Fig. 3.

Reduced synucleinopathy in mice carrying STI1 hypomorphic alleles (M83+/−::ΔTPR1 mice). a Representative images of psyn129 (red) immunolabelling with nuclear marker (blue) in coronal sections from male M83+/− and M83+/−::ΔTPR1 mice injected with PFFs. Images shown are near the coordinates of injection and were captured at 20× magnification. Ipsilateral and contralateral hemispheres are labelled as respective to right hemisphere intrastriatal injection. Scale bar = 1 mm. Quantification of immunolabelling is shown in Supplementary Fig. S4a&b. b–d Higher resolution imaging of co-localization analyzes in the Anterior Cingulate Cortex from M83+/− and M83+/−::ΔTPR1 PFF injected mice. Images taken at 63× with Leica Thunder microscope, with images post-processed to 2× zoom. Arrows illustrate areas where considerable co-localization was observed. Scale bars are 25 µm for all images. b Colocalization of ubiquitin with psyn129 (pSer129 antibody) c Colocalization of Hsp90 with psyn129 (pSer129 antibody) d Colocalization of STI1 with psyn129 (pSer129 antibody). e Representative immunoblot of STI1, psyn129, and human α-synuclein in whole lysates from the ipsilateral cortex. f Densitometric quantification of psyn129 (relative to human α-synuclein; (t(8) = 2.852, p = 0.0214). g Densitometric quantification of human α-synuclein (relative to actin; (t(8) = 0.9439, p = 0.3729)). N = 5 mice/genotype. h Representative immunoblot from the ipsilateral hippocampus of M83+/− and M83+/−::ΔTPR1 PFF injected mice. i Densitometric quantifications of psyn129 (relative to human α-synuclein; t(7) = 0.5526, p = 0.598) and j human α-synuclein (normalized to actin; t(7) = 0.4552, p = 0.6627). N = 5 M83+/− PFF, N = 4 M83+/−::ΔTPR1 PFF mice. k Immunoblot of STI1, psyn129, and human α-synuclein in ipsilateral cortical SDS/Urea lysates. l Densitometric quantification of psyn129 (relative to human α-synuclein; t(8) = 5.236, p = 0.0034) and m human α-synuclein (relative to amido black; t(8) = 1.139, p = 0.3063). N = 5 M83+/− PFF and N = 5 M83+/−::ΔTPR1. M83+/− PFF individual mice represented as blue circles, and M83+/−::ΔTPR1 individual mice identified as green triangles. Data represented as mean ± SEM and analyzed using unpaired t test. n Mouse body weight at 14–16 wpi, just prior to tissue collection (one-way ANOVA, genotype/group: F(3,38), 12.02, p < 0.0001; adjusted for multiple comparison’s and analyzed using Tukey’s post hoc test: M83+/− PFF vs. M83+/−::ΔTPR1 PFF p = 0.9146, M83+/− PFF vs. M83+/−:TgA PFF p = 0.999, M83+/− PFF vs. M83+/− PBS p < 0.0001, M83+/−::ΔTPR1 PFF vs. M83+/− PBS p = 0.0017, M83+/−:TgA PFF vs. M83+/− PBS p = 0.0002). N = 16 M83+/− PFF, N = 9 M83+/−::ΔTPR1 PFF, N = 9–10 M83+/−:TgA PFF and N = 15 M83+/− PBS for weight analyses. M83+/− PBS group includes STI1-WT, ΔTPR1 and TgA M83+/− PBS-injected mice as no significant differences were found between genotypes (one-way ANOVA, genotype/group: F(2,12) = 1.114, p = 0.3598). o Wire hang motor assessment. Mice were suspended upside down and the latency to fall was recorded across 3 trials spaced at least 10 min apart. Percentage of mice (across the three trials) that fell before 10 s ( <) or at and above ( ≥) 10 s. Percentages displayed on graph bars represent the genotype average. Data analyzed using one-way ANOVA with appropriate post hoc comparisons. There was a significant interaction of genotype x time (F(3,8) = 62.70, p < 0.0001, and data were adjusted for post hoc comparison’s using Tukey’s multiple comparisons test: M83+/− PFF vs. M83+/−::ΔTPR1 PFF p = 0.0004 (Cohen’s d, effect size = 6.20), M83+/− PFF vs. M83+/−:TgA PFF p = 0.176 (Cohen’s d, effect size = 1.94), M83+/− PFF vs. M83+/— PBS p < 0.0001 (Cohen’s d, effect size = 7.78), M83+/−::ΔTPR1 PFF vs. M83+/− PBS p = 0.3059 (Cohen’s d, effect size = 1.58), M83+/−:TgA PFF vs. M83+/− PBS p < 0.0001(Cohen’s d, effect size = 9.72). N = 17 M83+/− PFF (filled blue circles), N = 9 M83+/−::ΔTPR1 PFF (green triangles), N = 10 M83+/−:TgA PFF (purple squares) and N = 15 M83+/− PBS (gray open circles) for wire hang analyses. Data represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.0001