Figure 6

Monomeric Abeta was preferentially accumulated in isolated synaptosomal fraction, from aged PS1/APP mice, whereas APP-C99 fragment preferentially localized in microsomes. A) Microsomal and synaptosomal fractions were isolated from 6, 12 and 18 months WT and PS1/APP mice. Using these samples, we tested the distribution of LC3-II autophagosomal marker (a1), the APP-CTF fragments (a2) and the monomeric Abeta peptide (a3 and a4 for a longer exposure). ATP-synthase and b-actin were used as control of synapsomal integrity and protein loading. Quantitative analysis of western blots demonstrated the age-dependent accumulation of LC3-II (compared with the respective fraction in 6 month WT mice) (B) and C99 (as compared with 6-month-old PS1/APP microsomes) (C) in microsomal fractions. The monomeric Abeta was preferentially accumulated in the synaptosomal fractions (D). These experiments were repeated four times. E) Control experiments demonstrating the intrasynaptosomal origin of the monomeric Abeta. The tissue from a 12 month-old PS1/APP mice was divided, homogenized in absence (control) or in presence of 0.5% CHAPS + 0.5% DOC, and synaptosomes were isolated by Ficoll gradients. As shown, the disruption of cell membranes avoided the Abeta flotation on gradients (upper panels). On the other hand, tissue from 6 month-old WT mice were homogenized in absence (minus) of presence (plus) of synthetic Abeta42 and the synaptosomes were isolated. No Abeta were compartmentalized during homogenization.