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

ABSTRACT

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.