Plant poisoning leads to alpha-synucleinopathy and neuromelanopathy in kangaroos

Abstract

The pathogenesis of synucleinopathies, common neuropathological lesions normally associated with some human neurodegenerative disorders such as Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, remains poorly understood. In animals, ingestion of the tryptamine-alkaloid-rich phalaris pastures plants causes a disorder called Phalaris staggers, a neurological syndrome reported in kangaroos. The aim of the study was to characterise the clinical and neuropathological changes associated with spontaneous cases of Phalaris staggers in kangaroos. Gross, histological, ultrastructural and Immunohistochemical studies were performed to demonstrate neuronal accumulation of neuromelanin and aggregated α-synuclein. ELISA and mass spectrometry were used to detect serum-borne α-synuclein and tryptamine alkaloids respectively. We report that neurons in the central and enteric nervous systems of affected kangaroos display extensive accumulation of neuromelanin in the perikaryon without affecting neuronal morphology. Ultrastructural studies confirmed the typical structure of neuromelanin. While we demonstrated strong staining of α-synuclein, restricted to neurons, intracytoplasmic Lewy bodies inclusions were not observed. α-synuclein aggregates levels were shown to be lower in sera of the affected kangaroos compared to unaffected herd mate kangaroos. Finally, mass spectrometry failed to detect the alkaloid toxins in the sera derived from the affected kangaroos. Our preliminary findings warrant further investigation of Phalaris staggers in kangaroos, potentially a valuable large animal model for environmentally-acquired toxic synucleinopathy.

Figure 1

Macroscopic appearance of brain from a Phalaris-affected eastern grey kangaroo. Transverse section of the brain from case EGK89 displaying conspicuous bilateral and symmetrical, greenish, grey matter discoloration (arrows point to areas of pigmentation). (A) Shows the oculomotor nucleus and the red nucleus. Substantia nigra is evident but appears unaffected. (B) Shows the pontine nucleus. Representative of all affected Kangaroos.

Figure 2

Photomicrographs of the microscopic lesions in the central nervous system of a Phalaris-affected eastern grey kangaroo. (A) Normal appearance of the brain parenchyma in the healthy EGK (case 15-11801). (B–D) Are higher magnification of (A). (E) Intense neuromelanin-like brown pigments in neurons observed on routine H&E stained sections of brain of EGK89. (F–H) Are higher magnification of (E). Representative of all affected Kangaroos. (I) Silver stain reaction (Warthin Starry) did not display the presence of neuromelanin in the healthy EGK case 15–11801. (J–L) Are higher magnification of (I). Note absence of pigments in neurons. (M) Extensive intracytoplasmic melanosis in cerebral cortex derived from case EGK89, revealed by Warthin Starry reaction stain. (N–P) Are higher magnification of (K). Representative of all affected Kangaroos.

Figure 3

Photomicrographs of the microscopic and ultramicroscopic lesions in the spinal cord and enteric nervous system of a Phalaris-affected eastern grey kangaroo. (A) Intense neuromelanin-like brown pigments in neurons observed on routine H&E stained sections of intestine and (B) spinal cord from case EGK89. Representative of all affected Kangaroos. (C) Transmission electron microscopic (TEM) analysis of healthy EGK (case 15-11801) did not display electron-dense neuromelanin. (D) TEM analysis reveals intra-neuronal electron-dense neuromelanin (red arrows) in a Phalaris-affected EGK (EGK 92) with (E) lipid bulbs (arrow) attached to the granule.

Figure 4

Photomicrographs of the α-synucleinopathy in the central nervous system of a Phalaris-affected eastern grey kangaroo. (A) Immunohistochemical staining of healthy EGK (case 15–11801) with rabbit anti-human α-synuclein polyclonal IgG [97/8; 1:2000 dilution]. (B,C) Are higher magnification of (A). (D) Immunohistochemical staining with rabbit anti-human α-synuclein polyclonal IgG [97/8; 1:2000 dilution] of a Phalaris-affected EGK (EGK 92) which shows multi-shaped aggregates ranging from ovoid and fusiform to threadlike intensely stained structures. (E,F) Are higher magnification of (D). (G) Immunohistochemical staining with rabbit anti-human α-synuclein polyclonal IgG [97/8; 1:2000 dilution] of a Phalaris-affected EGK (EGK 59) which shows multi-shaped aggregates ranging from ovoid and fusiform to threadlike intensely stained structures. (H,I) Are higher magnification of (G). (J) Immunohistochemical staining with rabbit anti-human α-synuclein polyclonal IgG [97/8; 1:2000 dilution] of a Phalaris-affected EGK (EGK 42) which shows multi-shaped aggregates ranging from ovoid and fusiform to threadlike intensely stained structures. (K,L) Are higher magnification of (J). (M) Immunohistochemical staining with rabbit anti-human α-synuclein polyclonal IgG [MJRF1; 1:2000 dilution] of a Phalaris-affected EGK (EGK 92) which shows multi-shaped aggregates ranging from ovoid and fusiform to threadlike intensely stained structures (N,O). Are higher magnification of (M). (P) Immunohistochemical staining with rabbit anti-human α-synuclein polyclonal IgG [MJRF1; 1:2000 dilution] of a Phalaris-affected EGK (EGK 59) which shows multi-shaped aggregates ranging from ovoid and fusiform to threadlike intensely stained structures. (Q,R) Are higher magnification of (P). Representative of all affected kangaroos.

Figure 5

Immunofluorescence co-localisation of α-synuclein aggregates and neuron-specific nuclear protein, NeuN: Cortical co-staining with rabbit anti-human α-synuclein polyclonal IgG [97/8; 1:2000 dilution] (GREEN) and anti-mouse NeuN monoclonal IgG (Millipore, 1:2000 dilution) (RED) of phalaris-affected EGK92. DAPI (BLUE) was used to stain the nuclei. Representative of all affected kangaroos.

Figure 6

Quantitation of neuromelanin- and α-synuclein-laden neurons in Phalaris-affected and unaffected EGKs. Neuromelanin and α-synuclein staining levels of Phalaris-affected (n = 8) EGKs in cerebrum, mid-brain, cerebellum and spinal cord. The data are mean ± SEM from 5 randomly selected images per section. Similar regions from unaffected (n = 4) EGKs were used as a control. Neuromelanin and α-synuclein staining levels were quantified via ImageJ software as the relative intensity of neuromelanin (*p < 0.05) and α-synuclein (*p < 0.05) of all combined regions in Phalaris-affected over unaffected EGK’s.

Figure 7

Immunodetection of Phalaris-associated α-synuclein by ELISA. Serum α-synuclein Average levels of α-synuclein in Phalaris-affected kangaroos (EGK89; EGK90 and EGK91; n = 3) were compared with average levels of α-synuclein in control healthy EGKC (n = 5). Error bars represent the mean level derived from n = 3 wells.