Predes D et al. (2022), The Flavonol Quercitrin Hinders GSK3 Activity a...

XB-ART-59430

Int J Mol Sci 2022 Oct 11;2320:. doi: 10.3390/ijms232012078.

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The Flavonol Quercitrin Hinders GSK3 Activity and Potentiates the Wnt/β-Catenin Signaling Pathway.

Predes D , Maia LA , Matias I , Araujo HPM , Soares C , Barros-Aragão FGQ , Oliveira LFS , Reis RR , Amado NG , Simas ABC , Mendes FA , Gomes FCA , Figueiredo CP , Abreu JG .

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The Wnt/β-catenin signaling pathway dictates cell proliferation and differentiation during embryonic development and tissue homeostasis. Its deregulation is associated with many pathological conditions, including neurodegenerative disease, frequently downregulated. The lack of efficient treatment for these diseases, including Alzheimer's disease (AD), makes Wnt signaling an attractive target for therapies. Interestingly, novel Wnt signaling activating compounds are less frequently described than inhibitors, turning the quest for novel positive modulators even more appealing. In that sense, natural compounds are an outstanding source of potential drug leads. Here, we combine different experimental models, cell-based approaches, neuronal culture assays, and rodent behavior tests with Xenopus laevis phenotypic analysis to characterize quercitrin, a natural compound, as a novel Wnt signaling potentiator. We find that quercitrin potentiates the signaling in a concentration-dependent manner and increases the occurrence of the Xenopus secondary axis phenotype mediated by Xwnt8 injection. Using a GSK3 biosensor, we describe that quercitrin impairs GSK3 activity and increases phosphorylated GSK3β S9 levels. Treatment with XAV939, an inhibitor downstream of GSK3, impairs the quercitrin-mediated effect. Next, we show that quercitrin potentiates the Wnt3a-synaptogenic effect in hippocampal neurons in culture, which is blocked by XAV939. Quercitrin treatment also rescues the hippocampal synapse loss induced by intracerebroventricular injection of amyloid-β oligomers (AβO) in mice. Finally, quercitrin rescues AβO-mediated memory impairment, which is prevented by XAV939. Thus, our study uncovers a novel function for quercitrin as a Wnt/β-catenin signaling potentiator, describes its mechanism of action, and opens new avenues for AD treatments.

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Species referenced: Xenopus laevis
Genes referenced: gsk3b wnt3a wnt8a
GO keywords: Wnt signaling pathway

???displayArticle.disOnts??? Alzheimer's disease [+]
Phenotypes: Xla Wt + wnt8a (Fig. 2 abcgh) [+]

