Inoculation of α-synuclein preformed fibrils into the mouse gastrointestinal tract induces Lewy body-like aggregates in the brainstem via the vagus nerve

Abstract

Background: Intraneuronal α-synuclein (α-Syn) aggregates known as Lewy bodies (LBs) and the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) are the pathological hallmarks of Parkinson's disease (PD). Braak's hypothesis based on autopsy studies suggests that Lewy pathology initially occurs in the enteric nervous system (ENS) and then travels retrogradely to the dorsal motor nucleus of the vagus nerve (dmX), proceeding from there in a caudo-rostral direction. Recent evidence that α-Syn aggregates propagate between interconnected neurons supports this hypothesis. However, there is no direct evidence demonstrating this transmission from the ENS to the dmX and then to the SNpc.

Methods: We inoculated α-Syn preformed fibrils (PFFs) or phosphate-buffered saline (PBS) into the mouse gastric wall and analyzed the progression of the pathology.

Results: The mice inoculated with α-Syn PFFs, but not with PBS, developed phosphorylated α-Syn (p-α-Syn)-positive LB-like aggregates in the dmX at 45 days postinoculation. This aggregate formation was completely abolished when vagotomy was performed prior to inoculation of α-Syn PFFs, suggesting that the aggregates in the dmX were retrogradely induced via the vagus nerve. Unexpectedly, the number of neurons containing p-α-Syn-positive aggregates in the dmX decreased over time, and no further caudo-rostral propagation beyond the dmX was observed up to 12 months postinoculation. P-α-Syn-positive aggregates were also present in the myenteric plexus at 12 months postinoculation. However, unlike in patients with PD, there was no cell-type specificity in neurons containing those aggregates in this model.

Conclusions: These results indicate that α-Syn PFF inoculation into the mouse gastrointestinal tract can induce α-Syn pathology resembling that of very early PD, but other factors are apparently required if further progression of PD pathology is to be replicated in this animal model.

Keywords: Enteric nervous system; Lewy bodies; Parkinson’s disease; Propagation; Vagus nerve; α-synuclein.

Fig. 1

Inoculation of α-synuclein (α-Syn) preformed fibrils (PFFs) into the mouse gastric wall. a Coomassie brilliant blue (CBB) staining and western blot (WB) analysis of α-Syn. Smear bands are observed in PFFs. b Thioflavin T assay. High-intensity fluorescence at 535 nm is detected in PFFs (n = 3). A. U., arbitrary unit. c Transmission electron microscopy of α-Syn PFFs. Scale bar 200 nm. d-h α-Syn immunohistochemistry in the stomach at 45 days postinoculation. d α-Syn deposition in the submucosa (arrowheads). Scale bar 100 μm. e High-magnification image of α-Syn deposition in the submucosa. Scale bar 20 μm. f High-magnification image of neurons in the myenteric plexus. Scale bar 20 μm. g α-Syn deposition in the muscular layers. Neurons in the myenteric plexus (arrow). Scale bar 100 μm. h High-magnification image of α-Syn deposition in the muscular layers. Scale bar 20 μm. Data are the mean ± SEM

Fig. 2

Phosphorylated α-synuclein (p-α-Syn) pathology in the dmX 45 days after inoculation of α-Syn preformed fibrils. a Schematic of anatomy at bregma − 7.08 mm modified from [47]. dmX, dorsal motor nucleus of the vagus nerve; HG, hypoglossal nucleus; 4V, fourth ventricle. b P-α-Syn (EP1536Y) immunohistochemistry around bregma − 7.08 mm. Scale bar 100 μm. c High-magnification image of p-α-Syn–positive cells (arrows). P-α-Syn–positive intranuclear dots are occasionally observed (arrowhead). Scale bar 20 μm. d Schematic of anatomy at bregma − 7.48 mm. AP, area postrema; cc, central canal. e P-α-Syn (EP1536Y) immunohistochemistry around bregma − 7.48 mm. Scale bar 100 μm. f High-magnification image of p-α-Syn–positive cells. Scale bar 20 μm. g–i Immunohistochemistry assessing p-α-Syn (#64), p-α-Syn (81A), or nitrated α-Syn (Syn514) shows p-α-Syn–positive or nitrated α-Syn–positive cells in the dmX. Scale bar 20 μm

