Figure 4. Oligomeric Aβ42-mediated interruption of dynein-Snapin coupling and recruitment of dynein motors to amphisomes.
(A and B) Immunoisolation assays (A) and quantitative analysis (B) from three repeats showing reduced dynein attachment to amphisomes in sixteen-month mutant hAPP Tg mouse brains. Rab7-associated organelles were immunoisolated from light membrane fractions, followed by sequential immunoblotting on the same membranes with antibodies against the dynein intermediate chain (DIC), LC3, hAPP, Snapin, Rab7, and EEA1. Note that AD mouse brains exhibited reduced DIC and increased LC3-II levels in the purified amphisomal organelles. Data were quantified from three independent repeats of three pairs of mice. (C) Immunoprecipitation showing reduced Snapin-DIC coupling in COS7 cells expressing mutant hAPPswe, but not WT hAPP. (D) Direct interaction of Aβ1-42 with GST-DIC, but not GST-Snapin or GST. 6E10 antibody was used to detect Aβ. (E and F) GST-DIC specifically interacts with Aβ1-42, but not Aβ1-40 or scrambled Aβ. (G) Aβ1-42 interaction with DIC was increased as Aβ concentration was elevated from 0.5 μM to 4 μM in the presence of the same amount of GST-DIC. (H) Aβ1-42 interferes with DIC-Snapin coupling in a dose-dependent manner. Note that the DIC-Snapin interaction was competitively interrupted in the presence of as low as 0.2 μM Aβ1-42 when the same amount of Snapin and DIC was used. (I) The dot blot using anti-β amyloid antibody showed that GST-DIC bound to oligomeric Aβ. (J) Oligomeric Aβ1-42 interrupts dynein-Snapin coupling and the recruitment of dynein motors to LEs and amphisomes in mouse brains. Membranous organelles were pulled down from light membrane fractions (LMF) of mouse brains by GST-DIC in the presence or absence of 2 μM oligomeric Aβ1-42. Bead-bound membrane organelles were resolved by PAGE and sequentially detected with antibodies on the same membranes after stripping between applications of each antibody. Note that reduced tethering of dynein motors to LAMP-1 or Rab7-associated LEs and amphisomes in the presence of Aβ was specifically caused by impaired DIC-Snapin interaction because the dynein-dynactin (p150Glued) complex was not affected. The attachment of dynein motors to mitochondria showed no detectable change. TOM20: a mitochondrial outer membrane protein. The purity of the preparation pulled down by GST-DIC beads was confirmed by the absence of GAPDH. Results were representative from three independent repeats. (K) The DIC-Aβ complex was immunoprecipitated by anti-DIC antibody from mutant hAPP Tg mouse brains. Error bars represent SEM. Student’s t test: ***p<0.001, **p<0.01, *p<0.05.