Fig. 2: Accumulation of abnormally ELPVs is associated with spheroid expansion and cognitive decline.

a–h, Analyses in 5xFAD mice. a, Confocal image of spheroids labelled by LAMP1 (green) around an amyloid plaque (cyan). Bottom, magnified image (dashed box in the top image) of a large spheroid (white dotted outlines). The arrows indicate ELPVs. Scale bar, 10 μm. b, ELPV occurrence within spheroids at different ages in 5xFAD mice. n = 3 mice for each group. c, Electron microscopy images of spheroids showing diverse endolysosomal and autophagic vesicles (red shade). MVBs (i); an MVB, possibly fusing with an autophagosome (ii); an organelle with ILVs (iii); an autophagosome (iv); and the fusion of multiple MVBs (v) are shown. Scale bars, 500 nm. d, Spheroid size with the presence or absence of ELPVs. n = 3 mice from each group. e, Confocal images of spheroids (white dotted lines) with high or low levels of cathepsin D. Scale bar, 5 μm. f, Spheroid size as a function of cathepsin D levels. n = 6 mice for each group. g, Confocal image of spheroids expressing the pH sensor SEpHluorin-mCherry. Scale bar, 10 μm. h, Spheroid size as a function of pH (red–green fluorescence ratio). n = 4 mice for each group. i–o, Analyses of post-mortem brains of individuals with AD. i, Confocal image showing human spheroids around an amyloid plaque labelled by V0A1 (red). Scale bar, 10 μm. j, Confocal imaging of individual V0A1-labelled spheroids (white dotted lines). The arrowheads indicate enlarged vesicles. Scale bars, 2 μm. k, Spheroid size with the presence or absence of enlarged V0A1-labelled vesicles. n = 10 human individuals from each group. l, PLD3 (magenta) enrichment within spheroids. Scale bar, 10 μm. m,n, Confocal images of spheroids labelled with V0A1 in the brain of a human with AD with the PLD3(V232M) variant. Right, images of individual spheroids indicated by the orange boxes. Enlarged vesicles are indicated by arrows. The arrowheads indicate enlarged vesicles. Scale bars, 5 μm (m) and 2 μm (n). o, Occurrence of enlarged vesicle in PLD3(V232M) variant, AD and MCI post-mortem human brain tissues. n = 4 individuals with AD with the PLD3 variant, n = 6 individuals with AD and n = 4 individuals with MCI. Statistical analysis was performed using a Kruskal–Wallis test (b), two-tailed paired t-tests (d,f,h and k) and two-tailed Mann–Whitney U-tests (o). In all of the graphs, each pair of dots represents the average measurement from spheroids in the same mouse and data are mean ± s.e.m.