Journal: bioRxiv

Article Title: Toll-like receptor 9 contributes in microglial activation and lysosomal dysfunction to promote Alzheimer’s disease

doi: 10.64898/2026.01.16.699962

Figure Lengend Snippet: (A) Transcriptional analysis of lysosomal-related genes in WT, APP23 and A9KO microglia. n=3. Heatmap shows Z-Score (for genes). (B) Immunofluorescence images of purified microglia from 10 to 15 months old APP23 and A9KO mice stained for IBA-1 (green), cathepsin C (red), lysosomes (LAMP-1, blue) and nucleus (DAPI, yellow). Scale bar: 5 μm. (C) CTSC and LAMP-1 colocalization expressed as Mander’s coefficient. n=20 cells or more per condition for each experiment were analyzed. n=2. *p<0.05; two-tailed student’s t-test was performed. CTSC: cathepsin C. (D) Immunofluorescence images of purified microglia from 10 to 15 months old WT, APP23 and A9KO mice stimulated or not with 0.5 mM LLOMe for 2 h and stained for microglia (IBA-1, blue), LGALS1 (red) and lysosomes (LAMP-1, green). Scale bar: 5 μm. LLOMe: L-Leucyl-L-Leucine Methyl Ester, LGALS1: galectin 1. NS: non-stimulated. (E) Quantification of the number of LGALS1 + /LAMP-1 + puncta per cell (top) and of the percentage of cells with three or more puncta per field acquired (bottom). n= 20 cells or more per condition for each experiment was analyzed using IMARIS. n=3. NS: non-stimulated. ns: not significative, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA was performed with Tukey post-hoc test. 4 Immunofluorescence images of purified microglia from 10 to 15 months old APP23 and A9KO mice incubated with 4 μM of Aβ 1-42 fibrils coupled to HilexaFluor 488 (red) for 1, 2, and 6 hours and stained for microglia (IBA-1 green) and nucleus (DAPI, yellow). Scale bar: 5 μm. (F) Aβ 1-42 relative intracellular fluorescence intensity in APP23 and A9KO microglia. n= 20 cells or more per condition for each experiment was analyzed. n=3. *p<0.05, **p<0.01; one-way ANOVA was performed with Tukey post-hoc test.

Article Snippet: Cells were then incubated overnight at 4°C with a combination of the following primary antibodies diluted in blocking buffer (BB) (1X PBS, 2% BSA): anti-galectin 1 antibody (EPR3205) (Abcam, #ab108389), anti-mouse CD107a (LAMP-1) clone 1D4B (Invitrogen, #14-1071-85), anti-human CD107a (BD Pharmingen, #555789), anti-AIF/Iba1 Antibody (Biotechne #NB100-1028), anti-Iba1 antibody (for immunocytochemistry) (Fujifilm Wako chemicals #019-19741), anti-TFEB antibody (Proteintech, #13372-1-AP) and anti-cathepsin C/DPPI antibody (Biotechne, #AF1034).

Techniques: Immunofluorescence, Purification, Staining, Two Tailed Test, Incubation, Fluorescence

Journal: bioRxiv

Article Title: Toll-like receptor 9 contributes in microglial activation and lysosomal dysfunction to promote Alzheimer’s disease

doi: 10.64898/2026.01.16.699962

Figure Lengend Snippet: (A) Immunofluorescence images of human microglia cell line (HMC3) cultured with CpG-A (2 μg/ml) for 96 hours, pre-incubated or not for 1 h with 0.5 μM of AZP2006, treated or not with 1 mM LLOMe for 2 h and stained for LGALS1 (green), lysosomes (LAMP-1, red) and nucleus (DAPI, blue). Scale bar: 5 μm. Scale bar magnification: 2 μm. (B) Quantification of the number of LGALS1 + /LAMP-1 + puncta per cell (top) and of the percentage of cells with three or more puncta (bottom). n= 20 cells or more per condition for each experiment was analyzed. n=3. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA was performed with Tukey post-hoc test. (C) IL-6 production by HMC3 cells pre-incubated or not for 1 h with 0.5 μM or 1 μM of AZP2006, stimulated or not with 1 μg/ml of CpG-A or 100 ng/ml of LPS and measured by ELISA. n=3. ns: not significative, *p<0.05, ****p<0.0001, one-way ANOVA was performed with Tukey post-hoc test. NS: non-stimulated. (D) Immunofluorescence images of the pyramidal layer of the hippocampus (top) and entorhinal cortex region (bottom) in human brain samples from a healthy control (n=3, left) and an AD patient (n=3, right) stained for dsDNA (cyan) and nuclear envelope (Lamin B1, magenta). On the left is displayed whole tissue with regions analyzed highlighted. Scale bar whole region: 1 mm. Scale bar insets: 5 μm. PL: pyramidal layer, EC: entorhinal cortex. (E) Quantification of the percentage of dsDNA area in the nucleus (within Lamin B1, left) or total percentage of extranuclear dsDNA for the field acquired (right) in the PL and in the EC. Each dot represents a quantified image (n=3 for PL and n=4 for EC for each individual). ns: not significative, *p<0.05, ***p<0.001; two-tailed student’s t-test was performed.

Article Snippet: Cells were then incubated overnight at 4°C with a combination of the following primary antibodies diluted in blocking buffer (BB) (1X PBS, 2% BSA): anti-galectin 1 antibody (EPR3205) (Abcam, #ab108389), anti-mouse CD107a (LAMP-1) clone 1D4B (Invitrogen, #14-1071-85), anti-human CD107a (BD Pharmingen, #555789), anti-AIF/Iba1 Antibody (Biotechne #NB100-1028), anti-Iba1 antibody (for immunocytochemistry) (Fujifilm Wako chemicals #019-19741), anti-TFEB antibody (Proteintech, #13372-1-AP) and anti-cathepsin C/DPPI antibody (Biotechne, #AF1034).

Techniques: Immunofluorescence, Cell Culture, Incubation, Staining, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test