-function alleles. Similarly, RNAi-mediated knockdown of Tom70 in the developing eye causes roughening and synaptic transmission defect, common findings in neurodegenerative and mitochondrial disorders. These phenotypes were rescued by the reference, but not the variants, of TOMM70. Altogether, our data indicate that de novo loss-of-function variants in TOMM70 result in variable white matter disease and neurological phenotypes in affected individuals.

Salidroside protects dopaminergic neurons by regulating the mitochondrial MEF2D-ND6 pathway in the MPTP/MPP+ -induced model of Parkinson's disease.

Mitochondrial complex I damage and oxidative stress play critical roles in the degeneration of dopaminergic (DA) neurons during the progression of Parkinson's disease (PD). Our previous study showed that NADH dehydrogenase 6 (ND6), exclusively regulated by mitochondrial myocyte enhancer factor 2D (MEF2D), was critical for mitochondrial complex I assembly. Recently, we found that Salidroside (Sal), isolated from Rhodiola rosea L., protected DA neurons by regulating oxidative stress-related mitochondrial pathways. Here, we investigated whether the mitochondrial MEF2D-ND6 pathway was involved in the neuroprotective effects of Sal. Our results showed that in 1-methyl-4-phenylpyridinium (MPP+ )-injured SN4741 cells, Sal pretreatment improved cellular viability, inhibited apoptosis, and restored both the mitochondrial membrane potential and complex I activity. Similarly, the protective effects of Sal on mitochondrial complex I activity, DA neurons, and behavior were also confirmed in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-lesioned mice. Besides, Sal pretreatment restored the expression of mitochondrial MEF2D and ND6 in MPP+ -injured SN4741 cells and MPTP-lesioned mice. Finally and interestingly, the protective effects of Sal were not observed in cells transfected with Mt2Ddn, a specific blocker of mitochondrial MEF2D function, suggesting that Sal protects DA neurons primarily by regulating the mitochondrial MEF2D-ND6 pathway. Our study sheds light upon the protective role of Sal through targeting the mitochondrial MEF2D-ND6 pathway in regulations of mitochondrial function and DA neuronal viability, providing novel mechanistic insights into the neuroprotective effects of Sal against PD.

Conformational ensemble of an intrinsically flexible loop in mitochondrial import protein Tim21 studied by modeling and molecular dynamics simulations

BackgroundTim21, a subunit of a highly dynamic translocase of the inner mitochondrial membrane (TIM23) complex, translocates proteins by interacting with subunits in the translocase of the outer membrane (TOM) complex and Tim23 channel in the TIM23 complex. A loop segment in Tim21, which is in close proximity of the binding site of Tim23, has different conformations in X-ray, NMR and new crystal contact-free space (CCFS) structures. MD simulations can provide information on the structure and dynamics of the loop in solution. MethodsThe conformational ensemble of the loop was characterized using loop modeling and molecular dynamics (MD) simulations. ResultsMD simulations confirmed mobility of the loop. Multidimensional scaling and clustering were used to characterize the dynamic conformational ensemble of the loop. Free energy landscape showed that the CCFS crystal structure occupied a low energy region as compared to the conventional X-ray crystal structure. Analysis of crystal packing indicates that the CCFS provides larger conformational space for the motions of the loop. ConclusionsOur work reported the conformational ensemble of the loop in solution, which is in agreement with the structure obtained from CCFS approach. The combination of the experimental techniques and computational methods is beneficial for studying highly flexible regions of proteins. General significanceComputational methods, such as loop modeling and MD simulations, have proved to be useful for studying conformational flexibility of proteins. These methods in integration with experimental techniques such as CCFS has the potential to transform the studies on flexible regions of proteins.

Translocase of the Outer Mitochondrial Membrane 40 Is Required for Mitochondrial Biogenesis and Embryo Development in Arabidopsis.

