Lysosomal proteolysis inhibition selectively disrupts axonal transport of degradative organelles and causes an Alzheimer's-like axonal dystrophy - PubMed (original) (raw)

Biochemical and ultrastructural profiles of neurites after lysosomal clearance inhibition resemble AD dystrophic neurites. A–F, Swellings preferentially accumulate with lysosomal (proteolytic) vesicles after treatment with leupeptin (20 μ

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, 24 h). LysoTracker Red vesicles (A), Rab7 vesicles (B), and LC3 vesicles (C, arrows) are preferentially accumulated, whereas mitochondria (C, arrowheads), Rab5-positive early endosomes (D), neurofilament-light chain (E), or β-tubulin (F) are relatively evenly distributed along the axon. G–I, Swellings accumulate proteins that identify AD-dystrophic neurites including APP-containing autophagic vesicles (G; C1/6.1 antibody for C terminus of APP double labeled with LC3), ubiquitinated proteins (H), and phospho-neurofilaments (I; NF-M/H; double labeled with LC3). Phosphorylated neurofilament accumulation occurs in swollen regions containing accumulated LC3 vesicles. Scale bars, 5 μm. J, Time lapse of swelling with GFP-LC3 and DsRed-Mito cotransfection and treatment with leupeptin (20 μ

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, 5 h). The cell body is at the top. Although both GFP-LC3 vesicles and DsRed-mitochondria are accumulated, mitochondria occasionally resume transport (red arrowheads), whereas LC3 movement out from the swelling is not observed. K, Western blots with indicated antibodies in leupeptin-treated neurons compared with controls. Leupeptin increases the ratio of LC3-II/I, phospho-neurofilaments (SMI-31), and APP-CTFs, without increasing overall levels of neurofilaments or APP holoprotein (full length). Molecular mass is shown in kilodaltons. L, M, Representative electron micrographs of neurites after leupeptin (20 μ

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, 24 h; M) compared with controls (L). Leupeptin-induced accumulation of double-membrane, amorphous electron-dense AVs in neurites is shown. N, Quantification of morphometric analysis of organelles in dystrophic neurites from leupeptin-treated neurons (Leup Tx; n = 20) or AD mouse models (APP, n = 25; PSAPP, n = 25). O, P, Representative electron micrographs of dystrophic neurites from APP (O) and PSAPP (P) mouse brain. Organelles within dystrophic neurites are mostly double-membrane AVs. Values represents means ± SEM. Scale bars: A–J, 5 μm.