Role of LIPL-4 in lysosomal lipolysis and aging

描述(由申请人提供)：自噬/溶酶体途径的功能障碍在年龄相关疾病的发展中具有重要的病理学意义，例如神经退化。在线虫中，几种长寿模型依赖于增加的自噬来延长寿命，这表明自噬在衰老过程中发挥了关键作用。当营养传感器TOR--自噬的负调节因子--被抑制时，动物也可以享受更长的寿命。尽管如此，自噬是如何调节其有益影响的，人们却知之甚少。我们最近报道，过度表达溶酶体脂肪酶LIPL-4可以诱导自噬，导致寿命显著延长，脂肪分解增强，TOR信号改变，提示脂代谢、自噬和衰老之间存在联系。LIPL-4与人溶酶体酸性脂肪酶(LAL)有很强的同源性，LAL是通过自噬降解胆固醇的关键酶。值得注意的是，LAL介导的胆固醇处理受损与阿尔茨海默病的发展有关。我的新结果表明，过度表达lipl-4可以缓解线虫阿尔茨海默病模型的A？毒性。因此，这项提议将检验Lipl-4，类似于LAL，介导溶酶体脂水解的假说，并旨在阐明Lipl-4如何调节自噬和减轻A？毒性。在目标1中，我将确定Lipl-4在细胞内的作用部位，并确定其与TOR信号的关系。在目标2中，我将测试LIPL-4和LAL在线虫中是否在功能上可互换。LIPL-4诱导自噬和调节衰老的作用机制也将被阐明。在目标3中，我将研究Lipl-4是如何调节线虫阿尔茨海默病发病延迟的。我还将进行高通量筛选(HTS)，以发现激活LAL介导的脂解作用的新的和特定的候选基因，作为一种对抗神经退化的策略。通过确定溶酶体脂肪分解在衰老中的作用，我的提议将提供一个基础，在此基础上可以发现预防阿尔茨海默病的新药。为期2年的博士后K99阶段将包括对Lipl-4在溶酶体功能、脂代谢和衰老中的作用进行表征。与HTS兼容的基于细胞的检测报告系统将被用于寻找增强LAL表达的新药。为期3年的独立R00阶段将有助于进一步了解Lipl-4在溶酶体脂解、脂质信号和衰老中的作用，并扩展到脂质动力学、代谢和蛋白平衡的研究。LAL的主要候选激活剂将在线虫中使用阿尔茨海默病模型和细胞培养模型进行验证。这项建议包括前沿方法，如蛋白质组分析、CARS显微镜和HTS，并结合线虫疾病模型的创新使用。总而言之，K99/R00的资助对我来说是一个独特的机会，让我学习新技术和发展我的专业技能，成功地过渡到老龄研究领域的独立科学家。

DESCRIPTION (provided by applicant): Dysfunctions in the utophagy/lysosomal pathway are pathologically significant in the development of age-related diseases, such as neurodegeneration. In C. elegans, several longevity models rely on increased autophagy for lifespan extension, suggesting a critical role for autophagy in aging. Animals can also enjoy longer lifespan when the nutrient-sensor TOR, a negative regulator of autophagy, is inhibited. Nonetheless, how autophagy mediates its beneficial effects is poorly understood. We recently reported that autophagy could be induced by over-expressing the putative lysosomal lipase LIPL-4, which resulted in a significant lifespan extension, enhanced lipolysis and altered TOR signaling, suggesting a link between lipid metabolism, autophagy and aging. LIPL-4 displays strong homology with human lysosomal acid lipase (LAL), a key enzyme in the hydrolysis of cholesterol via autophagy. Notably, impaired LAL-mediated cholesterol processing has been linked to the development of Alzheimer's disease. My new results show that over-expressing LIPL-4 ameliorates A¿ toxicity in a C. elegans model of Alzheimer's disease. Therefore, this proposal will test the hypothesis that LIPL-4, similar to LAL, mediates lysosomal lipid hydrolysis and will aim to elucidate how LIPL-4 modulates autophagy and mitigates A¿ toxicity. In Aim 1, I will confirm the intracellular site of action of LIPL-4 and determine its relationship to TOR signaling. In Aim 2, I will test whether LIPL-4 and LAL are functionally interchangeable in C. elegans. The mechanism of action by which LIPL-4 induces autophagy and modulates aging will also be elucidated. In Aim 3, I will investigate how LIPL-4 mediates a delay in the onset of Alzheimer's disease in C. elegans. I will also perform a high-throughput screen (HTS) to discover novel and specific candidate that activates LAL-mediated lipolysis, as a strategy against neurodegeneration. By determining the role of lysosomal lipolysis in aging, my proposal will provide a basis on which novel drugs can be discovered to prevent Alzheimer's disease. The 2-year postdoctoral K99 phase will consist in the characterization of the role of LIPL-4 in lysosomal function, lipid metabolism and aging. Cell-based assay reporter systems compatible with HTS will be used to find novel drugs to enhance LAL expression. The 3-year independent R00 phase will serve to further understand the role of LIPL-4 in lysosomal lipolysis, lipid signaling and aging and expand into studies on lipid dynamics, metabolism and proteostasis. Lead candidate activators of LAL will be validated using Alzheimer's disease model in C. elegans and cell culture models. This proposal includes cutting-edge approaches, such as proteomic analyses, CARS microscopy and HTS combined with the innovative use of disease models in C. elegans. In summary, the K99/R00 grant represents a unique opportunity for me to learn new technologies and develop my professional skills to successfully transition into an independent scientist in aging research.