In eukaryotes, mitochondrion is an essential organelle which is surrounded by a double membrane system, including the outer membrane, intermembrane space and the inner membrane. The translocase of the outer mitochondrial membrane (TOM) complex has attracted enormous interest for its role in importing the preprotein from the cytoplasm into the mitochondrion. However, little is understood about the potential biological function of the TOM complex in Arabidopsis. The aim of the present study was to investigate how AtTOM40, a gene encoding the core subunit of the TOM complex, works in Arabidopsis. As a result, we found that lack of AtTOM40 disturbed embryo development and its pattern formation after the globular embryo stage, and finally caused albino ovules and seed abortion at the ratio of a quarter in the homozygous tom40 plants. Further investigation demonstrated that AtTOM40 is wildly expressed in different tissues, especially in cotyledons primordium during Arabidopsis embryogenesis. Moreover, we confirmed that the encoded protein AtTOM40 is localized in mitochondrion, and the observation of the ultrastructure revealed that mitochondrion biogenesis was impaired in tom40-1 embryo cells. Quantitative real-time PCR was utilized to determine the expression of genes encoding outer mitochondrial membrane proteins in the homozygous tom40-1 mutant embryos, including the genes known to be involved in import, assembly and transport of mitochondrial proteins, and the results demonstrated that most of the gene expressions were abnormal. Similarly, the expression of genes relevant to embryo development and pattern formation, such as SAM (shoot apical meristem), cotyledon, vascular primordium and hypophysis, was also affected in homozygous tom40-1 mutant embryos. Taken together, we draw the conclusion that the AtTOM40 gene is essential for the normal structure of the mitochondrion, and participates in early embryo development and pattern formation through maintaining the biogenesis of mitochondria. The findings of this study may provide new insight into the biological function of the TOM40 subunit in higher plants.

Aged endothelial cells exhibit a metabolic shift from anaerobic glycolysis to oxidative phosphorylation

Endothelial dysfunction is a hallmark of vascular aging and is associated with increased arterial oxidative stress. Mitochondria, via the electron transport chain, are thought to be an important source of cellular superoxide that suppresses endothelial function in older humans but surprisingly little is understood about the effect of aging on endothelial cell metabolic activity. Young, normally functioning endothelial cells typically utilize anaerobic glycolytic metabolism but with aging a shift toward a greater reliance on oxidative metabolism could explain enhanced superoxide production via the electron transport chain. Here we aimed to determine the impact of aging on endothelial cell metabolism. To do so, we utilized SeahorseTM technology to assess mitochondrial oxygen consumption and extracellular acidification rate as measures of oxidative and anaerobic glycolytic metabolism in primary lung endothelial cells (ECs) isolated from young (4–6M) and old (16–18M) C57BL/6 mice, as well as in early (P3) and late (P16) passage human umbilical vein endothelial cells (HUVECs), a cell culture model of aging. We found that old primary ECs demonstrate a 37% higher rate of oxygen consumption (p<0.01), 74% higher spare respiration (p<0.01) and 92% higher proton leak (p<0.01) compared with young primary ECs. In contrast, ATP production was 27% lower in old compared with young primary ECs (p<0.01). Total extracellular acidification rate (glycolytic shift) was 47% (p<0.01) lower and maximal glycolytic rate was 22% lower (p<0.01) in old compared with young primary ECs, indicating reduced glycolysis and increased oxidative phosphorylation in old ECs. Next, we assessed mitochondrial membrane potential, pyruvate kinase (PK) activity, lactate dehydrogenase (LDH) activity, as well as the expression of mitochondrial membrane complexes by western blot. Interestingly, we observed 62% lower mitochondrial membrane potential (p<0.05), and reduced expression of mitochondrial membrane complex II (34%, p<0.05), III (65%, p<0.01), IV (87%, p<0.01) and V (56%, p<0.01) in old compared with young ECs. PK activity was increased by 62% (p<0.01) and LDH activity was decreased by 72% (p<0.01) in old compared with young ECs. Similar differences were observed between early and late passage HUVECs. Taken together, these findings suggest that aging results in a shift away from anaerobic glycolysis and towards a greater reliance on oxidative phosphorylation in old ECs. This is despite less efficient mitochondrial coupling and lower protein expression of mitochondrial electron transport chain proteins. Overall, these results suggest that this age related metabolic shift in ECs may contribute to increased mitochondrial superoxide production and subsequent endothelial dysfunction.Support or Funding InformationSupported by awards from the National Institute of Health (R01 AG040297, R01 AG048366, K02 AG045339) and US Department of Veterans Affairs (1I01BX002151).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Wild-type and mutant (G2019S) leucine-rich repeat kinase 2 (LRRK2) associate with subunits of the translocase of outer mitochondrial membrane (TOM) complex

