Microglia in the developing retina couple phagocytosis with the progression of apoptosis via P2RY12 signaling

Abstract

Background: Microglia colonize the developing vertebrate central nervous system coincident with the detection of developmental apoptosis. Our understanding of apoptosis in intact tissue in relation to microglial clearance of dying cells is largely based on fixed samples, which is limiting given that microglia are highly motile and mobile phagocytes. Here, we used a system of microglial depletion and in vivo real-time imaging in zebrafish to directly address microglial phagocytosis of apoptotic cells during normal retinal development, the relative timing of phagocytosis in relation to apoptotic progression, and the contribution of P2RY12 signaling to this process. Results: The depletion of microglia resulted in accumulation of numerous apoptotic cells in the retina. Real-time imaging revealed precise timing of microglial engulfment with the progression of apoptosis, and dynamic movement and displacement of engulfed apoptotic cells. Inhibition of P2RY12 signaling delayed microglial clearance of apoptotic cells. Conclusions: Microglial engulfment of dying cells is coincident with apoptotic progression and requires P2RY12 signaling, indicating that microglial P2RY12 signaling is shared between development and injury response. Our work provides important in vivo insight into the dynamics of apoptotic cell clearance in the developing vertebrate retina and provides a basis to understand microglial phagocytic behavior in health and disease. K E Y W O R D S developmental apoptosis, microglia, p2ry12, phagocytosis, retina, zebrafish

The Alzheimer’s disease-associated protective Plcγ2-P522R variant promotes immune functions

“…2e), which is again in line with the finding that upon PLCγ2 KO in iMGs, the expression of P2RY12 is strongly diminished [6]. Although P2ry12 is a well-established homeostatic microglia marker [31], it has been considered also as a specific marker for healthy rodent CNS microglial cells [32] and is known to mediate microglia motility, phagocytosis of apoptotic cells, and protection of the bloodbrain-barrier [33][34][35]. Also, the expression pattern analysis of P2RY12 in response to β-amyloid in aging human brain has suggested that P2RY12 expression by microglia should not be considered solely as a marker of resting microglia [36].…”

Microglial Phagocytosis—Rational but Challenging Therapeutic Target in Multiple Sclerosis

“…Additionally, the extensive cell turnover through apoptosis during brain development is again an important step for creating homeostasis and generating a functional brain. For that to be accomplished, cellular apoptosis drives microglial colonization and establishment in the CNS for a coordinated and efficient system of clearance/removal of apoptotic bodies by microglia, whereas inefficient phagocytosis of apoptotic bodies results, instead, in subsequent cell death alongside with inflammatory and immune responses because of the released intracellular contents [ 73 , 74 ]. Microglia are essential to remove apoptotic neural progenitor cells within the neurogenic niches of the hippocampus-subgranular and subventricular zones-via activation of TAM receptor tyrosine kinases MerTK and AXL [ 75 ], but also mediated by the complement system, as the C1q complement protein was recently found expressed on microglial phagocytic pouches during hippocampal neurogenesis [ 76 ].…”

“…Microglia are essential to remove apoptotic neural progenitor cells within the neurogenic niches of the hippocampus-subgranular and subventricular zones-via activation of TAM receptor tyrosine kinases MerTK and AXL [ 75 ], but also mediated by the complement system, as the C1q complement protein was recently found expressed on microglial phagocytic pouches during hippocampal neurogenesis [ 76 ]. Apart from the hippocampus, microglia also phagocytosed apoptotic Purkinje neurons, for the proper maturation of rat cerebellum and correct development of the retina in zebrafish, a process that is surprisingly delayed after inhibition of microglial purinergic receptor P2RY12, thus unveiling an additional role of purinergic signaling in developmental phagocytosis of apoptotic cells [ 74 , 77 ]. On the other hand, current studies on microglia associate cell phagocytosis with the brain’s sexual differentiation.…”

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“…For instance, in the embryonic development of the eye, microglia entrain proximal cytosocial stimuli (such as complement cascade proteins, local chemokine milieu, and spontaneous neural activity) in the massive culling of excess neurons during the self‐organization of the crystal‐like regularity of the retina. [ 104,105 ] Postnatally, external photic stimuli rapidly drive further microglia‐mediated activity‐dependent organization of the retina, optic tracts, and primary visual cortex. [ 106,107 ] Then, as suggested by literature cited above, as the infant interacts with the environment in visually‐guided goal‐directed behavior, salient social stimuli (e.g., recognition of maternal face and expressions) begin the microglia‐mediated process of activity‐dependent sculpting of social behavior and memory circuits (presumed to be highly evolved and complex corticolimbic and corticohypothalamic pathways) [ 108 ] that preferentially and increasingly respond to psychosocial stimuli in infancy and early childhood.…”