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Hongman Song, Yong Zeng, Ronald A Bush, Ronald S Petralia, Ya Xian Wang, Robert N Fariss, Camasamudram Vijaysarathy, Paul A Sieving; The role of constitutively active RAC1 in rod out segment formation in mouse rhodopsin-null rods. Invest. Ophthalmol. Vis. Sci. 2019;60(9):570.
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© ARVO (1962-2015); The Authors (2016-present)
Dominant-active RAC1 rescues photoreceptor structure in Drosophila rhodopsin (Rho)-null mutants. This study is to investigate whether constitutively active (CA)-RAC1 can restore rod outer segment (ROS) formation in mouse Rho knockout (KO) rods.
We tested this hypothesis using two mouse models. Transgenic (Tg) CA-RAC1 Rho KO mice were generated by crossing Rho KO mice with transgenic CA-RAC1 mice created in our lab, and morphological features of the ROS were evaluated at P21 by transmission electron microscopy (TEM). AAV8-pOpsin-CA-RAC1-GFP vectors were injected subretinally into P3/P4 Rho KO retinas. Morphological features of CA-RAC1 expressing rods and non-CA-RAC1 expressing rods were compared by immunohistochemistry.
Transgenic (Tg) CA-RAC1 Rho KO mice expressed CA-RAC1 in Rho KO rods at early time of rod development (< P4). EM showed small and thin rudimentary OS membranes in Rho KO rods at P21. In contrast, EM images of Tg CA-RAC1 Rho KO rods showed a few enlarged ROS membrane sacs with long and curved tubular membranes emerging from ciliary plasma membranes. This suggests that CA-RAC1 could promote outward growth of OS membranes.Previous studies showed that CA-RAC1 disrupts rod morphology development. To minimize the adverse effects of CA-RAC1 during early-age rod development, here we expressed CA-RAC1 later during rod development by subretinal injection of AAV8-pOpsin-CA-RAC1 into the Rho KO retina at P3/P4, and we examined retinal morphology at P30. Differential interference contrast (DIC) images at P30 showed elongated fine string-like structures extending from IS regions towards the RPE in the presumptive OS space of CA-RAC1 injected Rho-/- retinas, whereas elongations were not seen in control Rho-/- retinas. These elongated fine string-like structures gave positive staining with peripherin/rds and CNGA1 (OS plasma membrane marker). These data indicate that CA-RAC1 increases outgrowth of OS domain membranes in Rho KO rods, consistent with the results observed in Tg CA-RAC1 Rho KO rods.
These data indicate that rhodopsin is not essential to initiate mouse ROS formation. Further, CA-RAC1 expression promoted outgrowth of both rudimentary OS disc membranes and plasma membrane in Rho KO rods, but it was not adequate to promote and/or maintain normal OS structure in Rho KO rods.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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