Proteomic Studies On Mechanisms Of Methylglyoxal-induced Neurotoxicity In SH-SY5Y Cells

Reactive carbonyl compounds are either products of glucose metabolism or products of oxidative damage to lipids. They contribute to protein cross-linking and thus present a toxic burden to a variety of cells, especially neurons, which have a high rate of oxidative metabolism. Therefore, dicarbonyl compounds such as methylglyoxal (MG) or glyoxal and unsaturated carbonyls such as hydroxynonenal or acrolein have been implicated as important neurotoxic mediators of oxidative damage in the pathogenesis of Alzheimer's disease (AD) and other neurodegenerative diseases.Among reactive carbonyl compounds, MG is unique in that it is produced predominantly as a non-enzymatic degradation product of triosephosphates. Notably, MG can yield advanced glycation end products (AGEs) via non-enzymatic glycation of proteins through a process that has been reported to cause free radical production. MG has been shown to be cytotoxic to macrophages, insulin-secreting cells, and neurons. Although several studies have revealed that MG can induce apoptosis of both non-neurons and neurons, possibly via the activation of the mitogen-activated protein kinase family or c-jun NH2-terminal kinase, the molecular mechanisms underling MG cytotoxicity remain poorly understood.Given the crucial role of MG in neurodegenerative diseases, it is imperative to understand the molecular and biochemical mechanisms of MG neurotoxicity. In principle, the proteomics approach offers a powerful tool to characterize the molecular events that occur during MG-induced neurodegeneration. Unfortunately, the successful application of proteomics to identify MG-induced protein changes in the central nervous system (CNS) remains a technical challenge due to the cellular heterogeneity of the CNS.To overcome this problem of sample heterogeneity we use a particular cell line for proteomic analysis of MG-induced neurotoxic effects. The human SH-SY5Y neuroblastoma cell line is well characterized and has been widely used as a model system to investigate the pathological effects of various neurotoxic compounds. In addition, SH-SY5Y cells constitutively express a receptor for AGEs. They also are highly sensitive to MG challenge due to a defect in their antioxidant and detoxifying abilities that prevents efficient scavenging and that elicits extensive caspase-9-dependent apoptosis.In the present study, SH-SY5Y cells were treated with MG and MTT assay, acridine orange/ethidium bromide (AO/EB) double staining,2-D fluorescence difference gel electrophoresis (2-D DIGE) coupled with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), were used to quantitatively examine the alteration in protein levels associated with MG treatment.The first step of this study was to verify that MG can induce cytotoxicity in SH-SY5Y cells. The MTT assay demonstrated that MG treatment led to a reduction of the viability of SH-SY5Y cells in a dose-dependent manner. The cell survival rate was 82.85%±2.1,71.08%±1.3,56.85%±1.6, and 44.50%±2.4 when cells were treated with MG at concentrations of 0.25 mM,0.50 mM,0.75 mM, and 1.00 mM, respectively. These results suggest that MG may reduce cell viability and induce cell death. The microscopic examination of AO/EB stained cells is a very reliable method for determining cell viability because it makes it possible to distinguish viable from early and late stage apoptotic and non-apoptotic death cells. AO/EB double fluorescent staining showed that MG treatment increased the cell death ratio in a dose-dependent manner. The cell apoptosis rate was 4.21%,12.32%,22.85%,23.09%, and 22.00% when SH-SY5Y cells were treated with MG at concentrations of 0 mM,0.25 mM, 0.50 mM,0.75 mM, and 1.00 mM, respectively. Taken together, these results indicate that MG induces decreased cell viability and increased cell death in SH-SY5Y cells.Having shown that MG treatment had cytotoxic effects on SH-SY5Y cells, next we performed 2D-DIGE analysis to examine the alteration in protein levels in MG-treated SH-SY5Y cells. After 2D fluorescent gel images were obtained via a Typhoon scanner, protein spots were detected, quantified, matched, and analyzed with DeCyder software. The use of the internal standard effectively eliminated gel-to-gel variation, allowing the detection of small differences in protein levels. Only the protein spots were detected at least three times on different gels were the proteins considered for further quantitative analysis. According to the statistics results,49 protein spots were found to be differentially expressed, of which 16 were increased and 33 decreased. To identify the differentially expressed proteins, these 49 protein spots of interest were excised and subjected to MALDI-TOF MS protein identification, which resulted in specific peptide mass data. The peptide mass data were first analyzed using in-house licensed ProFound by automatic searches of the NCBI protein database. Criteria for positive identification of proteins with MS were set as follows:match of molecular weight and isoelectric point, expectation< 0.05, and coverage> 20%. The protein identification was further verified by searching the Swiss-Prot protein database. Among them, totally 21 proteins were identified and only 8 proteins met the criteria of expectation< 0.05, and coverage> 20%.Further analysis of these 8 proteins showed that ACTB protein (beta actin) and sterile alpha motif domain containing 13 (SAMD13) protein were upregulated in MG versus vehicle samples, while six proteins including immunoglobulin V lambda/J lambda light chain, malignant cell expression-enhanced gene/tumor progression-enhanced gene, tight junction protein 2 (zona occludens 2 (ZO-2)), solute carrier family 9 (sodium/hydrogen exchanger)/ member 1 (antiporter, Na+/H+, amiloride sensitive), protein phosphatase 2 (PP2) catalytic subunit, and immunoglobulin heavy chain were downregulated in MG versus vehicle samples. We also found changes in protein levels of SAMD13, malignant cell expression-enhanced gene/tumor progression-enhanced gene, tight junction protein 2 (ZO-2), and solute carrier family 9 (sodium/hydrogen exchanger)/member 1 (antiporter, Na+/H+, amiloride sensitive). Their significance in neurotoxicity is not well defined yet, although ZO-2 is involved in the maintenance of the blood brain barrier.In summary, SH-SY5Y cells exposed to MG exhibited altered expression of multiple proteins that likely play a role in neurodegenerative diseases, especially in AD pathogenesis. To our knowledge this is the first proteomics study reporting that the protein level of ACTB was significantly upregulated and those of Ig lambda and PP2 were significantly downregulated in MG-treated SH-SY5Y cells. Thus, our results suggest that multiple pathways may be involved in MG-induced neurotoxicity.