Exposure to a mixture of BMAA and MCLR synergistically modulates behavior in larval zebrafish while exacerbating molecular changes related to neurodegeneration.

Abstract

Exposure to toxins produced by cyanobacteria (i.e., cyanotoxins) is an emerging health concern due to their increased occurrence and previous associations with neurodegenerative disease including amyotrophic lateral sclerosis (ALS). The objective of this study was to evaluate the neurotoxic effects of a mixture of two co-occurring cyanotoxins, {beta}-methylamino-L-alanine (BMAA) and microcystin leucine and arginine (MCLR), using the larval zebrafish model. We combined high-throughput behavior based toxicity assays with discovery proteomic techniques to identify behavioral and molecular changes following 6 days of exposure. While neither toxin caused mortality, morphological defects, or altered general locomotor behavior in zebrafish larvae, both toxins increased acoustic startle sensitivity in a dose-dependent manner by at least 40% (p<0.0001). Furthermore, startle sensitivity was enhanced by an additional 40% in larvae exposed to the BMAA/MCLR mixture relative to those exposed to the individual toxins. Supporting these behavioral results, our proteomic analysis revealed a 4-fold increase in the number of differentially expressed proteins (DEPs) in the mixture exposed group. Additionally, prediction analysis reveals activation and/or inhibition of 8 enriched canonical pathways (enrichment p-value<0.01; z-score[&ge;]|2|), including ILK, Rho Family GTPase, RhoGDI, and calcium signaling pathways, which have been implicated in neurodegeneration. We also found that expression of TDP-43, of which cytoplasmic aggregates are a hallmark of ALS pathology, was significantly upregulated by 5.7-fold following BMAA/MCLR mixture exposure. Together, our results emphasize the importance of including mixtures of cyanotoxins when investigating the link between environmental cyanotoxins and neurodegeneration as we reveal that BMAA and MCLR interact in vivo to enhance neurotoxicity.