Studies on BMAA-induced effects in SH-SY5Y and U-343 cells
Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
β-N-Methylamino-L-alanine (BMAA) is a neurotoxin produced by a wide range of cyanobacteria and it has been suggested to be involved in neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS). Different mechanism of BMAA toxicity has been suggested, such as an excitotoxic mechanism, as well as induction of oxidative stress and incorporation into proteins leading to misfolding and aggregation of proteins. In vitro metabolomics studies may provide a deeper understanding of the mechanism of action of BMAA. The general aim of this study was to examine the uptake and effects of BMAA in the human neuroblastoma cell line SH-SY5Y and the human neuronal glioblastoma cell line U-343. In addition, preliminary studies were performed to examine the amount of differentiated SH-SY5Y cells needed to obtain NMR spectra useful for in vitro metabolomics studies. These studies are necessary for the proper design of an in vitro metabolomics study of the effects of BMAA on intermediary metabolites in cultured brain cells. Caspase 3/7 activity in SH-SY5Y and U-343 cells were measured after 48h exposure of 0.1-1mM BMAA for determination of apoptotic cell death. The uptake of 14C-L-BMAA was investigated in U-343 cells during 15 minutes. Approximately 400 000 SH-SY5Y cells/dish were seeded as an initial study for the amount of cells needed for in vitro metabolomics studies. The results showed that BMAA concentrations up to 1 mM did not induce any increased caspase 3/7 activity neither in SH-SY5Y nor in U-343 cells in comparison to control cells. The results from the uptake study of BMAA into U-343 cells varied too much to draw any conclusions, but there was a tendency for an increased uptake over time. For the NMR analysis, the tested amount of cells (400 000 cells/dish) were too low to be able to detect a proper NMR signal for metabolites of interest, such as creatine. In conclusion, this report showed that concentrations up to 1 mM of the neurotoxin L-BMAA do not induce apoptotic cell death in SH-SY5Y and U-343 cells. Thus, BMAA concentrations up to 1 mM seem to be suitable for future metabolomics studies in cultured brain cells. Further studies are needed to investigate the mechanisms of cellular uptake for BMAA, as the studies in U-343 cells did show too high variance to be interpreted. Lastly, an increased number of cultured cells have to be used in the planned metabolomics studies of cultured cells. Such in vitro metabolomics studies may provide further information about mechanisms of toxicity of BMAA.
Place, publisher, year, edition, pages
2013. , 27 p.
β-N-Methylamino-L-alanine, BMAA, neurotoxin, cyanobacteria
Pharmacology and Toxicology
IdentifiersURN: urn:nbn:se:uu:diva-216526OAI: oai:DiVA.org:uu-216526DiVA: diva2:690285
Subject / course
Master of Science Programme in Pharmacy
Hellman, Björn, Prof.