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Identifying the regulators of vesicle fusion events in autophagy using human stem cells

Principal Supervisor: Dr. Sovan Sarkar - Institute of Cancer and Genomic Sciences

Co-supervisor: Prof. Jon Frampton

PhD project title: Identifying the regulators of vesicle fusion events in autophagy using human stem cells

University of Registration: University of Birmingham

Project outline:

Background: The proteolytic system plays a fundamental role in cellular functioning and survival, deregulation of which leads to various diseases including cancer and neurodegeneration. One central node implicated in diverse human physiological and pathological conditions is autophagy, an intracellular degradation pathway for aggregation-prone proteins and unwanted organelles that would otherwise cause cell death upon accumulation when this process malfunctions. Therefore, autophagy is critical for organismal health by maintaining cellular and energy homeostasis. This process involves multiple vesicle fusion events through the generation of double- membrane vesicles called autophagosomes that are destined to fuse with the lysosomes forming autolysosomes where the autophagic cargo is degraded. This generally occurs via two routes: (i) Multistep route where autophagosomes first fuse with endosomes to form amphisomes, which then fuse with lysosomes forming autolysosomes; (ii) Direct route where autophagosomes fuse with lysosomes to form autolysosomes1. Autophagic vesicle fusion is mediated by SNARE proteins on the target membranes, such as the autophagosomal STX17 forming a SNARE complex with late endosomal/lysosomal VAMP8 along with ATG14 and SNAP29 to facilitate autophagosome maturation2. However, this SNARE complex only mediates the multistep route since our published and new data point to the existence of yet-to-be- identified SNAREs mediating the direct route3.

Question: This proposal addresses a core issue related to the mechanistic control of autophagy: What are the mediators of direct autophagosome-lysosome fusion? We hypothesize the need to directly capture this vesicle fusion event, which can be achieved by using Niemann-Pick type C1 (NPC1) mutant cells where we have previously shown that the multistep route is blocked3. Stimulation of autophagy in this context restored autophagic flux by mediating autophagosome-lysosome fusion via the direct route independent of amphisome and STX17-VAMP8 complex formation.

Objectives: (1) Identify and characterize the novel SNAREs using NPC1 mutant mouse cells. (2) Validate their function via CRISPR/Cas9-mediated gene knockout in NPC1 human induced pluripotent stem cells4 (hiPSCs). (3) Investigate their role in autophagosome maturation in physiologically-relevant human cellular contexts using human embryonic stem cells (hESCs).

Methodology: We have undertaken genetic screens with 52 mammalian SNARE siRNAs in Npc1 mutant mouse cell lines expressing autophagy reporters. Our preliminary data has identified 2 potential genes regulating direct autophagosome- lysosome fusion. These will be further characterized through molecular, biochemical and cell biology techniques for their ability in membrane tethering and vesicle fusion. We will also identify their binding partners and the specific SNARE complex by mass spectrometry. We will then employ hESCs/hiPSCs for their ability to differentiate into adult cell types with isogenic background. Loss of SNARE function in abrogating the direct autophagosome maturation route will be validated in NPC1 hiPSC model via CRISPR/Cas9-mediated gene knockout. We will undertake similar knockout strategy in physiologically-relevant human context using genome-engineered, autophagy reporter hESC lines. Functional analysis will be done in these hESCs and hESC- derived neurons and hepatic cells on autophagosome-lysosome fusion by cell biological and biochemical methods including mathematical approaches to quantify autophagic flux.

Outcome: Understanding the regulators of autophagosome maturation will provide drug targets for improving defective autophagic flux in age-related pathologies like neurodegeneration. Our findings will thus be of basic and biomedical relevance.

References:

  1. Sarkar S. Biochem Soc Trans 41:1103 (2013);
  2. Itakura E. et al. Cell 151:1256 (2012);
  3. Sarkar S. et al. Cell Rep 5:1302 (2013);
  4. Maetzel D., Sarkar S. et al. Stem Cell Rep 2:866 (2014).

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

Culture of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs); differentiation of hESCs/hiPSCs into fibroblasts, neural precursors, neurons and hepatic cells; genome engineering using CRISPR/Cas9 system for creating gene knockout; gene knockdown by siRNA and lentiviral shRNA; autophagy assays; in vitro vesicle fusion assays; live-cell and confocal microscopy; high-content image-based screening and analysis; qPCR; immunofluorescence; immunoprecipitation; immunoblotting and Southern blot analyses; flow cytometry; molecular cloning; mutagenesis analysis; mass spectrometry and LUMIER assay; bioinformatics and mathematical approaches; statistical analyses; a range of molecular, biochemical and cell biological techniques.

Contact: Dr Sovan Sarkar, University of Birmingham