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: Internalized ANXA11 fibrils accumulate in lysosomes and trigger lysosomal membrane permeabilization (LMP) in neurons. a Representative confocal microscopy images of SH-SY5Y cells treated with Alexa Fluor 488-labeled ANXA11 preformed fibrils (PFFs-AF488, green) for 24 h. Cytoskeleton was stained with phalloidin (white) and nuclei with DAPI (blue). The images show intracellular localization of ANXA11 aggregates. b Flow cytometry analysis quantifying the uptake of ANXA11 PFFs by SH-SY5Y cells after 24 h treatment. c Confocal images showing the subcellular localization of internalized ANXA11 PFFs-AF647 (red) and the lysosomal marker LAMP1 (green). d Fluorescence intensity profile analysis along the white line drawn in ( c ), demonstrating the colocalization of ANXA11 PFFs with lysosomes. e Transmission electron microscopy images of SH-SY5Y cells treated with ANXA11 PFFs. Arrows indicate electron-dense aggregates within lysosomes and damaged mitochondria with disrupted cristae. f Western blot analysis of cathepsins (CSTA, CTSB, and CTSD) in cytosolic versus whole-cell lysate fractions. The presence of mature cathepsins in the cytosolic fraction upon PFFs treatment indicates lysosomal leakage. GAPDH was used as a loading control. g Representative images of Lysotracker Red staining in SH-SY5Y cells treated with or without ANXA11 PFFs, assessing lysosomal acidity and integrity. h Quantification of Lysotracker Red fluorescence intensity shown in ( g ). i Immunofluorescence images showing the recruitment of galectin-3 (GAL3, purple), a marker of lysosomal rupture, to LAMP1-positive lysosomes (green) containing ANXA11 PFFs-AF647 (red). j Fluorescence intensity profile analysis along the line in ( i ), showing the colocalization of GAL3 with ruptured lysosomes containing ANXA11 PFFs. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( h ). Exact P -values are indicated in the corresponding 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, Confocal Microscopy, Labeling, Staining, Flow Cytometry, Marker, Fluorescence, Transmission Assay, Electron Microscopy, Western Blot, Control, Immunofluorescence

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 PFFs trigger RB1CC1-dependent lysophagy and autophagic flux. a Western blot analysis of LC3 levels in SH-SY5Y cells treated with or without ANXA11 PFFs. GAPDH served as a loading control. b , c Quantification of the LC3-II/LC3-I ratio ( b ) and LC3-II/GAPDH ratio ( c ) from the blots in ( a ), indicating increased autophagic activity upon PFFs treatment. d Representative confocal images showing the recruitment of LC3 (purple) to LAMP1-positive lysosomes (green) containing ANXA11 PFFs-AF647 (red). Nuclei were stained with DAPI (blue). e Fluorescence intensity profile analysis along the white line in ( d ), confirming the colocalization of ANXA11 PFFs, LAMP1, and LC3. f Transmission electron microscopy images showing double-membrane autophagosomes engulfing electron-dense lysosomal contents (arrows), a hallmark of lysophagy, in cells treated with ANXA11 PFFs. g Western blot confirming the knockdown efficiency of RB1CC1 in SH-SY5Y cells. h Western blot analysis of LC3 turnover in Control KD and RB1CC1 KD cells treated with or without ANXA11 PFFs. i , j Quantification of LC3-II/I ( i ) and LC3-II/GAPDH ( j ) ratios from ( h ), showing that RB1CC1 knockdown prevents the PFF-induced LC3 lipidation. k Immunofluorescence staining of galectin-3 (GAL3, green) in Control KD and RB1CC1 KD cells treated with ANXA11 PFFs-AF647 (red). l Quantification of relative GAL3 fluorescence intensity in ( k ). The increase in GAL3 signal in RB1CC1 KD cells indicates an accumulation of ruptured lysosomes due to impaired clearance. m Analysis of autophagic flux using the mCherry-EGFP-LC3 reporter. Control KD cells treated with PFFs show red puncta (indicating autolysosomes, where GFP is quenched by acidity), whereas RB1CC1 KD cells retain yellow/green fluorescence. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( b, c ) or one-way ANOVA with Tukey’s post hoc test ( i, j, l ). Exact P -values are indicated in the corresponding 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: Western Blot, Control, Activity Assay, Staining, Fluorescence, Transmission Assay, Electron Microscopy, Membrane, Knockdown, Immunofluorescence

