Aberrant regulation of choline metabolism by mitochondrial electron transport system inhibition in neuroblastoma cells

Abstract

Anomalous choline metabolic patterns have been consistently observed in vivo using Magnetic Resonance Spectroscopy (MRS) analysis of patients with neurodegenerative diseases and tissues from cancer patient. It remains unclear; however, what signaling events may have triggered these choline metabolic aberrancies. This study investigates how changes in choline and phospholipid metabolism are regulated by distinct changes in the mitochondrial electron transport system (ETS). We used specific inhibitors to down regulate the function of individual protein complexes in the ETS of SH-SY5Y neuroblastoma cells. Interestingly, we found that dramatic elevation in the levels of phosphatidylcholine metabolites could be induced by the inhibition of individual ETS complexes, similar to in vivo observations. Such interferences produced divergent metabolic patterns, which were distinguishable via principal component analysis of the cellular metabolomes. Functional impairments in ETS components have been reported in several central nervous system (CNS) diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD); however, it remains largely unknown how the suppression of individual ETS complex function could lead to specific dysfunction in different cell types, resulting in distinct disease phenotypes. Our results suggest that the inhibition of each of the five ETS complexes might differentially regulate phospholipase activities within choline metabolic pathways in neuronal cells, which could contribute to the overall understanding of mitochondrial diseases.

Fig. 1

Changes in choline metabolite levels following ETS complex inhibition. (a) An ¹H-NMR spectrum of SH-SY5Y neuroblastoma cell metabolites in the absence of ETS inhibition. Metabolites were extracted with 12% perchloric acid. BCAA’s: branched chain amino acids; Lac: lactate; Ala: Alanine; Glu: Glutamate; Gln: Glutamine; Asp: Aspartate; Cr: Creatine; Cho: Choline; PC: Phosphorylcholine; GPC: Glycerophosphorylcholine; Tau: Taurine; m-Ino: Myo-inositol; Gly: Glycine. The chemical shifts were assigned according to Govindaraju et al. (2000). (b) Select regions within representative ¹H NMR spectra where Cho, PC and GPC were detected. Values are given as % over control, mean ± SD (n = 3, *P < 0.05, **P <0.01, ***P < 0.001). All ETS complex inhibitors resulted in a combined increase in total choline metabolites (tCho). (c) Quantitative analysis of NMR spectral changes in specific choline metabolites following each ETS complex inhibitor treatment. (d) A MS spectrum (m/z 50–300) of a control sample. The masses for Cho and PC are monoisotopic. (e) Quantitative analysis of Cho and PC by MALDI-TOF MS. Values are given as % over control, mean ± SD (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001)

Fig. 2

Principal component analysis of the metabolomes following all six treatments. (a) A PCA score plot distinguishes the metabolic profiles of SH-SY5Y neuroblastoma cells treated with: I: 1 mM MPTP; II: 5 mM 3-NP; III: 4 µg/ml antimycin-A; IV: 2 mM sodium azide and V: 1 µg/ml oligomycin. C: Control cells treated with saline only. The solid ellipse denotes the 95% significance limit (Hotelling T²) and the dotted ellipse indicates the differentially inhibited sample groups (the dotted lines do not have statistical significance). (b) Loading plot for principal component 1 (PC1). (c) Loading plot for principal component 2 (PC2). The PC1 calculated by this method represents the largest variation among the data sets and the subsequent PCs represent lesser variations by comparison. Usually, the first three PCs should be able to explain 50–80% of the total variation. The resulting PCA scores were subjected to one-way analysis of variance (ANOVA) in order to test the level of statistical difference among the sample groups. Differences were considered significant when P ≤ 0.05

Fig. 3

Western blot analysis of choline kinase following ETS Complex inhibition. Twenty micrograms of proteins derived from SH-SY5Y cells treated with each of five ETS complex inhibitors were loaded on a 12% SDS-PAGE gel and probed with a anti-choline kinase antibody (ab38290, Abcam). The results suggest that other than azide, choline kinase was induced by all of the other inhibitors of ETS complexes, albeit to a varying degrees compared to untreated SH-SY5Y cells

Fig. 4

Choline metabolic pathways. The scheme shows the structures of individual choline metabolic intermediates and the enzymes that take part in the choline metabolic flux. Metabolites observed in this study are highlighted