Leucine-rich repeat kinase 2 (LRRK2) is important in various cellular processes including mitochondrial homeostasis and mutations in this gene lead to Parkinson's disease (PD). However, the full spectrum of LRRK2's functions remain to be elucidated. The translocase of outer mitochondrial membrane (TOM) complex is essential for the import of almost all nuclear-encoded mitochondrial proteins and is fundamental for cellular survival. Using co-immunoprecipitation, super-resolution structured illumination microscopy (SR-SIM), and 3D virtual reality (VR) assisted co-localization analysis techniques we show that wild-type and mutant (G2019S) LRRK2 associate and co-localize with subunits of the TOM complex, either under basal (dimethyl sulfoxide, DMSO) or stress-induced (carbonyl cyanide m-chlorophenyl hydrazine, CCCP) conditions. Interestingly, LRRK2 interacted with TOM40 under both DMSO and CCCP conditions, and when the PD causing mutation, G2019S was introduced, the association was not altered. Moreover, overexpression of G2019S LRRK2 resulted in the formation of large, perinuclear aggregates that co-localized with the TOM complex. Taken together, this is the first study to show that both WT and mutant LRRK2 associate with the TOM complex subunits. These findings provide additional evidence for LRRK2's role in mitochondrial function which has important implications for its role in PD pathogenesis.

TOM7 silencing exacerbates focal cerebral ischemia injury in rat by targeting PINK1/Beclin1-mediated autophagy

Activated autophagy has been intensively observed in cerebrovascular diseases, including focal cerebral ischemia injury, but its molecular mechanisms remain unclear. TOM7, which is a component of the protein translocase of the outer mitochondrial membrane (TOM) complex, may modulate assembly of the TOM complex. However, an understanding of how TOM7 affects cerebral ischemia injury is limited. In this study, we demonstrate that the expression of TOM7 is up-regulated after a photothrombotic cerebral ischemic model in rats, peaking at 3 days. In addition, TOM7 knockdown may aggravate cerebral ischemic injury and inhibit autophagy after ischemic stroke. Mechanically, TOM7 may regulate autophagy through the PINK1/Beclin1 pathway after cerebral ischemia injury. These results demonstrate that TOM7 silencing may aggravate cerebral ischemia injury through inhibiting PINK1/Beclin1 pathway- mediated autophagy.

Abstract 419: Deep Sequencing Provides Evidence Of Mitochondrial Abnormalities In Right Ventricular Failure In Pulmonary Arterial Hypertension

Introduction: Right ventricular (RV) function is a critical predictor of both morbidity and mortality in pulmonary arterial hypertension (PAH). In PAH patients present either with adaptive remodelling RV and compensatory RV hypertrophy (RVH), or maladaptive remodeling that rapidly deteriorates to RV failure (RVF). While the molecular mechanisms underlying RVF remain poorly understood, acquired mitochondrial abnormalities including impaired mitochondrial electron transport chain activity, altered metabolic substrate utilization, and aberrant mitochondrial dynamics likely play a crucial role. We assessed the role of mitochondria in RVF using an unbiased assessment of gene expression in the RV of rats with maladaptive vs adaptive RVH and PAH. Method and result: Male rats were treated with either sugen+hypoxia (SuHx) or Monocrotaline (MCT). We determined RV status using non-invasive echocardiography and right heart catheterisation. Compared to control (Ctrl; n=15) both MCT (n=8) and SuHx (n=5) rats displayed severe PAH with increased RVSP, mPAP, total pulmonary resistance (TPR) and decreased PA acceleration time (PAAT) (p<0.05). MCT rats developed RVF characterized by increased RV end diastolic volume (EDV), decreased CO, TAPSE and ventricular-arterial decoupling (decreased Emax/Ea ratio) (p<0.05). SuHx rats had adaptive RVH. RNAsequencing revealed 2,547 differentially regulated transcripts. Functional Annotation analysis revealed multiple enriched GOterms, the most significant of which described mitochondrion (GO: 0005739; p=1.6E-53 which describes 414 genes). Significant GOterms also include mitochondrial inner membrane (GO: 0005743; p=3.9E-20) and mitochondrial respiratory chain complex I (GO: 0005747 ; p=4.9E-10). We validated 15 key dysregulated mitochondrial genes in maladaptive RVH (MCT; n=7) compared to both adaptive RVH (SuHx; n=4) and Ctrl (n=12) by qPCR, Finally, we validated these findings in human samples from PAH patients with RVH compared to controls (n=5 per group; p<0.05). Conclusion: We combined haemodynamic investigation and transcriptomics to identify specific mitochondrial-metabolic gene expression signatures associated with RVF in two different preclinical models and in human samples.

