SOD1 Overexpression Research Articles

Decreased glutathione levels cause overt motor neuron degeneration in hSOD1WT over-expressing mice

Mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. Several lines of evidence have shown that SOD1 mutations cause ALS through a gain of a toxic function that remains to be fully characterized. A significant share of our understanding of the mechanisms underlying the neurodegenerative process in ALS comes from the study of rodents over-expressing ALS-linked mutant hSOD1. These mutant hSOD1 models develop an ALS-like phenotype. On the other hand, hemizygous mice over-expressing wild-type hSOD1 at moderate levels (hSOD1WT, originally described as line N1029) do not develop paralysis or shortened life-span. To investigate if a decrease in antioxidant defenses could lead to the development of an ALS-like phenotype in hSOD1WT mice, we used knockout mice for the glutamate-cysteine ligase modifier subunit [GCLM(−/−)]. GCLM(−/−) mice are viable and fertile but display a 70–80% reduction in total glutathione levels. GCLM(−/−)/hSOD1WT mice developed overt motor symptoms (e.g. tremor, loss of extension reflex in hind-limbs, decreased grip strength and paralysis) characteristic of mice models over-expressing ALS-linked mutant hSOD1. In addition, GCLM(−/−)/hSOD1WT animals displayed shortened life span. An accelerated decrease in the number of large neurons in the ventral horn of the spinal cord and degeneration of spinal root axons was observed in symptomatic GCLM(−/−)/hSOD1WT mice when compared to age-matched GCLM(+/+)/hSOD1WT mice. Our results show that under conditions of chronic decrease in glutathione, moderate over-expression of wild-type SOD1 leads to overt motor neuron degeneration, which is similar to that induced by ALS-linked mutant hSOD1 over-expression.

Yeast polyubiquitin gene UBI4 deficiency leads to early induction of apoptosis and shortened replicative lifespan.

Ubiquitin is a 76-amino acid protein that is highly conserved among higher and lower eukaryotes. The polyubiquitin gene UBI4 encodes a unique precursor protein that contains five ubiquitin repeats organized in a head-to-tail arrangement. Although the involvement of the yeast polyubiquitin gene UBI4 in the stress response was reported long ago, there are no reports regarding the underlying mechanism of this involvement. In this study, we used UBI4-deletion and UBI4-overexpressing yeast strains as models to explore the potential mechanism by which UBI4 protects yeast cells against paraquat-induced oxidative stress. Here, we show that ubi4Δ cells exhibit oxidative stress, an apoptotic phenotype, and a decreased replicative lifespan. Additionally, the reduced resistance of ubi4Δ cells to paraquat that was observed in this study was rescued by overexpression of either the catalase or the mitochondrial superoxide dismutase SOD2. We also demonstrated that only SOD2 overexpression restored the replicative lifespan of ubi4Δ cells. In contrast to the case of ubi4Δ cells, UBI4 overexpression in wild-type yeast increases the yeast's resistance to paraquat, and this overexpression is associated with large pools of expressed ubiquitin and increased levels of ubiquitinated proteins. Collectively, these findings highlight the role of the polyubiquitin gene UBI4 in apoptosis and implicate UBI4 as a modulator of the replicative lifespan.

106 - DNMT3a Conditional Deletion in Neuro-epithelium Restores Maternal Diabetes-suppressed Neural Tube Closure Essential Genes Expression and Blocks Maternal Diabetes-activated ER Stress

Global DNA hypermethylation is induced in embryonic neuroepithelium by maternal diabetes, leading to neural tube defects (NTDs). However the underlying mechanism is not clear. Our previous study has demonstrated that the green tea polyphenal Epigallocatechin gallate (EGCG) inhibited DNA hypermethylation and ameliorated maternal diabetes-induced NTDs. To determine whether antioxidant enzymes can suppress DNA hypermethylation in diabetic embryos, SOD1 transgenic male mice are breed with diabetic wild type female mice. SOD1 overexpression reduces maternal diabetes-induced DNA hypermethylation leading to suppression of DNMT3a and DNMT3b, but not DNMT1. To further investigate whether maternal diabetes-increased DNA methyltransferase 3a (DNMT3a) is involved in the pathogenesis of diabetic embryopathy, the Dnmt3a gene was deleted using the Cre-Flox (f) approach by crossing DNMT3af/+ ;nestin-Cre male mice with DNMT3af/f female mice. Dnmt3a deletion specifically occurred in the developing neuroepithelium, and DNMT1 and DNMT3b expression were not affected. Conditional deletion of Dnmt3a significantly reduces the NTD rate from 27.2% in wild-type embryos to 4% in homozygous deletion embryos (DNMT3af/f ;nestin-Cre) under maternal diabetic conditions and ameliorates maternal diabetes-induced DNA hyper-methylation. Moreover, Dnmt3a deficiency in the neuroepithelium abrogated maternal diabetes-induced hyper-methylation in the promoters region of neural tube closure essential genes including Pax3, Tulp3 and GRHL3, leading to activation of these genes. Furthermore maternal diabetes-induced ER stress in neuroepithelium is blocked by conditional deletion of Dnmt3a, which suppresses neuroepithelium apoptosis. Taken together, our findings demonstrate that Dnmt3a conditional deletion in neuroepithelium restores maternal diabetes-suppressed neural tube closure essential genes expression and blocks maternal diabetes-activated ER stress.

