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.

Role of microglia in stroke

“…Some microglial cells may not utilize the LDs due to a putative dysfunction of lypophagy or decreased activity of cytoplasmic lipases, facilitating further LD accumulation that could eventually transform microglia into dysfunctional foamy cells (Figure 1). This phenomenon could be related to insufficient or defective autophagic flux leading to impaired phagocytosis (Beccari et al, 2023). Prolonged and exaggerated fat accumulation in the cells may be a sign of exhaustion and dysfunction.…”

“…Thus, signals from infiltrating macrophages may prompt immunometabolic changes in primed microglia. Impaired microglial phagocytic activity has been found in a model of transient ischemia where the MCA occlusion also induces hypoxia in the hippocampus causing reduced phagocytosis of apoptotic neurons in this region (Beccari et al, 2023; Rudolph et al, 2021). Phagocytosis of the damaged tissue imposes a tremendous metabolic load to the cells (Figure 3).…”

“…Autophagy is necessary to dispose and recycle components of the phagocytosed biological materials, as has been extensively studied in macrophages (Florey & Overholtzer, 2012; Oczypok et al, 2013; Puleston & Simon, 2014). Physiologic autophagy is also critical for microglia homeostasis (Beccari et al, 2023; Plaza‐Zabala et al, 2017). After stroke, autophagy seems to play complex roles in inflammation and phagocytosis (Peng et al, 2022; Plaza‐Zabala et al, 2017; Wang, Li, Zhang, et al, 2023).…”

“…After stroke, autophagy seems to play complex roles in inflammation and phagocytosis (Peng et al, 2022; Plaza‐Zabala et al, 2017; Wang, Li, Zhang, et al, 2023). Alterations in the microglia autophagic flux after ischemia may impair their phagocytic activity since the autophagy‐inducing agent rapamycin improved phagocytosis of apoptotic neurons (Beccari et al, 2023).…”

Myeloid cells in vascular dementia and Alzheimer's disease: Possible therapeutic targets?

“…In response to invading pathogens or after sensing adjacent cell damage, microglia get activated and undergo morphological changes such as expansion of their soma and shortening of their cellular processes (Town et al, 2005). Activated microglia are critical in pathogen phagocytosis and in the elimination of cellular debris and degenerating cells at the lesion site (Beccari et al, 2023). The secretory properties of microglia have been well characterized.…”