Functional analysis of BBS3 A89V that results in non-syndromic retinal degeneration

Abstract

Bardet-Biedl syndrome (BBS) is a syndromic form of retinal degeneration. Recently, homozygosity mapping with a consanguineous family with isolated retinitis pigmentosa identified a missense mutation in BBS3, a known BBS gene. The mutation in BBS3 encodes a single amino acid change at position 89 from alanine to valine. Since this amino acid is conserved in a wide range of vertebrates, we utilized the zebrafish model system to functionally characterize the BBS3 A89V mutation. Knockdown of bbs3 in zebrafish alters intracellular transport, a phenotype observed with knockdown of all BBS genes in the zebrafish, as well as visual impairment. Here, we find that BBS3 A89V is sufficient to rescue the transport delays induced by the loss of bbs3, indicating that this mutation does not affect the function of BBS3 as it relates to syndromic disease. BBS3L A89V, however, was unable to rescue vision impairment, highlighting a role for a specific amino acid within BBS3 that is necessary for visual function, but dispensable in other cell types. These data aid in our understanding of why patients with the BBS3 A89V missense mutation only present with isolated retinitis pigmentosa.

Figure 1.

BBS3 conservation and protein expression. (A) Multi-species alignment of BBS3 demonstrating the conservation among vertebrates. Shaded box highlights the location of the A89V mutation. Asterisks (*) indicate identical amino acids, while colons (:) and periods (.) represent conserved amino acids. (B) Schematic depicting the location of the A89V mutation in human BBS3 and BBS3L isoforms. Hatched box depicts the location of the P-loop motif and the grey box on the C-terminus of BBS3L denotes the region of difference between the two isoforms. (C) Western blot analysis of staged zebrafish embryos injected with either human BBS3L or BBS3L A89V myc-tagged RNA. Both proteins are present through 5 dpf. Actin served as a control.

Figure 2.

BBS3 A89V functions in melanosome transport. (A) Schematic illustrating the retrograde movement of melanosomes within the melanocyte from the periphery before epinephrine treatment to the perinuclear region after epinephrine treatment. (B) Dorsal view of a dark-adapted wild-type 6-day-old zebrafish embryo. Boxed head region is magnified below the full embryo. The left-hand image shows melanocytes prior to epinephrine treatment, while the right-hand panel depicts the same melanocytes at the endpoint of retrograde transport. (C) The graph summarizes the average epinephrine-induced response times in minutes for each experimental group. Both human BBS3 and BBS3 A89V RNAs are able to suppress the transport time seen with knockdown of bbs3. Sample size (n) is denoted on the X-axis. **P < 0.01, ANOVA with Tukey. Data presented as the mean ± SEM.

Figure 3.

The BBS3L A89V mutation is not functional in vision. (A) Still images of a 5-day-old zebrafish embryo illustrating the characteristic escape response elicited by a sudden change in light intensity. The white boxes below the stills indicate lights on, while the grey box indicates lights off. For visualization of the embryo in the dark, image contrast manipulation was performed in Adobe Photoshop. (B) Graphical representation of the vision startle response data. crx gene knockdown was used as a control for visual impairment. Human BBS3L can rescue the vision defect, whereas human BBS3L A89V is not able to rescue the vision defect in bbs3 knockdown embryos. The sample size (n) is noted on the X-axis. **P < 0.01, ANOVA with Tukey. Data presented as the mean ± SEM.