Fig 2: a-syn deposits in the skin nerves of PD.Confocal microscope (×400) study of a-syn deposits in the hand superficial dermal nerves which are stained with mouse protein gene product 9·5 (PGP, Bio-Rad Cat# MCA4750, RRID: AB_2210503). a. Nucleus marked with DAPI (Abcam Cat# ab104139, RRID: no) (blue), it is superficial derma (*); b. Skin nerves marked with PGP 9·5 (green); c. a-syn deposits marked with rabbit monoclonal a-syn (Abcam Cat# ab51253, RRID: AB_869973) (red); d. Merged images, there are a-syn deposits in the superficial dermal nerves (arrow). Scale bars: 50 µm. These figures are taken by Di Wu and Yanjuan Wang, and all authors confirm originality of it and retain copyright to it.

Fig 3: Immunofluorescence analysis on brain sections from mice injected with recombinant a-syn PFFs. (A) Adult C57Bl6 male mice were inoculated with 2.6 µg mouse PFF in the right dorsal striatum and were killed 1 (1WPI), 2 (2WPI), or 4 wk (4WPI) postinjection. Immunofluoresence images showing the 4B1 (green, aggregated WT-a-syn), Abcam ab51253 pS129 (red, phosphorylated a-syn) and DAT (white) immunoreactivity in the ipsilateral striatum at 1, 2, and 4 wk postinjection (40× magnification). (Scale bars, 50 µm.) The zoomed captions were obtained with 63× lens zoom 2.3. (Scale bars, 25 µm.) Graph showing the relative volume of the 4B1 and pS129 immunopositive accumulations in the ipsilateral dorsal striatum at 1, 2, and 4 wk post PFF inoculation. n = 3 mice per group, 3 serial striatal levels per mouse (**P < 0.01). (B) Immunofluoresence images showing the PK-resistant 4B1 (green) accumulations in the ipsilateral striatum at 2 wk post injection (40× magnification). (Scale bar, 50 µm.) The zoomed captions were obtained with 63× lens zoom 2.3. (Scale bar, 25 µm.) Graph showing the relative volume of the PK-resistant 4B1 immunopositive accumulations in the ipsilateral dorsal striatum at 2 wk post-PFF inoculation. n = 3 mice, 3 serial striatal levels per mouse (**P < 0.01).

Fig 4: Synucleinopathy in M83± mice following intravenous injection of assembled A53T a-synuclein. A Staining of midbrain periaqueductal grey (PAG) and lumbar spinal cord (ventral horn) for pS129 a-synuclein (Wako pSyn#64). Arrows denote perikaryal and arrowheads neuritic staining. Scale bar, 50 µm. B Immunoblots of sarkosyl-insoluble material from brain and spinal cord using an antibody specific for pS129 a-synuclein (Abcam ab51253). C Negative-stain immunoelectron microscopy of a-synuclein filaments from brain and spinal cord using an antibody specific for pS129 a-synuclein (Abcam ab51253). Scale bar, 50 nm. D Kaplan–Meier survival curves following intravenous injection of different amounts of assembled A53T a-synuclein or PBS. Gold, 1 mg; green, 100 µg; blue, 10 µg; magenta, 1 µg; purple, PBS

Fig 5: Synucleinopathy in M83± mice following oral (A–E) and nasal (F–I) administration of assembled A53T a-synuclein. A Staining of midbrain periaqueductal grey (PAG) and lumbar spinal cord (ventral horn) for pS129 a-synuclein (Wako pSyn#64). Arrows denote perikaryal and arrowheads neuritic staining. Scale bars, 50 µm. B Immunoblots of sarkosyl-insoluble material from brain and spinal cord using an antibody specific for pS129 a-synuclein (Abcam ab51253). C Negative-stain immunoelectron microscopy of filaments from brain and spinal cord using an antibody specific for pS129 a-synuclein (Abcam ab51253). Scale bars, 50 nm. D Kaplan–Meier survival curves following oral administration of assembled A53T a-synuclein (red) or PBS (purple). E Motor impairment on rotarod testing following oral administration of assembled A53T a-synuclein (red) compared to controls (purple). Unpaired one-tailed t-test for 6-month time-point. *p = 0.03. F Staining of midbrain periaqueductal grey (PAG) and lumbar spinal cord (ventral horn) for pS129 a-synuclein (Wako pSyn#64). Arrows denote perikaryal and arrowheads neuritic staining. Scale bars, 50 µm. G Immunoblots of sarkosyl-insoluble material from brain and spinal cord using an antibody specific for pS129 a-synuclein (Abcam ab51253). H Negative-stain immunoelectron microscopy of filaments from brain and spinal cord using an antibody specific for pS129 a-synuclein (Abcam ab51253). Scale bars, 50 nm. I Kaplan–Meier survival curves following nasal administration of assembled A53T a-synuclein (orange) or PBS (purple)