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The Role of Lysosomal Cholesterol Transport in Cellular Nutrient Sensing and Organelle Homeostasis

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The Role of Lysosomal Cholesterol Transport in Cellular Nutrient Sensing and Organelle Homeostasis The Role of Lysosomal Cholesterol Transport in Cellular Nutrient Sensing and Organelle Homeostasis by Oliver Brayer Davis A dissertation submitted in partial satisfaction of the requirement for the degree of Doctor of Philosophy in Molecular and Cell Biology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Roberto Zoncu, Chair Professor Jeremy W. Thorner Professor Elçin Ünal Professor Daniel K. Nomura Summer 2020 Abstract The Role of Lysosomal Cholesterol Transport in Cellular Nutrient Sensing and Organelle Homeostasis by Oliver Brayer Davis Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor Roberto Zoncu, Chair Lysosomes are the main catabolic organelles of a eukaryotic cell and are critical for maintenance of cellular homeostasis. Macromolecules, organelles captured via autophagy, and material taken up by endocytosis are degraded in lysosomes. Consequently, lysosomes concentrate, store, recycle, and distribute metabolites crucial for biosynthetic processes. The lysosome also functions as a platform for regulating and coordinating signaling pathways. In particular, a master regulator of cell growth and proliferation – the mechanistic target of rapamycin complex 1 (mTORC1), an evolutionarily conserved serine/threonine protein kinase – is activated at the surface of the lysosome when nutrients are plentiful (e.g., amino acids potently promote mTORC1 activity). Cholesterol, a key component of biomembranes, also stimulates mTORC1 recruitment and activation at the lysosome. Thus, by integrating its degradative and signaling roles, the lysosome serves as a hub for nutrient sensing. In diseases known as lysosomal storage disorders, pathogenic levels of a particular metabolite accumulate in the lysosome due to the loss of function of a human gene required for catabolism or transport of a substrate normally digested in the lysosome. In Neimann-Pick type C (NPC) disease, the lysosomal cholesterol exporter, NPC1, is inoperative, causing accumulation of cholesterol within lysosomes, resulting in disruption of lysosomal function, which is propagated to other organelles (e.g., mitochondria) also compromising their function. Ultimately, this damage leads to progressive neurodegeneration in patients. The accumulation of cholesterol caused by loss of NPC1 also chronically hyperactivates mTORC1, further interfering with the regulation of cellular homeostasis. To increase our understanding of the factors that cause lysosomal failure in NPC disease, I examined the compositional and functional alterations that occur in lysosomes lacking NPC1 activity. Likewise, I studied how NPC1 loss triggers aberrant mTORC1 signaling and how dysregulated mTORC1 contributes to organelle pathogenesis. I first used organelle immuno-isolation in conjunction with proteomic profiling to uncover a pronounced proteolytic impairment of NPC lysosomes that is compounded by depletion of luminal hydrolases and enhanced susceptibility to 1 membrane damage. I also tested a panel of NPC1 mutants in NPC1-deficient cells and demonstrated that, cholesterol transport by NPC1 is tightly linked to regulation of mTORC1 activity, indicating that lysosomal cholesterol accumulation is the primary underlying cause of mTORC1 hyperactivation in NPC disease. Next, I demonstrated that genetic and pharmacologic mTORC1 inhibition restores lysosomal proteolysis and lysosomal membrane integrity without correcting cholesterol accumulation, implicating mTORC1 hyperactivity as a main driver of downstream pathogenesis, including the loss of mitochondrial quality control and function. In agreement with those conclusions, I showed that mTORC1 inhibition reverses lysosomal and mitochondrial dysfunction in a neuronal model of NPC, extending my findings to a disease-relevant cellular context. Thus, targeting the cholesterol-mTORC1 signaling pathway may represent a novel therapeutic avenue in NPC that could complement or replace current approaches. 2 Table of Contents Abstract…………………………………………………………………………………………..1 Table of Contents……………………………………………………………………………….. i List of Figures…………………………………………………………………………………… iii Acknowledgements……………………………………………………………………………. iv Chapter 1: Introduction to Lysosome Structure, Function, and Role in Cellular Cholesterol Sensing by mTORC1 1.1 Lysosome structure and catabolic function in the cell………………………... 2 1.1.1 Structural organization of the lysosome………………………………………. 2 1.1.2 The lysosome is a catabolic hub of the cell…………………………………... 3 1.2 Lysosomes and mTORC1…………………………………………………………… 7 1.2.1 Regulation of cellular metabolism by mTORC1………………………………. 8 1.2.2 Cellular signaling upstream of mTORC1 activation………………………….. 