Abstract
Purpose :
Phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) is a minor lipid that plays important roles in membrane trafficking and signal transduction. The enzymes responsible for its production from the precursor PI(4)P are the catalytic subunits of the α, β, and γ isoforms of phosphatidylinositolphosphate-5-kinase (PIP5K1A, PIP5K1B, PIP5K1C). The phenotypes of mouse global knockouts of the α and β isoforms are relatively mild, whereas knockout of the γ isoform is embryonic lethal. The γ isoform has been reported to be important in ciliogenesis. Our purpose is to determine the role of the γ isoform in the retina.
Methods :
The phospholipase C-δ pleckstrin homology (PLC-δ-PH) domain was used to localize PI(4,5)P2 in photoreceptors using a plasmid encoding PLC-δ-PH under control of a mutant opsin promoter with reduced efficacy that was introduced by sub-retinal injection and electroporation of P0 WT mice. PI(4,5)P2 levels in the retina or photoreceptor outer/inner segments were determined by phosphoinositide ELISA. We bred mice to be homozygous for a “floxed” allele of the Pip5k1c gene and heterozygous for a Six3-driven Cre transgene to achieve knockout in early stages of retinal development. We used immunofluorescence and histology to assess the effects on retinal structure.
Results :
In photoreceptors, PI(4,5)P2 is located in the plasma membrane of outer and inner segments, and in the axon terminals. PIP5K1C immunostaining was observed in photoreceptor inner segments, and both outer and inner plexiform layers. Despite early expression of Cre, immunofluorescence revealed the presence of residual PIP5K1C protein at 2 months postnatal, whereas by 3 months the protein was virtually undetectable. The overall appearance of the retina was normal at 2 months, but by 5 months there was substantial thinning of the outer segment layer, and a reduction in the number of cones. PI(4,5)P2 level was remarkably increased in outer/inner segments of light-adapted WT mice compared to transducin alpha knockout mice. The PI(4,5)P2 level in PIP5K1C knockout mouse retina was not significantly different than in control mice.
Conclusions :
An intact Pip5k1c gene does not appear to be essential for early stages of retinal development, although the persistence of the protein after gene knockout renders this conclusion somewhat uncertain. At later stages, the enzyme is important for maintenance and health of photoreceptors.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.