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Abstract:Autophagy is emerging as a core regulator of Central Nervous System (CNS) aging and neurodegeneration. In the brain, it has mostly been studied in neurons, where the delivery of toxic molecules and organelles to the lysosome by autophagy is crucial for neuronal health and survival. However, we propose that the (dys)regulation of autophagy in microglia also affects innate immune functions such as phagocytosis and inflammation, which in turn contribute to the pathophysiology of aging and neurodegenerative diseases. Herein, we first describe the basic concepts of autophagy and its regulation, discuss key aspects for its accurate monitoring at the experimental level, and summarize the evidence linking autophagy impairment to CNS senescence and disease. We focus on acute, chronic, and autoimmunity-mediated neurodegeneration, including ischemia/stroke, Alzheimer's, Parkinson's, and Huntington's diseases, and multiple sclerosis. Next, we describe the actual and potential impact of autophagy on microglial phagocytic and inflammatory function. Thus, we provide evidence of how autophagy may affect microglial phagocytosis of apoptotic cells, amyloid-β, synaptic material, and myelin debris, and regulate the progression of age-associated neurodegenerative diseases. We also discuss data linking autophagy to the regulation of the microglial inflammatory phenotype, which is known to contribute to age-related brain dysfunction. Overall, we update the current knowledge of autophagy and microglia, and highlight as yet unexplored mechanisms whereby autophagy in microglia may contribute to CNS disease and senescence.

During adult hippocampal neurogenesis, most newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of intracellular contents. Here, we propose that phagocytosis is not merely passive corpse removal but has an active role in maintaining neurogenesis. First, we found that neurogenesis was disrupted in male and female mice chronically deficient for two phagocytosis pathways: the purinergic receptor P2Y12, and the tyrosine kinases of the TAM family Mer tyrosine kinase (MerTK)/Axl. In contrast, neurogenesis was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of the changes induced by phagocytosis in microglia in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro. Our data suggest that microglia act as a sensor of local cell death, modulating the balance between proliferation and survival in the neurogenic niche through the phagocytosis secretome, thereby supporting the longterm maintenance of adult hippocampal neurogenesis.

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words)During adult hippocampal neurogenesis, the majority of newborn cells undergo apoptosis and are rapidly phagocytosed by resident microglia to prevent the spillover of their intracellular contents. Here, we propose that phagocytosis is not merely a passive process of corpse removal but has an active role in maintaining adult hippocampal neurogenesis. First, we found that neurogenesis was disrupted in mice chronically deficient for two microglial phagocytosis pathways (P2Y12 and MerTK/Axl), but was transiently increased in mice in which MerTK expression was conditionally downregulated. Next, we performed a transcriptomic analysis of microglial phagocytosis in vitro and identified genes involved in metabolism, chromatin remodeling, and neurogenesis-related functions. Finally, we discovered that the secretome of phagocytic microglia limits the production of new neurons both in vivo and in vitro. Our data suggest that reprogrammed phagocytic microglia act as a sensor of local cell death, modulating the balance between cell proliferation and cell survival in the neurogenic niche, thereby supporting the long-term maintenance of adult hippocampal neurogenesis.

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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.

Microglial phagocytosis of apoptotic cells is an essential component of the brain regenerative response in neurodegenerative diseases. Phagocytosis is very efficient in physiological conditions, as well as during apoptotic challenge induced by excitotoxicity or inflammation, but is impaired in mouse and human mesial temporal lobe epilepsy (MTLE). Here we extend our studies to a genetic model of progressive myoclonus epilepsy type 1 (EPM1) in mice lacking cystatin B (CSTB), an inhibitor of cysteine proteases involved in lysosomal proteolysis. We first demonstrated that microglial phagocytosis was impaired in the hippocampus in Cstb knock-out (KO) mice when seizures arise and hippocampal atrophy begins, at 1 month of age. To test if this blockage was related to the lack of Cstb in microglia, we used an in vitro model of phagocytosis and siRNAs to acutely reduce Cstb expression but we found no significant effect in the phagocytosis of apoptotic cells. We then tested whether seizures were involved in the phagocytosis impairment, similar to MTLE, and analyzed Cstb KO mice before seizures begin, at postnatal day 14. Here, phagocytosis impairment was restricted to the granule neuron layer but not to the subgranular zone, where there are no active neurons. Furthermore, we observed apoptotic cells (both phagocytosed and not phagocytosed) in Cstb deficient mice at close proximity to active, cFos + neurons and used mathematical modeling to demonstrate that the physical relationship between apoptotic cells and cFos+ neurons was specific for Cstb KO mice. These results suggest a complex crosstalk between apoptosis, phagocytosis and neuronal activity, hinting that local neuronal activity could be related to phagocytosis dysfunction in Cstb KO mice. Overall, this data suggest that phagocytosis impairment is an early feature of hippocampal damage in epilepsy and opens novel therapeutic approaches for epileptic patients based on targeting microglial phagocytosis. METHODS Mice. Tissues ofCstb KO mice 7 (129S2/SvHsd5-Cstb tm1Rm ; Jackson Laboratory stock no. #003486) were obtained from the Lehesjoki´s lab at Folkhälsan Research Center and University of Helsinki. Wild-type litter mates with the same genetic background were used as controls. For the FACS experiments, fms-EGFP mice (B6.Cg-Tg(Csf1r-EGFP)1Hume/J; Jackson Laboratory stock #018549), where microglia constitutively express the GFP protein 25,26 were used. All procedures followed the European Directive 2010/63/EU and NIH guidelines, and were approved by the Animal Ethics Committee of the State Provincial Office of Southern Finland (decisions ) and Ethics Committees of the University of the Basque Country EHU/ UPV (Leioa, Spain; CEBA/205/2011, CEBA/206/2011, CEIAB/82/2011, CEIAB/105/2012). BV2 cell line. BV2 cells (Interlab Cell Line Collection San Martino-Instituto ScientificoTumori-Instituto Nazionale per la Ricerca sul Cancro), a cell line derived from raf/mycimmortalized rat neonatal microglia was used to perform the in vitro knock-down model of Cstb. BV2 cells were grown...