Molecular genetic regulation of autophagy in health and neurodegenerative disease

联系PD/PI：LA SPADA，ALBERT 自噬激活在细胞中基于营养可用性和细胞应激而受到严格调控。为 mTOR信号通路作为代谢信息和细胞应激整合的焦点， 自噬途径活性的主要仲裁者是mTORC 1复合物。鉴于许多重要作用， 细胞内稳态，我们假设自噬会受到复杂的调控， 在过去的5年里，我们已经定义了发生在上游和下游的关键监管节点， mTORC 1复合体当我们询问接受营养物质的初级神经元的转录组时， 在剥夺中，我们发现microRNA的let-7家族的成员表现出显著的上调。 然后，我们确定let-7通过抑制基因的表达来激活神经元自噬， 包括最近描述的氨基酸传感途径，其包括Ras相关的GTP结合蛋白家族， 蛋白质（RagA/B/C/D）、MAP激酶（MAP 4 K3）和5种锚mTORC 1的蛋白质（LAMTOR 1/2/3/4/5 到溶酶体。在定义自噬的转录调控方面的另一个重要进展是， Ballabio小组发现转录因子EB（TFEB）在促进自噬中的主要作用。 尽管TFEB的mTORC 1磷酸化已经成为控制TFEB功能的关键步骤， 自噬激活，我们已经发现了一个意想不到的关键作用MAP 4K 3作为一个主要的调节， 证明其对TFEB的磷酸化决定TFEB的定位和活性。正如我们也 我们的研究表明，TFEB调节对于CNS中的蛋白质稳态至关重要， 这个项目的一段时间已经推进了我们对控制自噬的调控网络的理解 通路及其与CNS稳态的潜在生理相关性。在这项重建计划中，我们建议 定义响应营养胁迫激活let-7并由此启动 自噬诱导基于将MAP 4K 3置于TFEB溶酶体上游的挑衅性初步数据 定位和mTORC 1磷酸化，我们将确定是否在特定的时间点MAP 4K 3磷酸化TFEB。 丝氨酸负责其定位于溶酶体和失活，我们将研究其意义 这种TFEB PTM在决定自噬激活状态中的作用。最后，我们将评估 调节let-7和MAP 4K 3表达以调节CNS中的自噬活化， 调节可以代表开发新的自噬诱导疗法的可行途径。 项目摘要/摘要第6页

Contact PD/PI: LA SPADA, ALBERT Autophagy activation is tightly regulated in the cell based upon nutrient availability and cell stress. As the mTOR signaling pathway serves as a focal point for integration of metabolic information and cell stress, the principal arbiter of autophagy pathway activity is the mTORC1 complex. In light of numerous essential roles in cellular homeostasis, we hypothesized that autophagy would be subject to sophisticated regulatory control, and over the last 5 years, we have defined key regulatory nodes that occur both upstream and downstream of the mTORC1 complex. When we interrogated the transcriptomes of primary neurons subjected to nutrient deprivation, we discovered that members of the let-7 family of microRNAs exhibited marked up-regulation. We then determined that let-7 activates neuronal autophagy by repressing the expression of genes that comprise a recently delineated amino acid sensing pathway that includes a family of Ras-related GTP-binding proteins (RagA/B/C/D), a MAP kinase (MAP4K3), and five proteins (LAMTOR 1/2/3/4/5) that anchor mTORC1 to the lysosome. Another important advance in defining the transcriptional regulation of autophagy was the Ballabio group's discovery of a principal role for transcription factor EB (TFEB) in promoting autophagy. Although mTORC1 phosphorylation of TFEB has emerged as a key step in control of TFEB function and autophagy activation, we have discovered an unexpectedly crucial role for MAP4K3 as a major regulator, by demonstrating that its phosphorylation of TFEB determines TFEB localization and activity. As we have also shown that TFEB regulation is critically important for proteostasis in the CNS, our studies during the initial period of this project have advanced our understanding of the regulatory network that controls the autophagy pathway and its potential physiological relevance for CNS homeostasis. In this renewal project, we propose to define the transcription regulatory network that responds to nutrient stress to activate let-7 and thereby initiate autophagy induction. Building on provocative preliminary data placing MAP4K3 upstream of TFEB lysosomal localization and mTORC1 phosphorylation, we will determine if MAP4K3 phosphorylation of TFEB at a specific serine is responsible for its localization to the lysosome and inactivation, and we will examine the significance of this TFEB PTM in dictating autophagy activation status. Finally, we will evaluate the potential utility of modulating let-7 and MAP4K3 expression to regulate autophagy activation in the CNS to establish if such modulation could represent a viable path to the development of novel autophagy-inducing therapies. Project Summary/Abstract Page 6