Role of autophagy and other downstream effectors in lifespan extension by teh GCN4/ATF4 pahtway

我们现在知道了多种延缓衰老的途径，并通过AS延长了一些动物的寿命 几乎是原来的10倍，然而我们并不完全了解延缓衰老的最终机制。 到目前为止。因此，我们不知道自然老化所涉及的潜在变化，可能是抵消的或延迟的 通过我们的干预。长期目标是了解延缓衰老的最下游变化。 这将允许一种合理的方法来开发干预措施，以广泛推迟多发性人类 老年性疾病，如阿尔茨海默病和癌症。这个应用程序的总体目标是下一个 追求长期目标步骤是了解延缓衰老的下游效应，特别是在 新的Gcn4途径。中心假设是保守的转录因子Gcn4的靶标很可能是 效应者，其中最相关的将由多个不同的依赖Gcn4的干预措施共享，以及 自噬和蛋白质周转增加可能起到关键作用。建议的理由是 研究有两个方面：1)一旦我们知道了Gcn4介导的延缓衰老的影响因素，它们可以直接 操纵，给出了预防和治疗人类衰老疾病的方法；2)功能上的 一组经过验证的效应器使我们能够询问这些效应器是否被其他路径共享，并将揭示 不完全自然老化的变化。在我们初步数据的指导下，这将通过以下三个方面进行测试 具体目标：1)确定线粒体翻译缺失对Gcn4的依赖；2)确定 自噬在Gcn4介导的延缓衰老中的作用；以及3)识别和验证功能上最相关的 GCN4/ATF-5的转录靶点。作为第二个目标的一部分，我们将使用UNM AIM科布雷核心阿姆尼斯 ImagestStream同时测量数以千计的年龄、自噬水平和Gcn4蛋白水平 酵母。申请人认为，这种方法是创新的，因为它代表了一种新的和实质性的 通过在多个不同的干预措施后评估转录输出来偏离现状 依赖于相同的转录因子，使用了一种新颖的区块设计RNASeq-ANOVA模型的变化 转录水平，以便特别关注那些与我们的延迟表型相关的转录变化 老化的同时排斥他人的。它还通过两个寿命测量实验进行验证 远亲模式生物，询问哪些变化最保守，因此最有可能 与人类生物学相关的。这项拟议的研究意义重大，因为对 延缓衰老的下游效应物将识别在预防中具有翻译重要性的药物靶点 以及治疗多种人类衰老疾病。这些发现的潜在影响从以下几个方面得到了强调 我们现在不能排除这样一种可能性，即多种已知的延缓衰老的途径，如 热量限制、胰岛素/IGF1信号转导和TOR都在很大程度上通过改变 自噬，我们也不能排除所有这些途径在某种程度上通过汇聚到Gcn4来实现这一点。

We now know multiple pathways that delay aging, and have extended the lifespan of some animals by as much as 10-fold, yet we do not fully understand the final mechanistic effectors of delayed aging in any example to date. Thus we do not know the underlying changes involved in natural aging, presumably offset or delayed by our interventions. The long-term goal is to is to understand the most downstream changes in delayed aging. This will allow a rational approach to developing interventions to broadly delay the onset of multiple human diseases of aging such as Alzheimer’s disease and cancer. The overall objective in this application, the next step in pursuit of that long-term goal, is to understand downstream effectors of delayed aging specifically in the novel Gcn4 pathway. The central hypothesis is that targets of conserved transcription factor Gcn4 are the likely effectors, that the most relevant of these will be shared by multiple distinct Gcn4-dependent interventions, and that autophagy and increased protein turnover are likely to play a key role. The rationale for the proposed research is two-fold: 1) once we know the effectors of Gcn4-mediated delayed aging, they can be directly manipulated, giving approaches for prevention and treatment of human diseases of aging; 2) A functionally validated set of effectors allows us to ask if these are shared by other pathways, and will also shed light on the changes that underly natural aging. Guided by our preliminary data, this will be tested by pursuing three specific aims: 1) Determine the Gcn4-dependence of mitochondrial translational deletions; 2) Determine the role of autophagy in Gcn4-mediated delayed aging; and 3) Identify and validate the most functionally relevant transcriptional targets of Gcn4 / ATF-5. In part of the second aim we will use the UNM AIM CoBRE Core Amnis Imagestream to simultaneously measure the age, autophagy level, and Gcn4 protein level of thousands of yeast. The approach is innovative, in the applicant’s opinion, because it represents a new and substantive departure from the status quo byassaying transcriptional output following multiple distinct interventions dependent on the same transcription factor, using a novel block design RNAseq-ANOVA model of changes in transcript levels, in order to focus specifically on those transcriptional changes tied to our phenotype of delayed aging while excluding others. It also uses validation through lifespan measurements experiments in two distantly related model organisms, to ask which changes are most conserved, and thus most likely to be relevant to human biology. The proposed research is significant, because a fuller understanding of the downstream effectors of delayed aging will identify drug targets with translational importance in the prevention and treatment of multiple human diseases of aging. The potential impact of these findings is underscored by the fact that we cannot now preclude the possibility that multiple known pathways that delay aging, such as caloric restriction, Insulin / IGF1 signaling, and TOR, all effect their phenotype largely through changes in autophagy, nor can we rule out that all of these pathways do this in some part by converging on Gcn4.