Discerning the functional role of plasmalogen at the synapse by shotgun lipidomics

Abstract

We seek to investigate the functional role of PEp in neurons with aims to better understand its involvement in neurodegeneration and the potential of clinical applications targeting its metabolism. To achieve this, we have developed a simple in vitro neuronal model to assay synapse function while modulating PEp levels. SH-SY5Y neuroblastoma cells are differentiated into neuron-like cells by serum starvation and incubation with neurotropic factors. These cells exhibit a highly polarised, neuron-like morphology, forming neural networks and co-localised pre- and post-synaptic markers. Plasmalogen levels and speciation are modulated by post-transcriptional gene silencing of PEp biosynthetic enzymes and supplementation with plasmalogen precursors and labelled PUFA. PEp levels are analysed by shotgun lipidomics in parallel with assays of synaptic assembly and protein expression including ICC/IF, PCR, and Western Blot. For targeted ESI-HRAM-MS/MS shotgun lipidomics, we utilised an LTQ-Orbitrap XL (ThermoFisher Scientific) coupled with a TriVersa NanoMate (Advion Biosciences), following established methods (Nielsen, 2017). Lipids are identified by product-ion scan MS n analyses, targeting PE, PEo, and PEp species for quantification against an internal standard. PEp specie identification was confirmed by MS n analysis (Hsu, 2018) and sensitivity to acid hydrolysis (Han, 2005). Our preliminary data demonstrates that PEp levels and fatty acid composition play a role in synapse formation, with further experiments being implemented to investigate underlying mechanisms. We are currently developing further our shotgun lipidomics approach to employ data-independent acquisition and incorporate a range of internal standards to enable identification and quantitation of the global lipidome.