Fig. 2: Progressive accumulation of reactive h-α-Syn protein in raphe nuclei.

Mice received 1 μl AAV5 construct containing a chicken-β-actin promoter to drive expression of h-α-Syn or vehicle into raphe nuclei and euthanized at 1, 4, and 8 weeks (W) post-injection. a Representative coronal midbrain sections showing progressive increases of h-α-Syn protein levels in the raphe nuclei assessed by immunohistochemistry procedures. Top: signal represents the optical density (OD) of autoradiograms as indicated at the right side of the image. Scale bar: 1 mm. Bottom: coronal midbrain sections showing immunostaining for h-α-Syn. Scale bar: 200 μm. b Increased h-α-Syn protein levels in AAV5-injected mice. c Representative photomicrographs showing gradual increases of phospho-S129-α-Syn (p-α-Syn) levels in the dorsal raphe nucleus (DR) of AAV5-injected mice. Frames indicate areas of highest magnification. Scale bars: 250 μm and 50 μm, respectively. d Number of p-α-Syn-positive cells in raphe nuclei. e Progressive increase in α-Syn oligomer levels in lysates of raphe nuclei of vehicle- and AAV5-injected mice assessed by ELISA. f Representative images of coronal midbrain sections showing specific signal for h-α-syn self-interaction detected by proximity ligand assay (PLA). Scale bar: 10 μm. Black arrowheads show the punctate brown staining likely represents accumulation of aggregated h-α-Syn. Values are presented as mean ± SEM. *p < 0.05, **p < 0.01, ****p < 0.0001 compared to vehicle- or AAV5-injected mice. See Supplementary Fig. 4.