Assessing Autophagy in Microglia: A Two-Step Model to Determine Autophagosome Formation, Degradation, and Net Turnover

Abstract

Autophagy is a complex process that encompasses the enclosure of cytoplasmic debris or dysfunctional organelles in membranous vesicles, the autophagosomes, for their elimination in the lysosomes. Autophagy is increasingly recognized as a critical process in macrophages, including microglia, as it finely regulates innate immune functions such as inflammation. A gold-standard method to assess its induction is the analysis of the autophagic flux using as a surrogate the expression of the microtubule-associated light chain protein 3 conjugated to phosphatidylethanolamine (LC3-II) by Western blot, in the presence of lysosomal inhibitors. Therefore, the current definition of autophagy flux actually puts the focus on the degradation stage of autophagy. In contrast, the most important autophagy controlling genes that have been identified in the last few years in fact target early stages of autophagosome formation. From a biological standpoint is therefore conceivable that autophagosome formation and degradation are independently regulated and we argue that both stages need to be systematically analyzed. Here, we propose a simple two-step model to understand changes in autophagosome formation and degradation using data from conventional LC3-II Western blot, and test it using two models of autophagy modulation in cultured microglia: rapamycin and the ULK1/2 inhibitor, MRT68921. Our two-step model will help to unravel the effect of genetic, pharmacological, and environmental manipulations on both formation and degradation of autophagosomes, contributing to dissect out the role of autophagy in physiology and pathology in microglia as well as other cell types.

Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia

“…There was no obvious upregulation of microtubule-associated protein light chain 3 (LC3)-II, an autophagy activation marker, 15 min after ATP stimulation (Figure S2G). These data were consistent with a previous finding that autophagy can only be activated via multiple signaling pathways after ATP stimulation, which would require up to 2 h ( Plaza-Zabala et al, 2020 ; Takenouchi et al, 2009 ). These results reflect the dynamic process of CD63 + EV release in this time window.…”

Traumatic brain injury in the presence of Aβ pathology affects neuronal survival, glial activation and autophagy

“…The results obtained in the presence and absence of the autophagic inhibitor Baf A1 can further determine the amount of autophagosomes that were formed and would have been degraded in the observed time frame 55 , 56 . The mean values of the results shown in Fig.…”

Siponimod Modulates the Reaction of Microglial Cells to Pro-Inflammatory Stimulation

“…Taken together, this finding suggests that although S1PR signalling is inhibited by siponimod treatment, sphingosine-1-phosphate metabolism remains intact. Furthermore, autophagy, another pathway enriched upon siponimod treatment, is thought to be one of the key regulators of innate immune responses [ 53 ]. Apart from that, we found downregulated DEG clusters in endocytosis, one of the key mechanisms of S1PR signalling and clusters in actin cytoskeletal organisation, which we gauge to be in line with our finding that siponimod modulates microglial morphology as shown in Figure 1 D.…”