Identification of key neuronal mechanisms triggered by dimethyl fumarate in SH-SY5Y human neuroblastoma cells through a metabolomic approach

Abstract

Dimethyl fumarate (DMF) is an old drug used for psoriasis treatment that has recently been repurposed to treat relapse-remitting multiple sclerosis, mostly due to its neuro- and immunomodulatory actions. However, mining of a pharmacovigilance database recently ranked DMF as the second pharmaceutical most associated with cognitive adverse events. To our best knowledge, the signaling mechanisms underlying its therapeutic and neurotoxic outcomes remain mostly undisclosed. This work thus represents the first-hand assessment of DMF-induced metabolic changes in undifferentiated SH-SY5Y human neuroblastoma cells, through an untargeted metabolomic approach using gas chromatography-mass spectrometry (GC-MS). The endometabolome was analyzed following 24 h and 96 h of exposure to two pharmacologically relevant DMF concentrations (0.1 and 10 μM). None of these conditions significantly reduced metabolic activity (MTT reduction assay). Our data showed that 24 h-exposure to DMF at both concentrations tested mainly affected metabolic pathways involved in mitochondrial activity (e.g., citric acid cycle, de novo triacylglycerol biosynthesis), and the synthesis of catecholamines and serotonin by changing the levels of their respective precursors, namely phenylalanine (0.68-fold decrease for 10 μM DMF vs vehicle), and tryptophan (1.36-fold increase for 0.1 μM DMF vs vehicle). Interestingly, taurine, whose levels can be modulated via Nrf2 signaling (DMF's primary target), emerged as a key mediator of DMF's neuronal action, displaying a 3.86-fold increase and 0.27-fold decrease for 10 μM DMF at 24 h and 96 h, respectively. A 96 h-exposure to DMF seemed to mainly trigger pathways associated with glucose production (e.g., gluconeogenesis, glucose-alanine cycle, malate-aspartate shuttle), possibly related to the metabolism of DMF into monomethyl fumarate and its further conversion into glucose via activation of the citric acid cycle. Overall, our data contribute to improving the understanding of the events associated with neuronal exposure to DMF.

Keywords: Endometabolome; Neuronal metabolic pathways; Neuroprotection; Nrf2 signaling.

Fig. 1

Metabolic activity, assessed by the MTT reduction assay, of SH-SY5Y cells exposed for 24 and 96 h to 0.1 μM and 10 μM dimethyl fumarate. Each bar represents the mean ± S.E.M., from at least three independent experiments, performed in duplicate. No statistically significant differences were found between the different concentrations tested and the respective vehicle control (0.1% DMSO), at each time point

Fig. 2

Partial least-squares discriminant analysis (PLS-DA) score scatter plots obtained for the DMF-exposed cells and controls (untreated and vehicle) after (A) 24 h and (B) 96 h. The ellipses indicate the 95% confidence limit of the model

Fig. 3

Bubble plot showing the magnitude and significance of alterations for each metabolite, according to the robust PLS-DA models obtained for dimethyl fumarate. Bubble size is proportionally related to the significance of the alteration, where smaller false discovery rate (fdr)-adjusted p values are associated with larger bubbles. The bubble color reflects the direction of the overall change (yellow for up-regulation and purple for downregulation)

Fig. 4

Overview of neuronal metabolic pathways altered by an acute exposure (24 h) with 0.1 μM (A) and 10 μM (B) DMF, and a prolonged exposure (96 h) with 10 μM (C) DMF. The colour of the scale is based on the p value, with yellow being associated with higher p values and red with lower p values. The red boxes highlight the significantly affected metabolic pathways (p < 0.05)