Bardet-Biedl syndrome (BBS) is a human ciliopathy characterized by the dysfunction of primary cilia and retinal degeneration via photoreceptor cell degradation in the affected retina. The BBS genes associated with the ciliopathy are all associated with the primary cilia, and 8 BBS proteins form a BBSome protein complex. Limited analysis of primary cilia dynamics in BBS mutant mice suggests that the BBSome is essential for transport of primary cilia cargo proteins, but beyond these preliminary findings, little is known of BBSome function in the primary cilia, including the primary cilia of rod photoreceptors in the retina. Notably, retinal gene replacement via AAV subretinal injection has yielded promising results in preliminary studies in BBS mutant moue models. In this proposal, I outline a series of experiments that utilize STORM and PALM super resolution imaging along with cryo-electron tomography (cryo-ET) ultra-structural analysis to address (1) the precise localization of the BBSome in rod photoreceptor cilia and (2) the subcellular outcome of retinal gene replacement therapy into rod photoreceptor cells. (1) STORM super resolution localization (to a resolution of ~20nm) will be used to localize the BBSome complex in rod photoreceptor cells of wild-type, as well as BBS4- /- and BBS1M390R mutant mice, via a series STORM immunostaining experiments with specific antibodies. (2) BBS1 and BBS4 fused to PALM protein tags will be cloned into an optimized AAV vector for validation and subsequent subretinal injection into the BBS4-/- and BBS1M390R mutant mouse retinas. Localization of treated rod photoreceptors cells will be assessed with a combination of STORM and PALM imaging, in addition to ultra-structural morphological analysis via cryo-Et. With the application of these new and powerful imaging techniques, I will assess, for the first time, the subcellular effects of retinal gene therapy, and accurately track the localization of the BBSome relative to markers of the rod cilium and the morphological defects associated with these BBS mutant models. Together, the results from these research aims will aid in determining the function of the wild-type BBSome, which is critical for rod cell viability, and will also demonstrate the feasibility for BBS gene replacement therapy into mutant photoreceptor cells as a possible treatment for BBS and other retinal degenerative defects.
The Bardet-Biedl syndrome (BBS) is a devastating human genetic disorder that causes early childhood retinal degeneration and leads to significant visual impairment and complete blindness. The BBS genes, which are mutated in human patients, are essential for the biological function of photoreceptor cells (rods and cones), the light sensing cells of the retina. In this fellowship proposal, I detail experiments which utilize novel super resolution microscopy techniques to uncover the function of BBS in normal mouse retinas and in mouse mutant models for BBS that have been treated with retinal gene therapy to detail the subcellular effects of this therapeutic approach.
| Dharmat, Rachayata; Eblimit, Aiden; Robichaux, Michael A et al. (2018) SPATA7 maintains a novel photoreceptor-specific zone in the distal connecting cilium. J Cell Biol 217:2851-2865 |
| Wensel, Theodore G; Zhang, Zhixian; Anastassov, Ivan A et al. (2016) Structural and molecular bases of rod photoreceptor morphogenesis and disease. Prog Retin Eye Res 55:32-51 |
