Phosphatidylinositol-3-phosphate is light-regulated and essential for survival in retinal rods

Abstract

Phosphoinositides play important roles in numerous intracellular membrane pathways. Little is known about the regulation or function of these lipids in rod photoreceptor cells, which have highly active membrane dynamics. Using new assays with femtomole sensitivity, we determined that whereas levels of phosphatidylinositol-3,4-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate were below detection limits, phosphatidylinositol-3-phosphate (PI(3)P) levels in rod inner/outer segments increased more than 30-fold after light exposure. This increase was blocked in a rod-specific knockout of the PI-3 kinase Vps34, resulting in failure of endosomal and autophagy-related membranes to fuse with lysosomes, and accumulation of abnormal membrane structures. At early ages, rods displayed normal morphology, rhodopsin trafficking, and light responses, but underwent progressive neurodegeneration with eventual loss of both rods and cones by twelve weeks. The degeneration is considerably faster than in rod knockouts of autophagy genes, indicating defects in endosome recycling or other PI(3)P-dependent membrane trafficking pathways are also essential for rod survival.

The Warburg Effect Mediator Pyruvate Kinase M2 Expression and Regulation in the Retina

“…The p85α subunit belongs to class I PI3K, and its deletion has no effect on rods. Deletion of class III PI3K in rods has been shown to result in age-related rod degeneration42. Studies to examine the effect of class III PI3K on PKM2 phosphorylation are currently underway in our laboratory.…”

Structural and molecular bases of rod photoreceptor morphogenesis and disease

“…Selective knockout of Atg5, a protein essential for normal autophagy, leads to progressive retinal degeneration (Rodriguez-Muela et al, 2013; Zhou et al, 2015) leading to the conclusion that basal autophagy, even in the absence of disease-induced stress, is essential for photoreceptor survival. Selective knockout of Vps34, and Type III phosphatidylinositol 3-kinase essential for synthesis of phosphatidyl inositol 3-phosphate and completion of autophagasome maturation, also leads to rod cell death (He et al, 2016). …”

“…Transmission electron micrograph showing the corresponding retinal layers from a mouse retina. Sample preparation and imaging were as described in (He et al, 2016). …”

“…Sample preparation and imaging were as described in (He et al, 2016). A. Longitudinal section showing connecting cilium and adjacent regions of outer and inner segments (Labels: d , disks; cc , connecting cilium; bb , basal body complex; a , axoneme; m , mitochondria).…”

A non‐canonical rhodopsin‐mediated insulin receptor signaling pathway in retinal photoreceptor neurons

“…Isolation of inner segment preparations is technically challenging. It has been shown previously that OptiPrep™ density centrifugation has yielded outer segments with attached inner segments of photoreceptor cells (Gilliam et al, 2012;He et al, 2016). OptiPrep™ is a non-ionic iodixanolbased medium with a density of 1.320 g/mL and has been extensively used to isolate cell organelles such as nuclei, mitochondria, peroxisomes, lysosomes, and endosomal subfractions (Graham, 2001;Van Veldhoven et al, 1996).…”