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β-N-methylamino-L-alanine (BMAA)-induced neurotoxicity: Studies in vitro and in vivo
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. (Toxikologi och läkemedelssäkerhet)
2020 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

β-N-methylamino-L-alanine (BMAA) is a neurotoxic non-proteinogenic amino acid produced naturally by cyanobacteria, diatoms and dinoflagellates and it has been detected in samples from fresh and marine water from all over the world. It can bioaccumulate in fish and shellfish, and has a potential to biomagnify in a terrestrial food chain. BMAA was first discovered in the search for a neurotoxin related to the amyotrophic lateral sclerosis/Parkinsonism-dementia complex (ALS/PDC) found among the Chamorro people in Guam. This non-proteinogenic amino acid has also been suggested to contribute to sporadic neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). BMAA can induce neurotoxicity via multiple mechanisms. It can act as an excitotoxin by activating glutamate receptors and to induce oxidative stress. It has also been suggested to be misincorporated into proteins leading to misfolded protein aggregates. Previous studies have demonstrated a specific damage in the hippocampus, including intracellular fibril formation, in adult rats following neonatal exposure to BMAA. In this thesis both in vitro and in vivo models were used to characterize the uptake, transport and effects of BMAA in cultured cell lines and in neonatal rodent brain tissue. The uptake of radiolabeled BMAA, as well as the effects of various amino acids and transporter antagonists on the uptake were studied in human mammary epithelial cells, intestinal epithelial cells, glioblastoma and neuroblastoma cells. Based on the obtained results a potential human mother-to-infant transfer of BMAA was suggested. BMAA-induced metabolic changes in a differentiated human neuroblastoma cell line were also characterized. The results revealed a plentitude of altered metabolites, many of them involved in amino acid metabolism and the TCA cycle. Of special interest were the perturbations in alanine, aspartate and glutamate metabolism as this pathway is involved in neurotransmission. The results revealed that BMAA can interfere with fundamental metabolic and neurotransmission pathways. Finally, the levels of free and protein-associated BMAA in the brain and peripheral tissues in neonatal rats exposed to BMAA were analysed. The results revealed high levels of free BMAA in some brain regions, thus demonstrating that the neonatal brain is not protected from BMAA by the blood-brain barrier. The results also revealed a protein-association of BMAA in the neonatal hypothalamus and hippocampus. Although the total amount of BMAA in the hippocampus was not high compared to other brain regions, the percentage of protein-associated BMAA was significantly higher. The results suggest that the protein-association of BMAA may play a role in the long-term effects in the hippocampus following neonatal exposure to BMAA. The studies in this thesis have demonstrated 1) a potential transfer of BMAA via breast milk to the brain of the nursing infant, 2) BMAA-induced metabolic alterations related to neurotransmission in human neuroblastoma cells and 3) that both free and protein-associated BMAA can be detected in the neonatal rat brain.

Place, publisher, year, edition, pages
Uppsala: Uppsala University, 2020.
Series
Licentiate thesis / Företagsekonomiska institutionen, Uppsala universitet, ISSN 1103-8462
Series
Licentiate Thesis from the faculty of Pharmacy ; 50
Keywords [en]
BMAA, neurotoxin, neurodegeneration, cellular transport, metabolic profiling, protein-association
National Category
Pharmaceutical Sciences Pharmacology and Toxicology
Research subject
Pharmaceutical Science
Identifiers
URN: urn:nbn:se:uu:diva-400222OAI: oai:DiVA.org:uu-400222DiVA, id: diva2:1380645
Presentation
2020-01-08, B7:101a, BMC Husargatan 3, Uppsala, 10:43 (English)
Supervisors
Available from: 2019-12-19 Created: 2019-12-19 Last updated: 2019-12-19Bibliographically approved
List of papers
1. Potential transfer of neurotoxic amino acid beta-N-methylamino-L-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines
Open this publication in new window or tab >>Potential transfer of neurotoxic amino acid beta-N-methylamino-L-alanine (BMAA) from mother to infant during breast-feeding: Predictions from human cell lines
2017 (English)In: Toxicology and Applied Pharmacology, ISSN 0041-008X, E-ISSN 1096-0333, Vol. 320, p. 40-50Article in journal (Refereed) Published
Abstract [en]

