Investigating the homeostatic landscape of autophagy in cell survival using human pluripotent stem cell-derived neuronal and cerebral organoid platforms

PROJECT OUTLINE: One of the commonly deregulated cellular processes in ageing and neurodegeneration is autophagy. It is involved in the removal of undesirable macromolecules (like protein aggregates) and damaged organelles (like mitochondria) from the cells, thereby acting as a vital homeostatic pathway pertinent for cellular survival and human health. Conversely, malfunction of autophagy is harmful to the cells, particularly for post-mitotic neurons causing neuronal cell death [1]. We recently described an evolutionarily conserved role of autophagy in promoting cell survival by maintaining the levels of a metabolite called nicotinamide adenine dinucleotide (NAD), whereas loss of autophagy compromises cell viability via depletion of NAD [2–4]. However, the mechanistic landscape underlying the causal link between dysfunctional autophagy and cytotoxicity is not fully understood. To undertake basic research in a manner relevant to human biology, we are harnessing the power of human embryonic stem cells (hESCs) for differentiating into isogenic human cell types and organoids, and have employed genome editing technologies to establish autophagy knockout genetic models [2].

QUESTION: This project addresses a core question in the field of autophagy – How malfunction of autophagy in age-related neurodegeneration mediates neuronal cell death and degeneration of the brain?

EXPERIMENTAL PLATFORM: We will utilize our hESC model of autophagy deficiency for differentiating into isogenic human cortical neurons (2D cultures) and cerebral organoids (3D min-brains) that exhibit increased cell death.

AIMS: To understand the homeostatic role of autophagy in neuronal survival and brain health, we aim to identify the perturbations in biological pathways and metabolic processes mediating cell death underlying autophagy deficiency.

METHODOLOGY: In this project, we will utilize wild-type and autophagy knockout hESC lines to generate cortical neurons and cerebral organoids. To underpin the cytotoxic mechanisms underlying autophagy deficiency, we will undertake unbiased multi-omics analyses using existing UoB facilities and established external collaborations. We will perform metabolomics, transcriptomics, and proteomics analyses to identify global changes in metabolites, gene expression and protein levels at cellular level arising due to loss of autophagy. We have recently carried out these methodologies coupled with computational approaches for studying the role of autophagy in cellular and metabolic homeostasis in hESCs. We will initially focus on cortical neurons and then on cerebral organoids to identify a common signature of cellular perturbations. Key changes will be confirmed by biochemical and cell biology techniques, and emphasis will be given to energetic deficits arising due to stress-related pathways or accumulation of autophagy substrates. Finally, key findings will be confirmed in patient iPSC-derived cortical neurons and cerebral organoids of two rare neurodegenerative diseases associated with defective autophagy that we are currently utilizing for biomedical studies.

OUTCOME: Gaining fundamental insights of the cytotoxic mechanisms underlying loss of autophagy will not only contribute to the basic understanding of the homeostatic role of autophagy in cell survival but will also have the potential for biomedical exploitation in age-related neurodegeneration. Moreover, this work could reveal novel autophagy substrates in the neuronal system. The outcome of this project will thus be of basic and biomedical relevance, along with multiple publications and possible patent applications