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Additional file 4. Quantification of phosphorylated ��-synuclein deposits in different brain regions of TgM83+/��� at 360 days after stroke. The number of phosphorylated ��-synuclein-positive cells per square millimeter was plotted for the ipsilateral and contralateral brain hemisphere for each quantified brain area of mice subjected to stroke. The coordinates of the quantified coronal tissue sections relative to the bregma were 0.74 mm (a), ���1.70 mm (b), ���2.92 mm (c), ���4.84 mm (d), and ���5.68 mm (e) as shown in the heat map in Fig. 7b. Data shown represent the mean �� standard deviation of four animals.

Additional file 1: Table S1. Antibodies used for immunofluorescence and immunohistochemistry.

Additional file 3. Spread of neuroinflammation to the contralateral brain hemisphere by 360 days after stroke. Staining of brain tissue sections of MCAO- and sham-treated TgM83+/��� mice with antibodies to Iba1 (a) and to GFAP (b) revealed that microgliosis and astrogliosis had spread to the contralateral brain hemisphere by 360 days after stroke. A higher amount of microglia was found throughout the entire contralateral brain hemisphere of MCAO-treated animals compared to sham-treated animals (a). Also, a higher amount of astrocytes was detected in the contralateral brain hemisphere of MCAO-treated animals compared to sham-treated animals, especially in the lower isocortical region and the upper part of the caudoputamen (b). Scale bars represent 1.0 mm.

Additional file 2. TgM83+/��� mice do not show any neuronal loss following sham surgery Immunohistochemical staining for NeuN showed that sham surgery did not cause any neuronal loss in the ipsilateral or contralateral brain hemispheres of TgM83+/��� mice at 14 (a), 30 (b), 90 (b), 180 (b), or 360 days (b) after sham surgery. The scale bar represents 0.5 mm.

Additional file 2 Supplementary Fig. S1. α-Syn expression enhances axonal growth in primary hippocampal, mesencephalic and cortical neurons. A. Primary hippocampal cultures from α-Syn−/− (C57BL/6JOlaHsd) mouse brains, virally transduced either with WT α-Syn, A53T α-Syn, K10,12E α-Syn, or a mock-GFP vector. Cells were fixed at 4 DIV and immunoreacted with antibodies against α-Syn (MJFR1, green), α-tubulin (white) and PI4,5P2 (red). Direct fluorescence was captured for GFP (green). Bar = 25 μm. B. Graph showing the average length of the main extension designated as axons (in μm); C. Total length of collaterals per axon (in μm); and D. PI4,5P2 levels within the main axon and its collaterals (per μm2 area) quantified by Fiji (Image J) program. Mean ± SE; n > 17 cells; *, P 15 cells; *, P 20 cells; *, P

Parkinson’s disease and related disorders are neuropathologically characterized by cellular deposits of misfolded and aggregated α-synuclein in the CNS. Disease-associated α-synuclein adopts a conformation that causes it to form oligomers and fibrils, which have reduced solubility, become hyperphosphorylated, and contribute to the spatiotemporal spreading of pathology in the CNS. The infectious properties of disease-associated α-synuclein, e.g., by which peripheral route and with which efficiency it can be transmitted, are not fully understood. Here, we investigated the potential of α-synuclein fibrils to induce neurological disease in TgM83+/− mice expressing the A53T mutant of human α-synuclein after oral or intravenous challenge and compared it to intraperitoneal and intracerebral challenge. Oral challenge with 50 µg of α-synuclein fibrils caused neurological disease in two out of eight mice in 220 days and 350 days, and challenge with 500 µg in four out of eight mice in 384 ± 131 days, respectively. Intravenous challenge with 50 µg of α-synuclein fibrils led to disease in 208 ± 20 days in 10 out of 10 mice and was in duration comparable to intraperitoneal challenge with 50 µg of α-synuclein fibrils, which caused disease in 10 out of 10 mice in 202 ± 35 days. Ten out of 10 mice that were each intracerebrally challenged with 10 µg or 50 µg of α-synuclein fibrils developed disease in 156 ± 20 days and 133 ± 4 days, respectively. The CNS of diseased mice displayed aggregates of sarkosyl-insoluble and phosphorylated α-synuclein, which colocalized with ubiquitin and p62 and were accompanied by gliosis indicative of neuroinflammation. In contrast, none of the control mice that were challenged with bovine serum albumin via the same routes developed any neurological disease or neuropathology. These findings are important, because they show that α-synuclein fibrils can neuroinvade the CNS after a single oral or intravenous challenge and cause neuropathology and disease.

Additional file 1. Table S1. Post mortem human brains. Table S2. list of antibodies.

Additional file 1. Table S1. Post mortem human brains. Table S2. list of antibodies.