Fig. 4: Inoculation of aSyn PFF-2, but not PFF-1, induce motor deficits within 16 weeks.

A Schematic of the forelimb grip force test, used to measure muscle strength. Subjects grasped a smooth, triangular pull bar with both forelimbs and the force exerted in Newtons (N) was measured. B aSyn PFF-2 but not PFF-1 inoculated mice displayed a significant reduction in forelimb muscle strength at 16 weeks post injection (WPI) relative to 8 (2-way RM ANOVA, Group x Session: main effect of group (F_2,72 = 4.409, p < 0.05), main effect of session (F_1,72 = 13.51, p < 0.001), and significant interaction (F_2,72 = 6.677, p < 0.01), in which Šídák’s multiple comparisons revealed to be driven by a significant difference within between 8 and 16 weeks within the PFF-2 group (adj. p < 0.0001). C Schematic of the accelerating Rota-rod, used to measure motor coordination and motor learning. Subjects were placed on a stationary rod which began to accelerate linearly from 5-36 revolutions per minute over 5 min, and latency to fall (s) was calculated automatically. D No significant difference between groups was found at 8 WPI (2-way RM ANOVA, Group x Session: main effect of session (F_9,675 = 15.41, p < 0.0001), but no main effect of group (p > 0.05). A significant interaction was first observed, (F_{18, 675} = 1.767, p < 0.05), but Šídák’s multiple comparisons revealed no significant differences between groups (adj. p > 0.05)). However, (E) aSyn PFF-2 mice exhibited a shorter latency to fall at 16 WPI relative to aSyn PFF-1 and PBS controls (2-way RM ANOVA, Group x Session: Main effect of group (F_2,71 = 7.436, p < 0.01), main effect of session (F_9,639 = 3.205, p < 0.001), but no interaction (p > 0.05). To examine this main effect further, Šídák’s multiple comparisons revealed a significant difference between aSyn PFF-2 and PBS controls (adj. p < 0.01) and aSyn PFF-2 and PFF-1 (adj. p < 0.01)). F Schematic of the catwalk XT Gait Analysis, used to measure gait and locomotion. Footprints were captured while subjects voluntarily traversed a glass-plated CatWalk runway, towards a dark goal box at the end. G A significant difference between 8 and 16 weeks was revealed in the stride length of aSyn PFF-2 inoculated mice (2-way RM ANOVA, Group x Session: no main effect of group (p > 0.05), but a main effect of session (F_1,24 = 4.493, p < 0.05), and significant interaction (F_{2,24= 3.646}, p < 0.05), in which Šídák’s multiple comparisons revealed a significant difference between timepoints within the aSyn PFF-2 group (adj. p < 0.01). H A significant difference between 8 and 16 weeks was revealed in the swing speed of aSyn PFF-2 inoculated mice (2-way RM ANOVA, Group x Session: no main effect of group (p > 0.05), but a main effect of session (F_{1,24= 6.537}, p < 0.05), and significant interaction (F_{2,24= 4.300}, p < 0.05) in which Šídák’s multiple comparisons revealed a significant difference between timepoints within the aSyn PFF-2 group (adj. p < 0.01). I A significant difference between 8 and 16 weeks was revealed in the step cycle of aSyn PFF-2 inoculated mice (2-way RM ANOVA, Group x Session: no main effect of group (p > 0.05), but a main effect of session (F_{1,24= 9.232}, p < 0.01) and significant interaction (F_{2,24= 4.035}, p < 0.05) in which Šídák’s multiple comparisons revealed a significant difference between timepoints within the aSyn PFF-2 group (adj. p < 0.01). Data presented as Mean + SEM, group x session Two-way RM ANOVA, ** p < 0.01, **** p < 0.0001. Graphics made with Biorender.com.