Mitochondrial protein import regulates cytosolic protein homeostasis and neuronal integrity

ABSTRACTNeurodegeneration is characterized by protein aggregate deposits and mitochondrial malfunction. Reduction in Tom40 (translocase of outer membrane 40) expression, a key subunit of the translocase of the outer mitochondrial membrane complex, led to accumulation of ubiquitin (Ub)-positive protein aggregates engulfed by Atg8a-positive membranes. Other macroautophagy markers were also abnormally accumulated. Autophagy was induced but the majority of autophagosomes failed to fuse with lysosomes when Tom40 was downregulated. In Tom40 RNAi tissues, autophagosome-like (AL) structures, often not sealed, were 10 times larger than starvation induced autophagosomes. Atg5 downregulation abolished Tom40 RNAi induced AL structure formation, but the Ub-positive aggregates remained, whereas knock down of Syx17, a gene required for autophagosome-lysosome fusion, led to the disappearance of giant AL structures and accumulation of small autophagosomes and phagophores near the Ub-positive aggregates. The protein aggregates contained many mitochondrial preproteins, cytosolic proteins, and proteasome subunits. Proteasome activity and ATP levels were reduced and the ROS levels was increased in Tom40 RNAi tissues. The simultaneous inhibition of proteasome activity, reduction in ATP production, and increase in ROS, but none of these conditions alone, can mimic the imbalanced proteostasis phenotypes observed in Tom40 RNAi cells. Knockdown of ref(2)P or ectopic expression of Pink1 and park greatly reduced aggregate formation in Tom40 RNAi tissues. In nerve tissues, reduction in Tom40 activity leads to aggregate formation and neurodegeneration. Rather than diminishing the neurodegenerative phenotypes, overexpression of Pink1 enhanced them. We proposed that defects in mitochondrial protein import may be the key to linking imbalanced proteostasis and mitochondrial defects.Abbreviations: AL: autophagosome-like; Atg12: Autophagy-related 12; Atg14: Autophagy-related 14; Atg16: Autophagy-related 16; Atg5: Autophagy-related 5; Atg6: Autophagy-related 6; Atg8a: Autophagy-related 8a; Atg9: Autophagy-related 9; ATP: adenosine triphosphate; Cas9: CRISPR associated protein 9; cDNA: complementary DNA; COX4: Cytochrome c oxidase subunit 4; CRISPR: clustered regularly interspaced short palindromic repeats; Cyt-c1: Cytochrome c1; DAPI: 4,6-diamidino-2-phenylindole dihydrochloride; Dcr-2: Dicer-2; FLP: Flippase recombination enzyme; FRT: FLP recombination target; GFP: green fluorescent protein; GO: gene ontology; gRNA: guide RNA; Hsp60: Heat shock protein 60A; HDAC6: Histone deacetylase 6; htt: huntingtin; Idh: Isocitrate dehydrogenase; IFA: immunofluorescence assay; Irp-1A: Iron regulatory protein 1A; kdn: knockdown; Marf: Mitochondrial assembly regulatory factor; MitoGFP: Mitochondrial-GFP; MS: mass spectrometry; MTPAP: mitochondrial poly(A) polymerase; Nmnat: Nicotinamide mononucleotide adenylyltransferase; OE: overexpression; Pink1/PINK1: PTEN-induced putative kinase 1; polyQ: polyglutamine; PRKN: parkin RBR E3 ubiquitin protein ligase; Prosα4: proteasome α4 subunit; Prosβ1: proteasome β1 subunit; Prosβ5: proteasome β5 subunit; Prosβ7: proteasome β7 subunit; ref(2)P: refractory to sigma P; RFP: red fluorescent protein; RNAi: RNA interference; ROS: reactive oxygen species; Rpn11: Regulatory particle non-ATPase 11; Rpt2: Regulatory particle triple-A ATPase 2; scu: scully; sicily: severe impairment of CI with lengthened youth; sesB: stress-sensitive B; Syx17: Syntaxin17; TEM: transmission electron microscopy; ttm50: tiny tim 50; Tom: translocase of the outer membrane; Tom20: translocase of outer membrane 20; Tom40: translocase of outer membrane 40; Tom70: translocase of outer membrane 70; UAS: upstream active sequence; Ub: ubiquitin; VNC: ventral nerve cord; ZFYVE1: zinc finger FYVE-type containing 1

17β-estradiol protects INS-1 insulinoma cells from mitophagy via G protein-coupled estrogen receptors and the PI3K/Akt signaling pathway.

