Journal: International Journal of Molecular Sciences

Article Title: Fluoxetine Repurposing Mitigates Alzheimer’s Disease Pathology via the GSK3β–CREB–ADAM10 Axis

doi: 10.3390/ijms27062676

Figure Lengend Snippet: Effect of fluoxetine on behavioral performance in wild-type and 5xFAD transgenic mice (AD Tg). ( A ) Open-field test assessing general locomotor activity and anxiety-like behavior based on total distance traveled and time spent in the center zone. ( B ) Y-maze test evaluating spontaneous alteration behavior as an index of spatial working. ( C ) Novel object recognition (NOR) test assessing recognition memory. Mice were exposed to two identical objects (blue square) during the training phase, and then to one familiar and one novel object (red triangle) during the testing phase conducted 24 h later. Data are represented as mean ± SEM. Sample sizes were as follows: WT + vehicle ( n = 6), WT + fluoxetine ( n = 5), AD Tg + vehicle ( n = 6), and AD Tg + fluoxetine ( n = 6). Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, **** p < 0.0001 vs. WT + vehicle; # p < 0.05, ## p < 0.01, #### p < 0.0001 vs. AD Tg + vehicle; ns, not significant.

Article Snippet: After 24 h, 10 μM fluoxetine was added to the culture dishes for 12 h. 1-Naphthyl PP1 (1-NA-PP1, HY-13941), a CAMKII inhibitor, and GSK3 inhibitor IX (HY-10580), a GSK3β inhibitor, were purchased from MedChem Express (Princeton, NJ, USA).

Techniques: Transgenic Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Fluoxetine Repurposing Mitigates Alzheimer’s Disease Pathology via the GSK3β–CREB–ADAM10 Axis

doi: 10.3390/ijms27062676

Figure Lengend Snippet: Fluoxetine is associated with reduced amyloid pathology in 5xFAD transgenic mice. ( A ) Representative images of Aβ immunostaining (6E10 antibody) in the hippocampus and whole brain hemisphere of wild-type and 5xFAD mice treated with fluoxetine (Fluo) or vehicle (Veh). ( B ) Quantification of Aβ-positive area as a percentage of total analyzed region in the hippocampus and whole brain hemisphere. Data are presented as mean ± SEM. Sample sizes for immunohistochemistry were as follows: WT + vehicle ( n = 5), WT + fluoxetine ( n = 5), AD Tg + vehicle ( n = 5), and AD Tg + fluoxetine ( n = 5). Individual data points represent biological replicates. ( C ) Representative Western blot analysis using 6E10 antibody to detect full-length APP (~110 kDa), C99 (~18 kDa), and Aβ (~4 kDa). ( D ) Quantitation of APP, C99 and Aβ protein levels normalized to β-actin. WT groups showed minimal background-level 6E10 immunoreactivity; therefore, statistical analysis was restricted to comparisons between AD Tg + vehicle and AD Tg + fluoxetine groups using an unpaired two-tailed Student’s t -test for ( B , D ). # p < 0.05, #### p < 0.0001 vs. AD Tg vehicle. ( E ) Representative Western blot analysis of ADAM10 and ADAM17 protein expression in hippocampal lysates. ( F ) Quantification of ADAM10 and ADAM17 levels normalized to β-actin. Data are presented as mean ± SEM. Sample sizes for Western blot analysis were as follows: WT + vehicle ( n = 4), WT + fluoxetine ( n = 4), AD Tg + vehicle ( n = 5), and AD Tg + fluoxetine ( n = 5). Individual data points represent biological replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. ### p < 0.001, vs. AD Tg + vehicle; ns, not significant.

Article Snippet: After 24 h, 10 μM fluoxetine was added to the culture dishes for 12 h. 1-Naphthyl PP1 (1-NA-PP1, HY-13941), a CAMKII inhibitor, and GSK3 inhibitor IX (HY-10580), a GSK3β inhibitor, were purchased from MedChem Express (Princeton, NJ, USA).