Combination of betulinic acid and chidamide inhibits acute myeloid leukemia by suppression of the HIF1α pathway and generation of reactive oxygen species.

Acute myeloid leukemia (AML) is a heterogeneous disorder of the hematopoietic system with no common genetic “Achilles heel” that can be targeted. Most patients respond well to standard therapy, while a majority relapse, and development of an effective therapy for AML patients is still urgently needed. In this study, we demonstrated that betulinic acid (BA) significantly increased Aryl hydrocarbon receptor (AHR) expression through demethylation on the AHR promoter in AML cells, and the increased AHR expression interacts with and sequesters ARNT, subsequently suppressing hypoxia-inducible factor-1α (HIF1α) pathway. We also found that histone deacetylase inhibitor chidamide (CDM) treatment significantly increased p300 over-acetylation in AML cells with dissociation of p300 with HIF1α, and subsequently suppressed the HIF1α pathway. Further investigation showed that BA/CDM combination additively increased generation of reactive oxygen species (ROS) with DNA damage, apoptosis and mitochondrial dysfunction. Also, BA/CDM combination additively suppressed the HIF1α pathway with decreased VEGF expression. in vivo mice study showed that BA/CDM combination significantly suppressed AML tumor growth, and overexpression of SOD2 and a constitutive HIF1α (HIF1C) completely diminished this effect. We conclude that a BA/CDM combination inhibits AML tumors through ROS over-generation and HIF1α pathway suppression. This is the first time we have shown the potential effect and possible mechanism of BA and CDM on the inhibition of AML tumor growth.

FUS Mutant Human Motoneurons Display Altered Transcriptome and microRNA Pathways with Implications for ALS Pathogenesis.

SummaryThe FUS gene has been linked to amyotrophic lateral sclerosis (ALS). FUS is a ubiquitous RNA-binding protein, and the mechanisms leading to selective motoneuron loss downstream of ALS-linked mutations are largely unknown. We report the transcriptome analysis of human purified motoneurons, obtained from FUS wild-type or mutant isogenic induced pluripotent stem cells (iPSCs). Gene ontology analysis of differentially expressed genes identified significant enrichment of pathways previously associated to sporadic ALS and other neurological diseases. Several microRNAs (miRNAs) were also deregulated in FUS mutant motoneurons, including miR-375, involved in motoneuron survival. We report that relevant targets of miR-375, including the neural RNA-binding protein ELAVL4 and apoptotic factors, are aberrantly increased in FUS mutant motoneurons. Characterization of transcriptome changes in the cell type primarily affected by the disease contributes to the definition of the pathogenic mechanisms of FUS-linked ALS.

SIRT3/SOD2 maintains osteoblast differentiation and bone formation by regulating mitochondrial stress

Recent studies have revealed robust metabolic changes during cell differentiation. Mitochondria, the organelles where many vital metabolic reactions occur, may play an important role. Here, we report the involvement of SIRT3-regulated mitochondrial stress in osteoblast differentiation and bone formation. In both the osteoblast cell line MC3T3-E1 and primary calvarial osteoblasts, robust mitochondrial biogenesis and supercomplex formation were observed during differentiation, accompanied by increased ATP production and decreased mitochondrial stress. Inhibition of mitochondrial activity or an increase in mitochondrial superoxide production significantly suppressed osteoblast differentiation. During differentiation, SOD2 was specifically induced to eliminate excess mitochondrial superoxide and protein oxidation, whereas SIRT3 expression was increased to enhance SOD2 activity through deacetylation of K68. Both SOD2 and SIRT3 knockdown resulted in suppression of differentiation. Meanwhile, mice deficient in SIRT3 exhibited obvious osteopenia accompanied by osteoblast dysfunction, whereas overexpression of SOD2 or SIRT3 improved the differentiation capability of primary osteoblasts derived from SIRT3-deficient mice. These results suggest that SIRT3/SOD2 is required for regulating mitochondrial stress and plays a vital role in osteoblast differentiation and bone formation.

