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.

Public Health Relevance

Autophagy performs numerous essential roles in cellular homeostasis, leading us to hypothesize that a sophisticated regulatory network likely controls autophagy activation status, and prompting us to pursue key regulators of autophagy in unbiased screens and in directed studies focusing on a recently uncovered master transcriptional regulator, TFEB. All of this work during the initial funding cycle for this grant yielded multiple provocative insights into autophagy regulation, including the discovery of let-7 induction as sufficient to promote autophagy activation by repressing the newly defined amino acid sensing pathway (which activates mTORC1), identification of MAP4K3 as the kinase that may be principally responsible for TFEB localization to the lysosome and its inactivation, and delineation of how TFEB transcription regulation is controlled and how TFEB dysregulation occurs in neurodegenerative disease. In this renewal project, we wish to continue these studies and define the transcriptional basis for let-7 induction in response to nutrient stress, determine f and how MAP4K3 phosphorylation of TFEB is a crucial step in autophagy regulation, and examine let-7 and MAP4K3 expression modulation as potential approaches for beneficial manipulation of autophagy in neurons, especially in the context of neurodegenerative disease.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
2R01AG033082-06
Application #
8973096
Study Section
Cellular and Molecular Biology of Neurodegeneration Study Section (CMND)
Program Officer
Wise, Bradley C
Project Start
2008-12-01
Project End
2020-05-31
Budget Start
2015-09-01
Budget End
2016-05-31
Support Year
6
Fiscal Year
2015
Total Cost
$412,722
Indirect Cost
$146,450
Name
University of California San Diego
Department
Pediatrics
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Dickey, Audrey S; Sanchez, Dafne N; Arreola, Martin et al. (2017) PPAR? activation by bexarotene promotes neuroprotection by restoring bioenergetic and quality control homeostasis. Sci Transl Med 9:
Tsai, Shih-Yin; Rodriguez, Ariana A; Dastidar, Somasish G et al. (2016) Increased 4E-BP1 Expression Protects against Diet-Induced Obesity and Insulin Resistance in Male Mice. Cell Rep 16:1903-14
Dickey, Audrey S; Pineda, Victor V; Tsunemi, Taiji et al. (2016) PPAR-? is repressed in Huntington's disease, is required for normal neuronal function and can be targeted therapeutically. Nat Med 22:37-45
Tsai, Shihyin; Sitzmann, Joanna M; Dastidar, Somasish G et al. (2015) Muscle-specific 4E-BP1 signaling activation improves metabolic parameters during aging and obesity. J Clin Invest 125:2952-64
Cortes, Constanza J; La Spada, Albert R (2015) Autophagy in polyglutamine disease: Imposing order on disorder or contributing to the chaos? Mol Cell Neurosci 66:53-61
Dubinsky, Amy N; Dastidar, Somasish Ghosh; Hsu, Cynthia L et al. (2014) Let-7 coordinately suppresses components of the amino acid sensing pathway to repress mTORC1 and induce autophagy. Cell Metab 20:626-38
Cortes, Constanza J; La Spada, Albert R (2014) The many faces of autophagy dysfunction in Huntington's disease: from mechanism to therapy. Drug Discov Today 19:963-71
McCormick, Mark A; Mason, Amanda G; Guyenet, Stephan J et al. (2014) The SAGA histone deubiquitinase module controls yeast replicative lifespan via Sir2 interaction. Cell Rep 8:477-86
Cortes, Constanza J; Miranda, Helen C; Frankowski, Harald et al. (2014) Polyglutamine-expanded androgen receptor interferes with TFEB to elicit autophagy defects in SBMA. Nat Neurosci 17:1180-9
Sekiyama, Kazunari; Waragai, Masaaki; Akatsu, Hiroyasu et al. (2014) Disease-Modifying Effect of Adiponectin in Model of ?-Synucleinopathies. Ann Clin Transl Neurol 1:479-489

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