Techniques: Transgenic Assay, Immunostaining, Immunohistochemistry, Western Blot, Quantitation Assay, Two Tailed Test, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Fluoxetine Repurposing Mitigates Alzheimer’s Disease Pathology via the GSK3β–CREB–ADAM10 Axis

doi: 10.3390/ijms27062676

Figure Lengend Snippet: Fluoxetine attenuates neuroinflammatory markers in 5xFAD mice. ( A ) Representative immunofluorescence staining image showing Aβ for (6E10, red) and microglia (Iba-1, green) staining in the hippocampus. Nuclei were counterstained with DAPI (blue). Scale bar = 200 μm. ( B ) Representative Western blot analysis of Iba-1 expression in hippocampal lysates. ( C ) Quantification of Iba-1 protein levels normalized to β-actin. Data are presented as mean ± SEM. Sample sizes for Western blot analysis were as follows: WT + vehicle (n = 4), WT + fluoxetine ( n = 4), AD Tg + vehicle ( n = 6), and AD Tg + fluoxetine ( n = 6). ( D ) mRNA expression of proinflammatory cytokines IL-6 and TNF-α in cortex tissue analyzed by quantitative RT-PCR. Data are presented as mean ± SEM. Sample sizes for qPCR were as follows: WT + vehicle ( n = 6), WT + fluoxetine ( n = 4), AD Tg + vehicle ( n = 6), and AD Tg + fluoxetine ( n = 6). Statistical analysis was performed using one-way ANOVA by Tukey’s post hoc test. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. WT + vehicle; # p < 0.05 vs. AD Tg + vehicle.

Article Snippet: After 24 h, 10 μM fluoxetine was added to the culture dishes for 12 h. 1-Naphthyl PP1 (1-NA-PP1, HY-13941), a CAMKII inhibitor, and GSK3 inhibitor IX (HY-10580), a GSK3β inhibitor, were purchased from MedChem Express (Princeton, NJ, USA).

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Fluoxetine Repurposing Mitigates Alzheimer’s Disease Pathology via the GSK3β–CREB–ADAM10 Axis

doi: 10.3390/ijms27062676

Figure Lengend Snippet: Fluoxetine is associated with enhanced phosphorylation of synaptic signaling molecules in the hippocampus. ( A ) Representative Western blot analysis of phosphorylated and total forms of CaMKII (Thr286), GSK3β (Ser9), and CREB (Ser133) in hippocampal lysates. ( B ) Quantification of phosphorylated proteins normalized to their corresponding total protein levels. ( C ) Representative immunohistochemistry images of p-CREB-positive cells the hippocampal region. Black squares represent the areas selected for enlarged image. Yellow arrows indicate immune-positive nuclei. Scale bar: 300 μm (upper panels), 25 μm (lower panels). ( D ) Quantification of p-CREB-positive cells per defined hippocampal field. Data are presented as mean ± SEM. Sample sizes were as follows: WT + vehicle ( n = 4), WT + fluoxetine ( n = 4), AD Tg + vehicle ( n = 6), and AD Tg + fluoxetine ( n = 6). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test ( B , D ). * p < 0.05 vs. WT + vehicle; # p < 0.05, ## p < 0.01 vs. AD Tg + vehicle; ns, not significant.

Article Snippet: After 24 h, 10 μM fluoxetine was added to the culture dishes for 12 h. 1-Naphthyl PP1 (1-NA-PP1, HY-13941), a CAMKII inhibitor, and GSK3 inhibitor IX (HY-10580), a GSK3β inhibitor, were purchased from MedChem Express (Princeton, NJ, USA).

Techniques: Phospho-proteomics, Western Blot, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Fluoxetine Repurposing Mitigates Alzheimer’s Disease Pathology via the GSK3β–CREB–ADAM10 Axis