Combination of betulinic acid and chidamide synergistically inhibits Epstein-Barr virus replication through over-generation of reactive oxygen species.

Epstein-Barr virus (EBV) has widely infected more than 90% of human populations. Currently, there is no efficient way to remove the virus because the EBV carriers are usually in a latent stage that allows them to escape the immune system and common antiviral drugs. In the effort to develop an efficient strategy for the removal of the EBV virus, we have shown that betulinic acid (BA) slightly suppresses EBV replication through SOD2 suppression with subsequent reactive oxygen species (ROS) generation and DNA damage in EBV-transformed LCL (lymphoblastoid cell line) cells. Chidamide (CDM, CS055), a novel histone deacetylase inhibitor (HDACi), could significantly switch EBV from the latent stage to the lytic stage with increased gene expression of BZLF1 and BMRF1, but has a small effect on EBV replication due to the suppression effect of CDM-mediated ROS generation. Interestingly, a combination of BA and CDM synergistically inhibits EBV replication with ROS over-generation and subsequent DNA damage and apoptosis. Overexpression of SOD2 diminishes this effect, while SOD2 knockdown mimics this effect. An in vivo xenograft tumor development study with the tail vein injection of EBV-transformed LCL cells in nude mice proves that the combination of BA and CDM synergistically increases superoxide anion release in tumor tissues and suppresses EBV replication and tumor growth, and significantly prolongs mouse survival. We conclude that the combination of BA and CDM could be an efficient strategy for clinical EBV removal.

P 142 - TRAMP mice overexpressing SOD2 develop poorly differentiated neuroendocrine tumors and lower survival

In addition of being a primary defense against O 2 •– SOD2, the mitochondrial antioxidant enzyme was claimed for years to have a tumor suppressor role. However, in the last few years numerous reports indicate that, rather on the contrary this enzyme is associated with poor patient outcome and a more aggressive disease in a wide variety of tumors. We used murine model C57BL/6-Tg (TRAMP) 8247Ng/J as well as Sod2+/- knockdown and Sod2+/++ overexpressing mice to generate 2 additional phenotypes TRAMP/Sod2+/- and TRAMP/Sod2+/++ to study the influence of systemic modification of expression levels of SOD2 in prostate cancer. We studied the tumor progression at different ages analyzing genitourinary system weight, tumor diagnosis, androgen receptor (AR), BCL-2, matrix metalloproteinases (MMPs), VEGF and redox enzymes levels. We found that an overexpression of SOD2 not only brought a more malignant tumor diagnosis (mostly poorly differentiated) but also compromised the survival of these mice. Moreover, castration at 12 weeks of age of the three phenotypes showed that TRAMP/Sod2+/++ mice had a relapse rate dramatically higher. These mice mostly developed poorly differentiated neuroendocrine tumors with multiple metastasis, and its survival rate was very low.

P62/SQSTM1 Cooperates with Hyperactive mTORC1 to Regulate Glutathione Production, Maintain Mitochondrial Integrity, and Promote Tumorigenesis.

p62/sequestosome-1 (SQSTM1) is a multifunctional adaptor protein and autophagic substrate that accumulates in cells with hyperactive mTORC1, such as kidney cells with mutations in the tumor suppressor genes tuberous sclerosis complex (TSC)1 or TSC2. Here we report that p62 is a critical mediator of TSC2-driven tumorigenesis, as Tsc2+/- and Tsc2f/f Ksp-CreERT2+ mice crossed to p62-/- mice were protected from renal tumor development. Metabolic profiling revealed that depletion of p62 in Tsc2-null cells decreased intracellular glutamine, glutamate, and glutathione (GSH). p62 positively regulated the glutamine transporter Slc1a5 and increased glutamine uptake in Tsc2-null cells. We also observed p62-dependent changes in Gcl, Gsr, Nqo1, and Srxn1, which were decreased by p62 attenuation and implicated in GSH production and utilization. p62 attenuation altered mitochondrial morphology, reduced mitochondrial membrane polarization and maximal respiration, and increased mitochondrial reactive oxygen species and mitophagy marker PINK1. These mitochondrial phenotypes were rescued by addition of exogenous GSH and overexpression of Sod2, which suppressed indices of mitochondrial damage and promoted growth of Tsc2-null cells. Finally, p62 depletion sensitized Tsc2-null cells to both oxidative stress and direct inhibition of GSH biosynthesis by buthionine sulfoximine. Our findings show how p62 helps maintain intracellular pools of GSH needed to limit mitochondrial dysfunction in tumor cells with elevated mTORC1, highlighting p62 and redox homeostasis as nodal vulnerabilities for therapeutic targeting in these tumors. Cancer Res; 77(12); 3255-67. ©2017 AACR.

