Rotenone and elevated extracellular potassium concentration induce cell‐specific fibrillation of α‐synuclein in axons of cholinergic enteric neurons in the guinea‐pig ileum

Abstract

Background Parkinson's disease is a progressive neurodegenerative disorder that results in the widespread loss of select classes of neurons throughout the nervous system. The pathological hallmarks of Parkinson's disease are Lewy bodies and neurites, of which α‐synuclein fibrils are the major component. α‐Synuclein aggregation has been reported in the gut of Parkinson's disease patients, even up to a decade before motor symptoms, and similar observations have been made in animal models of disease. However, unlike the central nervous system, the nature of α‐synuclein species that form these aggregates and the classes of neurons affected in the gut are unclear. We have previously reported selective expression of α‐synuclein in cholinergic neurons in the gut (J Comp Neurol. 2013; 521:657), suggesting they may be particularly vulnerable to degeneration in Parkinson's disease. Methods In this study, we used immunohistochemistry to detect α‐synuclein oligomers and fibrils via conformation‐specific antibodies after rotenone treatment or prolonged exposure to high [K+] in ex vivo segments of guinea‐pig ileum maintained in organotypic culture. Key Results Rotenone and prolonged raising of [K+] caused accumulation of α‐synuclein fibrils in the axons of cholinergic enteric neurons. This took place in a time‐ and, in the case of rotenone, concentration‐dependent manner. Rotenone also caused selective necrosis, indicated by increased cellular autofluorescence, of cholinergic enteric neurons, labeled by ChAT‐immunoreactivity, also in a concentration‐dependent manner. Conclusions & Inferences To our knowledge, this is the first report of rotenone causing selective loss of a neurochemical class in the enteric nervous system. Cholinergic enteric neurons may be particularly susceptible to Lewy pathology and degeneration in Parkinson's disease.

Enteric nervous system manifestations of neurodegenerative disease

“…Many enteric neurons, particularly those involved in peristalsis, are autonomous and probably continuously active, making them similarly vulnerable. Guinea-pig myenteric ganglia in organotypic explants exposed to rotenone or sustained high K + induced depolarization for several days, form and accumulate α-synuclein fibrils in axonal varicosities of cholinergic neurons 36 . Enteric neurons all have unmyelinated axons and many have multiple synaptic endings, features that could also increase their vulnerability in PD and could facilitate the release and reuptake of α-synuclein and its propagation through connected ganglia (Figure 1B).…”

Antibodies against alpha‐synuclein: tools and therapies

“…), guinea‐pig ileum (Sharrad et al . ), human skin biopsy of patients with idiopathic PD (Donadio et al . ), and in human post‐mortem brain tissues from PD, DLB, AD, and control cases (Vaikath et al .…”

“…2). Furthermore, the conformational specificity of these antibodies have been utilized to detect a-syn aggregates in biochemical studies (Leung et al 2015), in neurons of Caenorhabditis elegans (Kim et al 2016) and rats (Duffy et al 2018), guinea-pig ileum (Sharrad et al 2017), human skin biopsy of patients with idiopathic PD (Donadio et al 2018), and in human post-mortem brain tissues from PD, DLB, AD, and control cases (Vaikath et al 2018)…”

POSCAbilities: The Application of the Prion Organotypic Slice Culture Assay to Neurodegenerative Disease Research

“…Therefore, Sharrad et al cultured organotypic slices of guinea pig ileum, which were exposed to 10 µM of rotenone. α-syn fibrils were detected in the myenteric plexus after one day of exposure, the tertiary plexus after two days, and the circular muscle plexus after four days, supporting a prion-like spreading of α-syn [ 61 ].…”

“…Arguably, organotypic slice culture offers greater temporal resolution than in vivo models for tracking prionotypic spread of protein aggregates between cells and brain regions over short time periods. The propagation of α-syn aggregates both between subfields of the hippocampus and plexes of guinea pig ileum has been mapped over a period of days in slice culture [ 61 , 64 ]. Such precise timing of spread would be much more difficult in vivo and/or would require significantly more animals.…”