17β-estradiol (17β-E2) is a steroid hormone that is known to exert effects on blood glucose homeostasis. The Gprotein‑coupled estrogen receptor (GPER) has been identified as a non-genomic estrogenic receptor, and is involved in numerous physiological processes, including cell survival, energy provision and metabolism. 17β-E2 may decrease apoptosis by binding to the GPER. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is involved in physiological and pathological functions such as autophagy. The purpose of the present study was to investigate the role of the PI3K/Akt signaling pathway in the mediation of the effects of GPERs, and the effects of 17β-E2 on mitophagy in INS-1 cells, a rat insulin‑secreting β-cell line. Invitro, INS-1 cells were treated with different concentrations of 17β-E2 with and without pretreatment with a GPER antagonist (G15) or PI3K antagonist (LY294002) and compared with a negative control. An immunofluorescence assay demonstrated that GPERs are expressed in INS-1 cells. Western blot assays demonstrated that 17β-E2 increased GPER levels and the phosphorylation of Akt. Transmission electronic microscopy revealed that 17β-E2 reduced the formation of mitophagosomes and autophagosomes in INS-1 cells. An immunofluorescence staining assay indicated that the co-localization of translocase of mitochondrial outer membrane complex20(TOM20) with lysosomal-associated membrane protein2 (LAMP2) was decreased in INS-1 cells treated with 17β-E2 alone. Western blotting demonstrated that 17β-E2 reduced the protein levels of activated microtubule-associated protein-1 light chain3, and increased those of TOM20 and mitochondrial heat-shock protein60. Notably, the protective effects of 17β-E2 were significantly diminished by G15 or LY294002. In conclusion, the present study suggests that 17β-E2 activates the PI3K/Akt pathway via the GPER in INS-1 cells. Furthermore, 17β-E2 may be involved in mitophagy by the regulating the GPER/PI3K/Akt pathway.

Expression of the human TIMM23 and TIMM23B genes is regulated by the GABP transcription factor

The TIM23 protein is a key component of the mitochondrial import machinery in yeast and mammals. TIM23 is the channel-forming subunit of the translocase of the inner mitochondrial membrane (TIM23) complex, which mediates preprotein translocation across the mitochondrial inner membrane. In this paper, we aimed to characterize the promoter region of the highly similar human TIM23 orthologs: TIMM23 and TIMM23B. Bioinformatic analysis revealed putative sites for the GA-binding protein (GABP) and the recombination signal binding protein for immunoglobulin kappa J (RBPJ) transcription factors in both promoters. Luciferase reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation experiments showed three functional sites for GABP and one functional site for RBPJ in both promoters. Moreover, silencing of GABPA, the gene encoding the DNA-binding subunit of the GABP transcription factor, resulted in reduced expression of TIMM23 and TIMM23B. Our results show an essential role of GABP in activating TIMM23 expression. More broadly, they suggest that physiological signals involved in activating mitochondrial biogenesis and oxidative function also enhance the transcription but not the protein level of TIMM23, which is essential for maintaining mitochondrial function and homeostasis.

RhoA inhibits the hypoxia-induced apoptosis and mitochondrial dysfunction in chondrocytes via positively regulating the CREB phosphorylation.

Chondrocytes that are embedded within the growth plate or the intervertebral disc are sensitive to environmental stresses, such as inflammation and hypoxia. However, little is known about the molecular signalling pathways underlining the hypoxia-induced mitochondrial dysfunction and apoptosis in chondrocytes. In the present study, we firstly examined the hypoxia-induced apoptosis, mitochondrial dysfunction and the activation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) signalling in human chondrocyte cell line, C28/I2 and then investigated the regulatory role of RhoA, a well-recognized apoptosis suppressor, in such process, with gain-of-function strategy. Our results indicated that hypoxia induced apoptosis and inhibited CREB phosphprylation in chondrocytes, meanwhile, the dysfunctional mitochondria with up-regulated mitochondrial superoxide and reactive oxygen species (ROS) levels, whereas with a reduced mitochondrial membrane potential (MMP) and Complex IV activity were observed in the hypoxia-treated C28/I2 cells. However, the overexpressed RhoA blocked the hypoxia-mediated reduction in CREB phosphprylation and inhibited the apoptosis induction, along with an ameliorated mitochondrial function in the hypoxia-treated C28/I2 cells. In conclusion, the present study confirmed the reduced CREB phosphorylation, along with the apoptosis induction and mitochondrial dysfunction in the hypoxia-treated chondrocyte cells. And the overexpression of RhoA ameliorated the hypoxia-induced mitochondrial dysfunction and apoptosis via blocking the hypoxia-mediated reduction in CREB phosphorylation.