Cigarette smoke is associated with altered expression of antioxidant enzymes in granulosa cells from women undergoing in vitro fertilization.

This study was undertaken to evaluate whether cigarette smoke is associated with changes in the expression of antioxidant enzymes in granulosa cells of women undergoing IVF treatments. For this aim, the expression of three antioxidant enzymes (SOD1, SOD2 and catalase) in non-smokers (n = 20) and smokers (n = 20) was analyzed. There was a statistically significant overexpression of SOD2 and catalase mRNA levels in smokers in comparison with non-smokers. Cigarette smoking was associated with a lower fertilization rate, implantation rate and pregnancy rate in comparison with non-smokers. There was no effect on retrieved oocytes number, metaphase II oocytes number, quality of embryos transferred and live birth rate. These findings suggest that cigarette smoke initiates oxidative stress in granulosa cells.

Differential protein acetylation assists import of excess SOD2 into mitochondria and mediates SOD2 aggregation associated with cardiac hypertrophy in the murine SOD2-tg heart

SOD2 is the primary antioxidant enzyme neutralizing •O2− in mitochondria. Cardiac-specific SOD2 overexpression (SOD2-tg) induces supernormal function and cardiac hypertrophy in the mouse heart. However, the reductive stress imposed by SOD2 overexpression results in protein aggregation of SOD2 pentamers and differential hyperacetylation of SOD2 in the mitochondria and cytosol. Here, we studied SOD2 acetylation in SOD2-tg and wild-type mouse hearts. LC-MS/MS analysis indicated the presence of four acetylated lysines in matrix SOD2 and nine acetylated lysines in cytosolic SOD2 from the SOD2-tg heart. However, only one specific acetylated lysine residue was detected in the mitochondria of the wild-type heart, which was consistent with Sirt3 downregulation in the SOD2-tg heart. LC-MS/MS further detected hyperacetylated SOD2 with a signaling peptide in the mitochondrial inner membrane and matrix of the SOD2-tg heart, indicating partial arrest of the SOD2 precursor in the membrane during translocation into the mitochondria. Upregulation of HSP 70 and cytosolic HSP 60 enabled the translocation of excess SOD2 into mitochondria. In vitro acetylation of matrix SOD2 with Ac2O deaggregated pentameric SOD2, restored the profile of cytosolic SOD2 hyperacetylation, and decreased matrix SOD2 activity. As revealed by 3D structure, acetylation of K89, K134, and K154 of cytosolic SOD2 induces unfolding of the tertiary structure and breaking of the salt bridges that are important for the quaternary structure, suggesting that hyperacetylation and HSP 70 upregulation maintain the unfolded status of SOD2 in the cytosol and mediate the import of SOD2 across the membrane. Downregulation of Sirt3, HSP 60, and presequence protease in the mitochondria of the SOD2-tg heart promoted protein misfolding that led to pentameric aggregation.

Ionizing radiation induces long-term senescence in endothelial cells through mitochondrial respiratory complex II dysfunction and superoxide generation

