Studying Autophagy in Microglia: Overcoming the Obstacles.

In this chapter, we provide an overview of the main techniques and experimental approaches that can be used to analyze autophagy flux in microglia, the brain-resident macrophages. For this purpose, we first briefly introduce the main peculiarities of microglial biology, describe the basic mechanisms and functions of autophagy, and summarize the evidence accumulated so far on the role of autophagy in the regulation of microglial survival and functions, mainly phagocytosis and inflammation. Then, we highlight conceptual and technical aspects of autophagic recycling and microglial physiology that need to be taken into account for the accurate evaluation of autophagy flux in microglia. Finally, we describe the main assays that can be used to analyze the complete sequence of autophagosome formation and degradation or autophagy flux, mainly in cultured microglia and in vivo. The main approaches include indirect tracking of autophagosomes by autophagic enzymes such as LC3 by western blot and fluorescence-based confocal microscopy, as well as direct analysis of autophagic vesicles by electron microscopy. We also discuss the advantages and disadvantages of using these methods in specific experimental contexts and highlight the need to complement LC3 and/or electron microscopy data with analysis of other autophagic effectors and lysosomal proteins that participate in the initiation and completion of autophagy flux, respectively. In summary, we provide an experimental guide for the analysis of autophagosome turnover in microglia, emphasizing the need to combine as many markers and complementary approaches as possible to fully characterize the status of autophagy flux in microglia.