Quantitative Analysis of ELOVL4 Protein and Fatty Acid Products in Knock-out and Knock-in Mouse Tissues

Abstract

Purpose: : Mutations in the ELOVL4 gene cause STGD3 in humans and the ELOVL4 protein has been shown to elongate very long chain saturated (VLC-FA) and polyunsaturated fatty acids (VLC-PUFA) >26 carbons in length. It is not known whether the dominant pathology in the retina is due to reduction of VLC-FA and/or VLC-PUFA or due to a toxic effect exerted by the mutant ELOVL4 protein. Analysis of mouse models showed no pathology in heterozygous knock-out retinas having only one copy of ELOVL4, but a slowly developing pathology including photoreceptor cell loss and accumulation of lipofuscin-like material in the RPE was evident in heterozygous knock-in retinas having one copy of wild type ELOVL4 along with a mutant copy. Here, we analyzed the quantity of wild type ELOVL4 protein and its VLC-FA and VLC-PUFA products in both knock-in (KI) and knock-out (KO) heterozygous mouse tissues (retina and skin) to gain insight into the pathogenesis resulting from Elovl4 mutation.

Methods: : ELOVL4 protein (wild type) localization in the retina was determined by immunohistochemistry. Levels of wild type ELOVL4 protein in skin and retinas were determined by western blotting and densitometric analysis after normalization with actin. Total lipids from skin and retinas of 8-week-old mice were extracted and analyzed for VLC-FA and VLC-PUFA contents by GC-MS. The levels of these lipids were determined after normalization with either total fatty acid or total lipid phosphorus content.

Results: : Immunohistochemical analysis suggested that wild type ELOVL4 protein was reduced in both heterozygous KO and KI retinas. We did not detect any noticeable differences in the localization of wild type ELOVL4 protein in either KI-HET or KO-HET retinas by light (confocal) microscopy. The level of wild type ELOVL4 protein was only 37% in KO-HET and 28% in KI-HET retina compared to their wild type littermate controls. ELOVL4 protein was reduced to a similar magnitude in respective skin samples. ELOVL4 products (VLC-FA) were reduced by 50% in the skin of KO-HETs and by 60-65% in KI-HETs. A significant reduction in the level of VLC-PUFA was also detected in KI-HET retina.

Conclusions: : The reduction in the VLC-PUFA content in KI-Het retinas indicates partial loss of functional ELOVL4 in these mice, which may be due to the presence of a mutant copy of this protein.

Keywords: lipids • retinal degenerations: hereditary • photoreceptors