Ionizing radiation causes oxidative stress, leading to acute and late cellular responses. We previously demonstrated that irradiation of non-proliferating endothelial cells, as observed in normal tissues, induces early apoptosis, which can be inhibited by pretreatment with Sphingosine-1-Phosphate. We now propose to better characterize the long-term radiation response of endothelial cells by studying the molecular pathways associated with senescence and its link with acute apoptosis. First, senescence was validated in irradiated quiescent microvascular HMVEC-L in a dose- and time-dependent manner by SA β-galactosidase staining, p16Ink4a and p21Waf1 expression, pro-inflammatory IL-8 secretion and DNA damage response activation. This premature aging was induced independently of Sphingosine 1-Phosphate treatment, supporting its non-connection with acute IR-induced apoptosis. Then, senescence under these conditions showed persistent activation of p53 pathway and mitochondrial dysfunctions, characterized by O2·- generation, inhibition of respiratory complex II activity and over-expression of SOD2 and GPX1 detoxification enzymes. Senescence was significantly inhibited by treatment with pifithrin–α, a p53 inhibitor, or by MnTBAP, a superoxide dismutase mimetic, validating those molecular actors in IR-induced endothelial cell aging. However, MnTBAP, but not pifithrin–α, was able to limit superoxide generation and to rescue the respiratory complex II activity. Furthermore, MnTBAP was not modulating p53 up-regulation, suggesting that IR-induced senescence in quiescent endothelial cells is provided by at least 2 different pathways dependent of the mitochondrial oxidative stress response and the p53 activation. Further characterization of the actors involved in the respiratory complex II dysfunction will open new pharmacological strategies to modulate late radiation toxicity.

Overexpression of SOD2 in Myocytes Protects Mitochondrial Function from Myocardial Ischemia and Reperfusion Injury

Mitochondrial dysfunction in myocardial ischemia and reperfusion (IR) is caused by oxidative stress. Mitochondrial electron transport chain (ETC) is the major source of superoxide and superoxide‐derived reactive oxygen species during I/R injury. Ischemic defect and reperfusion‐induced injury to ETC and Krebs cycle components are critical in mitochondrial dysfunction and the related disease pathogenesis of post‐ischemic injury. SOD2 is the primary antioxidant enzyme in a network of detoxification enzymes that neutralizes superoxide to H2O2 in the mitochondrial matrix. Mouse model with transgenic overexpression of human SOD2 (SOD2‐tg) in myocytes develops supernormal cardiac function through enhanced bioenergetic efficiency and myocardial blood flow. To test the therapeutic potential of using human SOD2 for cardioprotection and the effect of human SOD2 overexpression in the mouse heart on the post‐ischemic injury, isolated mouse heart of the SOD2‐tg mouse model was subjected to global ischemia for 30 min followed by reperfusion for 45‐min. TCC (2,3,5‐triphenyltetrazolium chloride) staining indicated that transgenic overexpression of SOD2 in myocytes significantly decreased the infarct area of the post‐ischemic heart. Analysis of oxygen consumption rates (OCR) by the isolated mitochondria indicated that ADP‐dependent and uncoupling OCRs were preserved in the mouse heart of SOD2‐tg after I/R injury, and mitochondrial integrity was further protected from IR impairment due to transgenic overexpression of SOD2 in the mouse heart. Assessment of the electron transport activity (ETA) from the alamathicin‐permeabilized mitochondria further indicated that SOD2 overexpression significantly preserved the ETA of complexes I, II, III, and IV in the post‐ischemic heart. For the components of Krebs cycle in the isolated mitochondria, it was measured that the activities of aconitase and isocitrate dehydrogenase were impaired by IR in wild type heart, but preserved in the mouse heart of SOD2‐tg. However, the enzymatic activity of malate dehydrogenase and citrate synthase in wild type mouse heart was not affected after IR injury, suggesting accumulation of citrate for cytosolic fatty acid biosynthesis and malate for cytosolic gluconeogenesis under the physiological conditions of hypoxia and reoxygenation. Immunoblotting analysis indicated a downregulation of ETC components and aconitase was induced by IR heart in the wild type heart, but protein expression of ETC components and aconitase was preserved in the post‐ischemic heart of SOD2‐tg mice, suggesting that SOD2 overexpression maintains protein quality control in the mitochondria of post‐ischemic heart. Overexpression of SOD2 in myocytes results in a more reduced redox setting of mouse heart as determined by the redox activity of hydroxylamine oxidation using EPR measurement. However, SOD2 overexpression did not prevent myocardium from IR‐mediated increased redox activity of hydroxylamine oxidation, suggesting that IR mediated a more oxidized redox setting in myocardium independently of superoxide generation by mitochondria. In conclusion, transgenic overexpression of human SOD2 in the mouse myocyte protects myocardium from IR injury by preserving mitochondrial function via maintaining protein quality control of ETC and Krebs cycle components.Support or Funding InformationNIH/HL083237

Overexpression of manganese superoxide dismutase in mouse liver leads to defects in oxidative phosphorylation

