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Boris Busov, Mercy Pawar, Cagri Besirli; PI3K/Akt signaling is critical for photoreceptor survival and regulates multiple cell death and cell survival pathways. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5385. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To study the cell survival and death mechanisms regulated by the phosphatidylinositol 3-Kinase/Akt (PI3K) signaling in photoreceptor cells.
661W photoreceptor cells were treated with PI3K inhibitor LY294002 in combination with 3-methylamphetamine (3-MA), Bafilomycin (Baf-1), pan-caspase inhibitor (Z-VAD-FMK) and Necrostatin-1 (Nec-1) and cell viability, Caspase 3/7 and 8 activities were analyzed. Immunohistochemistry and annexin staining were performed to elucidate the pathways. Expression of proteins involved in the pathways were analyzed with Western blotting. The role of PI3K/Akt signaling in regulating photoreceptor neuroprotective protein Faim2 was evaluated using immunoprecipitation and immunohistochemistry. Microarray analysis of cell death-associated genes was done on LY294002 treated 661W cells using Mouse Cell Death Pathway Finder RT2 Profiler PCR Array. Microarray analysis of expression of Akt genes using PI3K-AKT Signaling PCR Array was done on detached and attached extract of wild type and Faim2 knockout mice.
Treatment of cells with various concentrations of LY294002 inhibitor demonstrated increased cell death in a dose-dependent manner. Combined treatment of inhibitor LY294002 with Z-VAD-FMK, Nec-1, Baf-1, and 3-MA did not prevent cell death. Increased levels of caspase 3/7 activity with high levels of cell death were seen in LY-treated cells but no caspase 8 activation was observed. LY-treated cells showed an increased LC3 II/LC3I ratio and decreased p62 levels in Westerns. The Cell Death Profiler Assay showed marked fold increases in the levels of many apoptotic and autophagy markers. Stress-induced Akt phosphorylation in the retina was diminished in Faim2 knockout animals. Direct association of Faim2 and Akt was detected by immunoprecipitation.
PI3K signaling is critical in the survival of photoreceptors. Inhibition of baseline PI3K activity led to increased apoptosis and autophagy, but blocking these pathways did not prevent cell death. In addition, blocking necroptosis did not provide protection. These results indicate that in addition to apoptosis, necroptosis, and autophagy, alternative cell death pathways are dis-inhibited in photoreceptors when baseline PI3K signaling is turned off. Faim2 is important for Akt activation in the retina and may be regulating PI3K-mediated Akt phosphorylation by direct association.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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