Purchase this article with an account.
Cindy Linn, Megan Stanchfield, Deborah Otteson, Mark Webster; Adult Mammalian Retinal Regeneration. Invest. Ophthalmol. Vis. Sci. 2018;59(9):559.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The experiments in this study test the hypothesis that PNU-282987 induces neurogenesis from Müller glia in adult mammals after activation of α7 nAChRs on retinal pigment epithelium (RPE).
PNU-282987 (1 mM) and 5-bromo-2'-deoxyuridine (BrdU) (1 mg/ml) were applied as eye drops once a day in adult mice for 1-14 days. BrdU incorporation and expression of progenitor and differentiated cell markers in all retinal layers were assayed using immunocytochemistry (IHC), transgenic lineage tracing and confocal imaging to determine if PNU-282987 induced adult neurogenesis from Müller glia. A rodent RPE cell line was also treated with PNU-282987 to determine if signaling molecules released from the RPE played a role in this neurogenic response. Reverse transcription polymerase chain reaction (RT-PCR) was used to assay gene expression from the RPE.
PNU-282987 and BrdU eye drops induced widespread incorporation of BrdU. IHC and transgenic lineage tracing demonstrated that new cells first appeared in the inner nuclear layer (INL) and were positive for BrdU, Pax6 and arose from Müller Glia. BrdU incorporation was observed in 20.5% (±2.3; SEM; n=6) of differentiated RGCs and in all retinal layers depending on the length of treatment. Culture media obtained from RPE cells 72 hours after exposure to 100 nM PNU-282987 induced BrdU incorporation in 50.1% (±7.4; SEM; n=6) of INL cells when injected intravitreally into adult mice compared to 28.8% (±3.6; SEM; n=8) of BrdU-incorporated INL cells when PNU-282987 was added as eye drops. To understand what signaling molecules may induce cell cycle reentry in Müller glia, candidate genes were assayed by RT-PCR on the RPE cell line. These data indicated Wnt2b expression only after PNU-282987 treatment. All responses were significantly reduced if eyes or cultured RPE cells were treated with the α7 nAChR antagonist, methyllycaconitine (MLA), before PNU-282987 and BrdU (ANOVA with Tukey's multiple comparison test P< 0.001).
The results from this study provide evidence that PNU-282987 causes cell cycle reentry in Müller glia and generates new retinal progenitor neurons in an adult mammal. PNU-282987 does not act on the Müller glia directly as they do not contain α7 nAChRs, but instead, act on α7 nAChRs on RPE to release signaling molecules capable of inducing cell cycle reentry in Müller glia and the genesis of new neurons.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
This PDF is available to Subscribers Only