Abstract
Purpose :
Age-related macular degeneration (AMD) is characterized by a progressive loss of photoreceptors and Retinal Pigment Epithelium (RPE) cells in the macula, leading to vision loss. Lipid deposits called drusen accumulate as the disease progresses and several genes involved in lipid metabolism have been shown to be associated with AMD. However, the role of lipids in AMD progression is unclear. In-house rare variant burden analysis identified four rare variants in CIDEC in patients with slow progressing AMD and better outcome after anti-VEGF therapy. CIDEC (Cell death-Inducing DFFA-like Effector C) has been shown to interact with effector molecules such as PLIN1, AS160 and Rab8a to facilitate lipid droplet (LD) fusion during adipogenesis. We tested the functional consequences of the slow progressing AMD CIDEC rare variants on LD fusion.
Methods :
3T3-L1 pre-adipocytes were transfected with CIDEC variants and the kinetics of LD fusion were assessed via live imaging. More than 100 LDs expressing both GFP tagged CIDEC and mCherry tagged PLIN1 were analyzed for wild-type (WT) and mutant CIDEC for six hours. CIDEC dimerization capability and functional interaction with PLIN1, AS160, and Rab8a were tested by immunoprecipitation analysis carried out using 293T cells transiently transfected with WT or mutant CIDEC.
Results :
All four CIDEC variants showed significant defects in LD fusion compared to WT (Student’s t-test, p<0.005). Over 6 hours, cells expressing CIDEC WT had 14.6%±1.9% of their LD achieving fusion. Cells expressing CIDEC V47I, Y61H and V161M had a severe loss of LD fusion with 0.3%±0.5%, 2.0%±1.8% and 1.2%±1.1% respectively, and cells expressing CIDEC Q220H had a total loss of LD fusion function (0% of LD fusion). We also found that CIDEC V47I and Y61H showed defective dimerization ability, whereas CIDEC V161M and Q220H were not affected. Finally, we found that all four CIDEC variants showed weaker interaction with PLIN1, AS160, and Rab8a compared to WT.
Conclusions :
Our results demonstrate that the cells expressing CIDEC rare variants have severe defect in LD fusion, which arise from defects in CIDEC dimerization and/or interaction with effector molecules. Future studies will focus on how disrupted LD fusion by mutations in CIDEC allow for slow progressing AMD and better outcome in anti-VEGF therapy.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.