Alteration in Lysophospholipids and Converting Enzymes in Glaucomatous Optic Nerves

Abstract

Purpose

To determine whether lysophospholipid (LPL) profiles and corresponding conversion enzymes in the LPL pathways are altered in the optic nerve (ON) between human control and glaucoma samples.

Methods

Lipids extracted from control ( n = 11) and glaucomatous ( n = 12) ON samples using the Bligh and Dyer method were subjected to high-resolution mass spectrometry on a Q-exactive mass spectrometer coupled with a high-performance liquid chromatography (Accela 600) system. Analysis was performed for LPLs (lysophosphatidylcholines, lysophosphatidylserines, lysophosphatidylethanolamines, lysophosphatidylinositols, and lysosphingomyelines) using LipidSearch v.4.1, MZmine v.2.0, and MetaboAnalyst v.4.0. LPL synthesis and degradation pathway maps, utilizing UniProt and BRENDA database entries as needed, were created using Kyoto Encyclopedia of Genes and Genomes (KEGG)–based tools. The mRNA expression level in normal and glaucomatous human ON were analyzed using Gene Expression Omnibus (GEO) entry GSE45570. Protein amounts were determined using PHAST gel and dot blot and were used for normalization of protein amounts across samples. Western blot, ELISA, and protein quantification were performed using established protocols.

Results

Principal component analysis of ON LPL profile placed control and glaucomatous ONs in two distinct separate groups. Mass spectrometric analysis of ON revealed decrease in lysophosphatidic acid, lysophosphatidylethanolamine, lysophosphatidylcholine, and significant increase in diacylglycerol in glaucomatous ON. Statistical analysis of LPL conversion enzymes revealed significant overexpression of phosphatidate phosphatase LPIN2, phospholipid phosphatase 3, phosphatidylcholine-sterol acyltransferase, and calcium-dependent phospholipase 2, and significant downregulation of glycerol-3-phosphate acyltransferase 4 at mRNA level in glaucomatous ON. Western blot and ELISA confirmed proteomic differences between normal and diseased ON.

Conclusions

Our analysis revealed alterations in specific LPL levels and corresponding select enzyme-level changes in glaucomatous ON.