Glenn Wilson - Academia.edu (original) (raw)

Papers by Glenn Wilson

Shock, Jul 1, 2017

Although studies in rat cultured pulmonary artery endothelial cells, perfused lungs, and intact m... more Although studies in rat cultured pulmonary artery endothelial cells, perfused lungs, and intact mice support the concept that oxidative mitochondrial (mt) DNA damage triggers acute lung injury (ALI), it has not yet been determined whether enhanced mtDNA repair forestalls development of ALI and its progression to multiple organ system failure (MOSF). Accordingly, here we examined the effect of a fusion protein construct targeting the DNA glycosylase, Ogg1, to mitochondria in a rat model intra-tracheal P. aeruginosa (strain 103; PA103)-induced ALI and MOSF. Relative to controls, animals given PA103 displayed increases in lung vascular filtration coefficient accompanied by transient lung tissue oxidative mtDNA damage and variable changes in mtDNA copy number without evidence of nuclear DNA damage. The approximate 40% of animals surviving 24h after bacterial administration exhibited multiple organ dysfunction, manifest as increased serum and tissue-specific indices of kidney and liver failure, along with depressed heart rate and blood pressure. While administration of mt-targeted Ogg1 to control animals was innocuous, the active fusion protein, but not a DNA repair-deficient mutant, prevented bacteriainduced increases in lung tissue oxidative mtDNA damage, failed to alter mtDNA copy number, and attenuated lung endothelial barrier degradation. These changes were associated with suppression of liver, kidney, and cardiovascular dysfunction and with decreased 24h mortality. Collectively, the present findings indicate that oxidative mtDNA damage in lung tissue initiates PA103-induced ALI and MOSF in rats.

American Journal of Physiology-Lung Cellular and Molecular Physiology, 2001

Mitochondrial (mt) DNA is damaged by free radicals. Recent data also show that there are cell typ... more Mitochondrial (mt) DNA is damaged by free radicals. Recent data also show that there are cell type-dependent differences in mtDNA repair capacity. In this study, we explored the effects of xanthine oxidase (XO), which generates superoxide anion directly, and menadione, which enhances superoxide production within mitochondria, on mtDNA in pulmonary arterial (PA), microvascular (MV), and pulmonary venous (PV) endothelial cells (ECs). Both XO and menadione damaged mtDNA in the EC phenotypes, with a rank order of sensitivity of (from most to least) PV > PA > MV for XO and MV = PV > PA for menadione. Dimethylthiourea and deferoxamine blunted menadione- and XO-induced mtDNA damage, thus supporting a role for the iron-catalyzed formation of hydroxyl radical. Damage to the nuclear vascular endothelial growth factor gene was not detected with either XO or menadione. PAECs and MVECs, but not PVECs, repaired XO-induced mtDNA damage quickly. Menadione-induced mtDNA damage was avidly re...

Cold Spring Harbor perspectives in biology, 2013

DNA molecules in mitochondria, just like those in the nucleus of eukaryotic cells, are constantly... more DNA molecules in mitochondria, just like those in the nucleus of eukaryotic cells, are constantly damaged by noxious agents. Eukaryotic cells have developed efficient mechanisms to deal with this assault. The process of DNA repair in mitochondria, initially believed nonexistent, has now evolved into a mature area of research. In recent years, it has become increasingly appreciated that mitochondria possess many of the same DNA repair pathways that the nucleus does. Moreover, a unique pathway that is enabled by high redundancy of the mitochondrial DNA and allows for the disposal of damaged DNA molecules operates in this organelle. In this review, we attempt to present a unified view of our current understanding of the process of DNA repair in mitochondria with an emphasis on issues that appear controversial.

American journal of physiology. Lung cellular and molecular physiology, 2002

In rat cultured pulmonary arterial (PA), microvascular, and venous endothelial cells (ECs), the r... more In rat cultured pulmonary arterial (PA), microvascular, and venous endothelial cells (ECs), the rate of mitochondrial (mt) DNA repair is predictive of the severity of xanthine oxidase (XO)-induced mtDNA damage and the sensitivity to XO-mediated cell death. To examine the importance of mtDNA damage and repair more directly, we determined the impact of mitochondrial overexpression of the DNA repair enzyme, Ogg1, on XO-induced mtDNA damage and cell death in PAECs. PAECs were transiently transfected with an Ogg1-mitochondrial targeting sequence construct. Mitochondria-selective overexpression of the transgene product was confirmed microscopically by the observation that immunoreactive Ogg1 colocalized with a mitochondria-specific tracer and, with an oligonucleotide cleavage assay, by a selective enhancement of mitochondrial Ogg1 activity. Overexpression of Ogg1 protected against both XO-induced mtDNA damage, determined by quantitative Southern analysis, and cell death as assessed by try...

