Cyanobacterial neurotoxin BMAA and brain pathology in stranded dolphins

Abstract

Dolphin stranding events occur frequently in Florida and Massachusetts. Dolphins are an excellent sentinel species for toxin exposures in the marine environment. In this report we examine whether cyanobacterial neurotoxin, β-methylamino-L-alanine (BMAA), is present in stranded dolphins. BMAA has been shown to bioaccumulate in the marine food web, including in the muscles and fins of sharks. Dietary exposure to BMAA is associated with the occurrence of neurofibrillary tangles and β-amyloid plaques in nonhuman primates. The findings of protein-bound BMAA in brain tissues from patients with Alzheimer's disease has advanced the hypothesis that BMAA may be linked to dementia. Since dolphins are apex predators and consume prey containing high amounts of BMAA, we examined necropsy specimens to determine if dietary and environmental exposures may result in the accumulation of BMAA in the brains of dolphins. To test this hypothesis, we measured BMAA in a series of brains collected from dolphins stranded in Florida and Massachusetts using two orthogonal analytical methods: 1) high performance liquid chromatography, and 2) ultra-performance liquid chromatography with tandem mass spectrometry. We detected high levels of BMAA (20-748 μg/g) in the brains of 13 of 14 dolphins. To correlate neuropathological changes with toxin exposure, gross and microscopic examinations were performed on cortical brain regions responsible for acoustico-motor navigation. We observed increased numbers of β-amyloid+ plaques and dystrophic neurites in the auditory cortex compared to the visual cortex and brainstem. The presence of BMAA and neuropathological changes in the stranded dolphin brain may help to further our understanding of cyanotoxin exposure and its potential impact on human health.

Fig 1. HPLC-FD detection of BMAA in the cerebral cortex of stranded dolphins.

(A) Separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acid standards tyrosine (Try), valine (Val), methionine (Met), Lysine (Lys), Leucine (Leu) and Phenylalanine (Phe). BMAA and BMAA structural isomers N-(2-aminoethyl)-glycine (AEG) & 2,4-diaminobutyric acid (DAB) standards are shown in red. (B) Representative chromatogram from the visual cortex (VCtx) of stranded dolphin IFAW12-201Dd with low-concentration spikes of BMAA and BMAA isomer standards in the same dolphin sample shown in blue. Chromatogram shows BMAA has a distinct peak with a retention time of 31.1 minutes. AEG and DAB have distinct retention times of 29.6 and 33.0 minutes, respectively.

Fig 2. Gross and microscopic evaluation of postmortem brains from stranded dolphins.

(A) External examination was performed on the cerebral cortex and cerebellum of formalin-fixed hemispheres from stranded dolphins (n = 7). (B) Following external examinations, brain hemispheres were cut into a series of coronal slices to investigate internal gray and white matter structures. Tissue blocks were sampled from anatomical regions in the dolphin cerebral cortex and brainstem involved with acoustico-motor navigation: auditory cortex (ACtx), visual cortex (VCtx), and the medulla oblongata (Md). (C) Digital pathology scans were obtained from routine histological stain. H&E stain shows hypoxic and eosinophilic changes in neurons of both upper and lower cortical layers. (D) Gliosis was also observed in the cerebral cortex. (E) Advanced age-related changes were observed including, corpora amylacea and (F) lipofuscin granules. (G) Karyorrhexis nuclear changes (black arrow) and chromatolysis (white arrow) were observed. (H) Representative scans of eosinophilic plaques and a rare hemosiderin deposits were observed in the ACtx of stranded dolphins. Representative scale bar: 5 cm (A, B), 1000 μm (C), 200 μm (D, F, G), 50 μm (E, H).

Fig 3. Aβ deposition in the cerbral cortex of stranded dolphins.

(A) Anti-Aβ IHC demonstrates Aβ⁺ plaques in the cerebral cortex of stranded dolphins. (B) Intraneuronal Aβ⁺ accumulation was aslo observed throughout upper and lower cortical layers. (C) High-resolution digital patholgy scans of pyramidal neurons in the ACtx containing dense intracellalur Aβ⁺ inclusions. (D) Large and sparse Aβ⁺ plaques were observed in the Md of stranded dolphins. (E-P) Aβ⁺ plaques were different in morphology as observed by H&E staining (E-H), MB silver staining (I-L) and Aβ⁺ IHC (M-P). Aβ⁺ plaques ranged from small focal with compact cores (E, I, M), primative immature cotton wool-like (F, J, N), to large and diffuse (G, K, O) and ill-defined (H, L, P). Representative scale bar: 500 μm (A, B & D), 100 μm (C), 50 μm (E-P).

Fig 4. Neurodegenerative changes observed in the brains of stranded dolphins.

(A) MB silver staining illustrates AD-like compact neuritic plaque in the ACtx of a stranded dolphin. (B) A representative digital scan of an ill-defined agrophyllic plaque containing dystrophic neurites. (C) Dense neuropil threads were observed in the VCtx. (D, E) agrophyllic neurons containing dystrophic neurites. (F) Coiled body in the ACtx. Representative scale bars: 50 μm.

Fig 5. Thioflavin-S⁺ pathology in the auditory cortex of stranded dolphins.

Thioflavin-S⁺ staining and IHC with GFAP and Neu-N was used to determine co-localization of plaques and intracellular inclusions with astroglia and neurons in the stranded dolphin brain. (A, E) Thioflavin-S⁺ plaques and intracellular staining was observed in the ACtx (arrows). (B, F) Activated GFAP⁺ astrocytes are demonstrated surrounding a cluster of dense thioflavin-S⁺ plaques. Neu-N⁺ neurons are shown with intracellular thioflavin-S⁺ inclusions. (C, G) DAPI staining highlights intact cellular nuclei. (D, H) The merged panels show the relative co-localization of thioflavin-S⁺ plaques, blood vessels, and intracellular inclusions within astrocytes and neurons. Panel inserts (dotted lined boxes) show high magnification detailing thioflavin-S⁺ structures. Representative scale bar: 150 μm.