Environmental toxins trigger PD-like progression via increased alpha-synuclein release from enteric neurons in mice

Francisco Pan-Montojo, Mathias Schwarz, Clemens Winkler, Mike Arnhold, Gregory A O'Sullivan, Arun Pal, Jonas Said, Giovanni Marsico, Jean-Marc Verbavatz, Margarita Rodrigo-Angulo, Gabriele Gille, Richard H W Funk, Heinz Reichmann, Francisco Pan-Montojo, Mathias Schwarz, Clemens Winkler, Mike Arnhold, Gregory A O'Sullivan, Arun Pal, Jonas Said, Giovanni Marsico, Jean-Marc Verbavatz, Margarita Rodrigo-Angulo, Gabriele Gille, Richard H W Funk, Heinz Reichmann

Abstract

Pathological studies on Parkinson's disease (PD) patients suggest that PD pathology progresses from the enteric nervous system (ENS) and the olfactory bulb into the central nervous system. We have previously shown that environmental toxins acting locally on the ENS mimic this PD-like pathology progression pattern in mice. Here, we show for the first time that the resection of the autonomic nerves stops this progression. Moreover, our results show that an environmental toxin (i.e. rotenone) promotes the release of alpha-synuclein by enteric neurons and that released enteric alpha-synuclein is up-taken by presynaptic sympathetic neurites and retrogradely transported to the soma, where it accumulates. These results strongly suggest that pesticides can initiate the progression of PD pathology and that this progression is based on the transneuronal and retrograde axonal transport of alpha-synuclein. If confirmed in patients, this study would have crucial implications in the strategies used to prevent and treat PD.

