Aging 167 Oxidative Stress Plays a Major Role in Mitochondrial Nucleoid Clustering Lukas Alan1, Jaroslav Zelenka1, Tomas Spacek1, and Petr Jezek1 1Institute of Physiology, Academy of Sciences, Prague Human mitochondrial (mt) DNA is present in hundreds to thousands of copies per cell assembled into nucleoprotein complexes called nucleoids. during oxidative stress and ageing, distortions in mtDNA such as point mutations or large scale deletions arise, including changes in nucleoid structure linked with inhibition of mt topoisomerase II (1) or down-regulation of mtDNA replication proteins (2). These changes were reported as enlargement and remodeling of mt nucleoids. We focused our research on similar changes in mt nucleoid structures upon ethidium bromide (EtBr) treatment. RT-PCR was employed to determine content of mtDNA per cell (mtDNA copy number), confocal and structural illumination microscopy (SIM) were used for mt nucleoid visualization, and spectrofluorimetry for semiquantification of membrane potential and ROS production. Two day EtBr treatment (100 ng/ml) led to a drastic reduction of mtDNA copy number down to 40 % and part of mt nucleoid population was clustered and coexisted with intact nucleoid population. Based on this finding we modify hypothesis on nucleoid remodeling to a simpler nucleoid clustering. We have also found that N-acetylcysteine (NAC) had no effect on mtDNA copy number in both control or EtBr treated cells but it acted on the nucleoid morphology: the size of nucleoid clusters was significantly reduced. We conclude that NAC antioxidant action disrupted nucleoid clustering, which occured during oxidative stress as adaptation to genotoxic stress. 1. Asley N and Poulton J. Oncogene. 2009 Nov 5; 28(44):3880-91. 2. Kasashima K, et al., Exp Cell Res. 2011 Jan 15; 317(2):210-20 Supported by grants No. 418811 (Grant Agency of Charles University), GACR303/07/0105, and KJB500110902.

doi:10.1016/j.freeradbiomed.2011.10.419

168 Disruption of the 12/15-Lipoxygenase Gene (Alox15) Protects Against Denervation-Induced Muscle Atrophy Arunabh Bhattacharya1, Amanda L Jernigan1, Marian Sabia1, Yiqiang Zhang1, Yan Li1, Wenbo Qi1, and Holly Van Remmen1,2 1Barshop Institute, University of Texas Health Science Center at San Antonio, 2South Texas Veterans Health Care System, San Antonio, Texas Aging is associated with a progressive decline in muscle mass and function which leads to significant increase in morbidity and mortality in the elderly. Loss of innervation or denervation is one of the important mechanisms of age-related muscle atrophy. Our recent study showed that muscle atrophy during aging and surgical denervation is associated with elevated levels of cytosolic phospholipase A2 (cPLA2) and the downstream pro-inflammatory 12/15-lipoxygenase (12/15-LO) pathway. Elevated cPLA2 would provide the substrate (arachidonic acid) for the generation of lipid mediators by 12/15-LO. Specifically, activation of 12/15-LO leads to increased generation of fatty acid hydroperoxides, 12- and 15-hydroperoxy-eicosatetraenoic acid (12-HpETE, 15-HpETE) which are converted by GPx4 to 12-hydroxyeicosatetranoic acid (12-HETE) and 15-HETE, respectively. We hypothesized that 12/15-LO and its downstream products play a causal role in muscle

atrophy. in support of this, we found that the expression of 12/15-LO protein and the level of 15(S)-HETE are significantly elevated in skeletal muscle during aging and in denervation-induced muscle atrophy. Furthermore, genetic ablation of 12/15-LO (12/15-LO null mice) and pharmacological inhibition (PD146176) of this pathway significantly attenuated denervation-induced muscle atrophy. Denervation resulted in an increase in NADPH oxidase activity and oxidative damage (HNE-bound protein adducts) and these parameters were significantly attenuated following genetic/ pharmacological inhibition of 12/15-LO. in addition, inhibition of 12/15-LO significantly reduced denervation-induced increase in the level of ubiquitinated proteins, E3 ubiquitin ligase, MuRF1 expression and 20S proteasome activity. Our results are the first to indicate that 1) activation of 12/15-LO plays an important role in muscle atrophy and 2) activation of 12/15-LO induces NADPH oxidase activity and oxidative damage in skeletal muscle and 3) interventions that target this pathway may potentially decrease denervation and age-related muscle atrophy. Our ongoing studies are focused on identifying the potential targets by which 15(S)-HETE can induce muscle atrophy. Supported by MDA grant 93020 and PO1 AG20591

doi:10.1016/j.freeradbiomed.2011.10.420 169 ER-Stress Drives the Increased Susceptibility To Lung Fibrosis in Aging Individuals Through a ROS-Mediated Mechanism Marta Bueno1,2, Patrick Joseph Pagano2,3, Mauricio Rojas1,4, and Ana Lucia Mora1,2 1Division of Pulmonary, Allergy and Critical Care Medicine. 2Vascular Medicine Institute 3Dept. of Pharmacology and Chemical Biology. 4Simmons Center, Dept. of Medicine, University of Pittsburgh Idiopathic pulmonary fibrosis (IPF) is a progressive, lethal form of interstitial lung disease with a prevalence that increases significantly with age. One of the most well-accepted theories of aging suggests that reactive oxygen species (ROS) produced during aerobic respiration cause cumulative oxidative damage resulting in cell death. in lung fibrosis the role of ROS and oxidative stress is still unclear but NADPH oxidases (Nox) have emerged as major players in its pathogenesis. We evaluated the role of oxidative stress in aging-associated susceptibility to lung fibrosis in two models of IPF: the well established bleomycin-induced lung fibrosis model and a novel mouse model of virus-induced lung fibrosis. in both models we observed increased susceptibility of aging mice to lung fibrosis associated with accumulation of oxidative damage, expression of ER stress markers (BiP, XBP-1, CHOP), and increased apoptosis. We analyzed the role of oxidases in lungs of infected mice and found higher expression of NADPH oxidase isoform Nox2 and? Nox4, in the naïve and injured aging lung. Mice deficient in Nox2 (a.k.a. gp91phox subunit) were protected from lung fibrosis. Using a targeted strategy to inhibit Nox2 signaling, a specific peptide inhibitor of p47phox binding to Nox2 (Nox2ds-tat), we show therapeutic efficacy in reduction of ROS formation, expression of ER stress markers and amelioration of fibrosis in both models. in conclusion, enhanced expression of Nox2 in the aging lung appears to elicit epithelial ER stress. These data are consistent with Nox2-derived ROS mediating epithelial apoptosis and fibrosis.

doi:10.1016/j.freeradbiomed.2011.10.421

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