Cone outer segment morphology and cone function in the Rpe65-/- Nrl-/- mouse retina are amenable to retinoid replacement
Kannan Kunchithapautham 1,
Beth Coughlin 2,
Rosalie K. Crouch 1,
and
Baerbel Rohrer 3*
1 Ophthalmology, Medical University of South Carolina, Charleston, South Carolina, United States
2 Neurosciences, Medical University of South Carolina, Charleston, South Carolina, United States
3 Ophthalmology, Medical University of South Carolina, Storm Eye Institute, Charleston, South Carolina, 29425, United States; Neurosciences, Medical University of South Carolina, Charleston, South Carolina, United States
* To whom correspondence should be addressed. E-mail: rohrer{at}musc.edu.
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Abstract |
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Purpose: RPE65, a major retinal pigment epithelium protein, is required to generate 11-cis retinal, the chromophore for all opsins. Without chromophore, cone opsins are mislocalized and cones degenerate rapidly (e.g., Rpe65-/- mouse). Function, survival and correct targeting of opsins is increased in Rpe65-/- cones upon supplying 11-cis retinal. Here, we determine the consequences of 11-cis retinal withdrawal and supplementation on cone development in the all-cone Nrl-/- retina. Methods: Rpe65-/- Nrl-/-, Nrl-/- and wild-type mice were examined. Cone structure was analyzed using TUNEL-labeling, electronmicroscopy and cone-specific antibodies; cone function was assessed using light-adapted single-flash ERGs. Results: (1) Rpe65-/- Nrl-/- mice have increased numbers of TUNEL-positive photoreceptors during programmed cell death compared to Nrl-/- in addition to accelerated age-related degeneration. (2) Cone function in Rpe65-/- Nrl-/- mice is minimal, and opsins are mislocalized. (3) Treatment with 11-cis retinal restored cone function, promoted outer segment formation and enabled opsin trafficking to outer segments. (4) Eliminating Rpe65 prevented rosette formation in Nrl-/- retinas; supplementation of Rpe65-/- Nrl-/- mice with 11-cis retinal resulted in their reoccurrence. Conclusions: Taken together, function and opsin trafficking in Nrl-/- and wild-type cones are comparable, confirming and extending our findings that cone maturation and outer segment development is dependent upon the presence of chromophore. The data on age-related cone death in Rpe65-/- Nrl-/- and the reintroduction of rosettes upon 11-cis retinal injections confirm that outer segments are required for cell survival, which for steric reasons appear to introduce rosettes in an all-cone retina. These results may have important consequences for understanding and treating chromophore-related cone dystrophies.
Key Words:
cones, photoreceptor dystrophy, visual pigment cycle, transgenic animals, cone pigment, electroretinography