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	Figure 1. Quercitrin potentiates the Wnt/β-catenin signaling pathway. (a) Reporter gene assay in RKO B/R cells treated with L-cell CM, Wnt3a CM or recombinant human Wnt3a. The cells were treated with the vehicle dimethyl sulfoxide (DMSO) or quercitrin for 24 h (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05, p < 0.01, * p < 0.001). Error bars denote mean ± SD. (b) TOPFLASH reporter gene assay in transfected HEK293T cells treated with L-cell CM or Wnt3a CM. The cells were treated with the vehicle DMSO or quercitrin for 24 h (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05, * p < 0.001). Error bars denote mean ± SD. (c) Reporter gene assay of RKO B/R cells treated for 6 h (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.001). Error bars denote mean ± SD. (d) Reporter gene assay of RKO B/R cells treated for 12 h (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.001). Error bars denote mean ± SD. (e) Immunoblot assay of RKO cells lysate after 4 h treatment. RKO cells were treated with L-cell CM (lane 1) or Wnt3a CM (lanes 2–4). (f) Densitometric analysis of (e) showing that quercitrin increases β-catenin protein levels (n = 4, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.001). Error bars denote mean ± SD. (g) Immunoblotting of RKO cells treated for 24 h. RKO cells were treated with L-cell CM (lane 1) or Wnt3a CM (lanes 2–4). (h) Densitometric analysis of (g) pLRP6/LRP6 protein levels revealing that quercitrin increases pLRP6 S1490 and decreases pβ-catenin S33/S37 protein levels (n = 4, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05, * p < 0.001). Error bars denote mean ± SD. (i) Densitometric analysis of (g) pβ-catenin/β-catenin protein levels revealing that quercitrin decreases pβ-catenin S33/S37 protein levels (n = 4, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.001). Error bars denote mean ± SD.
	Figure 2. Quercitrin potentiates the Wnt/β-catenin signaling pathway in Xenopus laevis. (a–f) Representative embryos injected with increasing concentrations of xWnt8 mRNA into the ventral blastomere, co-injected with DMSO or 1.6 pmol quercitrin. The injection induces the formation of an incomplete (arrow head) or a complete secondary axis (arrow). (g) Phenotype quantification of (a–f). The coinjection with quercitrin increases the complete secondary axis phenotype occurrence. The number of embryos in each condition is shown above the graph bars. (h) S01234 reporter gene assay. Quercitrin coinjection potentiates the S01234 reporter gene activation induced by Xwnt8 mRNA (n = 2, performed in triplicate, Unpaired t-test comparing the DMSO and quercitrin injected groups. ** p < 0.01). Error bars represent mean ± SD. See Figure S2 for the injection scheme.
	Figure 3. Quercitrin facilitates GSK3 S9 phosphorylation and hinders GSK3 activity. (a–c) TOPFLASH reporter gene assay of HEK293T cells transfected with (a) β-catenin S33A, (b) β-catenin WT, or (c) LRP6 (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, ns = not significant). (d) Reporter gene assay of HEK293T cells treated with 0.3, 0.5, 0.7, and 1.0 μM of BIO, a GSK3 inhibitor. (n = 3, performed in triplicate, Two-way ANOVA followed by Bonferroni’s multiple comparisons test, * p < 0.05). Error bars denote mean ± SD. (e) GSK3 biosensor scheme. The biosensor contains the GFP protein at the N-terminus and a FLAG-tag at the C-terminus. The phosphorylation of the biosensor by GSK3 triggers its degradation. (f) Immunoblotting assay of HEK293T transfected with the GSK3 biosensor after 6 h treatment. Cells were treated with L-cell CM (lane 1) or Wnt3a CM (lanes 2–5). (g) Densitometric analysis of β-catenin/α-tubulin protein levels (n = 4, Unpaired t-test, two-tailed, * p < 0.001). Error bars denote mean ± SD. (h) Densitometric analysis of GSK3 biosensor/α-tubulin protein levels showing that quercitrin treatment hinders GSK3 activity (n = 4, Unpaired t-test, two-tailed, * p < 0.001). Error bars denote mean ± SD. (i) Immunoblotting of RKO cells lysate after 24 h treatment. RKO cells were treated with L-cell CM (lane 1) or Wnt3a CM (lanes 2–4). (j) Densitometry analysis of pGSK3βS9/GSK3β protein levels demonstrating that quercitrin increases the pGSK3βS9/GSK3β ratio (n = 4, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05). Error bars denote mean ± SD. (k) Reporter gene assay of RKO B/R cells treated with L-cell CM or Wnt3a CM, quercitrin, and 5 μM XAV939 for 24 h. The treatment reveals that XAV939 impairs the quercitrin potentiation effect (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test considering Wnt3a CM + DMSO condition as control, * p < 0.05, *** p < 0.001, or considering Wnt3a CM + DMSO + XAV939 as the control condition, ‡‡‡ p < 0.001. An Unpaired t-test of DMSO and DMSO + XAV939 conditions was performed to assess the XAV939 effect on the reporter gene activity, †† p < 0.01). Error bars denote mean ± SD. (l) Scheme of quercitrin mechanism of action. Quercitrin hinders GSK3 activity, thus impairing β-catenin degradation and increasing its stabilization. The red arrow indicates β-catenin degradation. The black arrow denotes β-catenin translocation to the nucleus and Wnt target genes transcription.
