Deletion of the p85alpha regulatory subunit of phosphoinositide 3-kinase in cone photoreceptor cells results in cone photoreceptor degeneration

Abstract

Purpose: Downregulation of the retinal insulin/mTOR pathway in mouse models of retinitis pigmentosa is linked to cone cell death, which can be delayed by systemic administration of insulin. A classic survival kinase linking extracellular trophic/growth factors with intracellular antiapoptotic pathways is phosphoinositide 3-kinase (PI3K), which the authors have shown to protect rod photoreceptors from stress-induced cell death. The role of PI3K in cones was studied by conditional deletion of its p85α regulatory subunit.

Methods: Mice expressing Cre recombinase in cones were bred to mice with a floxed pi3k gene encoding the p85α regulatory subunit of the PI3K and were back-crossed to ultimately generate offspring with cone-specific p85α knockout (cKO). Cre expression and cone-specific localization were confirmed by Western blot analysis and immunohistochemistry (IHC), respectively. Cone structural integrity was determined by IHC using peanut agglutinin and an M-opsin-specific antibody. Electroretinography (ERG) was used to assess rod and cone photoreceptor function. Retinal structure was examined by light and electron microscopy.

Results: An age-related cone degeneration was found in cKO mice, evidenced by a reduction in photopic ERG amplitudes and loss of cone cells. By 12 months of age, approximately 78% of cones had died, and progressive disorganization of synaptic ultrastructure was noted in surviving cone terminals in cKO retinas. Rod viability was unaffected in p85α cKO mice.

Conclusions: The present study suggests that PI3K signaling pathway is essential for cone survival in the mouse retina.

Figure 1.

p85α protein levels in cone photoreceptor outer segments and the 661W cone cell line. Western blot analysis of total mouse retinal lysates, POS-enriched extracts from WT and Nrl^−/− mouse retinas, and cell extracts from the 661W cone cell line were used to assess expression levels of p85α (A, D) protein. M-cone opsin (B, E) and rhodopsin (C, F) were used as cone and rod photoreceptor markers, respectively. Immunocytochemical analysis of M-cone opsin (G) and p85α (H) expression were determined in the 661W cone cell line. For control, primary antibodies were omitted (I). Nuclei are counterstained with DAPI (blue). Scale bar, 50 μm for all panels.

Figure 2.

Generation of the cone-specific p85 KO mouse model. (A) Cone photoreceptor-specific deletion of Pik3r1, a pan-p85α regulatory subunit of PI3K, was made by cross-breeding floxed p85α mice to cone-specific Cre mice. (B) Expression levels of p85α in POS and Band II (retinal cells enriched with inner segments) from WT, WT-Cre+, p85α-flox, p85α-het Cre+, and p85α KO Cre+ mouse retinas were examined with anti–p85α antibody. (C) Immunohistochemical analysis of Cre recombinase immunolabeling in p85α cKO and WT control retinas harvested from littermates. Red: Cre-positive cone cells (arrowheads). Labeling of blood vessels (bv) is nonspecific. (D) Western blot analysis of duplicate p85α cKO and WT retinal extracts was used to determine the level of Cre expression; β-actin levels were used as a loading control. (E) Assessment of cone outer segment integrity using immunolabeling for M-cone opsin at 1 month of age. ROS, rod outer segment; ONL, outer nuclear layer; INL, inner nuclear layer; POS, photoreceptor outer segment. Scale bar, 100 μm for all panels.

Figure 3.

Morphology of cone-specific p85 KO retina and assessment of cone outer segment integrity. Morphologic examination of retinas from WT and p85α cKO mice at 1, 6, and 12 month of age.

Figure 4.

Function of the cone-specific p85α KO retina. Scotopic a- and b-wave (A, B) and photopic b-wave (C) electroretinographic analysis of WT and cKO mice at 1 month (WT p85α^flox/flox, n = 5; cKO, n = 5), 6 months (WT p85α^flox/flox, n = 14; cKO, n = 14), and 12 months (WT p85α^flox/flox, n = 10; WT Cre^±, n = 4; cKO, n = 10) of age. Values are mean ± SEM. *P < 0.05 for scotopic b-wave (B) for WT p85α^flox/flox vs. cKO at 6 and 12 months of age. P < 0.001 for photopic b-wave (C) for WT p85α^flox/flox compared with cKO at 6 and 12 months of age. (D) Western blot analysis of WT and p85α cKO littermate retinal extracts was used to assess M-cone opsin levels at 6 and 12 months of age; β-actin was used as a loading control.

Figure 5.

Loss of cone photoreceptors in p85α cKO retinas at 1, 6, and 12 months of age. (A) PNA (red) and anti–cone arrestin (cArr, green) immunofluorescence staining of retinal whole mounts from WT (a, d, g), and p85α cKO (b, e, h) mice (n = 4 each). For a control, primary antibodies were omitted (c, f, i). Scale bar, 100 μm for all panels. (B) Retinal sections of inferior and superior regions from WT and p85α cKO mice at 6 and 12 months of age were stained for M-cone opsin. (C) Quantitative analysis of M-cone opsin–positive cells at 6- and 12-month-old mice from inferior and superior regions of retina. Data are mean ± SEM; sample size is indicated on top of each bar. *P < 0.001, significance between WT and cKO. POS, photoreceptor outer segment; ONL, outer nuclear layer; INL, inner nuclear layer.

Figure 6.

Effect of conditional deletion of p85α on the organization of synaptic ultrastructure in surviving cone terminals. The synaptic terminals of cones in WT mouse retina show normal organization at 1 month (A), 6 months (B), and 12 months (C) of age, with multiple triadic ribbon synaptic complexes (arrows) and flat contacts along the base of the terminal (arrowheads). A progressive decline in organization of synaptic ultrastructure was evident in the terminals of surviving cones in the p85α cKO retina. Although cone terminals in the 1-month p85α cKO retina appeared ultrastructurally similar to that of the WT retina (D), in the p85α cKO retina at 6 months (E) and 12 months (F), fewer ribbon synaptic complexes were evident. Although flat contacts were present, they were sometimes inappropriately located in apposition to synaptic ribbons (F), indicating diminished ultrastructural organization. Scale bars, 0.5 μm for all panels.