Role of 12/15-lipoxygenase in mechanisms of muscle atrophy during aging

描述（由申请人提供）：aruth Bhattacharya博士的长期职业目标是通过在衰老领域，特别是肌肉生物学领域的持续基础和转化研究，使自己成为一名成功且资金充足的独立研究者。他在炎症性疾病和氧化应激领域的博士和博士后培训为本次职业发展奖的申请奠定了基础。他的研究生涯至今已在同行评议的期刊上发表了超过38篇论文。作为圣安东尼奥CSB/Barshop长寿与衰老研究所的教员，他一直在研究炎症途径产生的脂质介质在肌肉萎缩中的作用，这是UTHSCSA的一个新的研究方向。Barshop研究所拥有全国最大的衰老生物学项目之一，并为老年研究的初级教师提供极好的指导机会。arunh正在肌肉生物学领域进行长期的独立研究，K01职业发展奖将为他实现这一转变提供机制。他是在Barshop研究所的活跃教员Van Remmen博士和Musi博士的共同指导下提交这项提案的。Van Remmen博士是氧化应激及其在衰老中的作用领域的权威，Musi博士是埃里森基金会比森学者，研究骨骼肌炎症途径在胰岛素抵抗和衰老中的作用。本次K01奖概述的整体培训计划包括实践实验室培训与教学课程工作的结合，以及与导师/联合导师和咨询委员会其他成员的强化指导互动。此外，该培训计划旨在为研究的其他方面提供宝贵的经验，包括管理研究预算，独立运营实验室，建立合作关系，向国家资助机构（包括肌肉萎缩症协会）提交拨款申请，以及向国家老龄化研究所提交独立的R01申请。总之，这些因素将确保他成功过渡到一个独立的研究者在老龄化和老龄化相关的生活质量的变化领域。通过与他的导师/共同导师、咨询委员会成员以及同意协助arunh完成该提案的顾问的互动，K01奖将使他获得各种技术的专业知识，为他在肌肉生物学领域的持续进步提供坚实的基础。由于他对了解衰老过程中肌肉萎缩的机制很感兴趣，他设计了这个K01拨款申请来研究促炎的12/15-脂氧合酶（12/15-LO）途径在年龄相关的肌肉减少症中的作用。基于12/15-LO小鼠和衰老小鼠的初步数据，他提出了12/15-LO通过调节蛋白质降解的泛素-蛋白酶体和溶酶体自噬途径在年龄相关性肌肉萎缩中起重要作用的假设。这将使用体外和体内方法相结合的方法进行测试。在Specific Aim 1中，将使用体外原代肌管培养和离体肌肉制备来确定12/15- lo代谢物、12-和15-HETE对泛素-蛋白酶体和溶酶体自噬途径的调节作用。由于蛋白质降解途径的诱导是由PI3K/Akt信号和下游mTOR和FoxO3信号通路调节的，NF？B，这些途径将在野生型（含/不含PI3K/Akt抑制剂）和MISR小鼠(肌肉特异性抑制NF？B)确定PI3K/Akt和NF的相对作用？B信号通路。通过测量自噬蛋白（Atg-7, LC-3II/LC-3I, p62）的表达来确定下游溶酶体自噬途径的激活，通过测量蛋白酶体活性和蛋白酶体降解泛素标记的GFP底物的能力来确定该途径的自噬通量和泛素-蛋白酶体途径的激活。在Specific Aim 2中，将使用坐骨神经横断模型来确定抑制12/15-LO是否通过调节蛋白质降解途径来保护去神经支配诱导的肌肉萎缩。12-和15-HETE的水平，PI3K/Akt信号和下游mTOR和FoxO3通路，NF？B，在野生型和12/15-LO缺失小鼠的对照组和失神经肌肉中，溶酶体自噬诱导和泛素-蛋白酶体途径的标记物将在本特异性目的中进行测量。最后，该假设将在体内进行验证，以确定在衰老过程中抑制12/15-LO通路是否通过调节蛋白质降解途径来防止肌肉质量和功能的丧失。年龄相关性肌肉萎缩和功能丧失，12-和15-HETE水平，PI3K/Akt信号和下游mTOR和FoxO3通路，F？，以及野生型和12/15-LO敲除小鼠以及用12/15-LO药理学抑制剂治疗的小鼠中诱导溶酶体自噬和泛素-蛋白酶体途径的标记物将在本特异性Aim中进行测量。综上所述，本提案涉及的研究领域是在老龄化领域的高度相关。提出的研究将是第一个检查脂质介质（如HETEs）信号在肌肉萎缩中的潜在作用的研究；因此，在这个项目中有机会做出一些基本的新发现，这些发现可能对提高老龄化人口的生活质量具有长期影响。

DESCRIPTION (provided by applicant): The long-term career goal of Dr. Arunabh Bhattacharya is to establish himself as a successful and well-funded, independent investigator through sustained basic and translational research in the field of aging, in particular, in the area of muscle biology. His doctoral and postdoctoral training in the area of inflammatory diseases and oxidative stress laid the foundation for this career development award application. 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 Aging Studies in San Antonio, he has been studying the role of lipid mediators generated by the inflammatory pathways in muscle atrophy, which is a new direction of research at UTHSCSA. Barshop Institute has one of the largest Biology of Aging programs in the in the nation and provides excellent opportunities for junior faculty mentoring in aging research. Arunabh is on the path to a long-term independent research career in the field of muscle biology and the K01 career development award will provide the mechanism for him to make this transition. He is submitting this proposal under the joint mentorship of Dr. Van Remmen and Dr. Musi, active faculty members of the Barshop Institute. Dr. Van Remmen is a leading authority in the field of oxidative stress and its role in aging, and Dr. Musi, a funded Ellison Foundation Beeson Scholar, studies the role of inflammatory pathways in skeletal muscle in insulin resistance and aging. The overall training program that has been outlined in this K01 award includes a mixture of practical laboratory training with didactic course work and intensive