9 1.3 Lysosomes and Disease…………………………………………………………… 13 1.3.1 Lysosomal Storage Disorders………………………………………………… 13 1.3.2 Lysosomal Function, Autophagy, and Neurodegeneration………………... 13 1.4 Lysosomes and Cholesterol………………………………………………………. 14 1.4.1 The role and sources of cellular cholesterol………………………………… 14 1.4.2 NPC1 and NPC2-mediated cholesterol export from the lysosome………. 15 1.4.3 Other routes of lysosomal cholesterol transport……………………………. 16 1.4.4 Cholesterol sensing by mTORC1…………………………………………….. 18 Chapter 2: NPC1-mTORC1 Signaling Couples Cholesterol Sensing to Organelle Homeostasis and is a Targetable Pathway in Niemann-Pick Type C 2.1 Chapter Summary…………………………………………………………………… 21 2.2 Introduction…………………………………………………………………………... 21 2.3 Results………………………………………………………………………………… 23 2.3.1 NPC1-null lysosomes display extensive proteolytic defects……………… 23 2.3.2 NPC1-null lysosomes display increased susceptibility to membrane damage………………………………………………………………………………… 28 2.3.3 NPC1 regulates mTORC1 via its cholesterol-exporting function…………. 33 2.3.4 mTORC1 inhibition restores integrity and proteolytic function of NPC lysosomes downstream of cholesterol storage……………………………………. 36 2.3.5 mTORC1 inhibition restores lysosomal function in iPSC-derived NPC neuronal cultures……………………………………………………………………… 38 2.3.6 mTORC1 inhibition restores defective mitochondrial function in NPC1-null cells……………………………………………………………………………………... 40 2.4 Discussion……………………………………………………………………………. 43 2.5 Methods………………………………………………………………………………. 46 i 2.5.1 Mammalian Cell Culture……………………………………………………….. 46 2.5.2 Cloning and Generation of Stable Cell Lines………………………………... 47 2.5.3 Lysosome Immunoprecipitation (Lyso-IP)…………………………………… 47 2.5.4 Proteomics Analysis……………………………………………………………. 48 2.5.5 Immunofluorescence……………………………………………………………48 2.5.6 Microscopy……………………………………………………………………… 49 2.5.7 Image analysis………………………………………………………………….. 49 2.5.8 Measurement of GALC activity……………………………………………….. 49 2.5.9 Cholesterol starvation and replenishment…………………………………… 50 2.5.10 Cell lysis and immunoblotting……………………………………………….. 50 2.5.11 Cholesterol labeling in situ with D4H*-mCherry and filipin………………. 50 2.5.12 Measurement of lysosomal permeability…………………………………… 51 2.5.13 hIPSC generation and neuronal differentiation……………………………. 51 2.5.14 Generation of NPC1 knock-out hIPSCs……………………………………. 51 Chapter 3 Characterization of NPC2 function in regulation of mTORC1 activity 3.1 Chapter Summary…………………………………………………………………… 54 3.2 Introduction…………………………………………………………………………... 54 3.3 Results………………………………………………………………………………… 55 3.3.1 Loss of NPC2 causes cholesterol accumulation in the lysosomal limiting membrane and render mTORC1 cholesterol insensitive…………………………. 55 3.3.2 Lysosomal and mitochondrial perturbations caused by loss of NPC2 are remediated by inhibition of mTORC1………………………………………………..56 3.4 Discussion……………………………………………………………………………. 57 3.5 Methods………………………………………………………………………………. 59 References……………………………………………………………………………….. 60 ii List of Figures Figure 1.1 Structural organization of the lysosome and routes of macromolecular delivery………………………………………………………………………. 6 Figure 1.2 Logic of the mTORC1 signaling network and its regulation of cellular metabolism…………………………………………………………………………….. 8 Figure 1.3 Molecular signaling pathways upstream of mTORC1………………………... 11 Figure 1.4 Cholesterol transport pathways of the endolysosomal system…………….. 17 Figure 2.1 Lysosome proteomics reveals a proteolytic defect in NPC1-deficient lysosomes…………………………………………………………………... 24 Figure 2.2 Immunofluorescence reveals undigested autophagic material in lumen of NPC1-deficient lysosomes………………………………………………………...26 Figure 2.3 Cholesterol export by NPC1 mutants alleviates proteolytic block in NPC1-deficient cells………………………………………………………………………….. 27 Figure 2.4 NPC1-deficient lysosomes also have reduced levels of luminal hydrolases…………………………………………………………………………….. 28 Figure 2.5 NPC1-deficient cells have elevated levels of lysosome- associated ESCRT-III…………………………………………………………………………. 29 Figure 2.6 NPC1-deficient cells are more susceptible to lysosomal rupture…………... 31 Figure 2.7 Cholesterol export by NPC1 mutants restores lysosomal membrane integrity…………………………………………………………………………… 32 Figure 2.8 Transport by NPC1 controls mTORC1 activity in response to lysosomal cholesterol………………………………………………………………………… 34 Figure 2.9 Inhibition of mTORC1 activity alleviates lysosomal proteolysis defect associated with loss of NPC1……………………………………………………….. 35 Figure 2.10 Inhibition of mTORC1 activity reduces ESCRT-III accumulation on NPC1-deficient lysosomes……………………………………………………………….. 37 Figure 2.11 Validation of iPSC-derived neural lineage lines……………………………… 39 Figure 2.12 Inhibition of mTORC1 corrects lysosomal defects associated with loss of NPC1 in and iPSC-derived neuronal cell model…………………………….. 40 Figure 2.13 Mitochondrial
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