doi: 10.3390/ijms27062676

Figure Lengend Snippet: Fluoxetine modulates CaMKII-GSK3β-CREB signaling and ADAM10 expression in vitro. ( A ) Schematic diagram of the experimental design. SH-SY5Y cells were seeded and pretreated with fluoxetine (10 μM) for 12 h, followed by treatment with the CaMKII inhibitor 1-NA-PP1 (10 or 20 μM) or the GSK3β inhibitor IX (5 or 10 μM) for 1 h prior to cell harvest. ( B ) Representative Western blot analysis for phosphorylated and total forms of CaMKII (Thr286), GSK3β (Ser9), and CREB (Ser133). ( C ) Quantification of relative phosphorylation levels normalized to the corresponding total protein. ( D ) Representative Western blot analysis of signaling and APP-processing-related proteins following fluoxetine and/or GSK3β inhibitor treatment. ( E ) Quantification of protein expression levels normalized to β-actin (for protein) or to the corresponding total protein (for phosphorylated forms), as indicated. Data are presented as mean ± SEM ( n = 3 independent experiments). Individual data points represent independent biological replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. fluoxetine alone; ns, not significant. Created in BioRender. Son, Y. (2026) https://BioRender.com/s91n646 (accessed on 31 January 2026).

Article Snippet: After 24 h, 10 μM fluoxetine was added to the culture dishes for 12 h. 1-Naphthyl PP1 (1-NA-PP1, HY-13941), a CAMKII inhibitor, and GSK3 inhibitor IX (HY-10580), a GSK3β inhibitor, were purchased from MedChem Express (Princeton, NJ, USA).

Techniques: Expressing, In Vitro, Western Blot, Phospho-proteomics, Control

Journal: International Journal of Molecular Sciences

Article Title: Fluoxetine Repurposing Mitigates Alzheimer’s Disease Pathology via the GSK3β–CREB–ADAM10 Axis

doi: 10.3390/ijms27062676

Figure Lengend Snippet: Proposed model of the fluoxetine-mediated signaling pathway in AD. Chronic fluoxetine administration is associated with enhanced non-amyloidogenic processing of amyloid precursor protein (APP) through a GSK3β–CREB–ADAM10 signaling axis. Fluoxetine increases GSK3β phosphorylation at Ser9, which is associated with CREB activation and upregulation of the α-secretase ADAM10. This schematic summarizes the signaling interactions proposed based on the present findings. The model illustrates a potential relationship among GSK3β inhibition, CREB activation, ADAM10 upregulation, and reduced amyloid deposition; however, causal relationships among these events remain to be further established. Fluoxetine treatment was associated with reduced microglial activation and decreased pro-inflammatory cytokines. While these inflammatory changes may be influenced by altered amyloid burden, fluoxetine is also known to exert serotonergic and anti-inflammatory effects through mechanisms independent of APP processing. Therefore, the anti-inflammatory effects observed here may reflect both amyloid-dependent mechanisms and parallel signaling pathways. Created in BioRender. Son, Y. (2026) https://BioRender.com/s91n646 (accessed on 31 January 2026).

Article Snippet: After 24 h, 10 μM fluoxetine was added to the culture dishes for 12 h. 1-Naphthyl PP1 (1-NA-PP1, HY-13941), a CAMKII inhibitor, and GSK3 inhibitor IX (HY-10580), a GSK3β inhibitor, were purchased from MedChem Express (Princeton, NJ, USA).

Techniques: Phospho-proteomics, Activation Assay, Inhibition, Protein-Protein interactions

Journal: International Journal of Molecular Sciences

Article Title: Fluoxetine Repurposing Mitigates Alzheimer’s Disease Pathology via the GSK3β–CREB–ADAM10 Axis

doi: 10.3390/ijms27062676

Figure Lengend Snippet: Experimental design. Five-month-old wild-type (WT) and 5xFAD Transgenic mice were administered intraperitoneal injections of 10 mg/kg fluoxetine (Fluo) or vehicle (Veh), five times a week for 8 weeks. At 7 months of age, the animals underwent a series of behavioral tests, followed by sacrifice and brain collection for molecular and histopathologic analyses. Created in BioRender. Son, Y. (2026) https://BioRender.com/s91n646 (accessed on 31 January 2026).

Article Snippet: After 24 h, 10 μM fluoxetine was added to the culture dishes for 12 h. 1-Naphthyl PP1 (1-NA-PP1, HY-13941), a CAMKII inhibitor, and GSK3 inhibitor IX (HY-10580), a GSK3β inhibitor, were purchased from MedChem Express (Princeton, NJ, USA).

Techniques: Transgenic Assay