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

项目摘要/摘要：Arunabh Bhattacharya博士的长期职业目标是将自己确立为 通过持续的基础研究和翻译研究，成为一名成功且资金充足的独立调查员 衰老领域，特别是肌肉生物学领域。他在该地区接受的博士和博士后培训 炎症性疾病和氧化应激为这一职业发展奖奠定了基础 申请。到目前为止，他的研究生涯一直很有成效，在同行杂志上发表了38篇论文。 审阅了期刊。作为公务员事务局/Barshop长寿和老龄研究所系的教员 在圣安东尼奥的研究中，他一直在研究炎症所产生的脂质介质的作用 肌肉萎缩的途径，这是UTHSCSA的一个新的研究方向。Barshop Institute有一家 全国最大的老龄化生物学项目，为初级教师提供了极好的机会 在老龄化研究中提供指导。Arunabh正走在长期独立研究领域的道路上 肌肉生物学和K01职业发展奖将为他提供实现这一目标的机制 过渡。他是在Van Remman博士和Musi博士的联合指导下提交这份提案的 酒吧学院的教职员工。范·雷曼博士是氧化应激领域的权威人士 以及它在衰老中的作用，埃里森基金会比森学者穆西博士研究了 骨骼肌炎症途径在胰岛素抵抗和衰老中的作用。整个培训计划包括 在这个K01奖项中概述的包括实际实验室培训和教学课程工作的混合以及 与导师/共同导师和咨询委员会的其他成员进行密集的辅导互动。在……里面 此外，培训方案旨在提供其他方面的宝贵研究经验，包括 管理研究预算，独立运营实验室，建立合作伙伴关系和撰写赠款 向国家资助机构提出的申请，包括肌营养不良协会和独立的R01 申请国家老龄研究所。这些因素加在一起，将确保他成功过渡到 在老龄化和与老龄化相关的生活质量变化领域的独立研究员。 通过与他的导师/共同导师、咨询委员会成员和 已经同意在这项建议中协助阿鲁纳赫，K01奖将使他在各种 这些技术将为他在肌肉生物学领域的继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 K01拨款建议研究促炎12/15-脂氧合酶(12/15-LO)途径在老年性痴呆中的作用 相关的石棺减少症。基于12/15-LO小鼠和衰老小鼠的初步数据，他已经 提出了一个假说，即12/15-LO在年龄相关性肌肉萎缩中起着重要作用，它通过调节 泛素-蛋白酶体与蛋白质降解的溶酶体自噬途径。这将使用以下工具进行测试 体外和体内方法的结合。在特定目标1中，体外原代肌管培养和 分离的肌肉制剂将被用来确定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。因为蛋白质的诱导 降解途径受PI3K/Akt信号以及下游的mTOR和FOXO_3途径的调控。 核因子-B的激活，这些通路将在细胞和野生型的分离肌肉制剂中进行测量 (使用/不使用PI3K/Akt抑制剂)和MISR小鼠(肌肉特异性抑制核因子-kB的小鼠)确定 PI3K/Akt和核因子-βB信号通路的相对作用。下游溶酶体自噬的激活 途径将通过检测自噬蛋白(ATG-7，LC-3II/LC-3I，p62)和 通过检测泛素-蛋白酶体途径的自噬通量和激活 蛋白酶体的活性和降解泛素标记的GFP底物的能力。具体而言 目的：建立坐骨神经切断模型，以确定抑制12/15-LO是否具有保护作用 通过调节蛋白质降解途径来对抗失神经引起的肌肉萎缩。12岁的水平- 和15-HETE，PI3K/Akt信号通路和下游的mTOR和FOXO_3通路，核因子-B的激活，标志物 控制和失神经状态下溶酶体自噬和泛素-蛋白酶体通路的诱导 野生型和12/15 LO缺失型小鼠的肌肉将在这一特定目标下进行测量。最后，这一假设将 在体内进行测试，以确定在衰老过程中抑制12/15-LO通路是否可以防止 通过调节蛋白质降解途径来调节肌肉质量和功能。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FOXO_3通路， 核因子-βB的激活，以及诱导溶酶体自噬和泛素蛋白酶体的标志 野生型和12/15-LO基因敲除小鼠以及经药物抑制剂治疗的小鼠中的通路 12/15-LO将在此特定目标中进行测量。总而言之，这项建议涉及一个研究领域， 在老龄化领域具有很高的相关性。已经提出的研究将是第一个检查 HETE等脂质介质的信号在肌肉萎缩中的潜在作用；因此有机会 在这个项目中有一些基本的新发现，这些发现可能会对 提高老龄化人口的生活质量。

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.