Beyond LC3-associated phagocytosis: cross-talk between autophagy and efferocytosis during microglial corpse clearance
Date
2023
Authors
Singh, Sanjna
Editors
Advisors
Sargeant, Timothy J
Mäkinen, Ville-Petteri
Mäkinen, Ville-Petteri
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Thesis
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Abstract
Every day, billions of cells die in the human body, both as part of programmed cellular turnover and during injury or disease. These cells are engulfed and removed by specialised subsets of phagocytic immune cells, via a process termed efferocytosis. The efficient removal of apoptotic cells prevents necrosis and subsequent inflammatory damage to surrounding healthy tissue. Microglia are phagocytes that reside in the brain, where they serve as the primary executors of efferocytosis. However, corpse clearance in microglia is relatively under-studied, with most research performed in macrophages. Proper clearance of waste material and preventing inflammation is particularly crucial in the brain, where a tightly controlled environment must be maintained to ensure optimal function. Autophagy is another important waste disposal process in the brain; it involves the packaging and breakdown of intracellular cargo such as protein aggregates and organelles via double-membraned vesicles termed autophagosomes. Some the autophagy-related (ATG) machinery also facilitates the efficient clearance of apoptotic cells during efferocytosis in an autophagy-independent manner, via the conjugation of ATG8/LC3 to endolysosomal single membranes (CASM), also referred to as non-canonical autophagy. Importantly, both autophagy and efferocytosis shuttle their respective cargoes towards lysosomes for degradation and recycling of components. Defects in autophagy, efferocytosis, and CASM have been linked to pathological conditions such as stroke and neurodegeneration in the brain. However, beyond CASM, it is not known whether autophagy and efferocytosis interact with each other. Recent work connecting corpse clearance to amino acid metabolism and mammalian target of rapamycin complex 1 (mTORC1) signalling – two potent regulators of autophagy – hints at potential cross-talk between these pathways. Given the strong links between autophagy and nutrition, I hypothesised that microglial efferocytosis interacts with autophagy to co-ordinate cargo clearance in a nutrient-sensitive manner. During efferocytosis, apoptotic material is engulfed within phagosomes: these eventually mature, become acidic, and fuse with lysosomes for cargo breakdown. At present, fluorescent dye-based techniques to measure efferocytosis cannot adequately discriminate between changes to the engulfment and acidification of material. In this thesis, I generated the novel tool ‘epHero’, harnessing variations in the pH-sensitivity of red and green fluorescent proteins to simultaneously track the uptake and acidification of apoptotic cells. The epHero probe could be used to measure efferocytosis via flow cytometry and imaging in both microglia- and macrophage-like cells. Further, epHero was able to detect changes to phagolysosomal acidification in a mouse model of efferocytosis. Thus, epHero is a specific, pH-responsive reporter of corpse clearance and offers significant improvements over conventional techniques. Amino acid starvation is a well-established inducer of autophagy. Although metabolism of some apoptotic cell-derived amino acids has been implicated in corpse clearance, it is not known whether extracellular nutrient availability can modify efferocytosis. Here, the epHero reporter revealed that amino acid starvation increased acidification, but not uptake, of apoptotic material during microglial efferocytosis. Proteomic profiling of this phenomenon implicated proteins involved in arginine and fatty acid regulation, which have previously been linked to efferocytosis, as well as novel pathways such as branched chain amino acid metabolism, which will be explored in future work. ext, CASM was pharmacologically inhibited during microglial efferocytosis to determine whether autophagic and corpse clearance pathways interacted with each other beyond CASM. Efferocytosis reduced the lysosomal delivery of autophagic cargo. This defect could be partly rescued by stimulating autophagy via amino acid starvation or mTORC1 inhibition. Proautophagy stimuli exerted different effects on microglial corpse clearance: amino acid starvation boosted apoptotic cargo acidification whereas pharmacological mTORC1 inhibition reduced it. This occurred even though mTORC1 inhibition elevated lysosomal activity to a higher degree compared to starvation. These results indicate a role for mTORC1 in the efficient acidification of apoptotic material during efferocytosis. Residual levels of mTORC1 activity were observed in microglia during starvation, a stimulus that normally suppresses mTORC1. Efferocytosis further increased mTORC1 function in amino acid-starved microglia. In contrast, pharmacological inhibition completely blocked mTORC1 activity. Increasing the intracellular pool of amino acids via cycloheximide treatment reverted mTORC1 function to basal levels in starved microglia, consequently abolishing the increased corpse acidification response. Taken together, these data suggest that low levels of mTORC1 activity are, counterintuitively, required for the efficient clearance of apoptotic cargo. When mTORC1 is either fully functional or completely suppressed microglial efferocytosis is compromised. Collectively, findings from this thesis demonstrate cross-talk between corpse clearance and autophagic pathways in microglia, whereby efferocytosis reduces autophagy. Limiting amino acid availability promotes catabolism of both autophagic and efferocytic cargo via an mTORC1-dependent mechanism. Differences between corpse clearance in mTORC1- inhibited and amino acid-starved cells were detected using the newly generated epHero reporter, highlighting the importance of accurate measurement of efferocytosis dynamics.
School/Discipline
Adelaide Medical School
Dissertation Note
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2024
Provenance
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