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β - N -Methylamino- l -alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling

View Article: PubMed Central - PubMed

ABSTRACT

β-Methylamino-l-alanine (BMAA) is a non-proteinogenic amino acid that induces long-term cognitive deficits, as well as an increased neurodegeneration and intracellular fibril formation in the hippocampus of adult rodents following short-time neonatal exposure and in vervet monkey brain following long-term exposure. It has also been proposed to be involved in the etiology of neurodegenerative disease in humans. The aim of this study was to identify metabolic effects not related to excitotoxicity or oxidative stress in human neuroblastoma SH-SY5Y cells. The effects of BMAA (50, 250, 1000 µM) for 24 h on cells differentiated with retinoic acid were studied. Samples were analyzed using LC–MS and NMR spectroscopy to detect altered intracellular polar metabolites. The analysis performed, followed by multivariate pattern recognition techniques, revealed significant perturbations in protein biosynthesis, amino acid metabolism pathways and citrate cycle. Of specific interest were the BMAA-induced alterations in alanine, aspartate and glutamate metabolism and as well as alterations in various neurotransmitters/neuromodulators such as GABA and taurine. The results indicate that BMAA can interfere with metabolic pathways involved in neurotransmission in human neuroblastoma cells.

No MeSH data available.


PCA model (R2X = 0.79, Q2X = 0.63) displaying untreated control cells (open circles) vs BMAA exposed cells (low (50 µM), medium (250 µM) and high (1000 µM) concentration, filled circles) obtained from LC–MS analysis in positive mode. Data were pre-processed with XCMS, normalized to total signal intensity and subjected to Pareto scaling using the SIMCA-P+ software. Moreover, only features with CV values below 30% were included in the analysis
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Fig4: PCA model (R2X = 0.79, Q2X = 0.63) displaying untreated control cells (open circles) vs BMAA exposed cells (low (50 µM), medium (250 µM) and high (1000 µM) concentration, filled circles) obtained from LC–MS analysis in positive mode. Data were pre-processed with XCMS, normalized to total signal intensity and subjected to Pareto scaling using the SIMCA-P+ software. Moreover, only features with CV values below 30% were included in the analysis

Mentions: Multivariate modeling with PCA illustrates the complexity of biological variance observed in the data based on different passages of cells (Fig. 4). As seen, most samples originating from BMAA-exposed cells are found in the lower right and left side of the plot (Fig. 4, filled circles) while untreated control cell samples (Fig. 4, open circles) are found in the upper region which indicate an altered metabolome. Furthermore, all low concentration BMAA samples are found in close proximity to the untreated control cell samples demonstrating a lesser effect due to exposure. It is plausible to assume that a higher BMAA concentration generates a larger disturbance in the metabolome as compared to low BMAA concentration as shown in Fig. 4. In addition, this trend is consistent in data from negative ionization (data not shown). Moreover, samples also cluster according to the batch (passage) of the cells; hence a large contribution is observed which from now on will be referred to as inter-batch variation. To more adequately display the relevant difference according to BMAA concentration, representative examples of a PCA plots for each batch is given in Fig. 5a–d. As observed, SH-SY5Y cells treated with a medium (Fig. 5, filled squares) or high BMAA concentration (Fig. 5, filled inverted triangles) results in a more drastic alteration of the metabolome as compared to a low BMAA concentration (Fig. 5, filled upright triangles) and untreated control cells (Fig. 5, open circles). To study the metabolic alterations caused by BMAA in a concentration-dependent manner, OPLS-DA models were created for each treatment as compared to vehicle; all models gave sufficient predictive Q2 values (>0.5). Features that were important to describe the difference between vehicle and BMAA exposed cells were selected from the corresponding S-plots according to the criteria stated previously. These features were subsequently tested for significance with ANOVA with post hoc Tukey’s tests after which differentiating metabolites were annotated for each BMAA concentration group as compared to vehicle (Table 2). The annotated metabolites belong to a diverse set of metabolite classes, including numerous amino acids and their derivates as well as carnitines, nucleosides and smaller organic acids. No traces of the masses corresponding to the parent compound BMAA and known hepatic BMAA metabolites (methylamine, 2,3-diaminopropanoic acid) (Nunn and Ponnusamy 2009) were found upon examination of the raw data. The recorded data for the high BMAA concentration experiment is similar to the response observed in the medium and low BMAA concentration experiments as compared to vehicle (data not shown). As the response to BMAA exposure was readily observed in the high BMAA concentration, further pathway analysis focused upon this exposure.Fig. 4


