L-Methionine Protects against Oxidative Stress and Mitochondrial Dysfunction in an In Vitro Model of Parkinson's Disease

Abstract

Methionine is an aliphatic, sulfur-containing, essential amino acid that has been demonstrated to have crucial roles in metabolism, innate immunity, and activation of endogenous antioxidant enzymes, including methionine sulfoxide reductase A/B and the biosynthesis of glutathione to counteract oxidative stress. Still, methionine restriction avoids altered methionine/transmethylation metabolism, thus reducing DNA damage and possibly avoiding neurodegenerative processes. In this study, we wanted to study the preventive effects of methionine in counteracting 6-hydroxydopamine (6-OHDA)-induced injury. In particular, we analyzed the protective effects of the amino acid L-methionine in an in vitro model of Parkinson's disease and dissected the underlying mechanisms compared to the known antioxidant taurine to gain insights into the potential of methionine treatment in slowing the progression of the disease by maintaining mitochondrial functionality. In addition, to ascribe the effects of methionine on mitochondria and oxidative stress, methionine sulfoxide was used in place of methionine. The data obtained suggested that an L-methionine-enriched diet could be beneficial during aging to protect neurons from oxidative imbalance and mitochondrial dysfunction, thus preventing the progression of neurodegenerative processes.

Keywords: brain; dietary supplement; mitochondria; neurodegeneration; nutraceuticals; oxidative stress; reactive oxidative species.

Figure 1

Cell viability assay for differentiated SH-SY5Y pretreated with Met or Met-O and taurine before 6-OHDA treatment. Data are mean ± SD of 3 different experiments. * p < 0.05 vs. 6-OHDA; +++ p < 0.0001 vs. control (CTR).

Figure 2

On the left, heatmap of hierarchical clustering (Euclidean metrics) of the 3175 genes whose expression is significantly perturbed by 6-OHDA (adjusted p < 0.05). Color scale represents log2 ratios of the expression levels in the indicated condition versus control. Color scale limits are indicated in the box below the heatmap. On the right, MA plots show up- and down-regulated genes in the different treatments (in red, the significantly upregulated genes; while in green, the downregulated).

Figure 3

Heatmap of hierarchical clustering of the selected pathways. All the genes of the pathway that are significantly affected by 6-OHDA (adjusted p < 0.05) are reported in the heatmaps. Green boxes indicate the genes significantly affected by 6-OHDA. Color scale represents log2 ratios of the expression levels in the indicated condition versus control. Color scale limits are indicated in the lower box.

Figure 4

Apoptosis assayed by Annexin V in live-cell by Incucyte system and WB analysis for cleaved-caspase9 and p-JNK. A representative WB figure is shown. Data are mean ± SD of 3 different experiments. * p < 0.05, ** p < 0.005 vs. 6-OHDA; +++ p < 0.0001; ++ p < 0.005; + p < 0.05 vs. CTR.

Figure 5

WB analyses for neuroprotective pathways. A representative WB figure is shown. Data are mean ± SD of 3 different experiments. * p < 0.05, ** p < 0.005 vs. 6-OHDA; ++ p < 0.005 vs. CTR.

Figure 6

Antioxidant pathway analyzed by WB analyses, Oxyblot detection kit, and SOD activity assay. A representative WB figure is shown. Data are mean ± SD of 3 different experiments. *** p < 0.0001, ** p < 0.005, * p < 0.05 vs. 6-OHDA; +++ p < 0.0001; ++ p < 0.005; + p < 0.05 vs. CTR.

Figure 7

WB analyses for the Mrsb2 pathway. A representative WB figure is shown. Data are mean ± SD of 3 different experiments. ** p < 0.005, * p < 0.05 vs. 6-OHDA; +++ p < 0.0001; ++ p < 0.005; + p < 0.05 vs. CTR.

Figure 8

On the left, Mitotracker Green, Mitosox Red, and DAPI representative figures are shown; on the right, the normalized fluorescence intensity graph is shown. Scale bar: 50 µm. Data are mean ± SD of 3 different experiments. ** p < 0.005, * p < 0.05 vs. 6-OHDA; +++ p < 0.0001, + p < 0.05 vs. CTR.

Figure 9

TMRM in live cells by the Incucyte system at 0, 4, 12, and 24 h time points. Representative images and relative normalized fluorescence graph are shown. Data are mean of 3 different experiments ± SD, and the significance is reported in Table S1.

Figure 10

DAPI, Mitotracker deep red, and the masks generated by ImageJ representative images are reported. In the red circle the area is magnified to better appreciate the mitochondrial morphology. WB analyses for fission and fusion pathways. A representative WB figure is shown. Data are mean ± SD of 3 different experiments. *** p < 0.0001, ** p < 0.005 vs. 6-OHDA; +++ p < 0.0001; ++ p < 0.005; + p < 0.05 vs. CTR.

Figure 11

Mitochondrial bioenergetic profile in CTR and treated cells. (A) Seahorse XF Cell Mito stress test profile illustrated the key parameters of mitochondrial function upon the injection of the different drugs. (B) Graph relative to basal respiration, ATP production, maximal respiration, and non-mitochondrial respiration in control and treated cells. Data are mean ± SD of 3 different experiments. *** p < 0.0001, ** p < 0.005 vs. 6-OHDA; +++ p < 0.0001; ++ p < 0.005; + p < 0.05 vs. CTR.

Figure 12

Schematic representation of the underlying mechanisms of Met neuroprotective effects. Gclc: glutamate cysteine ligase catalytic subunit; Gclm: glutamate cysteine ligase regulatory subunit; GS: glutathione synthetase.