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.