Fig. 3

Characterization of p-α-Syn pathology in the dmX 45 days after inoculation of α-Syn preformed fibrils. a–d Double immunostaining for choline acetyltransferase (ChAT) (green) and p-α-Syn (EP1536Y, red). Scale bar 10 μm. e–h Thioflavin S (ThS) staining (green) and immunostaining for p-α-Syn (81A, red). Scale bar 10 μm. i–l Double immunostaining for p62 (green) and p-α-Syn (#64, red). Scale bar 10 μm. m–p Double immunostaining for ubiquitin (green) and p-α-Syn (#64, red). Scale bar 10 μm

Fig. 4

Vagotomy prevents appearance of p-α-Syn pathology in the dmX after inoculation of α-Syn preformed fibrils. a FluoroGold labeling of the dmX. Left panel: Labeling appears in the dmX bilaterally 5 days after intraperitoneal injection of FluoroGold in unvagotomized mice. Right panel: FluoroGold labeling is absent in the right dmX when right (Rt.) vagotomy was performed prior to intraperitoneal injection of FluoroGold. AP, area postrema; cc, central canal. Scale bar 200 μm. b choline acetyltransferase (ChAT) and p-α-Syn immunohistochemistry in right-vagotomized mice 23 days and 45 days after α-Syn PFF inoculation. The number of ChAT-positive neurons in the right dmX decreased at 23 days and further decreased at 45 days (arrowheads). P-α-Syn–positive aggregates are present only in the left dmX at both time points (arrows). Scale bar 200 μm. c–e High-magnification images of p-α-Syn–positive cells. Scale bars 20 μm. f Number of ChAT-positive neurons in the dmX of untreated mice (Control) and right-vagotomized mice 23 days and 45 days after α-Syn PFF inoculation (n = 3–4). One-way ANOVA with Tukey’s multiple-comparisons test was performed; **p < 0.01, ***p < 0.001. g Number of p-α-Syn–positive neurons in the dmX of unvagotomised mice 45 days after α-Syn PFF inoculation and right-vagotomized mice 23 days and 45 days after α-Syn PFF inoculation (n = 4). Data are the mean ± SEM

Fig. 5

Chronological analysis of p-α-Syn pathology after inoculation of α-Syn preformed fibrils. a Number of p-α-Syn–positive neurons in the dmX of mice bilaterally examined at 45 days, 4 months, 8 months, and 12 months postinoculation of α-Syn preformed fibrils (n = 5–8). One-way ANOVA with Tukey’s multiple-comparisons test was performed; *p < 0.05, ***p < 0.001, n.s. means not significant. b p-α-Syn (EP1536Y) immunohistochemistry in the dmX of a mouse at 12 months postinoculation. Dashed line outlines the approximate area of the dmX. dmX, dorsal motor nucleus of the vagus nerve; 4V, fourth ventricle. Scale bar 100 μm. A p-α-Syn–positive neuron (arrow) is enlarged in the inset. Scale bar 20 μm. c p-α-Syn (EP1536Y) immunohistochemistry in the myenteric plexus of a mouse at 12 months postinoculation. Scale bar 100 μm. A p-α-Syn–positive neuron (arrow) is enlarged in the inset. Scale bar 20 μm. Data are the mean ± SEM

Fig. 6

Analysis of types of neurons containing p-α-Syn–positive aggregates in the myenteric plexus. a-d Representative images of vasoactive intestinal peptide (VIP), nitric oxide synthase 1 (NOS1), choline acetyltransferase (ChAT), and substance P immunohistochemistry in the myenteric plexus. e–h Double immunostaining for VIP (green) and p-α-Syn (81A, red). Scale bar 10 μm. i–l Double immunostaining for NOS1 (green) and p-α-Syn (EP1536Y, red). Scale bar 10 μm. m–p Double immunostaining for ChAT (green) and p-α-Syn (EP1536Y, red). Scale bar 10 μm. q–t Double immunostaining for Substance P (green) and p-α-Syn (EP1536Y, red). Scale bar 10 μm