Fig. 2.

Progressive accumulation of reactive forms of h-α-syn protein in SNc/VTA. (a) Coronal brain sections showing progressive increases of h-α-syn protein levels in SNc/VTA and CPu assessed by immunohistochemistry procedures. Signal represents the relative optical density (ROD) of autoradiograms. Scale bar: 1 mm. (b) Increased h-α-syn protein levels in the ipsilateral (ipsi) SNc/VTA and CPu versus contralateral (contra) side (n = 3–5 mice/group; *P < 0.05, ***P < 0.001; two-way ANOVA and Tukey's multiple comparisons test). (c) Representative photomicrographs showing gradual increases of phospho-ser129-α-syn protein levels in the SNc/VTA of vehicle- and AAV5-injected mice. Scale bar: 250 μm. (d) Number of phospho-S129-α-syn⁺ cells in the SNc/VTA (n = 4 mice/group; *P < 0.05, **P < 0.01 versus AAV5-injected mice and sacrificed at 1 week (W) post-infection; one-way ANOVA and Tukey's multiple comparisons test). (e, g) Images of Western blot (e) and quantitative analysis (g) of monomeric α-syn protein in SNc/VTA lysates from mice injected with vehicle or AAV5 (4 W time point). For detection, immunoblots for α-syn were performed using different antibodies against: mouse and human α-syn, and specific human α-syn. β-actin used as loading control (n = 4–6 mice/group; **P < 0.01 versus vehicle-injected mice; two-tailed unpaired t-test). (f, h) Immunoblotting (f) and quantification (h) of α-syn oligomer sum in SNc/VTA lysates from the same mice using anti-α-syn antibody against mouse and human, and anti-β-actin antibody as loading control (n = 4–6 mice/group; **P < 0.01 versus vehicle-injected mice; two-tailed unpaired t-test). (i) Increased oligomer α-syn levels in SNc/VTA lysates of vehicle- and AAV5-injected mice at 4 W post-injection assessed by ELISA using anti-α-syn filament antibody (n = 9 mice/group; **P < 0.01 versus vehicle-injected mice, ^##P < 0.01 versus contralateral -contra- side; two-way ANOVA and Tukey's multiple comparisons test).