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

项目概要/摘要：Arunabh Bhattacharya博士的长期职业目标是成为 一个成功的，资金充足的，独立的调查员，通过持续的基础和转化研究，在 衰老领域，特别是肌肉生物学领域。他在该地区的博士和博士后培训 炎症性疾病和氧化应激为这个职业发展奖奠定了基础 应用程序.迄今为止，他的研究生涯一直是非常富有成效的，在同行中发表了超过38篇论文， 审查期刊。作为CSB/Barshop长寿和老龄化研究所系的教员 在圣安东尼奥的研究中，他一直在研究炎症产生的脂质介质的作用。 这是UTHSCSA的一个新的研究方向。巴肖普学院有一个 在全国最大的衰老生物学计划，并为初级教师提供了极好的机会 指导老龄化研究阿鲁纳布是在道路上的长期独立的研究生涯领域， 肌肉生物学和K 01职业发展奖将为他提供实现这一目标的机制。 过渡他是在货车Remmen博士和Musi博士的共同指导下提交这一建议的， Barshop Institute的成员。货车雷门博士是氧化应激领域的权威 以及它在衰老中的作用，而Musi博士是Ellison Foundation Beeson Scholar的资助者，研究了 胰岛素抵抗和衰老中骨骼肌的炎症途径。整个培训计划， 在这个K 01奖项中概述了包括实践实验室培训与教学课程工作的混合物， 与指导者/共同指导者和咨询理事会其他成员进行密集的指导互动。在 此外，培训计划旨在提供其他方面的研究，包括宝贵的经验， 管理研究预算，独立运行实验室，建立合作关系和撰写拨款 向国家资助机构申请，包括肌营养不良协会和独立的R 01 申请国家老龄化研究所。这些因素将共同确保他成功过渡到一个 老龄化和老龄化相关生活质量变化领域的独立研究者。 通过与他的导师/共同导师、咨询理事会成员和顾问的互动， 我已经同意协助Arunabh在这个建议，K 01奖将使他获得各种专业知识， 这些技术将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感兴趣，他设计了这个 K 01资助提案研究促炎性12/15-脂氧合酶（12/15-LO）通路在年龄增长中的作用 与肌肉减少症有关。根据12/15-LO缺失小鼠和衰老小鼠的初步数据，他 提出了一个假设，即12/15-LO通过调节肌肉萎缩，在与年龄相关的肌肉萎缩中起着重要作用。 泛素-蛋白酶体和蛋白质降解的溶酶体自噬途径。这将通过以下方式进行测试： 体外和体内方法的组合。在特定目标1中，体外原代肌管培养和 分离的肌肉制备物将用于确定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。由于蛋白质的诱导 降解途径受PI 3 K/Akt信号传导和下游mTOR和FoxO 3途径调节， NF-B的活化，这些途径将在细胞中和在来自野生型的分离的肌肉制备物中测量。 (with/没有PI 3 K/Akt抑制剂）和MISR小鼠（具有NF-κ B的肌肉特异性抑制的小鼠）来确定 PI 3 K/Akt和NF-B信号通路的相对作用。下游溶酶体自噬的激活 将通过测量自噬蛋白（Atg-7、LC-3 II/LC-3 I、p62）的表达来确定途径， 通过测量途径的自噬通量和泛素-蛋白酶体途径的活化， 蛋白酶体活性和蛋白酶体降解泛素标记的GFP底物的能力。在特定 目的2：利用坐骨神经切断模型，研究12/15-LO抑制是否能保护坐骨神经损伤。 通过调节蛋白质降解途径对抗去神经诱导的肌肉萎缩。12的水平- 和15-HETE，PI 3 K/Akt信号传导和下游mTOR和FoxO 3通路，NF-B的激活，标记物 诱导溶酶体自噬和泛素-蛋白酶体途径在控制和失神经支配 将在该特定目标中测量野生型和12/15-LO缺失小鼠的肌肉。最后，假设将 在体内进行测试，以确定在衰老过程中抑制12/15-LO途径是否可以防止 通过调节蛋白质降解途径来调节肌肉质量和功能。与肌肉萎缩相关的肌肉萎缩， 功能丧失，12-和15-HETE水平，PI 3 K/Akt信号传导和下游mTOR和FoxO 3通路， NF-B的激活，以及溶酶体自噬和泛素-蛋白酶体诱导的标志物 在野生型和12/15-LO敲除小鼠中以及在用药物抑制剂治疗的小鼠中， 12/15-LO将在此特定目标中进行测量。总而言之，这项建议涉及的研究领域， 在老龄化领域具有高度相关性。已经提出的研究将是第一个审查 来自脂质介质（如HETE）的信号传导在肌肉萎缩中的潜在作用;因此， 在这个项目中有一些基本的新发现，这些发现可能会对未来产生长期影响。 提高老龄人口的生活质量。

Project summary/abstract: 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-kB) 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 NF-¿B, 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.