Abstract
Purpose:
In the central nervous system, autophagy is a normal cellular process that prevents the formation of protein deposits from large protein aggregates that are inefficiently degraded by proteosomes. Autophagy-linked FYVE protein (Alfy) is a recently discovered, ubiquitously expressed protein that has an FYVE domain that binds to phosphoinositide 3-kinase (PI3K) -generated PI-3-P with high affinity. Overexpression of the C-terminal fragment of Alfy has been shown to increase aggregate clearance and neuroprotection in a Drosophila eye model of polyglutamine toxicity clearance. There are no studies available on the expression and role of Alfy in the retina, so we characterized Alfy in this study.
Methods:
Localization of Alfy was examined by immunohistochemistry performed on wild type and cone-dominant Nrl-/- retinal sections. The C-terminal fragments of Alfy and PI-3-P binding mutant were tested both in vitro (mammalian cells) and in vivo (lipoplex-mediated subretinal injection into mice). The outer nuclear thickness was used as a function of rod photoreceptor degeneration, and cone cell death was assessed by peanut agglutinin and cone-transducin labeling.
Results:
We found that Alfy was expressed in inner segments and the outer and inner plexiform layers, in both rod- and cone-dominant retina. The expression of Alfy-WT in HEK-293T cells did not change the morphology of the cells. However, the PI-3-P-binding mutant induced autophagosome-like vacuoles and increased cell death. We also found increased rod and cone cell death in mice injected with PI-3-P-binding mutant, but not in WT-injected mice.
Conclusions:
Our study shows that Alfy is expressed in the retina. Our research also suggests that the interaction between PI3K-generated PI-3-P and the FYVE domain of Alfy is indispensable for normal functions of autophagy in the retina.
Keywords: 657 protein modifications-post translational •
714 signal transduction •
615 neuroprotection