Conditional Targeting of the DNA Repair Enzyme hOGG1 into Mitochondria

Oxidative damage to mitochondrial DNA (mtDNA) has been suggested to be a key factor in the etiologies of many diseases and in the normal process of aging. Although the presence of a repair system to remove this damage has been demonstrated, the mechanisms involved in this repair have not been well defined. In an effort to better understand the physiological role of recombinant 8-oxoguanine DNA glycosylase/apurinic lyase (OGG1) in mtDNA repair, we constructed an expression vector containing the gene for OGG1 downstream of the mitochondrial localization sequence from manganese-superoxide dismutase. This gene construct was placed under the control of a tetracycline-regulated promoter. Transfected cells that conditionally expressed OGG1 in the absence of the tetracycline analogue doxycycline and targeted this recombinant protein to mitochondria were generated. Western blots of mitochondrial extracts from vector- and OGG1-transfected clones with and without doxycycline revealed that removal of doxycycline for 4 days caused an approximate 8-fold increase in the amount of OGG1 protein in mitochondria. Enzyme activity assays and DNA repair studies showed that the doxycycline-dependent recombinant OGG1 is functional. Functional studies revealed that cells containing recombinant OGG1 were more proficient at repairing oxidative damage in their mtDNA, and this increased repair led to increased cellular survival following oxidative stress

Mitochondrial DNA Damage and Dysfunction, and Oxidative Stress Are Associated with Endoplasmic Reticulum Stress, Protein Degradation and Apoptosis in High Fat Diet-Induced Insulin Resistance Mice

<div><h3>Background</h3><p>Recent studies showed a link between a high fat diet (HFD)-induced obesity and lipid accumulation in non-adipose tissues, such as skeletal muscle and liver, and insulin resistance (IR). Although the mechanisms responsible for IR in those tissues are different, oxidative stress and mitochondrial dysfunction have been implicated in the disease process. We tested the hypothesis that HFD induced mitochondrial DNA (mtDNA) damage and that this damage is associated with mitochondrial dysfunction, oxidative stress, and induction of markers of endoplasmic reticulum (ER) stress, protein degradation and apoptosis in skeletal muscle and liver in a mouse model of obesity-induced IR.</p> <h3>Methodology/Principal Findings</h3><p>C57BL/6J male mice were fed either a HFD (60% fat) or normal chow (NC) (10% fat) for 16 weeks. We found that HFD-induced IR correlated with increased mtDNA damage, mitochondrial dysfunction and markers of oxidative stress in skeletal muscle and liver. Also, a HFD causes a change in the expression level of DNA repair enzymes in both nuclei and mitochondria in skeletal muscle and liver. Furthermore, a HFD leads to activation of ER stress, protein degradation and apoptosis in skeletal muscle and liver, and significantly reduced the content of two major proteins involved in insulin signaling, Akt and IRS-1 in skeletal muscle, and Akt in liver. Basal p-Akt level was not significantly influenced by HFD feeding in skeletal muscle and liver.</p> <h3>Conclusions/Significance</h3><p>This study provides new evidence that HFD-induced mtDNA damage correlates with mitochondrial dysfunction and increased oxidative stress in skeletal muscle and liver, which is associated with the induction of markers of ER stress, protein degradation and apoptosis.</p> </div

HFD reduced protein content of transcription factors involved in mitochondrial biogenesis, mitochondrial antioxidants and mitochondrial markers.

<p>The average results ± SE are shown. (*p<0.05 vs corresponding NC, n = 6–9 mice per group).</p

Effect of HFD on content of some proteins involved in insulin signaling in skeletal muscle and liver.

<p>(A) and (C) displays data for pAkt, Akt and IRS-1. (B) a HFD reduced MHC protein expression in skeletal muscle. The average results ± SE are shown. (*p<0.05 vs corresponding NC, n = 6–9 mice per group).</p

HFD damaged mtDNA to a greater extent than nDNA in both skeletal muscle and liver.

<p>Representative autoradiograms from Southern blot analyses of (A) mtDNA and (B) nDNA are shown for skeletal muscle and liver from two NC and two HFD mice. (C) displays the graphs for mtDNA damage. (D) the graphs for nDNA damage are shown. (E) are representative autoradiograms from slot-blot analyses performed on the DNA from skeletal muscle and liver isolated from two NC and two HFD mice. (F) mtDNA copy number normalized to nDNA copy number in the gastrocnemius muscle and liver. Data represent the means ± SE. (*p<0.05 vs corresponding NC, n = 7–9 mice per group).</p

HFD induced markers of ER stress, protein degradation and increased apoptosis in skeletal muscle and liver.

<p>pPERK/PERK and CHOP levels were increased after a HFD in both skeletal muscle (A) and liver (B). (C) HFD increased protein ubiquitination (a marker of both ubiquitin-proteasome and autophagy-induced protein degradation) in both skeletal muscle and liver. The average results ± SE are shown. (* p<0.05 vs corresponding NC, n = 6–9 mice per group). (E) Caspase 3 and cleaved caspase 3 antibodies were used to recognize full length (35 kD) caspase 3 and cleaved caspase 3 large fragment (17 kD), respectively. (F) Western blot of cytochrome c release into cytosol. Cytosolic fractions isolated from skeletal muscle and liver from NC/HFD fed mice are shown. Equal loading was confirmed using anti actin antibody (n = 4–5 mice per group).</p

HFD induced oxidative stress, and decreased ATP levels in skeletal muscle and liver.

<p>(A) the GSH/GSSG ratio was significantly decreased in both gastrocnemius muscle and liver samples isolated from mice fed a HFD. (B) a HFD reduced the ATP levels in both skeletal muscle and liver. ATP concentrations were determined using the luciferase-based ATP-assay, values were normalized to mtDNA content. The average results ± SE are shown. (*p<0.05 vs corresponding NC, n = 7–9 mice per group).</p