Aging

Ablation of Nrf2 Sensitizes Skeletal Muscle to Cardiotoxin Induced Oxidative Stress and Impairs Regeneration Naser Abu-Rmaileh1, Sankarnarayanan Kannan2, Jennifer Hong1, Nancy Atieno1, Christopher J Davidson3, Russell Richardson3, Aldrin V Gomes4, John R Hoidal3, and Rajasekaran Namakkal Soorappan1 1Cardiac Aging & Redox Signaling Laboratory, Division of Cardiology, University of Utah, United States, 2M.D. Anderson Cancer Center, United States, 3University of Utah, United States, 4University of California, Davis, United States Transcriptional regulation of redox homoeostasis in skeletal muscle (SM) is predominantly controlled by nuclear erythroid-2-p45-related factor-2 (Nrf2). We recently demonstrated that age-associated stress impairs Nrf2-ARE transcriptional signaling. Here, we hypothesize that loss of Nrf2 will promote cardiotoxin (CTX) induced oxidative stress and thereby impairs myogenic progenitor cell differentiation and regeneration of SM. We have tested this hypothesis using C2C12 myoblasts (in vitro) and Nrf2-null mice (in vivo), and standard biochemical, molecular and cell biology techniques. First, we investigated whether Nrf2 signaling plays a critical role in proliferation vs. differentiation of myoblasts. Notably, upon differentiation, gene and protein levels of Nrf2/ARE-target antioxidants (i.e. glutathione reductase, NQO1 and GCLM) were significantly decreased in association with an increase in Pax7 (stem cells) and in MyoD (regeneration factor) when compared to proliferating myoblasts (Fig.1A), suggesting a mechanism for the development of oxidative stress due to down regulation of Nrf2 signaling upon differentiation. Next, we found that disruption of Nrf2 signaling resulted in altered intracellular ROS, redox state and antioxidant status as well as phenotypic changes in TA muscle of Nrf2-null mice. Upon CTX injury, Pax7 mRNA/protein were increased on day-2, peaked on day-4 and then gradually decreased on day-8, and returned to basal level on day-15 in WT mice. Whereas, the Pax7 expression was comparatively lower in Nrf2-null than WT mice in response to CTX injury (Fig 1B). Further, MyoD mRNA was significantly impaired in Nrf2-null when compared to WT mice. Conclusions: These results indicate that impaired activation of satellite cells in association with delayed muscle regeneration is largely depending on Nrf2-antioxidant signaling and intracellular redox state.

The Role of Peroxiredoxin5 in Aging and Immunity Marziyeh Badinloo1, Vladimir Klichko1, William Orr1, Erwin Xia1, Judith Benes1, Olena Odnokoz1, and Svetlana Radyuk1 1Southern Methodist University, United States Recent findings suggest functional links between cellular redox homeostasis and the innate immunity responses, which tend to be excessive and pro-inflammatory with age. Using the Drosophila model, we have found that activation of the effectors of immunity depends on enzymes, called peroxiredoxins (Prxs), which are known to control redox-sensitive signaling. Specifically, one member of the Prx gene family, dPrx5, which is expressed in

different sub-cellular compartments, protects flies from oxidative stress and apoptosis and extends life span but negatively regulates immune response in Drosophila. To elucidate a role for each dPrx5 isoform in regulating immune response in flies during aging, three different types of transgenic flies have been generated, which express dPrx5 in mitochondria, cytosol or nuclei in the absence of endogenous dPrx5 expression. Ectopic high-level expression of Prx5 in various subcellular compartments caused a significant dampening of production of the Drosophila humoral immunity factors, antimicrobial peptides (AMPs), which are normally chronically induced in older flies. Moreover, these effects of Prx5 on AMP production were redox-dependent, as the redox-negative Prx5 variant failed to reduce AMP levels. We have also found that dPrx5 may function via control of the stress-sensing JNK pathway, as activation of the Drosophila JNK kinase Basket, regulated via its phosphorylation, was increased in older flies but significantly suppressed in flies overexpressing dPrx5. Together, the results suggest that 1) links between humoral immunity and senescence are controlled by redox-sensitive components and 2) dPrx5 may play an important role in balancing the immune and stress responses, which have a tendency to be over-activated during aging.

Glutathione Compartmentalization and Exchange between the Cytosol and Mitochondria Maxwell Darch1 1University of South Carolina, United States Control of thiol redox homeostasis within eukaryotic cells is critical for maintaining proper cellular function. in particular, mitochondrial dysfunction related to redox imbalance has been linked to several neurodegenerative diseases and the overall aging process. an important redox buffer component, glutathione (GSH), also plays a significant role in detoxifying carcinogens and the control of cancer-related genes. Characterization of mitochondrion redox control is our primary interest given the variety of essential thiol redox pathways in this organelle. GFP-based sensors targeted to the intermembrane space (IMS) and matrix of yeast mitochondria were developed in our lab to characterize the compartments individually. Our previous studies using these sensors have shown that the IMS is more oxidizing than the cytosol and matrix, and redox control is maintained separately in subcellular compartments. There is also evidence showing direct exchange of GSH:GSSG between the cytosol and IMS. Current studies are aimed to elucidate the effects of GSH depletion/overaccumulation on redox balance as well as pathways for GSH:GSSG subcellular compartmental exchange using Saccharomyces cerevisiae as a model system. to examine the effects of extreme redox imbalance, a yeast strain overexpressing a plasma membrane GSH:GSSG transporter in conjunction with the GFP-based cytosolic, IMS, and matrix sensors is currently being studied in collaboration with Dr. Michel Toledano. GSH and GSSG overaccumulation were both found to directly impact GSH:GSSG pools and thiol redox pathways in the IMS, while having little effect on the matrix. to further elucidate exchange of GSH:GSSG pools in subcellular compartments and identify sources of GSSG production in the cell, a strain has been studied. When compared to WT, the studies suggest that GSH is selectively imported (or GSSG exported) in the IMS, while matrix pools are largely isolated from both GSH and GSSG fluxes in the cytosol and IMS. in addition, production of GSSG in the IMS was found to impact cytosolic GSH:GSSG pools.

doi: 10.1016/j.freeradbiomed.2013.10.675

doi: 10.1016/j.freeradbiomed.2013.10.676