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis.

We have found that granzyme B (GB)-induced apoptosis also requires reactive oxygen species resulting from the alteration of mitochondrial complex I. How GB, which does not possess a mitochondrial targeting sequence, enter this organelle is unknown. We show that GB enters the mitochondria independently of the translocase of the outer mitochondrial membrane complex, but requires instead Sam50, the central subunit of the sorting and assembly machinery that integrates outer membrane β-barrel proteins. Moreover, GB breaches the inner membrane through Tim22, the metabolite carrier translocase pore, in a mitochondrial heat-shock protein 70 (mtHsp70)-dependent manner. Granzyme A (GA) and caspase-3 use a similar route to the mitochondria. Finally, preventing GB from entering the mitochondria either by mutating lysine 243 and arginine 244 or depleting Sam50 renders cells more resistant to GB-mediated reactive oxygen species and cell death. Similarly, Sam50 depletion protects cells from GA-, GM- and caspase-3-mediated cell death. Therefore, cytotoxic molecules enter the mitochondria to induce efficiently cell death through a noncanonical Sam50-, Tim22- and mtHsp70-dependent import pathway.

Melatonin attenuates postmyocardial infarction injury via increasing Tom70 expression

Mitochondrial dysfunction leads to reactive oxygen species (ROS) overload, exacerbating injury in myocardial infarction (MI). As a receptor for translocases in the outer mitochondrial membrane (Tom) complex, Tom70 has an unknown function in MI, including melatonin-induced protection against MI injury. We delivered specific small interfering RNAs against Tom70 or lentivirus vectors carrying Tom70a sequences into the left ventricles of mice or to cultured neonatal murine ventricular myocytes (NMVMs). At 48h post-transfection, the left anterior descending coronary arteries of mice were permanently ligated, while the NMVMs underwent continuous hypoxia. At 24h after ischemia/hypoxia, oxidative stress was assessed by dihydroethidium and lucigenin-enhanced luminescence, mitochondrial damage by transmission electron microscopy and ATP content, and cell apoptosis by terminal deoxynucleotidyl transferase dUTP nick-end labeling and caspase-3 assay. At 4weeks after ischemia, cardiac function and fibrosis were evaluated in mice by echocardiography and Masson's trichrome staining, respectively. Ischemic/hypoxic insult reduced Tom70 expression in cardiomyocytes. Tom70 downregulation aggravated post-MI injury, with increased mitochondrial fragmentation and ROS overload. In contrast, Tom70 upregulation alleviated post-MI injury, with improved mitochondrial integrity and decreased ROS production. PGC-1α/Tom70 expression in ischemic myocardium was increased with melatonin alone, but not when combined with luzindole. Melatonin attenuated post-MI injury in control but not in Tom70-deficient mice. N-acetylcysteine (NAC) reversed the adverse effects of Tom70 deficiency in mitochondria and cardiomyocytes, but at a much higher concentration than melatonin. Our findings showed that Tom70 is essential for melatonin-induced protection against post-MI injury, by breaking the cycle of mitochondrial impairment and ROS generation.

