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. 2021 Aug 11;16:54. doi: 10.1186/s13024-021-00471-2

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© The Author(s) 2021

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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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