Fig. 1

Organotypic mouse hippocampal slice cultures as a model to study seeded α-syn aggregation in the region between DG and CA3. a Diagram showing the synaptic connections of granule cells of DG (where S129A PFFs were injected) to pyramidal neurons in CA3 that subsequently connect to the pyramidal neurons of the CA1 region. b OHSCs from mouse pups were cultivated on an air-liquid interface. c Progressive accumulation of total (i) and pS129-α-syn (ii, 11A5) in cultures from wild type mouse pups after 0, 7, 14, and 21 DIV analyzed by immunoblotting. d Experimental flow showing time of PFF microinjection at 7 DIV and tissue collection for analysis at 3, 5, 7, and 14 dpi. e pS129-positive α-syn structures (D1R1R) imaged at DG, following PFF injection at DG. Aggregates are first recognizable at 3 dpi as short serpentine aggregates (i) that coalesce into longer aggregates by 5 dpi (ii) and at 7 dpi occur as fibrillar aggregates around neuronal nuclei (iii). Scale bars: 20 μm. f MJF-14-positive serpentine aggregates co-localize with the axonal marker neurofilament light chain (NF-L). Scale bar: 20 μm. g pS129-positive cell body inclusions (D1R1R) are located in NeuN-positive neurons. Scale bar: 20 μm, inset: 5 μm. h Thread-like cell body inclusion detected by MJF-14 and reconstructed in 3D by IMARIS software. i Cell body pS129 α-syn pathology (D1R1R) in the hindbrain of end-stage h-A53T-α-syn transgenic mice (M83) resembles inclusions in the slice model (panels e,iii & g). j dSTORM image reconstruction of pS129-positive axonal processes (D1R1R) within the OHSC. Scale bar: 1 μm. k Progressive accumulation of insoluble pS129-positive mouse α-syn (11A5) in PFF-injected slices. Western blots in c & k are representative of 2–3 separate experiments. Images in e are examples from 2 to 6 individual experiments with 9–17 slices in total. Images in f & g are representative of 4–5 experiments/15–16 slices in total