Figure 3.

Delayed degradation and accumulation of LC3-II and Aβ within autophagic-lysosomal compartments in the brains of TgCRND8. Subcellular fractions containing the autophagic-lysosomal compartments AV10, AV20 and lysosomes were isolated from brains of 12-month-old TgCRND8 and wild-type (WT) (n = 6 per genotype), and equal amounts of protein from each fraction were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis and processed for western blotting (WB) with an antibody directed against cathepsin D (CatD) (A, B, top panels), LC3 (A) or 6E10 (B). Immunoblot for cathepsin D showing that the AV10, AV20 and lysosome fractions exhibit strongest cathepsin D signals, confirming the autophagic-lysosomal origin of these fractions. Corresponding ponceau S stained blots are shown below LC3 antibody-probed blots as loading controls (A). (C) Double immunofluorescent labelling showing that most giant autolysosomes and some smaller lysosomes were co-immunolabelled (C3) by RU2 (C1) and 4G8 (C2) in the CA1 sector of a 6-month-old TgCRND8. Granules displaying only RU2 signal or 4G8 signal are labelled with arrows and arrowheads, respectively. (D) Detection of giant autolysosomes in the CA1 sector by additional antibodies directed against various regions of the Aβ domain, including JRF/AβN/25, JRF/Aβtot/17, 4G8 and JRF/cAβ42/26. The images are representative of the results from three 12-month-old mice per genotype. Scale bars: 10 µm (C); 20 µm (D). AV10 = early autophagic vacuole/autophagosome; AV20 = late autophagic vacuole/autolysosome; CRND8 = TgCRND8; Cyto = cytosol; ER = endoplasmic reticulum-enriched; Homo = homogenate; Lyso = lysosome; Mito = mitochondria.