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: The FTLD/ALS-linked ANXA11 D40G mutant exhibits enhanced lysosomal disruption and seeding capacity. a Representative immunofluorescence images showing the accumulation of GAL3 (purple) puncta in SH-SY5Y cells treated with WT or D40G mutant ANXA11 PFFs-AF647 (red). Lysosomes were labeled with LAMP1 (green) and nuclei with DAPI (blue). b Quantification of relative GAL3 fluorescence intensity from ( a ). The results indicate that D40G fibrils induce more severe lysosomal membrane rupture compared to WT fibrils. c Representative confocal images displaying the recruitment of LC3 (purple) to LAMP1-positive lysosomes (green) containing internalized WT or D40G ANXA11 PFFs (red). d Quantification of relative LC3 fluorescence intensity from ( c ), showing enhanced autophagic response to D40G-induced damage. e Representative images of LysoTracker Red staining in cells treated with WT or D40G ANXA11 PFFs to assess lysosomal integrity. f Quantification of LysoTracker Red fluorescence intensity from ( e ). The D40G mutant caused a more pronounced reduction in lysosomal acidity. g Representative images of HEK293T cells expressing ANXA11-GFP treated with WT or D40G ANXA11 seeds, showing induced intracellular aggregation. h Quantification of the percentage of cells containing GFP-positive inclusions from ( g ). Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( b, d ) or one-way ANOVA with Tukey’s post hoc test ( f, h ). Exact P -values are indicated in the corresponding 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: Mutagenesis, Disruption, Immunofluorescence, Labeling, Fluorescence, Membrane, Staining, Expressing

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: Lysosomal damage by ANXA11 fibrils activates the p38/MK2/HSP27 signaling axis to promote lysophagy. a Western blot analysis of phosphorylated HSP27 (p-HSP27) and total HSP27 expression in SH-SY5Y cells treated with or without ANXA11 PFFs. GAPDH was used as a loading control. b , c Quantification of p-HSP27 levels normalized to total HSP27 ( b ) and GAPDH ( c ) from ( a ). d Representative immunofluorescence images showing the formation of HSP27 puncta (purple) and their colocalization with ANXA11 PFFs-AF647 (red). Phalloidin (green) stains F-actin and DAPI (blue) stains nuclei. Arrows indicate colocalization sites. e Western blot analysis of phosphorylated p38 MAPK (p-p38, T180/Y182) and total p38 MAPK levels. f , g Quantification of p-p38 levels normalized to total p38 ( f ) and GAPDH ( g ) from ( e ). h Western blot analysis of p-HSP27 and p-p38 MAPK levels in cells treated with ANXA11 PFFs in the presence or absence of the p38 MAPK inhibitor SB203580. i – l Quantification of relative protein levels from ( h ), showing that p38 inhibition effectively blocks the phosphorylation of HSP27. m Western blot analysis of phosphorylated MK2 (p-MK2, Thr334) and total MK2 levels. n , o Quantification of p-MK2 levels normalized to total MK2 ( n ) and GAPDH ( o ) from ( m ). p Western blot analysis of the p38/MK2/HSP27 pathway in cells treated with ANXA11 PFFs in the presence or absence of the MK2 inhibitor PF-3644022. q–v Quantification of phosphorylation levels from ( p ). Inhibition of MK2 significantly reduces the phosphorylation of HSP27 and upstream signaling components. w Representative immunofluorescence images of galectin-3 (GAL3, green) in cells treated with ANXA11 PFFs-AF647 (red) in the presence or absence of the p38 inhibitor PF-3644022. The increase in GAL3 puncta upon inhibition indicates exacerbated lysosomal damage due to impaired protective signaling. Data are presented as mean ± SEM. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t -test ( b, c, f, g, i, j, k, l, n, o, q, r, s, t, u, v ). Exact P -values are indicated in the corresponding 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: Western Blot, Expressing, Control, Immunofluorescence, Inhibition, Phospho-proteomics

