An in vitro study investigating the combined toxicity of the cyanotoxins β-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (2,4-DAB)
- Publication Type:
- Thesis
- Issue Date:
- 2022
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Sporadic motor neurone disease (MND) is a neurodegenerative disorder with poorly understood aetiology and accounts for up to 90% of all MND cases with the remaining cases being familial. Several factors may contribute to the development of sporadic MND, one of which is exposure to the cyanobacterial toxin, BMAA. While research has primarily focused on BMAA as a potential trigger for MND, several other isomers including 2,4-DAB have largely been ignored despite being produced concurrently with BMAA in cyanobacteria. BMAA is a non-protein amino acid with similar structure to canonical amino acid L-serine where the latter has been shown to prevent BMAA induced toxicity. The aims of this thesis are to investigate the toxic mechanisms behind the two isomers and how they may contribute to the development of sporadic neurodegenerative disorders.
Cell viability assays performed on SH-SY5Y neuroblastoma cells revealed equimolar concentrations of BMAA and 2,4-DAB being the most toxic combination out of all combinations used and more than the isomers individually. Proteomic analysis on cells treated with equimolar concentrations of BMAA and 2,4-DAB revealed significant enrichment in pathways involved with energy production (fatty acid β-oxidation and glycolysis) and L-serine biosynthesis. The L-serine biosynthesis pathway interlinks with energy production as it uses metabolites derived from the glycolytic pathway. The expression levels of the enzymes involved in L-serine biosynthesis were further looked at with RT qPCR. Interestingly, the first enzyme involved in the L-serine biosynthesis pathway (PHGDH), was significantly decreased in cells exposed to 2,4-DAB alone and even more so in combination with BMAA. Supplementation of glycolytic metabolite, pyruvate was able to protect and prevent the impact of the toxins on the PHGDH gene expression. These results highlight the importance of the contribution to energy dysfunction which may parallel those seen in some neurodegenerative diseases.
Furthermore, LC-MS/MS was used to quantify the level of amino acids and antioxidant capacity of cells exposed to the toxins. 2,4-DAB exposure showed evidence of oxidative stress which was increased when combined with BMAA. Intracellular L-alanine levels were significantly decreased following treatment with BMAA and 2,4-DAB alone. The decreases in L-alanine levels in cells support existing studies that have demonstrated the affinity of BMAA for alanyl-tRNA synthetase.
The results of these studies contribute to the ever-growing knowledge of BMAA and its role in neurodegenerative diseases and highlight the importance of studying the toxin in combination with its isomers that are found concurrently in nature.
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