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Identifying autophagy regulators in human cellular platforms using human pluripotent stem cell models

Primary Supervisor: Dr Sovan Sarkar, Institute of Cancer and Genomic Sciences

Secondary supervisor: Professor Timothy Barrett

PhD project title: Identifying autophagy regulators in human cellular platforms using human pluripotent stem cell models

University of Registration: University of Birmingham

Project outline:

Background: Autophagy is an intracellular degradation pathway essential for cellular homeostasis and survival, and human health. Impairment of autophagy reduces cell viability and contributes to the pathology of diverse diseases like cancer and neurodegeneration, as well as ageing1; whereas stimulating autophagy promotes longevity and is beneficial in various transgenic disease models including neurodegenerative disorders1,2. Autophagy is regulated by mTOR (mechanistic target of rapamycin) and mTOR-independent pathways that are amenable to chemical perturbations2,3. Despite the growing need of modulating this process for therapeutic benefits, its precise regulation in the human system is not well elucidated. Emerging data including ours indicate that there is cell-type specificity of autophagy regulation4,5. In order to undertake human-relevant biology, we are harnessing the power of human embryonic stem cells (hESCs) for differentiating into isogenic human cell types, and have employed genome editing technologies to establish genetic models6. This project will utilize these human cellular platforms to study the landscape of autophagy in the human system at a physiological level, and translate some of the findings for future biomedical applications.

Question: This project addresses important issues in the field of autophagy: What are the cell-type specific regulators of autophagy in physiologically-relevant human cellular platforms, and can they be utilized for biomedical applications to combat ageing and neurodegeneration? This can be achieved via our human pluripotent stem cell models of autophagy that can be differentiated into the desired human cell-types having isogenic background to compare cell-specific effects.

Aims: Identify the regulators and pathways governing autophagy in hESCs and hESC-derived neurons, and evaluate their efficacy to combat ageing and neurodegeneration.

Methodology: The initial goal is to identify the molecular regulators of autophagy in autophagy reporter hESCs wherein a high-content image-based screen will be undertaken using a chemogenomics set of 225 compounds with known cellular targets. The top significant hits will be characterized by additional autophagy assays and their effects on mTOR activity. The high-confidence hits will then be genetically validated by lentiviral shRNA knockdown or gene knockout by CRISPR/Cas9, and further assessed for their pathway-specific effects in autophagy-deficient cells. Subsequently, we will investigate novel mechanisms of autophagy regulation in hESCs by cell biology and biochemical techniques, and extend this experimental paradigm in elucidating the autophagy-regulating pathways in hESC-derived neurons. The druggable autophagy inducers identified in human neurons will be evaluated for their therapeutic efficacy in human induced pluripotent stem cell models of ageing and neurodegeneration. Overall, we aim to gain mechanistic insights for human cell-specific regulation of autophagy that will contribute to the basic understanding of this process in the human system and have the potential for biomedical exploitation.

Outcome: Elucidating the regulators of autophagy in the human system will provide fundamental insights into this vital biological process, and will also reveal potential drug targets for improving defective autophagic flux in ageing and neurodegeneration. The outcome of this project will thus be of basic and biomedical relevance. In addition, multiple publications, possible patent applications and industrial collaboration avenues are likely outcomes of this project.

References:

1Mizushima N. et al. Nature 451:1069-1075, 2008; 2Sarkar S. Biochem. Soc. Trans. 41:1103-1130, 2013; 3Sarkar S. et al. Nat. Chem. Biol. 3:331-338, 2007; 4Maetzel D., Sarkar S. et al. Stem Cell Rep. 2:866-880, 2014; 5Seranova E. et al. J. Mol. Biol., 432:2754-2798, 2020; 6Murry C.E. & Keller G. Cell 132:661-680, 2008.

Further details: https://www.sovansarkarlab.com/

BBSRC Strategic Research Priority: Understanding the Rules of Life: Stem Cells

      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 into neural precursors and neurons; gene knockdown by lentiviral shRNA or gene knockout by CRISPR/Cas9; autophagy assays; confocal microscopy; high-content image-based screening and analysis; qPCR; immunofluorescence; immunoprecipitation; immunoblotting; flow cytometry; cell death assays; neuronal assays; molecular cloning; cell signalling pathway analysis; cheminformatics and mathematical approaches; statistical analyses; a range of molecular, biochemical and cell biological techniques.

      Contact: Dr Sovan Sarkar, University of Birmingham