Müller glia, the principal glial cell type in the retina, have the potential to reenter the cell cycle after retinal injury. In mammals, proliferation of Müller glia is followed by gliosis, but not regeneration of neurons. Retinal injury is also accompanied by phagocytic removal of degenerated cells. We here investigated the possibility that proliferation and gliosis of Müller glia and phagocytosis of degenerated cells may be regulated by the same molecular pathways. After N-methyl-N-nitrosourea-induced retinal injury, degenerated photoreceptors were eliminated prior to the infiltration of microglia/macrophages into the outer nuclear layer, almost in parallel with cell cycle reentry of Müller glia. Inhibition of microglia/macrophage activation with minocycline did not affect the photoreceptor clearance. Accumulation of lysosomes and rhodopsin-positive photoreceptor debris within the cytoplasm of Müller glia indicated that Müller glia phagocytosed most photoreceptor debris. Pharmacological inhibition of phosphatidylserine and Rac1, key regulators of the phagocytic pathway, prevented cell cycle reentry, migration, upregulation of glial fibrillary acidic protein, and phagocytic activity of Müller glia. These data provide evidence that phosphatidylserine and Rac1 may contribute to the crosstalk between different signaling pathways activated in Müller glia after injury.Müller glia, the principal glial cells in the retina, possess a variety of functions to support retinal neurons and act to maintain retinal homeostasis under physiological as well as pathological conditions 1,2 . In lower vertebrates like fish, retinal injury induces Müller glia to proliferate and dedifferentiate to neuronal progenitor cells that are capable of regenerating retinal neurons 3,4 . In mammals, however, such regenerative capacity of Müller glia is extremely limited. In rats, for example, Müller glia proliferate in response to injury, but they quickly exit the cell cycle and many undergo cell death possibly by DNA damage response 5 . In addition, Müller glia in mammals show a set of injury-induced responses called reactive gliosis, including cellular hypertrophy, migration, and upregulation of intermediate filaments such as glial fibrillary acidic protein (GFAP) and vimentin 6 . Although gliosis may be neuroprotective, it may hamper tissue repair if the reaction is prolonged 6 .Previous evidence has indicated that the injury-induced responses of Müller glia are mediated by growth factors or cytokines secreted from damaged neurons, microglia, or Müller glia themselves 6-8 . A pioneering study by Rattner and Nathans 9 showed that damaged photoreceptors produce endothelin2 (Edn2), which signals onto Müller glia and induces their reactive responses such as GFAP upregulation. However, the Edn2-mediated interaction between damaged photoreceptors and Müller glia seems to be initiated by Leukemia inhibitory factor (LIF) release from Müller glia 10 . Upregulation of LIF has been observed in a variety of retinal injury models 10-13 , and TNFα may be i...

Our findings suggest that the DNA damage response induced by unscheduled cell cycle reentry may be one of the mechanisms that limit the proliferative and regenerative capacity of Müller glia in the mammalian retina.

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Mammalian Müller glia express transcription factors and cell cycle regulators essential for the function of retinal progenitors, indicating the latent neurogenic capacity; however, the role of these regulators remains unclear. To gain insights into the role of these regulators in Müller glia, we analyzed expression of transcription factors (Pax6, Vsx2 and Nfia) and cell cycle regulators (cyclin D1 and D3) in rodent Müller glia, focusing on their age- and cell cycle-related expression patterns. Expression of Pax6, Vsx2, Nfia and cyclin D3, but not cyclin D1, increased in Müller glia during development. Photoreceptor injury induced cell cycle-associated increase of Vsx2 and cyclin D1, but not Pax6, Nfia, and cyclin D3. In dissociated cultures, cell cycle-associated increase of Pax6 and Vsx2 was observed in Müller glia from P10 mice but not from P21 mice. Nfia levels were highly correlated with EdU incorporation suggesting their activation during S phase progression. Cyclin D1 and D3 were transiently upregulated in G1 phase but downregulated after S phase entry. Our findings revealed previously unknown links between cell cycle progression and regulator protein expression, which likely affect the cell fate decision of proliferating Müller glia.