Our research objective is to study the link between Rds (a retinal tetraspanin) and the maintenance of the cone and rod outer segments in order to promote a better understanding of the debilitating retinal degenerations that accompany Rds mutations. In this proposal, I hypothesize that Rds forms an intricate complex of multiple proteins which interact to regulate the outer segment cytoskeleton. The goal of this application is to characterize the differences between Rds complexes in rods vs. cones and examine the way those complexes interact with the cytoskeleton by using cone and rod-dominant mouse models expressing different amounts of Rds.
Two aims are proposed:
in Aim 1 I will determine what differences exist in the protein composition of Rds complexes in rods and cones. I will use radiolabeling and affinity purification to identify Rds interacting proteins and palmitoylation status in rods (using the rod only Crx-Nrl mouse) and cones (using the Nrl""""""""'"""""""" mouse). Further, I will employ biochemical and structural methods such as reciprocal co-IP, velocity sedimentation, and immunohistochemistry/confocal microscopy to characterize Rds binding partners identified by preliminary proteomic studies on affinity purified Rds complexes from bovine outer segments. The goal of Aim 2 is to determine whether Rds complexes participate in regulation of the outer segment cytoskeleton. I hypothesize that Rds is needed for the proper formation of the outer segment rim microdomain and that this microdomain is necessary for the proper localization of proteins that regulate the cytoskeleton and adhesion complexes.
This aim will include biochemical and structural experiments using several unique mouse models which express varying amounts of Rds in rods (Crx-Nrl, Crx-Nrl/rc/s+A, Crx-Nrl/rctev') and cones (Nrl""""""""'', Nrl^/rcfe* Nrf/L/rcfe-/). In addition to the core scientific aims of this project, an additional purpose of this work is to give me an experimental framework within which I can develop into an independent researcher. In keeping with the goals of the National Eye Institute, this application is directly relevant to public health. Rds mutations can cause severe visual defects and there are no curative treatments for inherited retinal degenerations currently available. The project will provide important insight into the cell biology of outer segment formation particularly regarding the role of Rds in that process. Further understanding of outer segment morphogenesis, particularly in the less well-studied cone, is critical both for understanding the disease process in the human retina and for developing new treatments for macular degenerations.
|Conley, Shannon M; Al-Ubaidi, Muayyad R; Han, Zongchao et al. (2014) Rim formation is not a prerequisite for distribution of cone photoreceptor outer segment proteins. FASEB J 28:3468-79|
|Conley, Shannon M; Stuck, Michael W; Burnett, Justin L et al. (2014) Insights into the mechanisms of macular degeneration associated with the R172W mutation in RDS. Hum Mol Genet 23:3102-14|
|Koirala, Adarsha; Conley, Shannon M; Makkia, Rasha et al. (2013) Persistence of non-viral vector mediated RPE65 expression: case for viability as a gene transfer therapy for RPE-based diseases. J Control Release 172:745-52|
|Koirala, Adarsha; Conley, Shannon M; Naash, Muna I (2013) A review of therapeutic prospects of non-viral gene therapy in the retinal pigment epithelium. Biomaterials 34:7158-67|
|Chakraborty, Dibyendu; Conley, Shannon M; Naash, Muna I (2013) Overexpression of retinal degeneration slow (RDS) protein adversely affects rods in the rd7 model of enhanced S-cone syndrome. PLoS One 8:e63321|
|Han, Zongchao; Guo, Junjing; Conley, Shannon M et al. (2013) Retinal angiogenesis in the Ins2(Akita) mouse model of diabetic retinopathy. Invest Ophthalmol Vis Sci 54:574-84|
|Chakraborty, Dibyendu; Rodgers, Karla K; Conley, Shannon M et al. (2013) Structural characterization of the second intra-discal loop of the photoreceptor tetraspanin RDS. FEBS J 280:127-38|
|Koirala, Adarsha; Makkia, Rasha S; Conley, Shannon M et al. (2013) S/MAR-containing DNA nanoparticles promote persistent RPE gene expression and improvement in RPE65-associated LCA. Hum Mol Genet 22:1632-42|
|Stuck, Michael W; Conley, Shannon M; Naash, Muna I (2012) Defects in the outer limiting membrane are associated with rosette development in the Nrl-/- retina. PLoS One 7:e32484|
|Conley, Shannon M; Stuck, Michael W; Naash, Muna I (2012) Structural and functional relationships between photoreceptor tetraspanins and other superfamily members. Cell Mol Life Sci 69:1035-47|
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