Abstract
Purpose :
The P23H mutation in the rhodopsin gene is associated with adRP and sector RP phenotypes. Pathogenic mechanisms in both the inner segment (IS) and outer segment (OS) have been implicated, including altered OS morphology and ER retention of the mutant rhodopsin. Heat shock (HS) response has been reported to protect against RD caused by P23H rhodopsin. Here we investigated the effects of HS on P23H rhodopsin biosynthesis and trafficking to the OS.
Methods :
We generated transgenic X. laevis expressing a rhodopsin-GFP fusion protein (GFPrhoCT44) under control of the HSP70 heat shock promoter. Heterozygous animals from a transgenic line were crossed with a line expressing bovine P23H rhodopsin (bP23H rho) under control of the X. laevis rod opsin promoter. Tadpoles positive for GFP expression were raised in darkness; these rearing conditions prevent bP23H rho induced RD. At two weeks of age, the animals were subjected to a 1 hr 30C heat shock. Tadpole eyes were fixed, cryosectioned and labeled with antibodies to detect bP23H rho, and GFP and bP23H rho were imaged by confocal microscopy.
Results :
HS treatment induced transient expression of GFPrhoCT44, creating a marker within the rod OS that allowed us to differentiate disks synthesized prior to and subsequent to heat shock. There were no significant differences in the migration of GFPrhoCT44 in dark reared P23H+ versus P23H- animals, indicating that expression of bP23H rho did not significantly alter disk synthesis rates. The band of GFPrhoCT44 was reproducibly associated with lack of staining for bP23H rho, indicating dramatic reduction in delivery of bP23H rho to the OS immediately following HS. This region of negative staining also appeared at the equivalent location in bP23H rho+ rods that did not express GFPrhoCT44, indicating it was associated with the HS, and not due to interference of GFPrhoCT44 with bP23H rho biosynthesis or trafficking. Preliminary results suggest that HS also causes a transient reduction in the levels of bP23H rho in rod IS.
Conclusions :
GFP-rhoCT44 expression driven by a HS promoter can be used to track OS disk synthesis rates, confirm HS responses, and distinguish OS disks synthesized prior to and following HS. We determined that bP23H rho expression is compatible with normal rates of OS disk synthesis in dark-reared animals, and that HS causes a transient reduction in bP23H rho trafficking to the OS.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.