Investigating the homeostatic role and biomedical implications of the autophagy–NAD axis using human pluripotent stem cell-based platforms

Sun, Congxin ORCID: https://orcid.org/0000-0001-7575-9132 (2025). Investigating the homeostatic role and biomedical implications of the autophagy–NAD axis using human pluripotent stem cell-based platforms. University of Birmingham. Ph.D.

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Abstract

Autophagy is a homeostatic process critical for cell survival and human health. It is an intracellular degradative process that removes undesirable macromolecules (like protein aggregates) and damaged organelles (like mitochondria) from the cells. Due to the vital role of autophagy in maintaining cellular homeostasis, malfunction of this process contributes to cell death and tissue degeneration. This is particularly detrimental for post-mitotic cells like neurons. Indeed, dysfunction of autophagy has been implicated in diverse human pathological conditions including a range of neurodegenerative diseases. However, the mechanistic understanding of how impairment in autophagy contributes to cell death remains elusive. Elucidating the cytotoxic mechanism will be of biomedical interest for developing therapeutic interventions to combat neurodegeneration.
To undertake human-relevant biology in physiological and preclinical in vitro experimental platforms, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) were utilised in this study to generate neuronal cells. A genetic hESC model with autophagy knockout (ATG5−/−) was used for generating human neurons with autophagy deficiency in order to study how loss of autophagy affects neuronal survival. Autophagy-deficient hESCs and hESC-derived neurons exhibited higher cell death at basal level accompanied by metabolic defects. Depletion of a metabolite called nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes, such as SIRTs and PARPs, was found to trigger cell death via mitochondrial depolarisation in ATG5−/− hESC-derived neurons. Elevating intracellular NAD levels by supplementation with NAD precursors promoted the survival of ATG5−/− hESC-derived neurons by restoring mitochondrial bioenergetics and proteostasis. These findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative diseases associated with autophagic defects.
The biomedical relevance of this mechanism was further investigated in patient-derived iPSC model of a rare, early-onset neurodegenerative disease called Wolfram syndrome (WS). WS patient iPSC-derived neurons exhibited higher cell death that was associated with defective autophagy and NAD depletion, along with mitochondrial dysfunction and impairment in proteostasis. Targeting the defective autophagy–NAD axis in WS by autophagy and NAD enhancers ameliorated these disease phenotypes and improved the survival of WS patient iPSC-derived neurons, thereby highlighting a potential therapeutic strategy for the treatment of WS.
For future clinical translation by drug repurposing, a human neuronal platform was established using an autophagy reporter (mCherry-EGFP-LC3) hESC line in order to identify FDA-approved, CNS-penetrant, autophagy-inducing drugs via image-based chemical screen. Based on the efficacy in inducing autophagy at clinically-relevant dose, paediatric use, oral deliverability, and mTOR independence, four high-confidence hits were selected that include carbamazepine, nimodipine, sodium valproate and verapamil. All these FDA-approved autophagy inducers were shown to rescue disease phenotypes and improve cell survival in WS patient iPSC-derived neurons, thus informing potential drug candidates for clinical trials in future.
In a nutshell, defective autophagy–NAD axis was found to contribute to neuronal cell death, whilst targeting this cytotoxic cascade by autophagy and NAD enhancers exerted therapeutic benefits in patient-derived neuronal cells of a rare neurodegenerative disease. Since many rare and common neurodegenerative conditions are associated with autophagy dysfunction and NAD depletion, the findings from this work has the potential to be generalisable to some of these disease contexts.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):