Abstract
Purpose :
Microglia, the innate immune cells of the central nervous system (CNS), are pivotal in the development, maintenance, and degeneration of the neural parenchyma. We previously showed that microglia in embryonic retina phagocytose a subset of non-apoptotic retinal ganglion cells (RGCs), and that this is in part mediated by complement recognition. Why microglia target these viable RGCs and whether additional recognition pathways contribute is unclear. We hypothesize that these embryonic RGCs are stressed, and present “eat-me” cues that promote clearance by microglia through recognition receptors, Mertk and CR3.
Methods :
B6.Mertk-/- and CR3 (Cd11b-/-) mice were used to assess microglial receptor contribution to RGC elimination. RGC density was assessed by immunostaining for RBPMS at P0 (when excess RGCs persist). To evaluate whether RGCs are stressed, immunostaining for phosphorylated-cJUN (P-cJUN) was performed at e14.5 (peak RGC loss). Differences between control and mutant mice were quantified and evaluated using Student’s t-test. To evaluate externalization of “eat-me” cue phosphatidylserine, ex-vivo e14.5 eyes were incubated in psVue643.
Results :
A subset of newborn RGCs showed upregulation of P-cJUN at e14.5, which persisted in Mertk-/-.CR3-/- mice until e16.5. Ex-vivo culture of enucleated embryonic eyes (e14.5), showed that a subset of RGCs externalized phosphatidylserine, further suggesting RGCs are stressed. Genetic ablation of Mertk partially inhibited phagocytosis of RGCs and led to an 8.95% (n=12; p=0.005) increase in RGC density at birth compared to wildtype controls. Loss of both CR3 and Mertk, inhibited phagocytosis of embryonic RGCs and resulted in excess RGCs at birth (19.78%; n=8; p<0.0001), similar to what we previously showed with depletion of microglia (20%), suggesting a cooperative role in assuring that unfit RGCs are eliminated.
Conclusions :
Shortly after RGCs are generated, a subset is eliminated by phagocytic microglia. These non-apoptotic RGCs show signs of stress and externalize the phospholipid phosphatidylserine, which along with opsonins like C1q we previously showed, can signal microglia to phagocytose. We further show that microglial receptors Mertk and CR3 are essential in recognizing these cells for elimination. Microglia are capable of phagocytosing viable cells, which may have implications in glaucoma, where RGCs are lost.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.