Fig. 3

Application I. Demonstrating trans-synaptic spreading as a route for spreading of α-syn-aggregate pathology from DG via CA3 to the CA1 region using surgical and viral transgene methods. a Illustration of PFF injection in CA1 in WT OHSCs to test the efficiency of the retrograde route of spreading. 1 Composite image 14 dpi of S129A PFFs at CA1. Scale bar: 200 μm. MJF-14-positive aggregates are seen at the CA1 region (i, ii), but there is no spreading to DG (iii). Scale bar i & iii: 50 μm, ii: 20 μm. b Diagram showing transection of axonal projections between DG and CA3, which blocks spreading of α-syn aggregate pathology from DG to CA1. The surgical destruction of the tissue is demonstrated by the absence of nuclei (1), axonal marker NF-L (2), and MJF-14 staining (3). Scale bars: 200 μm. Magnified images from 3 show aggregates at DG (i) and proximal to the cut (ii), but not distal to the lesion (ii, iii). Scale bars: 50 μm. c Diagram showing expression of WT-α-syn in α-syn KO slices by AAV vectors injected in DG, CA3, and CA1. 1 α-syn expression in DG, CA3, and CA1 supports spreading of aggregated pS129 α-syn (11A5) to CA1 7 dpi of PFFs in DG, as seen from the magnified panels i-iiii. Scale bar: 200 μm, i & iiii: 20 μm, ii & iii: 10 μm. Note the strong AAV-dependent expression of pS129 in some neuronal nuclei. d Illustration of WT-α-syn expression in DG and CA1 only of α-syn KO slices. 1 Absence of α-syn expression in the CA3 abolishes spreading of aggregation to CA1 at 7 dpi. Scale bar: 200 μm. i, ii pS129-positive aggregates are detectable at DG. iii No pS129-positive aggregates are found at the CA1 region. Iiii A few neurons show nuclear expression of pS129-α-syn at CA3. Scale bars: i: 20 μm, ii, iii & iiii: 10 μm. Data in a are illustrative of 12 slices divided over 3 experiments. Images in b are representative of 4 experiments with 18 slices in total, while c & d representative of 3 separate experiments/18–21 slices in total per condition