Abstract
Purpose: :
Inherited retinal degenerations are highly heterogeneous disorders, with mutations in over 160 genes identified to date. Recently, an increasing number of new disease genes have been found to encode components of the photoreceptor sensory cilium (PSC), such as SDCCAG8, AHI1, CEP290, FAM161A, IMPG2 and PCDH2. Based on these observations, we hypothesize that genes which encode novel PSC proteins will be good candidate disease genes for inherited retinal degenerations. To identify novel PSC proteins required for photoreceptor formation and function, we are currently investigating the locations and functions of 192 novel PSC proteins selected from the PSC proteome.
Methods: :
We prepared expression plasmids of the selected PSC proteins using a pCAG-V5-cDNA-IRES-EGFP vector, and transfected the plasmids into the retinas of 1-2 day old rats using in vivo electroporation. We assessed the location of the recombinant proteins in the photoreceptor cells of the transfected retinas 21 days following transfection by immunofluorescence analysis.
Results: :
To date, we have analyzed 125/192 selected proteins. EGFP and V5 signals were detected from 113 plasmids. 12 plasmids expressed only EGFP, but not the V5-tagged proteins. Through these analyses, we have identified 26 novel proteins that are located in the outer segments and/or axoneme of PSC. For example, proteins PSC253, PSC258 and PSC272 were located exclusively in outer segments, and PSC269, PSC304 and PSC382 are located exclusively in the transition zone. Over 30 proteins were detected in inner segment. The majority of the remaining proteins were soluble with the V5 signals detected in the water space of PSCs. Immunofluorescence analyses using antibodies to several of the novel PSC proteins confirmed the reliability of the location of the recombinant proteins.
Conclusions: :
Our results indicate that the PSC proteome contains novel photoreceptor proteins that appear to be required for photoreceptor formation and function. We are hopeful that the identification of new photoreceptor proteins will lead to discovery of new disease genes for inherited retinal degenerations. Screening of the human genes which encode the novel PSC proteins for mutations in patients with inherited retinal degenerations and related cilia disorders has been initiated.
Keywords: retinal degenerations: hereditary • photoreceptors • proteins encoded by disease genes