Microglial phagocytosis dysfunction in stroke is driven by energy depletion and induction of autophagy.

Microglial phagocytosis of apoptotic debris prevents buildup damage of neighbor neurons and inflammatory responses. Whereas microglia are very competent phagocytes under physiological conditions, we report their dysfunction in mouse and preclinical monkey models of stroke (macaques and marmosets) by transient occlusion of the medial cerebral artery (tMCAo). By analyzing recently published bulk and single cell RNA sequencing databases, we show that the phagocytosis dysfunction was not explained by transcriptional changes. In contrast, we demonstrate that the impairment of both engulfment and degradation was related to energy depletion triggered by oxygen and nutrient deprivation (OND), which led to reduced process motility, lysosomal exhaustion, and the induction of a protective macroautophagy/autophagy response in microglia. Basal autophagy, in charge of removing and recycling intracellular elements, was critical to maintain microglial physiology, including survival and phagocytosis, as we determined both in vivo and in vitro using pharmacological and transgenic approaches. Notably, the autophagy inducer rapamycin partially prevented the phagocytosis impairment induced by tMCAo in vivo but not by OND in vitro, where it even had a detrimental effect on microglia, suggesting that modulating microglial autophagy to optimal levels may be a hard to achieve goal. Nonetheless, our results show that pharmacological interventions, acting directly on microglia or indirectly on the brain environment, have the potential to recover phagocytosis efficiency in the diseased brain. We propose that phagocytosis is a therapeutic target yet to be explored in stroke and other brain disorders and provide evidence that it can be modulated in vivo using rapamycin.Abbreviations: AIF1/IBA1: allograft inflammatory factor 1; AMBRA1: autophagy/beclin 1 regulator 1; ATG4B: autophagy related 4B, cysteine peptidase; ATP: adenosine triphosphate; BECN1: beclin 1, autophagy related; CASP3: caspase 3; CBF: cerebral blood flow; CCA: common carotid artery; CCR2: chemokine (C-C motif) receptor 2; CIR: cranial irradiation; Csf1r/v-fms: colony stimulating factor 1 receptor; CX3CR1: chemokine (C-X3-C motif) receptor 1; DAPI: 4',6-diamidino-2-phenylindole; DG: dentate gyrus; GO: Gene Ontology; HBSS: Hanks' balanced salt solution; HI: hypoxia-ischemia; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MCA: medial cerebral artery; MTOR: mechanistic target of rapamycin kinase; OND: oxygen and nutrient deprivation; Ph/A coupling: phagocytosis-apoptosis coupling; Ph capacity: phagocytic capacity; Ph index: phagocytic index; SQSTM1: sequestosome 1; RNA-Seq: RNA sequencing; TEM: transmission electron microscopy; tMCAo: transient medial cerebral artery occlusion; ULK1: unc-51 like kinase 1.

Microglial phagocytosis dysfunction in the dentate gyrus is related to local neuronal activity in a genetic model of epilepsy.

OBJECTIVE: Microglial phagocytosis of apoptotic cells is an essential component of the brain regenerative response during neurodegeneration. Whereas it is very efficient in physiological conditions, it is impaired in mouse and human mesial temporal lobe epilepsy, and now we extend our studies to a model of progressive myoclonus epilepsy type 1 in mice lacking cystatin B (CSTB). METHODS: We used confocal imaging and stereology-based quantification of apoptosis and phagocytosis of the hippocampus of Cstb knockout (KO) mice, an in vitro model of phagocytosis and siRNAs to acutely reduce Cstb expression, and a virtual three-dimensional (3D) model to analyze the physical relationship between apoptosis, phagocytosis, and active hippocampal neurons. RESULTS: Microglial phagocytosis was impaired in the hippocampus of Cstb KO mice at 1 month of age, when seizures arise and hippocampal atrophy begins. This impairment was not related to the lack of Cstb in microglia alone, as shown by in vitro experiments with microglial Cstb depletion. The phagocytosis impairment was also unrelated to seizures, as it was also present in Cstb KO mice at postnatal day 14, before seizures begin. Importantly, phagocytosis impairment was restricted to the granule cell layer and spared the subgranular zone, where there are no active neurons. Furthermore, apoptotic cells (both phagocytosed and not phagocytosed) in Cstb-deficient mice were at close proximity to active cFos+ neurons, and a virtual 3D model demonstrated that the physical relationship between apoptotic cells and cFos+ neurons was specific for Cstb KO mice. SIGNIFICANCE: 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, these 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.

Microglia Actively Remodel Adult Hippocampal Neurogenesis through the Phagocytosis Secretome.

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 long-term maintenance of adult hippocampal neurogenesis.SIGNIFICANCE STATEMENT Microglia are the brain professional phagocytes and, in the adult hippocampal neurogenic niche, they remove newborn cells naturally undergoing apoptosis. Here we show that phagocytosis of apoptotic cells triggers a coordinated transcriptional program that alters their secretome, limiting neurogenesis both in vivo and in vitro In addition, chronic phagocytosis disruption in mice deficient for receptors P2Y12 and MerTK/Axl reduces adult hippocampal neurogenesis. In contrast, inducible MerTK downregulation transiently increases neurogenesis, suggesting that microglial phagocytosis provides a negative feedback loop that is necessary for the long-term maintenance of adult hippocampal neurogenesis. Therefore, we speculate that the effects of promoting engulfment/degradation of cell debris may go beyond merely removing corpses to actively promoting regeneration in development, aging, and neurodegenerative diseases.

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

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.

Autophagy and Microglia: Novel Partners in Neurodegeneration and Aging.

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-ß, 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.