Non‐alcoholic fatty liver disease (NAFLD) is a chronic disease that is characterized by inflammation in the liver. This inflammation is due primarily to an increase in reactive oxygen species, and therefore antioxidant therapy can reduce the severity of the disease. However, these antioxidants have also been met with negative side effects, which warrants further investigation. Manganese superoxide dismutase, or MnSOD, is a mitochondrial antioxidant enzyme that converts then reduces superoxide levels by converting them into hydrogen peroxide. Hydrogen peroxide is then detoxified by a number of mitochondrial antioxidant enzymes. Although genetic overexpression of SOD2 could be beneficial to the treatment of NAFLD, preliminary data has shown that mice with SOD2 overexpression actually demonstrate defects in oxidative phosphorylation. These defects include reductions in levels of complex II and V measured by blue native PAGE, and reductions in levels of complex II, III and IV measured by SDS‐PAGE. Western blot results for reduction in levels of complex I, II, and III are under investigation. These data indicate that antioxidant therapy may have unexpected complications that should be considered before administration of these drugs.

In Vivo Characterization of I91T Sod2 Polymorphism of Saccharomyces cerevisiae.

The mitochondrial antioxidant enzyme Mn-Superoxide Dismutase (Sod2) is essential for mammalian survival. I82T mutation in human Sod2 has been linked to a wide variety of diseases, including Alzheimer's and Parkinson's diseases as well as some types of cancers. Yeast wild-type (WT) Sod2 and the mutant Sod2 I91T, which corresponds to the human mutant Sod2 I82T, were cloned in sod2Δ strain. Residue I82 is conserved among a variety of species, showing that it has a biological importance. To assess the functionality of Sod2 I91T under oxidative stress, yeast cells were shifted from glucose (fermentative metabolism) to glycerol growth medium (respiratory metabolism). Overexpression of both Sod2 WT and Sod2 I91T increased Sod activity, but in long-term, the mutation brought impairment to Sod function. Aconitase, a sensor of superoxide radical production in vivo, had its activity preserved by overexpressions of both Sod2, in lesser extent in sod2ΔSod2I91T. In respiratory metabolism, sod2ΔSod2WT and sod2ΔSod2I91T showed high viability; although, sod2ΔSod2I91T showed high percentage of cells with mitochondrial function compromised. Moreover, the fitness analysis of mixed cultures showed that sod2ΔSod2I91T was less robust than WT cells. Although overexpression of Sod2 containing I91T mutation allows higher cell viability, longevity of cells is hampered, showing that in long-term this mutation is not neutral. J. Cell. Biochem. 118: 1078-1086, 2017. © 2016 Wiley Periodicals, Inc.

Sirtuin 3 is required for osteogenic differentiation through maintenance of PGC-1ɑ-SOD2-mediated regulation of mitochondrial function.

Osteogenic differentiation is crucial for the maintenance of bone homeostasis. Sirtuin 3 (SIRT3), a member of sirtuins family, functions as a critical deacetylase that regulates many key proteins. In the current study, we aimed to clarify the role of SIRT3 in osteogenic differentiation and the possible mechanisms, using mouse pre-osteoblastic MC3T3-E1 cells. Expression of SIRT3 was substantially increased in differentiated MC3T3-E1 cells. Knock down of SIRT3 significantly decreased alkaline phosphatase (ALP) staining, and mRNA expression of runt-related transcription factor 2 (Runx2) and collagen type I ɑ 1 (Col1ɑ1), and osteocalcin in differentiated MC3T3-E1 cells. Overexpression of wild type but not mutant SIRT3 could reverse SIRT3 knockdown-resulted decrease of ALP staining. Complex I, II, III, IV, and V activities, oxygen consumption and mitochondrial membrane potential were significantly decreased by SIRT3 knockdown. Moreover, SIRT3 knockdown reduced mitochondrial density, increased mitochondrial size and decreased the expression of NRF1 and TFAM. Knock down of SIRT3 decreased mRNA and protein expression of SOD2 and increased ROS level. Overexpression of SOD2 significantly suppressed SIRT3 knockdown-induced decrease of mitochondrial function and osteogenic differentiation. SIRT3 knockdown resulted in a significant decrease of PGC-1ɑ protein expression but not mRNA expression. Overexpression of wild type but not mutant SIRT3 could reverse SIRT3 knockdown-resulted decrease of PGC-1ɑ protein expression. Moreover, we detected a direct interaction between SIRT3 and PGC-1ɑ and SIRT3 knockdown reduced SIRT3 and PGC-1ɑ interaction, resulting in a reduction of PGC-1ɑ protein stability and PGC-1ɑ-binding in the promoters of SOD2. Overexpression of PGC-1ɑ blocked SIRT3 knockdown-induced decrease of SOD2 expression, increase of ROS level, and decrease of mitochondrial function and biogenesis, leading to improvement of osteogenesis. Overall, the data provide a better understanding of the role of SIRT3 in osteogenic differentiation.

