Recruitment of LC3⁺ vesicles to enlarged pSER129-lysosomal aggregates. a, Confocal microscopy (merged and single-spectra images) of brain sections quadruple-labeled for pSer129 (red), LAMP-1 (magenta), LC3-II (green), and nuclei (DAPI, blue). Bottom, 8-bit (gray) images of the staining highlight the increase of of LC3⁺ puncta within the large pSer129 aggregates. Bottom panels show 15G7 hu aS immunogold labeling (arrow) at round, abnormal lysosomal inclusions (black asterisks) in cortex of young (4 months old) homozygous 3K mice. The smaller inclusions are close to a larger round electron-dense perinuclear inclusion (N, nucleus) (white asterisk). Arrow points to hu aS immunogold labeling. b, Colocalized points of the respective dual-color confocal images were analyzed using ImageJ colocalization highlighter and then estimated for their size by the ImageJ particle analyzer plugin. Quantification for colocalized aggregate sizes (pSER129-LAMP1) and LC3⁺ counts in n = 10 fields each of sections from each genotype. c, Quantitative WBs of autophagy-lysosomal marker quantified in d, show reduced LAMP1 accumulation, increase in LC3-II, and lesser membrane-associated αS in 3K female and by DHED treatment. Data are mean ± SEM. One-way ANOVA post Tukey: *p < 0.05; **p < 0.01. Scale bars: a, top, 20 μm; bottom, 500 nm.