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Martin-Paul G. Agbaga, Jenny S. Wong, Robert E. Anderson, Orson L. Moritz; Mutant ELOVL4 Protein is Misrouted to Rod Outer Segments of X. laevis Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1368.
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© ARVO (1962-2015); The Authors (2016-present)
Autosomal dominant Stargardt-like macular dystrophy (STGD3) caused by mutations in the Elongation of Very Long Chain fatty acids (ELOVL4) gene results in early onset juvenile macular degeneration. Animal models expressing the mutant protein develop progressive photoreceptor degeneration and accumulate lipofuscin and A2E in the retina and retinal pigment epithelium. However, the exact mechanism by which the mutation causes retinal degeneration remains unclear; dominant negative mechanisms and reduction in retinal very long chain polyunsaturated fatty acids have been suggested. To better understand the molecular mechanisms involved in the disease progression in vivo, we addressed the hypothesis that the disease-linked C-terminal truncation mutants of ELOVL4 exert a dominant negative effect on wild type ELOVL4, altering its subcellular localization and contributing to STGD3.
We generated transgenic X. laevis that over-expressed murine ELOVL4 variants in rod photoreceptors, including untagged wild type (WT), HA-tagged wild type, an HA-tagged disease-linked 5-bp deletion mutant, and HA tagged full-length ELOVL4 containing a mutation in the C-terminal dilysine ER retention motif. In addition, we co-expressed the untagged WT and HA-tagged 5-bp deletion mutant proteins. Expression, subcellular protein localization, and photoreceptor integrity were assessed by Western and dot blots, immunolabeling, and confocal microscopy.
Tagged or untagged WT ELOVL4 proteins localized to rod inner segments of X. laevis, as previously shown in rodents and human retinas, while the mutant protein was localized to both inner and outer segments. Tissue culture experiments have shown that the mutant can interact with the WT protein to form aggregates. However, co-expression of the mutant and WT proteins in rods did not result in mislocalization of the WT protein to outer segments or in formation of aggregates. Full-length HA-tagged ELOVL4 protein lacking the dilysine motif (K308R/K310R), necessary for targeting the WT ELOVL4 protein to the endoplasmic reticulum, was similarly mislocalized to rod outer segments.
Our results suggest that mutant ELOVL4 does not cause retinal degeneration via a dominant negative mechanism; rather, our preliminary results suggest a gain of function mechanism. It is possible that outer segment mislocalization of the 5-bp deletion mutant ELOVL4 protein alters photoreceptor structure and function, thereby resulting in subsequent retinal degeneration.
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