320 - Differential Protein Acetylation Mediates Mitochondrial Localization and Pentameric Aggregation of SOD2 in the Murine Heart of SOD2-Tg

Cardiac-specific overexpression of SOD2 (mitochondrial manganese superoxide dismutase, mouse model of SOD2-tg) induces supernormal and physiologically hypertrophic phenotypes in the mouse heart. However, the reductive stress imposed by SOD2 overexpression also results in protein aggregation of SOD2 pentamer and differential acetylation of SOD2 in the compartments of mitochondrial matrix, mitochondrial inner membrane, and cytosol. We studied SOD2 acetylation in the matrix, submitochondrial particle (SMP), and cytosol from the SOD2-tg mouse heart. LC/MS/MS analysis revealed that K68, K75, K89, K114, K122, K130, K132, K134, and K154 residues of the cytosolic SOD2 were acetylated. LC/MS/MS analysis further indicated that K68, K122, and K130 of SOD2 in SMP and K68, K122, K 130, K202 of matrix SOD2 were acetylated. LC/MS/MS also detected a signaling peptide, 12QLAPVLGYLGSR23, in the SMP SOD2. The results implicated that mitochondrial localization of SOD2 resulted in specific deacetylation of K75, K89, K114, K132, K134, K154 residues, and site-specific acetylation of K202 residue in the matrix. In vitro acetylation of matrix SOD2 with acetic anhydride de-aggregated pentameric SOD2, restored the profile of hyperacetylation observed in cytosolic SOD2, and decreased the enzymatic activity of matrix SOD2. As indicated by the 3D structure of SOD2, specific acetylation of K89, K134, and K154 detected in cytosolic SOD2 induces unfolding tertiary structure and breaking salt bridges for stabilizing the quaternary structure. Stable isotope labeling of both wild type and SOD2-tg matrix proteins followed by LC/MS analysis indicated significant up-regulation of stress-induced mitochondrial hsp70 (GRP75) and down-regulation of presequence protease (PREP), thus supporting increased reductive stress, compromised mitochondrial localization, and weakening correct folding of the nascent SOD2 in the SOD2-tg murine heart. In conclusion, hyperacetylation maintains the unfolded cytosolic SOD2 and mediates importation of SOD2 across the mitochondrial membrane. Activation of the matrix SOD2 is subsequently initiated by deacetylation that further induces SOD2 misfolding for pentameric aggregation.

MnTE-2-PyP modulates thiol oxidation in a hydrogen peroxide-mediated manner in a human prostate cancer cell

To improve the treatment of advanced prostate cancer, the development of effective and innovative antitumor agents is needed. Our previous work demonstrated that the ROS (reactive oxygen species) scavenger, MnTE-2-PyP, inhibited human prostate cancer growth and also inhibited prostate cancer migration and invasion. We showed that MnTE-2-PyP treatment altered the affinity of the histone acetyltransferase enzyme, p300, to bind to DNA. We speculate that this may be one mechanism by which MnTE-2-PyP inhibits prostate cancer progression. Specifically, MnTE-2-PyP decreased p300/HIF-1/CREB complex (p300/hypoxia-inducible factor-1/cAMP response element-binding protein) binding to a specific hypoxia-response element (HRE) motif within the plasminogen activator inhibitor-1 (PAI-1) gene promoter region, and consequently, repressed PAI-1 expression. However, it remains unclear how MnTE-2-PyP reduces p300 complex binding affinity to the promoter region of specific genes. In this study, we found that overexpression of Cu/ZnSOD (superoxide dismutase 1, SOD1) significantly suppressed PAI-1 gene expression and p300 complex binding to the promoter region of PAI-1 gene, just as was observed in cells treated with MnTE-2-PyP. Furthermore, catalase (CAT) overexpression rescued the inhibition of PAI-1 expression and p300 binding by MnTE-2-PyP. Taken together, the above findings suggest that hydrogen peroxide (H2O2) is likely the mediator through which MnTE-2-PyP inhibits the PAI-1 expression and p300 complex binding in PC3 cells. To confirm this, we measured the production of H2O2 following overexpression of SOD1 or catalase with MnTE-2-PyP treatment in the presence or absence of radiation. We found that MnTE-2-PyP increased the intracellular steady-state levels of H2O2 and increased nuclear H2O2 levels. As expected, catalase overexpression significantly decreased the levels of intracellular H2O2 induced by MnTE-2-PyP. We then determined if this increased H2O2 production could result in oxidized protein thiol groups. In the presence of MnTE-2-PyP, there was a significant increase in oxidized thiols in PC3 cell lysates and this was reversed with catalase overexpression. Specifically, we showed that p300 was oxidized after MnTE-2-PyP treatment, indicating that MnTE-2-PyP is creating a more oxidizing environment and this is altering the oxidation state of p300 thiol residues. Our data provide an in depth mechanism by which MnTE-2-PyP regulates gene transcription through induced H2O2 mediated oxidation of particular proteins, supporting an important role for MnTE-2-PyP as an effective and innovative antitumor agent to enhance treatment outcomes in prostate cancer radiotherapy.

