The inhibitor of phagocytosis, O-phospho-L-serine, suppresses Muller glia proliferation and cone cell regeneration in the light-damaged zebrafish retina

The damaged zebrafish retina replaces lost neurons through a regenerative response that initiates with the asymmetric cell division of Muller glia to produce neuronal progenitor cells that proliferate and migrate to the damaged retinal layer, where they differentiate into the lost neuronal cell types. Because Muller glia are known to phagocytose apoptotic retinal cells during development, we tested if Muller glia engulfed apoptotic rod cell bodies in light-damaged retinas. After 24 h of constant intense light, damaged retinas revealed both a strong nuclear TUNEL signal in photoreceptors and a weak cytoplasmic TUNEL signal in Muller glia, although Muller glial apoptosis is not observed in the light-damaged retina. Light damage of a rod-specific transgenic reporter line, Tg(XlRho:EGFP)(f11), resulted in some Muller glia containing both TUNEL signal and EGFP, which indicated that this subset of Muller glia engulfed apoptotic photoreceptor cell bodies. To determine if phagocytosis induced the Muller glial proliferative response in the light-damaged retina, we utilized O-phospho-L-serine (L-SOP), a molecule that mimics the phosphatidylserine head group and partially blocks microglial phagocytosis of apoptotic cells. Intravitreal injection of L-SOP immediately prior to beginning constant intense light treatment: i) did not significantly reduce light-induced photoreceptor cell death, ii) significantly reduced the number of PCNA-positive Muller glia, and iii) significantly reduced the number of cone photoreceptors in the regenerated retina relative to control retinas. Because L-SOP is also a specific group III metabotropic glutamate receptor (mGluR) agonist, we also tested if the more potent specific group III agonist, L-2-amino-4-phosphonobutyrate (L-AP4), the specific group III antagonist (RS)-alpha-Methylserine-O-phosphate (MSOP) or the specific group I antagonist, L-2-amino-3-phophonopropanoic acid (L-AP3) affected Muller glial proliferation. We found no changes with any of these factors compared to control retinas, revealing that metabotropic glutamate receptors were not necessary in the Muller glia proliferative response. Furthermore, ascl1a and stat3 expression were unaffected in either the L-SOP or MSOP-injected retinas relative to controls, suggesting L-SOP disrupts Muller glia proliferation subsequent to or in parallel with ascl1a and stat3 activation. This implies that at least one signaling mechanism, in addition to the process disrupted by L-SOP, is required to activate Muller glia proliferation in the light-damaged retina. (C) 2010 Elsevier Ltd. All rights reserved.