β-N-methylamino-alanine (BMAA) is a non-protein amino acid produced by cyanobacteria, diatoms and dinoflagellates. BMAA has potential to biomagnify in a terrestrial food chain, and to bioaccumulate in fish and shellfish. We have reported that administration of [14C]l-BMAA to lactating mice and rats results in a mother to off-spring transfer via the milk. A preferential enantiomer-specific uptake of [14C]l-BMAA has also been demonstrated in differentiated murine mammary epithelium HC11 cells. These findings, together with neurotoxic effects of BMAA demonstrated both in vitro and in vivo, highlight the need to determine whether such transfer could also occur in humans. Here, we used four cell lines of human origin to examine and compare the transport of the two BMAA enantiomers in vitro. The uptake patterns of [14C]l- and [14C]d-BMAA in the human mammary MCF7 cell line were in agreement with the results in murine HC11 cells, suggesting a potential secretion of BMAA into human breast milk. The permeability coefficients for both [14C]l- and [14C]d-BMAA over monolayers of human intestinal Caco2 cells supported an efficient absorption from the human intestine. As a final step, transport experiments confirmed that [14C]l-and [14C]d-BMAA can be taken up by human SHSY5Y neuroblastoma cells and even more efficiently by human U343 glioblastoma cells. In competition experiments with various amino acids, the ASCT2 specific inhibitor benzylserine was the most effective inhibitor of [14C]l-BMAA uptake tested here. Altogether, our results suggest that BMAA can be transferred from an exposed mother, via the milk, to the brain of the nursed infant.

Place, publisher, year, edition, pages
Elsevier, 2017
Keywords
BMAA, Cellular transport, Amino acid transporters, Breast milk, Neurodegeneration
National Category
Cell Biology Developmental Biology
Research subject
Biology with specialization in Environmental Toxicology
Identifiers
urn:nbn:se:uu:diva-265857 (URN)10.1016/j.taap.2017.02.004 (DOI)000396798200006 ()28174119 (PubMedID)
Funder
Swedish Research Council Formas
Available from: 2015-11-03 Created: 2015-11-03 Last updated: 2019-12-19Bibliographically approved
2. β-N-Methylamino-L-alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling
Open this publication in new window or tab >>β-N-Methylamino-L-alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling
Show others...
2017 (English)In: Amino Acids, ISSN 0939-4451, E-ISSN 1438-2199, Vol. 49, no 5, p. 905-919Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
BMAA, Global metabolite profiling, MS, Metabolism, NMR, Neurotoxin
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-322142 (URN)10.1007/s00726-017-2391-8 (DOI)000399176200006 ()28161796 (PubMedID)
Funder
Swedish Research Council Formas
Available from: 2017-05-16 Created: 2017-05-16 Last updated: 2019-12-19Bibliographically approved
3. Environmental neurotoxin interaction with proteins: Dose-dependent increase of free and protein-associated BMAA (beta-N-methylamino-L-alanine) in neonatal rat brain
Open this publication in new window or tab >>Environmental neurotoxin interaction with proteins: Dose-dependent increase of free and protein-associated BMAA (beta-N-methylamino-L-alanine) in neonatal rat brain
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2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 15570Article in journal (Refereed) Published
Abstract [en]

beta-Methylamino-L-alanine (BMAA) is implicated in the aetiology of neurodegenerative disorders. Neonatal exposure to BMAA induces cognitive impairments and progressive neurodegenerative changes including intracellular fibril formation in the hippocampus of adult rats. It is unclear why the neonatal hippocampus is especially vulnerable and the critical cellular perturbations preceding BMAA-induced toxicity remains to be elucidated. The aim of this study was to compare the level of free and protein-associated BMAA in neonatal rat brain and peripheral tissues after different exposures to BMAA. Ultra-high performance liquid chromatography-tandem mass spectrometry analysis revealed that BMAA passed the neonatal blood-brain barrier and was distributed to all studied brain areas. BMAA was also associated to proteins in the brain, especially in the hippocampus. The level in the brain was, however, considerably lower compared to the liver that is not a target organ for BMAA. In contrast to the liver there was a significantly increased level of protein-association of BMAA in the hippocampus and other brain areas following repeated administration suggesting that the degradation of BMAA-associated proteins may be lower in neonatal brain than in the liver. Additional evidence is needed in support of a role for protein misincorporation in the neonatal hippocampus for long-term effects of BMAA.

National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-267198 (URN)10.1038/srep15570 (DOI)000363397500001 ()26498001 (PubMedID)
Funder
Swedish Research Council Formas
Available from: 2015-11-19 Created: 2015-11-19 Last updated: 2019-12-19Bibliographically approved

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