Peripheral Blood Mitochondrial DNA Damage as a Potential Noninvasive Biomarker of Diabetic Retinopathy.

PurposeIn the development of diabetic retinopathy, retinal mitochondria become dysfunctional, and mitochondrial DNA (mtDNA) is damaged. Because retinopathy is a progressive disease, and circulating glucose levels are high in diabetes, our aim was to investigate if peripheral blood mtDNA damage can serve as a potential biomarker of diabetic retinopathy.MethodsPeripheral blood mtDNA damage was investigated by extended-length PCR in rats and mice, diabetic for 10 to 12 months (streptozotocin-induced, type 1 model), and in 12- and 40-week-old Zucker diabetic fatty rats (ZDF, type 2). Mitochondrial copy number (in gDNA) and transcription (in cDNA) were quantified by qPCR. Similar parameters were measured in blood from diabetic patients with/without retinopathy.ResultsPeripheral blood from diabetic rodents had significantly increased mtDNA damage and decreased copy numbers and transcription. Lipoic acid administration in diabetic rats, or Sod2 overexpression or MMP-9 knockdown in mice, the therapies that prevent diabetic retinopathy, also ameliorated blood mtDNA damage and restored copy numbers and transcription. Although blood from 40-week-old ZDF rats had significant mtDNA damage, 12-week-old rats had normal mtDNA. Diabetic patients with retinopathy had increased blood mtDNA damage, and decreased transcription and copy numbers compared with diabetic patients without retinopathy and nondiabetic individuals.ConclusionsType 1 diabetic rodents with oxidative stress modulated by pharmacologic/genetic means, and type 2 animal model and patients with/without diabetic retinopathy, demonstrate a strong relation between peripheral blood mtDNA damage and diabetic retinopathy, and suggest the possibility of use of peripheral blood mtDNA as a noninvasive biomarker of diabetic retinopathy.

SOD3 Ameliorates Aβ25-35-Induced Oxidative Damage in SH-SY5Y Cells by Inhibiting the Mitochondrial Pathway.

This study was designed to investigate the protective effects of extracellular superoxide dismutase (SOD3) against amyloid beta (Aβ25-35)-induced damage in human neuroblastoma SH-SY5Y cells and to elucidate the mechanisms responsible for this beneficial effect. SH-SY5Y cells overexpressing SOD3 were generated by adenoviral vector-mediated infection and Aβ25-35 was then added to the cell culture system to establish an in vitro model of oxidative stress. Cell viability, the generation of intracellular reactive oxygen species (ROS), the expression and activity of antioxidant enzymes, the levels of lipid peroxidation malondialdehyde (MDA), the expression of mitochondrial apoptosis-related genes and calcium images were examined. Following Aβ25-35 exposure, SOD3 overexpression promoted the survival of SH-SY5Y cells, decreased the production of ROS, decreased MDA and calcium levels, and decreased cytochrome c, caspase-3, caspase-9 and Bax gene expression. Furthermore, SOD3 overexpression increased the expression and activity of antioxidant enzyme genes and Bcl-2 expression. Together, our data demonstrate that SOD3 ameliorates Aβ25-35-induced oxidative damage in neuroblastoma SH-SY5Y cells by inhibiting the mitochondrial pathway. These data provide new insights into the functional actions of SOD3 on oxidative stress-induced cell damage.