mentoring interactions with the mentor/co-mentor and other members of the advisory council. In addition, the training program is designed to provide valuable experience in other aspects of research including managing a research budget, running a laboratory independently, forging collaborations and writing grant applications to national funding agencies including Muscular Dystrophy Association and an independent R01 application to National Institute of Aging. Together these factors will ensure his successful transition to an independent 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 who have agreed to assist Arunabh in this proposal, the K01 award will allow him to gain expertise in a variety of techniques 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 this K01 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 has developed a hypothesis that 12/15-LO plays a significant role in age-related muscle atrophy by modulating the ubiquitin-proteasome and the lysosomal autophagy pathways of protein degradation. This will be tested using a combination of in vitro and in vivo approaches. In Specific Aim 1, in vitro primary myotube culture and isolated muscle preparation will be used to determine the effect of 12/15-LO metabolites, 12- and 15-HETE on the regulation of the ubiquitin-proteasome and lysosomal autophagy pathways. Since induction of protein degradation pathways is modulated by PI3K/Akt signaling and downstream mTOR and FoxO3 pathways, and activation of NF?B, 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?B) to determine the relative role of PI3K/Akt and NF?B signaling pathways. Activation of downstream lysosomal autophagic pathway will be determined by measuring the expression of autophagic proteins (Atg-7, LC-3II/LC-3I, p62) and the autophagic flux through the pathway and activation of ubiquitin-proteasome pathway by measuring proteasome activity and ability of the proteasome to degrade an ubiquitin-labeled GFP substrate. In Specific Aim 2, the sciatic nerve transection model will be used to determine whether the inhibition of 12/15-LO protects against 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?B, markers of the induction of the lysosomal autophagy and ubiquitin-proteasome pathways in control and denervated muscle from wild-type and 12/15-LO null mice will be measured in this Specific Aim. Finally, the hypothesis will be tested in vivo to determine whether inhibition of the 12/15-LO pathway during aging protects against loss of muscle mass and function by modulating the protein degradation pathways. Age-related muscle atrophy and loss of function, level of 12- and 15-HETE, PI3K/Akt signaling and downstream mTOR and FoxO3 pathways, activation of ?F??, and markers of the induction of the lysosomal autophagy and ubiquitin-proteasome pathways in wild-type and 12/15-LO knockout mice and in mice treated with a pharmacological inhibitor of 12/15-LO will be measured in this Specific Aim. In summary, this proposal addresses an area of research that is of high relevance in the field of aging. The studies that have been proposed will be the first to examine the potential role of signaling from lipid mediators such as the HETEs in muscle atrophy; thus the opportunity exists to make some fundamental novel discoveries in this project which may have long-term implications in improving 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.