Figure 1. Autophagic accumulation in the distal axons of mutant hAPP Tg mice.
(A and B) Representative images (A) and quantitative analysis (B) showing accumulation of LC3-labeled autophagic vacuoles (AVs) in the hippocampal mossy fibers of eight-month mutant hAPP Tg mice. (C and D) Quantitative analysis (C) and representative images (D) showing amphisome retention in the hippocampal mossy fibers of hAPP mice. Note that LC3-labeled AV clusters were co-localized with cation-independent mannose 6-phosphate receptor (CI-MPR), a late endosome (LE) marker, suggesting that those AVs are amphisomes in nature following fusion with LEs. (E and F) Representative TEM images (E) and quantitative analysis (F) showing abnormal retention of AVd-like organelles within enlarged neurites in the hippocampal regions of mutant hAPP Tg mouse brains. Note that dystrophic/swollen neurites contained predominantly AVd-like structures marked by arrows, which was not readily observed in wild-type (WT) mice. The average number of AVd per EM field was quantified. (G and H) Quantitative analysis (G) and representative TEM images (H) showing aberrant accumulation of AVd-like structures (black arrows) at presynaptic terminals in hAPP mice. AVd-like structures, indicated by arrows, were not readily observed in WT mouse brains. Percentage of presynaptic terminals containing AVd was quantified. (I and J) Abnormal synaptic retention of LC3-II and p62 (autophagy markers), APP, and Aβ in mutant hAPP Tg mouse brains. Equal amounts (15 μg) of synapse-enriched synaptosomal preparations (Syn) and post-nuclear supernatants (PNS) from WT and hAPP mice were sequentially immunoblotted on the same membrane after stripping between each antibody application. The purity of synaptosomal fractions was confirmed by the absence of EEA1 and GAPDH. The synaptosome/PNS ratio in AD mice was compared to those in WT littermates. Data were quantified from three independent repeats. Stx1: syntaxin 1; SYP: synaptophysin Scale bars: 25 μm (A and D), 100 nm (E), and 200 nm (H). Data were quantified from a total number of imaging slice sections indicated on the top of bars (B and C) from three pairs of mice. The average numbers of AV clusters per section (320 μm × 320 μm) and per EM field (10 μm × 10 μm) were quantified (B and C). Error bars represent SEM. Student’s t test: ***p<0.001, **p<0.01, *p<0.05.
Figure 1—figure supplement 1. Axonal autophagic stress in mutant hAPP Tg mouse brains.