Reduction of apoptosis and preservation of mitochondrial integrity under ischemia/reperfusion injury is mediated by estrogen receptor β

BackgroundEstrogen improves cardiac recovery after ischemia/reperfusion (I/R) by yet incompletely understood mechanisms. Mitochondria play a crucial role in I/R injury through cytochrome c-dependent apoptosis activation. We tested the hypothesis that 17β-estradiol (E2) as well as a specific ERβ agonist improve cardiac recovery through estrogen receptor (ER)β-mediated mechanisms by reducing mitochondria-induced apoptosis and preserving mitochondrial integrity.MethodsWe randomized ovariectomized C57BL/6N mice 24h before I/R to pre-treatment with E2 or a specific ERβ agonist (ERβA). Isolated hearts were perfused for 20min prior to 30min global ischemia followed by 40min reperfusion.ResultsCompared with controls, ERβA and E2 treated groups showed a significant improvement in cardiac recovery, i.e. an increase in left ventricular developed pressure, dP/dtmax and dP/dtmin. ERβA and E2 pre-treatment led to a significant reduction in apoptosis with decreased cytochrome c release from the mitochondria and increased mitochondrial levels of anti-apoptotic Bcl2 and ACAA2. Protein levels of mitochondrial translocase inner membrane (TIM23) and mitochondrial complex I of respiratory chain were increased by ERβA and E2 pre-treatment. Furthermore, we found a significant increase of myosin light chain 2 (MLC2) phosphorylation together with ERK1/2 activation in E2, but not in ERβA treated groups.ConclusionsActivation of ERβ is essential for the improvement of cardiac recovery after I/R through the inhibition of apoptosis and preservation of mitochondrial integrity and can be a achieved by a specific ERβ agonist. Furthermore, E2 modulates MLC2 activation after I/R independent of ERβ.Electronic supplementary materialThe online version of this article (doi:10.1186/s13293-016-0104-8) contains supplementary material, which is available to authorized users.

The Import of Proteins into the Mitochondrion of Toxoplasma gondii

Outside of well characterized model eukaryotes, relatively little is known about the translocons that transport proteins across the two membranes that surround the mitochondrion. Apicomplexans are a phylum of intracellular parasites that cause major diseases in humans and animals and are evolutionarily distant from model eukaryotes such as yeast. Apicomplexans harbor a mitochondrion that is essential for parasite survival and is a validated drug target. Here, we demonstrate that the apicomplexan Toxoplasma gondii harbors homologues of proteins from all the major mitochondrial protein translocons present in yeast, suggesting these arose early in eukaryotic evolution. We demonstrate that a T. gondii homologue of Tom22 (TgTom22), a central component of the translocon of the outer mitochondrial membrane (TOM) complex, is essential for parasite survival, mitochondrial protein import, and assembly of the TOM complex. We also identify and characterize a T. gondii homologue of Tom7 (TgTom7) that is important for parasite survival and mitochondrial protein import. Contrary to the role of Tom7 in yeast, TgTom7 is important for TOM complex stability, suggesting the role of this protein has diverged during eukaryotic evolution. Together, our study identifies conserved and modified features of mitochondrial protein import in apicomplexan parasites.

CHANGES OF ULTRASTRUCTURAL ORGANIZATION OF CELLS OF ESOPHAGEAL MUCOSA OF RATS WITH THE THIRD DEGREE ESOPHAGEAL STRICTURE IN EXPERIMENT

Electronic microscopy examination of the submicroscopic organization of epithelial cells of multilayered epithelium, smooth myocytes of a muscular plate and contractile elements of the muscle layer of the esophagus in rats with the third degree esophageal stricture in experiment was conducted. The focal lysis of nuclear membrane, mitochondrial membrane, endoplasmic reticulum and lamellar cytoplasmic Golgi complex was revealed. Catabolic processes develop in esophageal cells with the third-degree stricture and lead to the disintegration of organelles.

Ceramide-tamoxifen regimen targets bioenergetic elements in acute myelogenous leukemia

The objective of our study was to determine the mechanism of action of the short-chain ceramide analog, C6-ceramide, and the breast cancer drug, tamoxifen, which we show coactively depress viability and induce apoptosis in human acute myelogenous leukemia cells. Exposure to the C6-ceramide-tamoxifen combination elicited decreases in mitochondrial membrane potential and complex I respiration, increases in reactive oxygen species (ROS), and release of mitochondrial proapoptotic proteins. Decreases in ATP levels, reduced glycolytic capacity, and reduced expression of inhibitors of apoptosis proteins also resulted. Cytotoxicity of the drug combination was mitigated by exposure to antioxidant. Cells metabolized C6-ceramide by glycosylation and hydrolysis, the latter leading to increases in long-chain ceramides. Tamoxifen potently blocked glycosylation of C6-ceramide and long-chain ceramides. N-desmethyltamoxifen, a poor antiestrogen and the major tamoxifen metabolite in humans, was also effective with C6-ceramide, indicating that traditional antiestrogen pathways are not involved in cellular responses. We conclude that cell death is driven by mitochondrial targeting and ROS generation and that tamoxifen enhances the ceramide effect by blocking its metabolism. As depletion of ATP and targeting the "Warburg effect" represent dynamic metabolic insult, this ceramide-containing combination may be of utility in the treatment of leukemia and other cancers.

