Environmental neurotoxin interaction with proteins: Dose-dependent increase of free and protein-associated BMAA (β-N-methylamino-L-alanine) in neonatal rat brain

Abstract

β-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.

Figure 1. Male neonatal rats were given subcutaneous injections of BMAA.

Twenty-four hours after the last injection the animals were killed and selected tissues were rapidly frozen for subsequent UHPLC-MS/MS analysis of protein-associated BMAA. The figure shows mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the group administered 2 × 40 mg/kg BMAA (ANOVA followed by Student's t-test test. Chi-square test was used when groups had not detectable levels of protein-associated BMAA).

Figure 2. Male neonatal rats were given subcutaneous injections of BMAA.

Twenty-four hours after the last injection the animals were killed and selected tissues were rapidly frozen for subsequent UHPLC-MS/MS analysis of free and protein-associated BMAA. The figure shows the percentage of protein-associated BMAA of total BMAA (protein-associated BMAA/total levels of BMAA) in neonatal rat tissues. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the group administered 2 × 40 mg/kg BMAA (ANOVA followed by Student's t-test test. Chi-square test was used when groups had not detectable levels of protein-associated BMAA).

Figure 3

The sample preparation workflow for the detection of free and protein-associated BMAA separately using UHPLC-MS/MS analysis in tissues of neonatal rats administered BMAA.