Fig 2: 3D STED images of APP fragments in early endosomes in soma. Immunolabeling and 3D 3-channel STED microscopy was used to visualize AβPP and AβPP fragments in early endosomes in hippocampal neurons. Scale bars for A, C and E: 500 nm; scale bars for B, D and F: 100 nm. A, B) APP-CT and EEA1. C, D) APP-CT and APP-NT. E, F) APP-NT and EEA1. Using Huygens analysis software, a filter that can remove all non-overlapping vesicles was applied to show only the early endosomes that contain AβPP or AβPP fragments in A, C, and, E. Zoomed-in images of the regions marked by a yellow square in the left panel of A, C, and, E are shown in the middle panel and the selected vesicles marked by a yellow square in the middle panel are shown from two directions (xy, xz) in the right panel (B, D, and F). APP-CT budding from the early endosomes are marked by white arrows. APP-NT in the same early endosomes are marked by magenta arrows. All analyses were based on 3D data, which means that voxel-based practical colocalization analysis was applied. G) Schematic picture showing the sorting of AβPP fragments in early endosome.

Fig 3: STED images of Aβ42 and subcellular markers in soma. A) 3-color STED image of Aβ42 (red), EEA1 (green), and Rab9 (cyan). (B-G) 2-channel STED imaging combined with actin staining (white) in a confocal channel. The zoomed-in pictures are shown to the right. B) Aβ42 (red) and Rab7 (green). C) Aβ42 (red) and LC3A (green). D) Aβ42 (red) and Rab26 (green). E) Aβ42 (red) and flotillin-1 (FLOT1, green). Yellow arrows point at areas rich in flotillin-1 containing vesicles. F) Aβ42 (red) and clathrin (green). The white line marks the edge of soma. G) Colocalization between Aβ42 and clathrin is shown in yellow. Scale bars for all pictures: 500 nm. H) Quantification of Aβ42-containing vesicles in soma (n = 20 for each subcellular marker). Data were quantified from 3 different preparations of hippocampal neurons for each subcellular marker. All error bars represent mean±sd. Analysis was done by “particle analyze” in ImageJ. Threshold “Moments” was applied for the Aβ42 channel. Threshold “Triangle” was applied for subcellular markers channel. I) Quantification of the size (diameter) of vesicles containing Aβ42 at the rim in soma. Data were quantified from 3 different batches of hippocampal neurons. Vesicles containing Aβ42 at the rim were selected manually and 41 vesicles were quantified in total.

Fig 4: STED images of Aβ42 and subcellular markers in neurites. 2-channel STED imaging combined with actin staining (white) in a confocal channel. The colocalization between Aβ42 and subcellular markers is shown to the right in yellow. A) Aβ42 (red) and clathrin (green). B, C) Aβ42 (red) and EEA1 (green). D) Staining of Aβ42 (red) and Rab9 (green). E) Aβ42 (red) and Rab7 (green). F) Aβ42 (red) and Rab26 (green). Scale bars for all pictures: 500 nm. G) Distribution of Aβ42 staining in neurites. Data were quantified from 3 different batches of hippocampal neurons for each subcellular marker. All error bars represent mean±sd. Analysis was done by “particle analyze” in ImageJ. Threshold “Moments” was applied for the Aβ42 channel. Threshold “Triangle” was applied for the subcellular markers channel.

Fig 5: Scheme of subcellular localization of full-length AβPP, CTFs, and/or Aβ42 in hippocampal neurons. The subcellular localization of full-length AβPP (FL-APP), APP-C-terminal fragments (APP-CTF), and Aβ42 in the endocytic-lysosomal system, based on STED imaging of hippocampal neurons, is shown. Early endosome antigen 1 (EEA1); clathrin coated vesicle (CCV); Early endosome (EE); Multi-vesicular body (MVB); Late endosome (LE); Autophagosome (AP); Synaptic vesicle (SV).