The p110α isoform of phosphoinositide 3-kinase is essential for cone photoreceptor survival

Abstract

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylates the 3'OH of the inositol ring of phosphoinositides (PIs). They are responsible for coordinating a diverse range of cellular functions. Class IA PI3K is a heterodimeric protein composed of a regulatory p85 and a catalytic p110 subunit. In this study, we conditionally deleted the p110α-subunit of PI3K in cone photoreceptor cells using the Cre-loxP system. Cone photoreceptors allow for color vision in bright light (daylight vision). Cone-specific deletion of p110α resulted in cone degeneration. Our studies suggest that PI3K signaling is essential for cone photoreceptor functions.

Keywords: Cone photoreceptors; Cre-loxP system; Phosphoinositide 3-kinases; Phosphoinositides; Retina; Vision.

Figure 1. Generation of the cone-specific p110α KO mouse model

Cone photoreceptor-specific deletion of PIK3CA, a pan-p110α catalytic subunit of PI3K, was performed by cross-breeding floxed p110α mice to cone-specific Cre mice (A). PCR diagnostic for cone-opsin cre (B), and floxed p110α and WT genes (C) using mouse tail DNA samples. Immunohistochemical analysis of Cre recombinase immunolabeling in p110α KO and WT control retinas harvested from littermates. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. Red indicates Cre-positive cone cells. Labeling of blood vessels (*) is non-specific.

Figure 2. Reduced expression of p110α in cones of cone-conditional p110α knockout mice

PNA (red), anti-p110α (green), and DAPI (blue) immunofluorescence staining of retinal sections from two-month-old p110α wild-type littermates (A) and p110α KO mice (B). The images shown are representative of four retinas examined from WT and KO mice. Panel C represents the omission of p110α antibody. A region of the photoreceptor layer was enlarged in both WT and KO mice (H, I). Scale bar = 50 µm for panels A-C, 20 µm for panels H - I.

Figure 3. Flat mounts of cone photoreceptors in p110α KO retinas at 3 months of age

PNA (red) and anti-cone arrestin (cArr, green) immunofluorescence staining of retinal whole mounts from WT and p110α KO mice (A, C, E, G, I). The images shown are representative of four retinas examined from WT and KO mice. Scale bar = 100 µm for all panels. A region (□) on the flat mount was imaged at a higher magnification (B, D, F, H, J). Photopic b-wave electroretinographic (ERG) analysis of WT and p110α KO mice at 3 months of age (K). Values are mean ± SEM, n= 10. * P<0.05.

Figure 4. Quantification of cone cell loss in cone photoreceptors in p110α KO retinas

PNA-labelled retinal whole mounts from WT and p110α KO mice were subjected to imageJ analysis on selected areas (□) of dorsal, ventral, nasal and temporal regions of the retina (A). Combined PNA-labeled positive cones were expressed as percentage and the wild-type was set as 100 percent (B). Data are mean ± SEM, n = 3, *p<0.001.