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Feng He, Yan Yin Tse, Melina A Agosto, Samuel M Wu, Theodore G Wensel; Aggressive photoreceptor cell degeneration in PI3KC3/Vps34 conditional knockout mice. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5393.
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© ARVO (1962-2015); The Authors (2016-present)
The type III phosphoinositide 3-kinase (PI3KC3/Vps34) participates in various cellular functions, including intracellular trafficking and survival. The biological significance of Vps34 in retina is not fully understood. In this study, we investigated Vps34 functions in photoreceptors using rod-specific Vps34 conditional knockout mice.
A mouse line with a conditional functional deletion of Vps34 in rods was generated. Functional and structural changes in the retina were determined by morphological analysis, western blotting and electroretinography (ERG). An ultrasensitive ELISA-based assay with luminescence detection was used to measure phosphatidylinositol 3-phosphate (PI(3)P) levels. Electroporation of plasmids encoding a PI(3)P binding domain-EGFP fusion was used to localize PI(3)P within rod cells.
PI(3)P localized to discrete puncta of various sizes in the inner segment. Light exposure induced a massive increase of PI(3)P levels in rods, with a slow time course spanning hours. Deletion of Vps34 in rods decreased light-induced PI(3)P levels by more than 94% and caused aggressive retinal degeneration. In contrast, deletion of the type I PI-3 kinase (conditional KO of P85a) had no effect on PI(3)P levels or retinal structure. The number of photoreceptor cells in Vps34 KO mice was decreased by about 50% at 1.5 months and almost none remained at 3 months, while there was no significant change in the inner nuclear or ganglion cell layer. The retinal degeneration was not significantly affected by light. Western blotting of autophagy markers showed dramatic increases in the levels of LC3-II, Rab7, p62, LAMP-1, and LAMP-2 in Vps34 KO mice. Although both LC3 and LAMP-1 accumulated in the inner segments of Vps34 KO mice, they did not co-localize as determined using immunofluorescence microscopy. ERG was normal at 1 month, but exhibited greatly reduced amplitudes of both a- and b-waves at 2 months. Rhodopsin trafficking was examined using Vps34 KO mice which were also heterozygous for a rho-EGFP knock-in. Our results suggest PI(3)P does not significantly contribute to rhodopsin trafficking, as rho-EGFP did not accumulate in the inner segments and ONL in vps34 KO mice.
The type III PI-3 kinase but not the type I kinase, contributes to light-induced synthesis of PI(3)P in rods, and is essential for rod survival. The mechanisms may involve disruption of autophagy and/or related pathways.
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