β - N -Methylamino- l -alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling
PCA model (R2X = 0.79, Q2X = 0.63) displaying untreated control cells (open circles) vs BMAA exposed cells (low (50 µM), medium (250 µM) and high (1000 µM) concentration, filled circles) obtained from LC–MS analysis in positive mode. Data were pre-processed with XCMS, normalized to total signal intensity and subjected to Pareto scaling using the SIMCA-P+ software. Moreover, only features with CV values below 30% were included in the analysis
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Related In: Results  -  Collection

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Fig4: PCA model (R2X = 0.79, Q2X = 0.63) displaying untreated control cells (open circles) vs BMAA exposed cells (low (50 µM), medium (250 µM) and high (1000 µM) concentration, filled circles) obtained from LC–MS analysis in positive mode. Data were pre-processed with XCMS, normalized to total signal intensity and subjected to Pareto scaling using the SIMCA-P+ software. Moreover, only features with CV values below 30% were included in the analysis
Mentions: Multivariate modeling with PCA illustrates the complexity of biological variance observed in the data based on different passages of cells (Fig. 4). As seen, most samples originating from BMAA-exposed cells are found in the lower right and left side of the plot (Fig. 4, filled circles) while untreated control cell samples (Fig. 4, open circles) are found in the upper region which indicate an altered metabolome. Furthermore, all low concentration BMAA samples are found in close proximity to the untreated control cell samples demonstrating a lesser effect due to exposure. It is plausible to assume that a higher BMAA concentration generates a larger disturbance in the metabolome as compared to low BMAA concentration as shown in Fig. 4. In addition, this trend is consistent in data from negative ionization (data not shown). Moreover, samples also cluster according to the batch (passage) of the cells; hence a large contribution is observed which from now on will be referred to as inter-batch variation. To more adequately display the relevant difference according to BMAA concentration, representative examples of a PCA plots for each batch is given in Fig. 5a–d. As observed, SH-SY5Y cells treated with a medium (Fig. 5, filled squares) or high BMAA concentration (Fig. 5, filled inverted triangles) results in a more drastic alteration of the metabolome as compared to a low BMAA concentration (Fig. 5, filled upright triangles) and untreated control cells (Fig. 5, open circles). To study the metabolic alterations caused by BMAA in a concentration-dependent manner, OPLS-DA models were created for each treatment as compared to vehicle; all models gave sufficient predictive Q2 values (>0.5). Features that were important to describe the difference between vehicle and BMAA exposed cells were selected from the corresponding S-plots according to the criteria stated previously. These features were subsequently tested for significance with ANOVA with post hoc Tukey’s tests after which differentiating metabolites were annotated for each BMAA concentration group as compared to vehicle (Table 2). The annotated metabolites belong to a diverse set of metabolite classes, including numerous amino acids and their derivates as well as carnitines, nucleosides and smaller organic acids. No traces of the masses corresponding to the parent compound BMAA and known hepatic BMAA metabolites (methylamine, 2,3-diaminopropanoic acid) (Nunn and Ponnusamy 2009) were found upon examination of the raw data. The recorded data for the high BMAA concentration experiment is similar to the response observed in the medium and low BMAA concentration experiments as compared to vehicle (data not shown). As the response to BMAA exposure was readily observed in the high BMAA concentration, further pathway analysis focused upon this exposure.Fig. 4

View Article: PubMed Central - PubMed

ABSTRACT

β-Methylamino-l-alanine (BMAA) is a non-proteinogenic amino acid that induces long-term cognitive deficits, as well as an increased neurodegeneration and intracellular fibril formation in the hippocampus of adult rodents following short-time neonatal exposure and in vervet monkey brain following long-term exposure. It has also been proposed to be involved in the etiology of neurodegenerative disease in humans. The aim of this study was to identify metabolic effects not related to excitotoxicity or oxidative stress in human neuroblastoma SH-SY5Y cells. The effects of BMAA (50, 250, 1000 µM) for 24 h on cells differentiated with retinoic acid were studied. Samples were analyzed using LC–MS and NMR spectroscopy to detect altered intracellular polar metabolites. The analysis performed, followed by multivariate pattern recognition techniques, revealed significant perturbations in protein biosynthesis, amino acid metabolism pathways and citrate cycle. Of specific interest were the BMAA-induced alterations in alanine, aspartate and glutamate metabolism and as well as alterations in various neurotransmitters/neuromodulators such as GABA and taurine. The results indicate that BMAA can interfere with metabolic pathways involved in neurotransmission in human neuroblastoma cells.

No MeSH data available.