Lysophagy protects against propagation of α-synuclein aggregation through ruptured lysosomal vesicles

The neuron-to-neuron propagation of misfolded α-synuclein (αSyn) aggregates is thought to be key to the pathogenesis of synucleinopathies. Recent studies have shown that extracellular αSyn aggregates taken up by the endosomal–lysosomal system can rupture the lysosomal vesicular membrane; however, it remains unclear whether lysosomal rupture leads to the transmission of αSyn aggregation. Researchers at the Osaka University, Graduate School of Medicine applied cell-based αSyn propagation models to show that ruptured lysosomes are the pathway through which exogenous αSyn aggregates transmit aggregation, and furthermore, this process was prevented by lysophagy, i.e., selective autophagy of damaged lysosomes. αSyn aggregates accumulated predominantly in lysosomes, causing their rupture, and seeded the aggregation of endogenous αSyn, initially around damaged lysosomes. Exogenous αSyn aggregates induced the accumulation of LC3 on lysosomes. This LC3 accumulation was not observed in cells in which a key regulator of autophagy, RB1CC1/FIP200, was knocked out and was confirmed as lysophagy by transmission electron microscopy. Importantly, RB1CC1/FIP200-deficient cells treated with αSyn aggregates had increased numbers of ruptured lysosomes and enhanced propagation of αSyn aggregation. Furthermore, various types of lysosomal damage induced using lysosomotropic reagents, depletion of lysosomal enzymes, or more toxic species of αSyn fibrils also exacerbated the propagation of αSyn aggregation, and impaired lysophagy and lysosomal membrane damage synergistically enhanced propagation. These results indicate that lysophagy prevents exogenous αSyn aggregates from escaping the endosomal–lysosomal system and transmitting aggregation to endogenous cytosolic αSyn via ruptured lysosomal vesicles. These findings suggest that the progression and severity of synucleinopathies are associated with damage to lysosomal membranes and impaired lysophagy.

Lysophagy protects against seeded αSYN aggregation by sequestering damaged lysosomes

(A) Immunofluorescent images of LC3 in PFF550-treated cells. (B) Number of LC3-positive dots per cell in αSyn monomer- or PFF-treated cells for 24 h. Unpaired Student’s t test, ****P < 0.0001. (C) Immunofluorescent images of LC3 and LAMP1 in cells treated with PFFs for 24 h. (D) Immunofluorescent images of LC3 in RB1CC1-KO cells treated with PFF550. (E) Number of LC3-positive dots per cell in WT and RB1CC1-KO cells treated with PFF550. Two-way ANOVA with post hoc analysis, *P = 0.0372 WT vs. RB1CC1-KO at 0 h, **P = 0.0026 WT vs. RB1CC1-KO at 4 h, and ****P < 0.0001 WT vs. RB1CC1-KO at 24 h. n = 30 images for each condition, more than 300 cells for each condition, from three independent experiments. (F) Immunofluorescent images of LC3 in WT or penta-KO cells treated with PFF550. (G) Number of LC3-positive dots per cell in WT and penta-KO cells treated with PFF550 for 24 h. Unpaired Student’s t test, ****P < 0.0001. n = 40 images for each condition, more than 600 cells for each condition, from four independent experiments. Representative TEM images of (H) WT cells treated with PFFs for 24 h and (I) WT cells treated with PFFs for 2 h followed by bafilomycin A1 for 2 h. Asterisks indicate electron-dense lysosome-like structures, and arrowheads indicate membrane structure. (J) Ratio of lysophagy-like structure, electron-dense lysosomes surrounding double-membranes, per lysosome-like structure of TEM images of WT or RB1CC1-KO cells treated with PFFs with or without bafilomycin A1. One-way ANOVA with Tukey post hoc analysis, ****P < 0.0001, WT+bafilomycin A1 vs. WT+PFFs+bafilomycin A1 and WT+PFFs+bafilomycin A1 vs. RB1CC1-KO+PFFs+bafilomycin A1, n = 30 cells per treatment. (K) Number of lysosomes per cell in TEM images of the cells with each treatment. n = 30 cells per treatment. (L) Immunofluorescent images of Gal3 in WT and RB1CC1-KO cells treated with PFFs for 24 h. Arrows indicate Gal3-positive dots. (M) Percentage of Gal3 dot-positive cells in WT and RB1CC1-KO cells treated with PFFs for 24 h. Unpaired Student’s t test, ****P < 0.0001. n = 9 wells for each condition from three independent experiments. (N) Immunofluorescent images of p-αSyn in WT and RB1CC1-KO cells treated with PFFs for 48 h. (O) Percentage of p-αSyn-positive cells in WT and RB1CC1-KO cells treated with PFFs for 48 h. Two-way ANOVA with post hoc analysis, ****P < 0.0001 WT vs. RB1CC1-KO at 1 µM or 2 µM. n = 8 wells for each condition from two independent experiments. (P) Mean intensity of cytosolic p-αSyn in p-αSyn-positive cells, relative to WT. Unpaired Student’s t test, ****P < 0.0001. n = 8 wells for each condition from two independent experiments. (A), (C), (D), (F), (L), and (N), Scale bar, 10 µm. (H) and (I), Scale bar, 200 nm. (B), (E), (G), (M), (O), and (P), Bars represent the mean ± SEM. (J) and (K), Bars represent the mean ± 95 % CI.

Kakuda K, Ikenaka K, Kuma A, Doi J, Aguirre C, Wang N, Ajiki T, Choong CJ, Kimura Y, Badawy SMM, Shima T, Nakamura S, Baba K, Nagano S, Nagai Y, Yoshimori T, Mochizuki H. (2024) Lysophagy protects against propagation of α-synuclein aggregation through ruptured lysosomal vesicles. PNAS 121(1):e2312306120. [article]

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