R953C Mutation in SORL1 Gene Causes Functional Abnormality of the Protein

Abstract

AbstractBackgroundThe SORL1 (Sortilin Related Receptor 1) gene encodes for an endosomal sorting receptor, SORLA, and recent genetic studies have shown that it is the gene most frequently affected in patients with Alzheimer’s disease (AD). In the current study, we have identified a variant, R953C, (residing in the YWTD‐domain of SORLA) in a family where it segregates with early‐onset AD. We used cell biological assays to evaluate maturation and sorting defects of the mutant receptor.MethodTo investigate molecular consequences of R953C mutation, we transiently expressed SORLA‐WT and SORLA‐R953C in HEK293 and N2a cell lines. We analyzed the maturation and shedding of the receptor by Western blotting. Using flow cytometry, we determined the cell surface expression of the mutant compared to WT. Finally, the intracellular localization of the mutant was assessed by immunocytochemistry and confocal microscopy.ResultWe observed a significant decrease in the maturation and shedding of the mutant receptor, leading to an overall 80% reduction in soluble SORLA present in the culture media of cells transfected with R953C construct compared to WT. Our flow cytometry analysis demonstrated that in 80% of the cells expressing the mutant receptor, the SORLA protein is retrained intracellularly. In comparison, only 10‐15% of cells expressing the WT receptor showed intracellular retention. Within the cells, the R953C mutant receptor was primarily localized to the endoplasmic reticulum (ER) whereas the WT was mainly localized to the endosomes, suggesting the retention of the mutant in the ER and uncovering possible defects in entering the endosomal sorting pathway.ConclusionIn conclusion, we have elucidated some of the molecular mechanisms that appear to have a role in pathogenicity of R953C mutation.