Figures

Figure 1. Hemivagotomy and partial sympathectomy delay…
Figure 1. Hemivagotomy and partial sympathectomy delay the appearance of motor deficits but do not induce gastrointestinal functional alterations.
(A) Performance in the rotarod test using an acceleration protocol (0.3 rpm/sec) shows a delay in the appearance of symptoms in hemivagotomized or partially sympathectomized mice when compared to rotenone treated non-operated mice. (B) Bar graph showing the results of the 1-hour stool collection test one week after the operation. Hemi-vagotomized and partially sympathectomized mice show an acute transient decrease in gut motility just after the operation. (C) Bar graph showing the results of 1-hour stool test during rotenone treatment. Gut motility recovered and was equal to control until the first month of treatment. Rotenone treatment induces a progressive decrease in gut motility. CONT: control, NORT: non-operated rotenone treated, SRT: sympathectomized rotenone treated and HRT: hemivagotomized rotenone treated mice. Error bars in all graphs represent s.e.m. * is P<0.05.
Figure 2. Sympathectomy of the nervus mesentericus…
Figure 2. Sympathectomy of the nervus mesentericus inferior inhibits alpha-synuclein accumulation in the lumbal IML nuclei.
(A-D) Confocal images of thorax superior (A), thorax medium (B), thorax inferior (C) and lumbal (D) fluorescence intensity color-coded images of spinal cord sections stained with rabbit anti-alpha-synuclein or goat anti-ChAT antibodies from SRT mice 4 months after treatment. Scale bar: 20 μm. (E) Quantification of alpha-synuclein MFI rate between ChAT+ and ChAT− areas in different regions of the spinal cord after 2 (n = 8) and 4 months (n = 8) of rotenone treatment. CONT: control, NORT: non-operated rotenone treated and SRT: sympathectomized rotenone treated mice. Values for CONT. 4 months (CONT.4M), NORT 4 months (NORT.4M) and SRT 2 (SRT.2M) and 4 (SRT.4M) months mice are grouped within the thoracic medium (Thorax Med.), thoracic inferior (Thorax Inf.) and the thoracolumbal (Lumbal) regions of the spinal cord. All groups for each region are compared with the CONT.4M group. Error bars represent (s.e.m.) values, n.s. is non-significant, *P<0.05, **P<0.005, ***P<0.001.
Figure 3. Hemivagotomy prevents alpha-synuclein accumulation in…
Figure 3. Hemivagotomy prevents alpha-synuclein accumulation in the ipsilateral DMV.
(A-C) Confocal images (single plane) of DMV sections stained with rabbit anti-alpha-synuclein (green) and goat anti-ChAT antibodies (red) and with DAPI (blue) obtained from (A) control (CONT) (n = 10), (B) non-operated rotenone-treated (NORT) (n = 9) and (C) hemivagotomized rotenone-treated (HRT) (n = 9) mice 4 months after treatment. L, R, A and P are left, right, anterior and posterior. Scale bar: 20 μm. (D) Bar graph showing ratio between right and left alpha-synuclein MFI in the DMV of CONT, NORT, and HRT 2 (2M) and 4 (4M) months after treatment. MFI intensity values were normalized to alpha-synuclein MFI of a neutral region. (E) Bar graph showing the quantification of left to right difference in the number of ChAT+ neurons inside the DMV in HRT mice after 2 and 4 months compared to CONT mice after 4 months. Whiskers represent max.-min. values, n.s. is non-significant, * P<0.05 and ** P<0.01.
Figure 4. Hemivagotomy prevents dopaminergic cell death…
Figure 4. Hemivagotomy prevents dopaminergic cell death in the ipsilateral SNc.
(A-C) Stitched fluorescence microscope images of 40 μm brain mesencephalic sections from 4 months CONT. 4M: control (A), NORT. 4M: non-operated rotenone treated (B) and HRT. 4M: hemivagotomized rotenone treated (C) mice. Brain sections were stained with sheep anti-tyrosine hydroxylase (TH) antibody (red). L, R, Cr and Cd are left, right, cranial and caudal. Scale bar represents 200 μm. TH+ neurons density in the SNc is diminished in NORT mice (B) when compared to CONT (A) and HRT (C) mice. In NORT mice, a clear reduction can be observed in the lateral (arrowheads in B) and medial (arrows in B) SNc when compared to CONT mice (arrows in A). A reduction in the density of TH+ neurons can be observed in the lateral part of the right SNc (arrows in (C)) when compared to the contralateral SNc (arrowheads in (C)). (D-F) Bar graphs showing the quantification of the total number of TH+ neurons in the SNc (D), the left to right ratio in the number of TH+ neurons within the SNc (E) and the amount of neurons in the left or right SNc (F) in CONT, NORT and HRT mice. Error bars represent (s.e.m.), n.s. (non-significant), *P<0.05, **P<0.01.
Figure 5. Rotenone induces alpha-synuclein release in…
Figure 5. Rotenone induces alpha-synuclein release in sympathetic and enteric primary neuronal cultures.
(A-C) Confocal images (maximum projection) of enteric cell culture expressing endogenous alpha-synuclein after control (A) or rotenone ((B) and (C)) treatment and subsequently stained with rabbit anti-protein related peptide 9.5 (PGP 9.5) (green) and mouse anti-alpha-synuclein (red) antibodies and DAPI (blue). All scale bars in this figure represent 20 μm. Alpha-synuclein inclusions in non-neuronal cells can be observed only after rotenone treatment (arrows in (B) and (C)). They are usually localized around the nucleus inside non-neuronal cells surrounding enteric somas (B) and neurites (C). Rotenone treatment also increases enteric intraneuronal alpha-synuclein (arrowheads in (B)). (D) Bar graph showing quantification of alpha-synuclein inclusions per non-neuronal cells after Ethanol (Vehicle), 10, 50 and 100 nM rotenone treatment. Error bars represent (s.e.m.), n.s. (non-significant), *P<0.05, **P<0.01. (E) Confocal images (maximum projection) of enteric cells stained with rabbit anti-Smooth Muscle Actin (SMA, green) and mouse anti-alpha-synuclein (red). Alpha-synuclein can be observed inside SMA+ cells (arrows in (E)).
Figure 6. Alpha-synuclein is secreted inside and…
Figure 6. Alpha-synuclein is secreted inside and outside exosomes upon rotenone treatment.
(A) Western-blot of 10,000 × g (non-exosomal) and 100,000 × g (exosomal) fraction isolated from the medium collected from control (MA), vehicle (Veh), 0.1 μM, 1 μM and 5 μM rotenone treated primary sympathetic neuronal cultures. Blots were probed with a rabbit anti-alpha-synuclein antibody. (B) Size distribution of exosomal structures in exosomal (Pellet 4) and non-exosomal (Pellet 3) centrifugation fractions treated with rotenone (Rotenone 0.1 μM) and vehicle (Vehicle). (C) Electron microscopy images of the exosomal fraction. Scale bar: 100 nm. Arrows in (C) show exosome-like structures. (D) Graph showing the number of exosomes per field in control (vehicle) and treated (Rotenone 0.1 μM) samples corresponding to the exosomal and non-exosomal fractions. Error bars represent (s.e.m.), n.s. (non-significant), **P<0.01. (E-G) Electron microscopy images of the exosomal (E-F) and non-exosomal (G) fractions labeled with gold against alpha-synuclein. Immunogold labeling can be observed on exosomal structures (arrow in (E)) and on lipid membranes (arrowheads in (E)). Alpha-synuclein seems to be enhanced on lipid membranes (black circles in (F)) and gold particles can also be observed on extracellular fibrillar structures in the non-exosomal fraction (arrows in (G)).
Figure 7. Released enteric alpha-synuclein can be…
Figure 7. Released enteric alpha-synuclein can be up-taken and retrogradely transported by sympathetic neurons.
Scale bars are 20 μm in all images. (A) Fluorescence microscope images of co-cultured ENS cells expressing mCherry-alpha-synuclein (red) and sympathetic neurons stained with a rabbit anti-TH antibody (green) (n = 3). Sympathetic neurons (arrows in the upper left image in A are in contact with mCherry-alpha-synuclein enteric neurons (*). Upper right image in panel A shows another image of the sympathetic neuron marked with an arrow in the upper left image highlighting the somal accumulation of mCherry-alpha-synuclein (arrow). Lower left image in panel A shows a zoomed in region of TH+ neurites in contact with mCherry-alpha-synuclein expressing enteric neurons framed in the upper left image . mCherry-alpha-synuclein can be observed inside sympathetic neurites (arrowheads). (B) Schematics of the Campenot chamber with sympathetic neurons in green and enteric neurons in red. (C) Montages of microscopy low magnification images from Campenot chambers corresponding to the region underneath the divider and the proximal neurite compartment. Sympathetic neurons were stained with rabbit anti-TH antibody (green) and enteric neurons express cherry-alpha-synuclein (red). Each row of images represents different channels (from top to bottom: cherry-alpha-synuclein, TH, bright-field (BF)) and a merge. Sympathetic TH+ neurons can be observed under the divider and forming a dense network around cherry-alpha-synuclein expressing enteric neurons. (D) Zoomed in region of the sympathetic neurites framed in (C). Cherry-alpha-synuclein can be observed along sympathetic neurites under the divider. (E) Confocal images showing cherry-alpha-synuclein (red) accumulating inside sympathetic TH+ neurons in grey (BF) and green in F.

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