Mouse husbandry and inoculations

All studies involving animals were approved by the animal protection committee of the North Rhine-Westphalia State Environment Agency (LANUV). All mice were housed under environmentally controlled standard conditions with a 12 h light/dark cycle and free access to food and water. B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J mice (short: TgM83^+/− mice, The Jackson Laboratory) were crossed to wild-type C57BL/6 J mice and their progeny genotyped by real-time PCR for the presence of transgenes encoding human α-synuclein with the familial A53T mutation [17]. For challenge with α-synuclein fibrils or bovine serum albumin (BSA), we used 6–8-week-old male and female TgM83^+/− mice. Oral challenge was done by way of oral gavage, intravenous and intraperitoneal challenges by injection with a 29-gauge disposable hypodermic needle into the tail vain or peritoneum with 50 µg of sonicated α-synuclein fibrils in 12 µL phosphate-buffered saline (PBS, Sigma) or 50 µg BSA in 25 µL 0.9% (w/v) saline solution (Pierce). A high-dose oral challenge was performed with 500 µg of α-synuclein fibrils in 120 µL or 500 µg BSA in 250 µL. For intracerebral challenge, TgM83^+/− mice were anaesthetized with isoflurane and stereotactically injected with 10 µg (2.3 µL) or 50 µg (12 µL) of sonicated α-synuclein fibrils or 50 µg BSA (25 µL) into the right striatum (coordinates: + 0.2 mm relative to the bregma, + 2.0 mm relative to the midline, and 2.6 mm below the dura). For stereotactic delivery, we used a rate of 1.4 µL/min, whereafter we left the syringe in place for five additional minutes before slowly retracting the needle. Challenged animals were monitored daily for health and three times weekly for signs of neurological disease, such as reduced grooming, ataxia, tremor, bradykinesia, akinesia, lethargy, circling, tail rigidity, paraparesis, paralysis, kyphosis, and more. Diseased mice were narcotized with ketamine/xylazine and then transcardially perfused with 0.9% (w/v) saline followed by 10% (v/v) formalin neutral buffer solution (Sigma). Dissected brain and spinal cord samples were fixed overnight with 10% formalin neutral buffer solution for immunohistochemistry. Alternatively, diseased mice were killed and their brains and spinal cords snap-frozen on dry ice and stored at − 80 °C for biochemical analysis.