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Autophagy is the process by which a cell sends cytosolic material to the lysosome for digestion. In this work, I investigated the importance of autophagy to lipid droplet formation and protein turnover in the neural stem cells, neurons, and glia of the larval Drosophila brain. The role of the endomembrane system in autophagy in neurons and neural stem cells was also considered, as was the movement of acid-resistant protein from neural stem cell lineage to the surrounding cortex glia. The location of various proteins involved in nutrient sensing and autophagic induction was also determined, and the reproducibility of manual image classification attempts was assessed using deep learning.

Macroautophagy (hereby autophagy) is a catabolic pathway whereby a double membrane organelle called an autophagosome, engulfs cytoplasmic components in order to degrade them via the lysosome. While autophagy is induced in response to various stresses to promote homeostasis and cell survival, a key mechanistic step in initiation of autophagosome formation is thought to be regulated by the serine/threonine kinases ULK1/2. ULK1/2 kinase activity is required for amino acid starvation-induced and other types of autophagy. Our lab recently showed that inhibition of ULK1 with the potent compound MRT68921, results in formation of stalled autophagosomes that are positive for early and late autophagy markers. These data suggested that ULK1 is not only regulating initiation of autophagosome formation but also later stages, such as elongation and fusion with lysosomes. Here, I compared the most selective and potent ULK1 inhibitors available to date as to what autophagosomal defects they cause. From this work it is concluded that ULK1 inhibition may block autophagosome flux by affecting dissociation of autophagy-initiating protein complexes from the forming autophagosome and influencing its closure. In order to understand the underlying mechanisms regulating this process I performed a proximity ligation assay (BioID) using MRT68921 as a tool to enrich for autophagy regulators trapped on stalled autophagosomes. With this approach ATG2B was identified, among others, as a protein that is enriched on autophagosomal structures upon ULK1 inhibition. I here show that ULK1 phosphorylates ATG2B upon autophagy induction in response to amino acid starvation. It is known that loss of ATG2A/B results in accumulation of arrested autophagosomes, consistent with that seen by ULK1 kinase inhibition. Thus, ATG2B and its homologue ATG2A might be key substrates of ULK1, the phosphorylation of which is required for proper autophagosome formation.

Macroautophagy (‘autophagy’ hereafter) mediates the capture of aberrant cytoplasmic material into vesicles called autophagosomes, which then shuttle to lysosomes for degradation. Autophagy is implicated in numerous diseases, largely in a pro-survival role. However, autophagy has also been suggested as a form of programmed cell death (PCD), from cases of dying cells showing autophagosome accumulations. Debate occurs between whether these vesicles drive the lethality, or are instead a failing rescue attempt. This study aimed to provide clarity on this issue. Via the use of chemical and genetic strategies of inducing autophagosome accumulations, we found combining stimulators of autophagosome biogenesis with lysosomal degradation inhibitors gave rise to toxicity. Notably, this effect was dependent on the autophagy machinery and independent of other PCD routes. Research into the underlying mechanisms revealed an energy deficit under these conditions. Since autophagosomes cannot be recycled at lysosomes here, their continued synthesis affords no survival benefits, and instead just serves to deplete cellular energy further. For this reason, we designate this event ‘Futile Autophagosome Synthesis’ (FAS) toxicity. Other contributors to this toxicity include the persistence of harmful agents like Reactive Oxygen Species (ROS). Having established our FAS model, we explored its relevance in both cancer and neurodegeneration. Importantly, we found FAS inducing strategies to be effective in tumour treatment. Also, inhibiting FAS reduced the toxicity seen in neurodegenerative disease. Therefore, not only does this study improve our knowledge of autophagy in PCD, but also indicates it may have important medical implications.