Alterations of outer mitochondrial membrane translocases in Alzheimer's disease

Event Abstract Back to Event Alterations of outer mitochondrial membrane translocases in Alzheimer's disease Yuek Ling Chai1, Huayang Xing1, Paul T. Francis2, Clive G. Ballard2, Christopher P. Chen1 and Mitchell K. Lai1* 1 National University of Singapore, Department of Pharmacology, Yong Loo Lin School of Medicine, Singapore 2 King's College London, Wolfson Centre for Age-Related Diseases, United Kingdom The translocases of the outer mitochondrial outer membrane (Tom) complex is an important transport system facilitating the import of almost all mitochondrial proteins into the mitochondria. While mitochondrial dysfunction is evident in Alzheimer’s disease (AD), it remains unclear whether the Tom complex is affected and involved in the pathogenesis of AD. Thus, we aim to quantify the expression level of various Tom subunits in the post mortem brains of AD patients. Brodmann area 40 from post mortem brains of AD patients (n=12) and non-demented age-matched controls (n=19) were processed and subjected to subcellular fractionation. Immunoblotting was performed using the mitochondrial fraction with Tom40, Tom22, Tom20 and Tom70 antibodies. The expression levels of the surface receptor subunits Tom20 (p<0.001) and Tom70 (p=0.016) were significantly reduced in AD as compared to controls. Meanwhile, there was a non-significant trend towards a decrease of Tom40 (p=0.080) and Tom22 (p=0.325) subunits in AD. Reduction in Tom subunits suggests impairment of the mitochondrial transportation system, which may then lead to mitochondrial dysfunction and subsequent oxidative stress in AD. Keywords: Brain, neurodegeneration, Alzheimer’s disease, Transport system, Mitochondrial membrane Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Chai Y, Xing H, Francis PT, Ballard CG, Chen CP and Lai MK (2016). Alterations of outer mitochondrial membrane translocases in Alzheimer's disease. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00174 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Mitchell K Lai, National University of Singapore, Department of Pharmacology, Yong Loo Lin School of Medicine, Singapore, Singapore, mitchell.lai@dementia-research.org Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Yuek Ling Chai Huayang Xing Paul T Francis Clive G Ballard Christopher P Chen Mitchell K Lai Google Yuek Ling Chai Huayang Xing Paul T Francis Clive G Ballard Christopher P Chen Mitchell K Lai Google Scholar Yuek Ling Chai Huayang Xing Paul T Francis Clive G Ballard Christopher P Chen Mitchell K Lai PubMed Yuek Ling Chai Huayang Xing Paul T Francis Clive G Ballard Christopher P Chen Mitchell K Lai Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Hypoxia-induced apoptosis and mitochondrial dysfunction in chondrocytes arising from CREB phosphorylation reduction.

Chondrocytes, which are embedded within the growth-plate or the intervertebral disc, are sensitive to environmental stresses, such as inflammation and hypoxia. However, little is known about the molecular signaling pathways underlying hypoxia-induced mitochondrial dysfunction and apoptosis in chondrocytes. We first examined the apoptosis, caspase-3 activity, and apoptosis-associated markers in human chondrocyte cell line C28/I2 under normoxia or hypoxia. We then investigated mitochondrial dysfunction and the activation of cyclic adenosine monophosphate response element-binding protein (CREB) signaling in the same human chondrocyte cell line. Our results indicated that hypoxia induced apoptosis and reduced CREB phosphorylation in chondrocytes. Upregulated mitochondrial superoxide and reactive oxygen species levels, and reduced mitochondrial membrane potential and complex IV activity were observed in hypoxia-treated C28/I2 cells. In conclusion, the present study confirmed reduced CREB phosphorylation, apoptosis induction, and mitochondrial dysfunction in the hypoxia-treated chondrocyte cells. This implies the key role played by CREB signaling in hypoxia-induced mitochondrial dysfunction and apoptosis in chondrocytes.