Abstract
Purpose :
Peripherin-2/rds (PRPH2), a photoreceptor-specific tetraspanin protein, is essential for the morphogenesis of the outer segment (OS). Mutations in the PRPH2 gene are responsible for a variety of inherited retinal diseases. Recently, it was reported that late endosomes/lysosomes play a crucial role in the ciliary transport of PRPH2. However, the mechanism associated with this specific pathway has not been fully understood. Homozygous mutations in the progranulin (PGRN) gene (GRN) cause neuronal ceroid lipofuscinosis 11, a lysosomal storage disease, and Grn-deficiency causes retinal degeneration in mice. This study tested the hypothesis that PGRN is involved in the intracellular transport of tetraspanins including PRPH2 in murine photoreceptors.
Methods :
cDNA containing either human PRPH2 with c-Myc-tag or human CD63 with 3xHA-tag was transferred into the pCAG-IRES-GFP background. The targeting sequences of shRNA against murine Grn gene (Grn-sh) were inserted into pCAG-IRES-tdTomato to be identified by the co-expressed tdTomato. Embryonic retinal transfection was performed in Slc:ICR mice (Japan SLC, Hamamatsu, Japan) at gestational day 14.5 as reported previously with minor modifications. Animals were maintained in a 12-h light/12-h dark cycle until postnatal 21 days, and the harvested retinas were subjected to immunostaining. All animal manipulations were performed after the approval by the Animal Experimental Committee of Gifu Pharmaceutical University. The images were obtained by Olympus confocal microscope FV3000.
Results :
Transfected PRPH2 reporters displayed a banding pattern at the OS in murine rods as reported previously, whereas the signal of CD63 exhibited the cell borders and the vesicular pattern at the inner segments (IS). Interestingly, Grn-sh transfection resulted in the distribution change of CD63 reporter to the banding pattern at the OS, similar to PRPH2. In the transfected cones, both PRPH2 and CD63 reporters were found at the OS. Grn-sh caused the morphological defects of cone IS/OS; thinned OS and swollen IS, in which the signal of CD63 reporter was found as enlarged vacuoles, suggesting that the ciliary transport could be impaired by the deficiency of PGRN.
Conclusions :
Progranulin may play an important role in the proper ciliary targeting of tetraspanins in photoreceptor. Cones would be more susceptible to lysosomal dysfunction and subsequent defects in the OS targeting than rods.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.