Additional file 1 of Phosphodiesterase 5 inhibitor mirodenafil ameliorates Alzheimer-like pathology and symptoms by multimodal actions
Creators: name: Kang, Byung Woo
name: Kim, Fred
name: Cho, Joon-Yong
name: Kim, SangYun
name: Rhee, Jinseol
name: Choung, Jai Jun
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Cancer
subject: 111714 Mental Health
subject: FOS: Health sciences

description: Additional file 1: Tables S1-S3. Figure S1. Semiquantitative measurements for western blot analysis. (A) Tau phosphorylated at S199/202 and S396 in SH-SY5Y cells after co-treatment with okadaic acid and mirodenafil (n=4). (B) Phosphorylated GSK-3β and phosphorylated Akt in SH-SY5Y cells after co-treatment with Aβ42, mirodenafil, and the PI3K inhibitor LY-294002 (n=4). (C) APP and BACE1 expression after co-treatment with Aβ42 and mirodenafil in HT-22 cells (n ≥ 5). (D) Phosphorylated AMPK and Aβ after co-treatment with Aβ42 and mirodenafil in SH-SY5Y cells (n=4). (E) The autophagy markers p62 and LC3B after co-treatment with Aβ42, mirodenafil, and autophagy inhibitor 3-MA (n=4) (F) GR after co-treatment with Aβ42 and mirodenafil in HT-22 cells. Data are presented as the mean ± SEM; *P &lt; 0.05, **P &lt; 0.01. All statistical comparisons were performed relative to Aβ42-treated cells. Statistical significance was assessed by one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. Figure S2. A. Mirodenafil impedes nuclear localization of GR homodimers after treatment with dexamethasone. HEK293 cells were transfected with Myc-tagged GR and GFP-tagged GR constructs. Following incubation for 24 h, Myc-tagged GR and GFP-tagged GR were detected by immunofluorescence confocal microscopy after co-treatment with dexamethasone and PDE5 inhibitors for 6 h. B. Quantification of nuclear localization of Myc-tagged GR and GFP-tagged GR after co-treatment with dexamethasone and PDE5 inhibitors. Immunofluorescence confocal images were obtained using LSM 800 confocal laser scanning microscopy (Zeiss). Mean fluorescence intensity in the nuclei and whole cells was measured using Zen Blue Edition software (Zeiss). Nuclear GR intensity was divided by total GR intensity to indicate relative distribution of Myc-GR and GFP-GR in nuclei and cytoplasm of HT-22 cells. (n&gt;12). Data are presented as the mean ± SEM; *P &lt; 0.05. All statistical comparisons were performed relative to Aβ42-treated cells. Statistical significance was assessed by one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. Figure S3. Real-time qRT-PCR for Wnts (Wnt1, Wnt3a, Wnt5a, and Wnt7a) after treatment with Aβ42 and mirodenafil for 6 h in HT-22 cells (n ≥ 5). Data are presented as the mean ± SEM; *P &lt; 0.05, **P &lt; 0.01. All statistical comparisons were performed relative to Aβ42-treated cells. Statistical significance was assessed by one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. Figure S4. Mirodenafil ameliorates AD pathology through multiple mechanisms of action. Representative schematic illustration of the possible mechanisms through which mirodenafil improves cognition and ameliorates AD pathology. Mirodenafil enhances neuronal survival through the activation of the cGMP/PKG/CREB signaling pathway, which upregulates the expression of neurotrophic factors, promotes the maintenance of the mitochondrial membrane potential, and prevents apoptotic cell death. The downregulation of GSK-3β activity contributes to the reduction of phosphorylated tau, whereas the amyloid-targeting properties of mirodenafil decrease the Aβ burden by modulating the production, aggregation, and clearance of Aβ. Additionally, mirodenafil activates Wnt/β-catenin signaling by downregulating the expression of the Wnt antagonist Dkk1 or repressing GR transcriptional activity.