Thesis Seminar: Pamela Pretorius
Advisers: Diane Slusarski and Val Sheffield
Place: Braley Auditorium (Pomerantz Family Pavilion)
Elucidating a role for BBS3 in syndromic and non-syndromic retinal disease
Bardet-Biedl Syndrome (BBS, OMIM) is a syndromic form of retinitis pigmentosa, characterized by retinal degeneration, obesity, learning disabilities, congenital abnormalities and increased incidences of hypertension and diabetes. Individuals with BBS are blind by the third decade of life. At least fourteen genes are reported to individual cause BBS. This thesis focuses on BBS3, with the overall goal of characterizing the function of BBS3 in terms of both syndromic and non-syndromic retinal degeneration.
A member of the Ras family of small GTP-binding proteins, BBS3 is postulated to play a role in vesicular transport. A second highly conserved transcript of BBS3, BBS3L, is expressed in the mouse and zebrafish eye. Histological analysis of Bbs3L knockout mice at 9 months reveals disorganization of the inner segments, indicative of retinal degeneration. To further evaluate the functional effects of BBS3 deficiency in the eye, an antisense oligonucleotide (Morpholino) approach was utilized to knockdown bbs3 gene expression in zebrafish. Consistent with an eye specific role, knockdown of bbs3L results in mislocalization of the photopigment green cone opsin and reduced visual function, but not abnormalities of the Kupffer’s vesicle or delays in intracellular trafficking of melanosomes, both cardinal features of BBS in the zebrafish. To dissect the individual functions of BBS3 and BBS3L, BBS3 or BBS3L RNA was co-injected with the bbs3 morpholinos. BBS3L RNA, but not BBS3 RNA, restores green opsin localization and vision. Moreover, only BBS3 RNA is sufficient to rescue melanosome transport. Together these data demonstrate that BBS3L is necessary and sufficient for retinal function and organization.
This work was extended to humans by characterizing the A89V missense mutation in BBS3 identified in retinitis pigmentosa patients. To evaluate the in vivo function of the A89V missense mutation in non-syndromic retinal degeneration and BBS, rescue experiments were performed in the zebrafish. Unlike BBS3L RNA, BBS3L A89V RNA does not rescue the vision defect seen with loss of bbs3 in zebrafish; however, BBS3 A89V RNA suppresses melanosome transport delays. These data demonstrate that the mutation identified in patients with non-syndromic retinal degeneration is critical and specific for the vision defect.