Abstract
Purpose :
The BBSome is a stable network of 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.
Methods :
BBS5 knock-out (KO) mice were generated with ES cells from Eucomm. Electroretinography (ERG) scotopic and photopic tests were performed on 2-month old mice using the HMsERG LAB system from OcuScience. Immunohistochemistry (IHC) was performed on 12 μm retinal cryosections taken from light- or dark-adapted animals that were labeled with antibodies against key proteins. Sections were also stained with wheat germ agglutinin (WGA), peanut agglutinin (PNA) and DAPI (nuclei). TUNEL staining was performed to analyze fragmented DNA as a hallmark of apoptosis. Transmission electron microscopy (TEM) was performed to monitor ultrastructure of photoreceptor outer segments. One-way ANOVA was used for quantification of the average fluorescence intensity.
Results :
At 2 months of age, mouse cone ERG responses were severely diminished in BBS5 KO mice. IHC revealed mislocalization of arrestin-1 in light-adapted animals, whereas arrestin-4, M-opsin, S-opsin GNAT2 and CNGA3 have abnormal staining. Rhodopsin, transducin and peripherin-2 all have normal localization. Labeling with WGA showed that outer segments are shortened significantly by 9 months, with 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.
Conclusions :
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 arrestin-1 and abnormal staining of cone photoreceptor markers such as arrestin-4, M-opsin, S-opsin GNAT2 and CNGA3 were found as well. Photoreceptors contain abnormal disk morphology. These data support the hypothesis that BBS5 plays a vital functional role within cone photoreceptors and protein trafficking.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.