Case Report: Synucleinopathy Associated With Phalaris Neurotoxicity in Sheep

Abstract

Chronic intoxication with tryptamine-alkaloid-rich Phalaris species (spp.) pasture plants is known colloquially as Phalaris staggers syndrome, a widely occurring neurological disorder of sheep, cattle, horses, and kangaroos. Of comparative interest, structurally analogous tryptamine-alkaloids cause experimental parkinsonism in primates. This study aimed to investigate the neuropathological changes associated with spontaneous cases of Phalaris staggers in sheep with respect to those encountered in human synucleinopathy. In sheep affected with Phalaris staggers, histological, immunohistochemical, and immunofluorescence analysis revealed significant accumulation of neuromelanin and aggregated α-synuclein in the perikaryon of neurons in the cerebral cortex, thalamus, brainstem, and spinal cord. Neuronal intracytoplasmic Lewy bodies inclusions were not observed in these cases of ovine Phalaris staggers. These important findings established a clear link between synucleinopathy and the neurologic form of Phalaris plant poisoning in sheep, demonstrated in six of six affected sheep. Synucleinopathy is a feature of a number of progressive and fatal neurodegenerative disorders of man and may be a common endpoint of such disorders, which in a variety of ways perturb neuronal function. However, whether primary to the degenerative process or a consequence of it awaits clarification in an appropriate model system.

Keywords: Phalaris; neuromelanopathy; neurotoxicity; parkinsonism; sheep; synucleinopathy; α-synuclein.

Figure 1

Photomicrographs of microscopic lesions in central nervous system of Phalaris-affected sheep. (A) Normal appearance of brain parenchyma of brainstem of an unaffected sheep (case 11,054). (B,C) Areas a higher magnification of (A). (D) Intense neuromelanin brown pigments in several neurons observed on routine H&E-stained sections of brainstem of a Phalaris-affected sheep (case 18,659). (E,F) Higher magnification of (D) with an inset showing a perinuclear cap of pigment (white arrows). Representative of all affected and unaffected sheep.

Figure 2

Photomicrographs of GFAP staining in central nervous system of Phalaris-affected sheep. (A) Staining for fibrillary astrocyte in brainstem brain sections of an unaffected sheep (case 11,054). (B) Staining for fibrillary astrocyte (black arrows) in brainstem brain sections of a Phalaris-affected sheep (case 18,659). Representative of all affected and unaffected sheep.

Figure 3

Photomicrographs of neuromelanin in central nervous system of Phalaris-affected sheep. (A) Warthin–Starry reaction did not display presence of neuromelanin in neurons in control unaffected sheep (case 11,054). Note absence of pigments in neurons. (B) Higher magnification of (A). (C) Abundant neuronal intracytoplasmic melanin pigmentation in cerebral cortex (black arrows) revealed by Warthin–Starry reaction stain (case 18,659). (D) Higher magnification of (C) and shows a perinuclear distribution (black arrows). Representative of all affected and unaffected sheep. Red arrow points to a healthy neuron.

Figure 4

Quantification of GFAP and neuromelanin in Phalaris-affected sheep. GFAP and neuromelanin in cerebral cortex were quantified in six Phalaris-affected sheep. Signal intensity of both GFAP and neuromelanin were correlated (p < 0.0001; r = 0.5211). Data represent mean signal intensity. One-way analysis of variance with Dunnett's post-test was performed using GraphPad Prism version 7.00 for Windows (GraphPad, San Diego, CA, USA) for statistical analysis.

Figure 5

Photomicrographs of immunohistochemical demonstration of α-synuclein in central nervous system of a Phalaris-affected sheep. (A) 97/8 anti-α-synuclein antibody applied to brainstem sections of a Phalaris-affected sheep (case 18,659), which shows densely immunoreactive cytoplasmic aggregates, ranging from ovoid and fusiform to thread-like intensely stained structures. (B) Higher magnification of (A). There is a perinuclear pattern and neurons with seemingly solid deposits (black arrows). (C) MJFR1 anti-α-synuclein antibody of brainstem sections of Phalaris-affected sheep (case 18,659), which shows scattered, strongly immunoreactive neurons and clusters of axons in sweeping, somewhat curved bundles widespread cytosolic distribution of aggregate structures. (D) Higher magnification of (C) showing α-synuclein aggregate structures (black arrows). (E) 97/8 anti-α-synuclein antibody applied to brainstem sections of an unaffected sheep (case 11,054), which does display α-synuclein cytoplasmic aggregates. (F) Higher magnification of (E) showing normal neurons (black arrows). Representative of all affected and unaffected sheep.

Figure 6

Immunofluorescence co-localization of α-synuclein and GFAP in central nervous system of a Phalaris-affected sheep. (A) Cerebral co-staining with 97/8 polyclonal anti-α-synuclein IgG antibody (white arrow) and anti-GFAP monoclonal IgG antibody (blue arrow) in brainstem sections of Phalaris-affected sheep (case 18,659) show perinuclear α-synuclein deposition. (B) Higher magnification of (A). (C) Cerebral staining with 97/8 polyclonal anti-α-synuclein IgG antibody (red) and anti-GFAP monoclonal IgG antibody (green) of brain sections of control unaffected sheep (case 11,054). Representative of all affected and unaffected sheep. (D) Higher magnification of (C).