Basic research in cardiology, 2015

Recent reports indicate that elevating DNA glycosylase/AP lyase repair enzyme activity offers mar... more Recent reports indicate that elevating DNA glycosylase/AP lyase repair enzyme activity offers marked cytoprotection in cultured cells and a variety of injury models. In this study, we measured the effect of EndoIII, a fusion protein construct that traffics Endonuclease III, a DNA glycosylase/AP lyase, to the mitochondria, on infarct size in a rat model of myocardial ischemia/reperfusion. Open-chest, anesthetized rats were subjected to 30 min of occlusion of a coronary artery followed by 2 h of reperfusion. An intravenous bolus of EndoIII, 8 mg/kg, just prior to reperfusion reduced infarct size from 43.8 ± 1.4 % of the risk zone in control animals to 24.0 ± 1.3 % with no detectable hemodynamic effect. Neither EndoIII's vehicle nor an enzymatically inactive EndoIII mutant (K120Q) offered any protection. The magnitude of…

Nucleic Acids Research, 2009

Mitochondrial DNA (mtDNA) is located in close proximity of the respiratory chains, which are the ... more Mitochondrial DNA (mtDNA) is located in close proximity of the respiratory chains, which are the main cellular source of reactive oxygen species (ROS). ROS can induce oxidative base lesions in mtDNA and are believed to be an important cause of the mtDNA mutations, which accumulate with aging and in diseased states. However, recent studies indicate that cumulative levels of base substitutions in mtDNA can be very low even in old individuals. Considering the reduced complement of DNA repair pathways available in mitochondria and higher susceptibility of mtDNA to oxidative damage than nDNA, it is presently unclear how mitochondria manage to maintain the integrity of their genetic information in the face of the permanent exposure to ROS. Here we show that oxidative stress can lead to the degradation of mtDNA and that strand breaks and abasic sites prevail over mutagenic base lesions in ROS-damaged mtDNA. Furthermore, we found that inhibition of base excision repair enhanced mtDNA degradation in response to both oxidative and alkylating damage. These observations suggest a novel mechanism for the protection of mtDNA against oxidative insults whereby a higher incidence of lesions to the sugar-phosphate backbone induces degradation of damaged mtDNA and prevents the accumulation of mutagenic base lesions.

Neonatology, 2013

Background: Numerous studies in cultured cells indicate that damage to mitochondrial DNA (mtDNA) ... more Background: Numerous studies in cultured cells indicate that damage to mitochondrial DNA (mtDNA) dictates cellular responses to oxidant stress, yet the consequences of mtDNA damage have not been studied directly in the preterm lung. Objective: We sought to determine whether hyperoxia-induced fetal lung dysmorphogenesis is linked to mtDNA damage and establish mtDNA repair as a potential therapeutic approach for treating lung dysplasia in the preterm neonate. Methods: Hyperoxia-induced mtDNA damage was assessed by quantitative alkaline gel electrophoresis in normoxic (3% O2) and hyperoxic (21% O2) fetal rat lung explants. A fusion protein construct targeting the DNA repair enzyme endonuclease III (Endo III) to the mitochondria was used to augment mtDNA repair. Fetal lung branching and surfactant protein C (SFPTC) were assessed in these tissues. Results: Hyperoxia induced mtDNA damage in lung explants and was accompanied by impaired branching morphogenesis and decreased SFPTC mRNA expr...

Molecular Biology Reports, 2007

In this study, we report that the partitioning between mitochondria and cytoplasm of two variants... more In this study, we report that the partitioning between mitochondria and cytoplasm of two variants, mCherry and DsRed Express (DRE), of the red fluorescent protein, DsRed, fused to one of the six matrix targeting sequences (MTSs) can be affected by both MTS and amino acid substitutions in DsRed. Of the six MTSs tested, MTSs from superoxide dismutase and DNA polymerase gamma failed to direct mCherry, but not DRE to mitochondria. By evaluating a series of chimeras between mCherry and DRE fused to the MTS of superoxide dismutase, we attribute the differences in the mitochondrial partitioning to differences in the primary amino acid sequence of the passenger polypeptide. The impairment of mitochondrial partitioning closely parallels the number of mCherry-specific mutations, and is not specific to mutations located in any particular region of the polypeptide. These observations suggest that both MTS and the passenger polypeptide affect the efficiency of mitochondrial import and provide a rationale for the observed diversity in the primary amino acid sequences of natural MTSs.

