Role of 12/15-Lipoxygenase in Mechanisms of Muscle Atrophy During Aging

Project summary/abstract: The long-term career goal of Dr. Arunabh Bhattacharya is to establish himself asa successful and well-funded, independent investigator through sustained basic and translational research inthe field of aging, in particular, in the area of muscle biology. His doctoral and postdoctoral training in the areaof inflammatory diseases and oxidative stress laid the foundation for this career development awardapplication. His research career to date has been highly productive with more than 38 publications in peer-reviewed journals. As a faculty member of the Department of CSB/Barshop Institute for Longevity and AgingStudies in San Antonio, he has been studying the role of lipid mediators generated by the inflammatorypathways in muscle atrophy, which is a new direction of research at UTHSCSA. Barshop Institute has one ofthe largest Biology of Aging programs in the in the nation and provides excellent opportunities for junior facultymentoring in aging research. Arunabh is on the path to a long-term independent research career in the field ofmuscle biology and the K01 career development award will provide the mechanism for him to make thistransition. He is submitting this proposal under the joint mentorship of Dr. Van Remmen and Dr. Musi, activefaculty members of the Barshop Institute. Dr. Van Remmen is a leading authority in the field of oxidative stressand its role in aging, and Dr. Musi, a funded Ellison Foundation Beeson Scholar, studies the role ofinflammatory pathways in skeletal muscle in insulin resistance and aging. The overall training program that hasbeen outlined in this K01 award includes a mixture of practical laboratory training with didactic course work andintensive mentoring interactions with the mentor/co-mentor and other members of the advisory council. Inaddition, the training program is designed to provide valuable experience in other aspects of research includingmanaging a research budget, running a laboratory independently, forging collaborations and writing grantapplications to national funding agencies including Muscular Dystrophy Association and an independent R01application to National Institute of Aging. Together these factors will ensure his successful transition to anindependent investigator in the field of aging and aging-associated changes in quality of life. Through interactions with his mentor/co-mentor, members of the advisory council and the consultants whohave agreed to assist Arunabh in this proposal, the K01 award will allow him to gain expertise in a variety oftechniques that will provide a strong basis for his continued advancement in the field of muscle biology.Because of his interest in understanding the mechanisms of muscle atrophy during aging, he has designed thisK01 grant proposal to study the role of the pro-inflammatory 12/15-lipoxygenase (12/15-LO) pathway in age-related sarcopenia. Based on the preliminary data in mice null for 12/15-LO and in aging mice, he hasdeveloped a hypothesis that 12/15-LO plays a significant role in age-related muscle atrophy by modulating theubiquitin-proteasome and the lysosomal autophagy pathways of protein degradation. This will be tested usinga combination of in vitro and in vivo approaches. In Specific Aim 1, in vitro primary myotube culture andisolated muscle preparation will be used to determine the effect of 12/15-LO metabolites, 12- and 15-HETE onthe regulation of the ubiquitin-proteasome and lysosomal autophagy pathways. Since induction of proteindegradation pathways is modulated by PI3K/Akt signaling and downstream mTOR and FoxO3 pathways, andactivation of NF-øB, these pathways will be measured in cells and in isolated muscle preparation from wild-type(with/without PI3K/Akt inhibitor) and MISR mice (mice with muscle-specific inhibition of NF-kB) to determinethe relative role of PI3K/Akt and NF-øB signaling pathways. Activation of downstream lysosomal autophagicpathway will be determined by measuring the expression of autophagic proteins (Atg-7, LC-3II/LC-3I, p62) andthe autophagic flux through the pathway and activation of ubiquitin-proteasome pathway by measuringproteasome activity and ability of the proteasome to degrade an ubiquitin-labeled GFP substrate. In SpecificAim 2, the sciatic nerve transection model will be used to determine whether the inhibition of 12/15-LO protectsagainst denervation-induced muscle atrophy by modulating the protein degradation pathways. The level of 12-and 15-HETE, PI3K/Akt signaling and downstream mTOR and FoxO3 pathways, activation of NF-øB, markersof the induction of the lysosomal autophagy and ubiquitin-proteasome pathways in control and denervatedmuscle from wild-type and 12/15-LO null mice will be measured in this Specific Aim. Finally, the hypothesis willbe tested in vivo to determine whether inhibition of the 12/15-LO pathway during aging protects against loss ofmuscle mass and function by modulating the protein degradation pathways. Age-related muscle atrophy andloss of function, level of 12- and 15-HETE, PI3K/Akt signaling and downstream mTOR and FoxO3 pathways,activation of NF-øB, and markers of the induction of the lysosomal autophagy and ubiquitin-proteasomepathways in wild-type and 12/15-LO knockout mice and in mice treated with a pharmacological inhibitor of12/15-LO will be measured in this Specific Aim. In summary, this proposal addresses an area of research thatis of high relevance in the field of aging. The studies that have been proposed will be the first to examine thepotential role of signaling from lipid mediators such as the HETEs in muscle atrophy; thus the opportunityexists to make some fundamental novel discoveries in this project which may have long-term implications inimproving the quality of life in the aging population. PUBLIC HEALTH RELEVANCE: Aging is associated with progressive loss of muscle mass (termed as sarcopenia) which leads to functional impairments in the aging population, significantly worsens the quality of life and increases the risk of morbidity and mortality in the elderly. This proposal will help identify novel targets for therapeutic intervention to prevent or delay sarcopenia.