Journal: Translational Neurodegeneration

Article Title: Lysophagy protects against ANXA11 amyloid fibril toxicity and propagation in FTLD

doi: 10.1186/s40035-026-00561-5

Figure Lengend Snippet: ANXA11 fibril-induced lysosomal rupture triggers a cathepsin B-dependent cascade of mitochondrial dysfunction, oxidative stress, and apoptosis. a Schematic illustration of the proposed cytotoxic mechanism: lysosomal membrane permeabilization (LMP) induced by ANXA11 fibrils leads to the cytosolic release of cathepsin B, which subsequently triggers mitochondrial dysfunction, ROS accumulation, and Caspase-3-mediated apoptosis. b Representative transmission electron microscopy images of SH-SY5Y cells. Control cells exhibit healthy mitochondria with intact cristae, whereas ANXA11 PFFs-treated cells display swollen mitochondria with disrupted cristae (black arrows). c Representative confocal images showing mitochondrial morphology. Cells were stained for the mitochondrial outer membrane marker TOM20 (green), F-actin (Phalloidin, white), and nuclei (DAPI, blue). ANXA11 PFFs (red) induce mitochondrial fragmentation and network disruption. d Flow cytometric analysis of intracellular reactive oxygen species (ROS) levels using the DCFH-DA probe. Treatment with the specific Cathepsin B inhibitor CA-074Me attenuates the ROS surge induced by ANXA11 PFFs. e Representative fluorescence images of ROS generation in SH-SY5Y cells and iPSC-derived neurons. The green signal represents the fluorescence emitted by the oxidized DCF probe, visually indicating the levels of intracellular ROS. These images confirm the induction of oxidative stress upon PFFs exposure and its mitigation by lysosomal protection. f Assessment of mitochondrial membrane potential using JC-1 staining. In healthy cells, JC-1 forms red aggregates; in depolarized mitochondria, it exists as green monomers. ANXA11 PFFs induce a shift toward green fluorescence (depolarization), which is prevented by CA-074Me. g Quantification of the JC-1 aggregate-to-monomer fluorescence ratio from ( f ). h Western blot analysis of apoptotic signaling. ANXA11 PFFs increase levels of cleaved Caspase-3 (Asp175). This activation is partially blocked by inhibiting cathepsin B (CA-074Me) or autophagic flux (Bafilomycin A1). GAPDH served as a loading control. i, j Densitometric quantification of total Caspase-3 ( i ) and cleaved Caspase-3 ( j ) levels normalized to GAPDH. k Flow cytometric quantification of apoptosis using Annexin V-PE/Propidium Iodide (PI) double staining. The proportion of early (Q3) and late (Q2) apoptotic cells was significantly increased by ANXA11 PFFs but rescued by CA-074Me treatment. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test ( g , i , j ). Exact P -values are indicated in the graphs

Article Snippet: For flow cytometric analysis, cells treated with ANXA11 PFFs and CA-074Me (MedChemExpress, HY-100201, Monmouth Junction, NJ) were trypsinized and scraped from the culture plate.

Techniques: Membrane, Transmission Assay, Electron Microscopy, Control, Staining, Marker, Disruption, Fluorescence, Derivative Assay, Western Blot, Activation Assay, Double Staining