Mitochondrial DNA, 2014

In a living cell, oxidative stress resulting from an external or internal insult can result in mi... more In a living cell, oxidative stress resulting from an external or internal insult can result in mitochondrial DNA (mtDNA) damage and degradation. Here, we show that in HeLa cells, mtDNA can withstand relatively high levels of extracellular oxidant H 2 O 2 before it is damaged to a point of degradation, and that mtDNA levels in these cells quickly recover after removal of the stressor. In contrast, mtDNA degradation in mouse fibroblast cells is induced at eight-fold lower concentrations of H 2 O 2 , and restoration of the lost mtDNA proceeds much slower. Importantly, mtDNA levels in HeLa cells continue to decline even after withdrawal of the stressor thus marking the "slow" mode of mtDNA degradation. Conversely, in mouse fibroblasts maximal loss of mtDNA is achieved during treatment, and is already detectable at 5 min after exposure, indicating the "fast" mode. These differences may modulate susceptibility to oxidative stress of those organs, which consist of multiple cell types.

Journal of Biological Chemistry, 2009

Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular vi... more Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.

Journal of Biological Chemistry, 2013

Background: Nonviability of cells lacking mitochondrial DNA ligase suggests essential function of... more Background: Nonviability of cells lacking mitochondrial DNA ligase suggests essential function of this enzyme. Results: We report the isolation of viable Lig3 Ϫ/Ϫ cells, which lack mtDNA. Conclusion: The lethality of the Lig3 knockout is mediated by the 0 phenotype. Significance: This is definitive proof that the essential function of LIG3 in mitochondria is limited to DNA transactions. Multiple lines of evidence support the notion that DNA ligase III (LIG3), the only DNA ligase found in mitochondria, is essential for viability in both whole organisms and in cultured cells. Previous attempts to generate cells devoid of mitochondrial DNA ligase failed. Here, we report, for the first time, the derivation of viable LIG3-deficient mouse embryonic fibroblasts. These cells lack mtDNA and are auxotrophic for uridine and pyruvate, which may explain the apparent lethality of the Lig3 knockout observed in cultured cells in previous studies. Cells with severely reduced expression of LIG3 maintain normal mtDNA copy number and respiration but show reduced viability in the face of alkylating and oxidative damage, increased mtDNA degradation in response to oxidative damage, and slow recovery from mtDNA depletion. Our findings clarify the cellular role of LIG3 and establish that the loss of viability in LIG3deficient cells is conditional and secondary to the 0 phenotype. In mammalian cells, there are two DNA-containing organelles, the nucleus and the mitochondrion. Human mitochondrial DNA (mtDNA) is a 16,568-bp circular molecule (1) crucial for proper mitochondrial function and cellular ATP production. It encodes 13 proteins as follows: 11 polypeptide subunits of the mitochondrial electron transport chain complexes I, III, and IV and two subunits of the ATP synthase, complex V. These polypeptides are encoded using a genetic code distinct from that used in the nucleus and therefore require a separate translational apparatus, some components of which (22 tRNAs and 2 rRNAs) also are encoded in mtDNA (2). Therefore, it comes as no surprise that the loss of mtDNA integrity either through mutation or depletion may lead to serious, often fatal diseases (3, 4). The mitochondrial electron transport chain is a major cellular source for the production of reactive oxygen species. Although the exact rates of mitochondrial reactive oxygen

Gene Therapy, 2008

Mitochondrial diseases are not uncommon, and may result from mutations in both nuclear and mitoch... more Mitochondrial diseases are not uncommon, and may result from mutations in both nuclear and mitochondrial DNA (mtDNA). At present, only palliative therapies are available for these disorders, and interest in the development of efficient treatment protocols is high. Here, we demonstrate that in cells heteroplasmic for the T8993G mutation, which is a cause for the NARP and MILS syndromes, infection with an adenovirus, which encodes the mitochondrially targeted R.XmaI restriction endonuclease, leads to selective destruction of mutant mtDNA. This destruction proceeds in a timeand dose-dependent manner and results in cells with significantly increased rates of oxygen consumption and ATP production. The delivery of R.XmaI to mitochondria is accompanied by improvement in the ability to utilize galactose as the sole carbon source, which is a surrogate indicator of the proficiency of oxidative phosphorylation. Concurrently, the rate of lactic acid production by these cells, which is a marker of mitochondrial dysfunction, decreases. We further demonstrate that levels of phosphorylated P53 and gH2ax proteins, markers of nuclear DNA damage, do not change in response to infection with recombinant adenovirus indicating the absence of nuclear DNA damage and the relative safety of the technique. Finally, some advantages and limitations of the proposed approach are discussed.

