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Katie Bales, Mandy J Croyle, Adriana Reyes Moon, Bradley K Yoder, Alecia K Gross; Congenital knock-out of transition zone protein BBS5 reveals cone-rod dystrophy with light induced protein mislocalization. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2361.
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© ARVO (1962-2015); The Authors (2016-present)
The BBSome is a stable network of eight proteins that has been found necessary for normal cilia function and is involved in vesicular trafficking to the ciliary membrane. Mutations occurring in BBSome components result in retinal degeneration-associated ciliopathies. In photoreceptors the precise role of BBSome remains elusive and the function of the protein Bardet-Biedl Syndrome 5 (BBS5) is currently unknown.
BBS5 knock-out (KO) mice were generated with ES cells from Eucomm. Electroretinography (ERG) scotopic and photopic tests were performed on 2 month and 9 month-old mice using the HMsERG LAB system from OcuScience. For immunohistochemistry (IHC) animals were either light- or dark-adapted, eyes were enucleated, fixed in 4% paraformaldehyde, embedded and cut into 12-micron sections. Sections were then labeled with antibodies against rhodopsin (1D4), transducin (SC-389, Santa Cruz), arrestin-1 (SC-67130, Santa Cruz), arrestin-4 and M-Opsin (Cheryl Craft, USC) and nuclei (DAPI). TUNEL staining was performed to analyze cell death markers. Transmission electron microscopy (TEM) was performed on 3 month-old animals to monitor ultrastructure of photoreceptor outer segments.
At 2 months of age, mouse cone ERG responses were completely diminished in BBS5 KO mice, while scotopic responses were reduced. IHC revealed mislocalization of rhodopsin, arrestin-1 in light-adapted animals, whereas arrestin-4 and M-opsin have abnormal staining in light- and dark-adapted animals. Labeling with WGA showed that outer segments are shortened significantly by 9 months, while interestingly there is only a reduction of 2 rows of nuclei in the outer nuclear layer by 9 months. TUNEL staining revealed a significant increase of cell death by 9 months. TEM showed the appearance of abnormal membranes in 3 month-old BBS5 KO photoreceptors.
Based on these results we find BBS5 plays a critical role in cone and rod photoreceptor function and outer segment protein localization. By 2 months of age, BBS5 KO mice lose cone function entirely, accompanied by loss of rod function via ERG analyses. Mislocalization of rhodopsin, arrestin-1 and abnormal staining of arrestin-4 and M-opsin are found as well. Photoreceptors contain abnormal disk morphology. These data support the hypothesis that BBS5 plays a vital functional role within photoreceptors and protein trafficking.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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