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: Lysosomal rupture and autophagy deficiency synergistically promote the cytoplasmic seeding and neuronal propagation of ANXA11 aggregates. a Representative confocal microscopy images of SH-SY5Y cells stably expressing ANXA11-GFP. Cells were treated with ANXA11 PFFs (5 µg/mL) for 24 h, followed by exposure to the lysosomotropic agent LLOMe (1 mM) to induce lysosomal membrane permeabilization. Nuclei were counterstained with DAPI (blue). Note the marked increase in intracellular ANXA11-GFP inclusions in the combined treatment group. b Quantification of the percentage of cells containing ANXA11-GFP positive inclusions from the experiment in ( a ). c , d Dot blot analysis of cell lysates fractionated into Triton X-100 soluble and insoluble fractions. Membranes were probed with the conformation-dependent antibodies OC (targeting amyloid fibrils) ( c ) and A11 (targeting prefibrillar oligomers) ( d ). e , f Densitometric quantification of the relative signal intensity of OC ( e ) and A11 ( f ) in the insoluble fractions, normalized to controls. g Schematic illustration of the neuron-to-neuron transmission assay. Donor iPSC-derived neurons were loaded with AF647-labeled ANXA11 PFFs, washed to remove extracellular fibrils, and the conditioned medium/lysate was transferred to recipient neurons. Recipient cells were subsequently treated with LLOMe or BafA1 to assess the impact of lysosomal integrity and autophagic flux on aggregate propagation. h Representative confocal immunofluorescence images of recipient iPSC-derived neurons stained for the neuronal marker MAP2 (green). Internalized intracellular ANXA11 PFFs-AF647 are shown in red. The confocal imaging ensures that the red fluorescent signals represent genuine intracellular aggregates rather than extracellular membrane-bound particles. i Quantification of the percentage of recipient neurons containing ANXA11 PFFs-positive aggregates. The data demonstrate that both lysosomal rupture (LLOMe) and autophagy inhibition (BafA1) significantly enhance the accumulation of pathological seeds in recipient neurons. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test ( b, e, f, i ). 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: Confocal Microscopy, Stable Transfection, Expressing, Membrane, Dot Blot, Transmission Assay, Derivative Assay, Labeling, Immunofluorescence, Staining, Marker, Imaging, Inhibition

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

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: The FTLD/ALS-linked D40G mutation exacerbates lysosomal rupture and drives a hyperactive lysophagic response in human cerebral organoids. a Schematic timeline illustrating the generation of human iPSC-derived cerebral organoids and the experimental window for ANXA11 PFFs treatment (Day 40 +). b Multiplex immunofluorescence characterization of Day 40 organoids. The tissue exhibits a complex cytoarchitecture containing PAX6 + neural progenitors (red), TUJ1 + early neurons (purple), MAP2 + mature neurons (green), TBR1 + deep-layer cortical neurons (orange), and NeuN + neuronal nuclei (white/cyan). Nuclei are counterstained with DAPI (blue). c Representative confocal images showing the internalization of AF647-labeled ANXA11 PFFs (red) into the organoid parenchyma and their specific colocalization with LAMP1 + lysosomes (green). d Assessment of lysosomal membrane permeabilization (LMP). Organoids were treated with WT or D40G ANXA11 PFFs. Note the widespread accumulation of galectin-3 (GAL3, purple) puncta on lysosomes in the D40G group, indicating severe membrane rupture. e , f Fluorescence intensity profile analyses along the white lines in ( d ), demonstrating the recruitment of GAL3 to lysosomes containing WT ( e ) and D40G ( f ) fibrils. The D40G profile shows a higher degree of signal overlap. g Evaluation of the lysophagic response. Immunostaining revealed the recruitment of the autophagy marker LC3 (purple) to LAMP1 + lysosomes (green) containing ANXA11 aggregates (red). h , i Fluorescence intensity profile analyses along the white lines in ( g ) , confirming the colocalization of LC3 with ruptured lysosomes in WT ( h ) and D40G ( i ) treated organoids. j Western blot analysis of the p38/MK2/HSP27 signaling axis in organoid lysates following treatment with WT or D40G ANXA11 PFFs. GAPDH served as a loading control. k – p Densitometric quantification of the phosphorylation levels of HSP27 ( k , l ), p38 MAPK ( m , n ), and MK2 ( o , p ). The data indicate that the D40G mutant induces a significantly more robust activation of this protective stress response pathway compared to WT fibrils. Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test ( k , l , m , n , o , p ). 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: Mutagenesis, Derivative Assay, Multiplex Assay, Immunofluorescence, Labeling, Membrane, Fluorescence, Immunostaining, Marker, Western Blot, Control, Phospho-proteomics, Activation Assay