Environmental and Molecular Mutagenesis, 2010

Over the past decade a large volume of research data has accumulated which has established a fund... more Over the past decade a large volume of research data has accumulated which has established a fundamental role for mitochondria in normal cellular functioning, as well as in various pathologies. Mitochondria play a pivotal role in metabolism and energy production, and are one of the key players involved in programmed cell death. On the other hand, mitochondrial dysfunction is implicated, directly or indirectly in numerous pathological conditions including inherited mitochondrial disorders, diabetes, cardiovascular and neurodegenerative diseases, and a variety of malignancies. The ability to modulate mitochondrial function by altering the diverse protein component of this organelle may be of great value for developing future therapeutic interventions. This review will discuss approaches used to introduce proteins into mitochondria. One group of methods utilizes strategies aimed at expressing proteins from genes in the nucleus. These include overexpression of nuclear‐encoded mitochondr...

DNA Repair, 2013

Considerable progress has been made recently toward understanding the processes of mitochondrial ... more Considerable progress has been made recently toward understanding the processes of mitochondrial DNA (mtDNA) damage and repair. However, a paucity of information still exists regarding the physiological effects of persistent mtDNA damage. This is due, in part, to experimental difficulties associated with targeting mtDNA for damage, while sparing nuclear DNA. Here, we characterize two systems designed for targeted mtDNA damage based on the inducible (Tet-ON) mitochondrial expression of the bacterial enzyme, exonuclease III, and the human enzyme, uracil-N-glyosylase containing the Y147A mutation. In both systems, damage was accompanied by degradation of mtDNA, which was detectable by six hours after induction of mutant uracil-N-glycosylase and by twelve hours after induction of exoIII. Unexpectedly, increases in the steady-state levels of single-strand lesions, which led to degradation, were small in absolute terms indicating that both abasic sites and single-strand gaps may be poorly tolerated in mtDNA. mtDNA degradation was accompanied by the loss of expression of mtDNA-encoded COX2. After withdrawal of the inducer, recovery from mtDNA depletion occurred faster in the system expressing exonuclease III, but in both systems reduced mtDNA levels persisted longer than 144h after doxycycline withdrawal. mtDNA degradation was followed by reduction and loss of respiration, decreased membrane potential, reduced cell viability, reduced intrinsic reactive oxygen species production, slowed proliferation, and changes in mitochondrial morphology (fragmentation of the mitochondrial network, rounding and "foaming" of the mitochondria). The mutagenic effects of abasic sites in mtDNA were low, which indicates that damaged mtDNA molecules may be degraded if not rapidly repaired. This study establishes, for the first time, that mtDNA degradation can be a direct and immediate consequence of persistent mtDNA damage and that increased ROS production is not an invariant consequence of mtDNA damage.

American Journal of Physiology-Lung Cellular and Molecular Physiology, 2006

American Journal of Physiology-Lung Cellular and Molecular Physiology, 2012

This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage trigger... more This study tested the hypothesis that oxidative mitochondrial-targeted DNA (mtDNA) damage triggered ventilator-induced lung injury (VILI). Control mice and mice infused with a fusion protein targeting the DNA repair enzyme, 8-oxoguanine-DNA glycosylase 1 (OGG1) to mitochondria were mechanically ventilated with a range of peak inflation pressures (PIP) for specified durations. In minimal VILI (1 h at 40 cmH2O PIP), lung total extravascular albumin space increased 2.8-fold even though neither lung wet/dry (W/D) weight ratios nor bronchoalveolar lavage (BAL) macrophage inflammatory protein (MIP)-2 or IL-6 failed to differ from nonventilated or low PIP controls. This increase in albumin space was attenuated by OGG1. Moderately severe VILI (2 h at 40 cmH2O PIP) produced a 25-fold increase in total extravascular albumin space, a 60% increase in W/D weight ratio and marked increases in BAL MIP-2 and IL-6, accompanied by oxidative mitochondrial DNA damage, as well as decreases in the total ...

Cell transplantation, Nov 1, 2017

Long-term graft survival is an ongoing challenge in the field of islet transplantation. With the ... more Long-term graft survival is an ongoing challenge in the field of islet transplantation. With the growing demand for transplantable organs, therapies to improve organ quality and reduce the incidence of graft dysfunction are of paramount importance. We evaluated the protective role of a recombinant DNA repair protein targeted to mitochondria (Exscien I-III), as a therapeutic agent using a rodent model of pancreatic islet transplantation. We first investigated the effect of therapy on isolated rat islets cultured with pro-inflammatory cytokines (interleukin-1 β, interferon γ, and tumor necrosis factor α) for 48 h and documented a significant reduction in apoptosis by flow cytometry, improved viability by immunofluorescence, and conserved functional potency in vitro and in vivo in Exscien I-III-treated islets. We then tested the effect of therapy in systemic inflammation using a rat model of donor brain death (BD) sustained for a 6-h period. Donor rats were allocated to 4 groups: (non-...

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