Fig. 1
Induction of αS inclusions in mouse hippocampal slice cultures. (A) Treatment scheme for hippocampal slice cultures (HSCs). Hippocampi of postnatal day 4–6 (P4–6) of Tg-Thy1-h[A53T]αS (Tg), wildtype (Wt) or Snca−/− mice were sectioned into 350 μm thick slices and plated onto semi-permeable culture membranes. After 10 days in vitro (DIV-10), 1 μl of either Wt or Tg brain homogenate, or αS preformed fibrils (αS pff) at 35 μM were added on top of each culture. All three cultures within one well received the same treatment. Five weeks later, the cultures were collected for analysis. (B) Shown is a Tg culture treated with αS pff. Anti-pS129 immunofluorescence revealed a myriad of inclusions in both somas and processes of cells with a neuronal appearance. In contrast, ThioS staining revealed mainly small wool-like inclusions. Shown is also the merged triple staining with DAPI. Scale bars represent 500 μm (overview), and 20 μm (closeup). (C) Immunofluorescence double staining for pS129 (red) and NeuN or pFTAA (green) as well as for iba1 (red) and ThioS or pFTAA (green). Note that pS129-positive inclusions are predominantly present in neurons (NeuN), while ThioS-positive aggregates appeared mainly to be associated with microglia (iba1) consistent with previous work [36]. pFTAA stained both pS129-positive neuritic inclusions and microglial inclusions. Scale bars represent 50 μm, and 10 μm for inserts. (D) Quantification of pS129- and ThioS-positive inclusions (in percentage of culture area) in dependence of the seeding agent (αS pff, Tg brain homogenate) and host (Tg, Wt, Snca−/−) 5 weeks after seeding. Mean ± SEM is shown; n = 5–6 HSCs per group; − = no inclusions. (E) Immunoblot using an antibody against pS129 of sarkosyl-insoluble material of seeds and HSC homogenates. Monomeric αS phosphorylated at S129 was present as a band at around 15 kDa in brain extract as well as tg HSCs seeded with αS pff or Tg brain homogenate, but not αS pff. Multimeric species were also present as higher molecular bands; n = 16 pooled HSCs per group