	Figure 4. Quercitrin potentiates the canonical Wnt synaptogenic effect and rescues AβO-induced memory impairment in mice. (a–d) Synaptophysin and PSD-95 immunocytochemistry of E16 13DIV cultured hippocampal neurons treated with XAV939 and/or quercitrin for 24 h. Scale bars: 20 μm. (e) Synaptophysin/PSD-95 colocalized puncta quantification of (a–d), showing that quercitrin potentiates the recombinant Wnt-mediated synaptogenesis in vitro, which is impaired by XAV939 (n = 3, performed in duplicate, one-way ANOVA statistical analysis followed by Newman–Keuls multiple comparisons test. ** p < 0.01, comparing hWnt3a + DMSO, and hWnt3a + quercitrin conditions. †† p < 0.01, comparing hWnt3a + quercitrin and hWnt3a + quercitrin + XAV939 conditions). Scale bars denote 20 μm. (f) Immunoblotting analysis of injected mice hippocampus lysate staining for β-catenin, synaptophysin, and PSD-95. β-actin was used as a cytoskeleton loading control. GAPDH was used as a metabolic loading control. Each lane represents a different animal. The animals were injected with vehicle (lanes 1–4), vehicle + AβO (lanes 5–8), or quercitrin + AβO (lanes 9–12). (g–l) Densitometric quantification of (f) showing that quercitrin rescues the decreased β-catenin, synaptophysin, and PSD-95 protein levels caused by AβO ICV injection (n = 4, Unpaired t-test, two-tailed, considering the AβO + vehicle as the control condition, * p < 0.05, p < 0.01, * p < 0.001). Error bars denote mean ± SEM. Each white circle denotes one animal. (m) Workflow scheme of the i.c.v. injections, object recognition training, and testing. Red square represents a familiar object, and blue object represents a novel object. (n) Novel object recognition (NOR) test, 24 h after i.c.v. injection. Quercitrin reverses the memory impairment caused by AβO injection, which is impaired by XAV939 (n = 10, One sample t-test, theoretical mean value of 50%, * p < 0.05, ** p < 0.01). F: familiar object, N: novel object. Error bar represents mean ± SEM. See also Figure S5 for NOR training and open field arena test data.
	Figure S1. Quercitrin potentiates the signaling after Wnt ligand stimulation. TOPFLASH assay of HEK293T transfected with increasing amounts (1, 10, 30 ng/well) of hWnt3a plasmid. HEK293T cells were treated overnight with quercitrin or quercetin (a Wnt signaling inhibitor). (n=3, performed in triplicate, two-way ANOVA analysis considering the DMSO as the control condition followed by a Dunnett multiple comparison test. p<0.05, *p<0.01). Error bars represent mean ± SD
	Figure S2. Injection scheme of 4-cell Xenopus laevis embryos. (a) For the Axis Duplication Assay, the ventral blastomeres were equatorially injected. (b) For the S01234 reporter gene assay, we injected one ventral and one dorsal blastomeres.
	Figure S3. Quercitrin does not potentiate the Wnt signaling in the SW480 tumoral cell line. (a) RKO B/R cell line was treated with L-cell CM, Wnt3a CM, the vehicle DMSO, or increasing concentrations of quercitrin (n=3, performed in triplicate, one-way ANOVA followed by Dunnett's multiple comparisons test, p < 0.01, p < 0.001). Error bars denote mean ± SD. (b) SW480 B/R reporter gene cell lines were treated with DMSO or increasing concentrations of quercitrin (n=3, performed in triplicate, one-way ANOVA followed by Dunnett's multiple comparisons test, p < 0.05, **p < 0.01). Error bars denote mean ± SD.
	Figure S4. Quercitrin per se does not induce synaptogenesis in vitro. (a) Protocol illustration of in vitro hippocampus neuronal culture and treatment. (b-c) Synaptophysin and PSD-95 immunostaining of hippocampal neuronal culture. (d) Colocalized puncta quantification shows that 10 μM quercitrin per se does not induce synaptogenesis (n=3, performed in duplicate, Welch t-test statistical analysis). Error bars denote mean ± SEM. Scale bars denote 10 μm
	Figure S5. ICV injection does not alter the experimental exploration time of injected mice. (a) Total exploration time (s) of the left and right objects in the novel object recognition (NOR) training. (b) Exploration time (% of total time) in both objects under training and testing conditions. (c) Distance travelled (m) in the Open Field analysis. (d) Time (s) in center during the Open Field analysis.

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	Figure 1. Quercitrin potentiates the Wnt/β-catenin signaling pathway. (a) Reporter gene assay in RKO B/R cells treated with L-cell CM, Wnt3a CM or recombinant human Wnt3a. The cells were treated with the vehicle dimethyl sulfoxide (DMSO) or quercitrin for 24 h (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05, p < 0.01, * p < 0.001). Error bars denote mean ± SD. (b) TOPFLASH reporter gene assay in transfected HEK293T cells treated with L-cell CM or Wnt3a CM. The cells were treated with the vehicle DMSO or quercitrin for 24 h (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05, * p < 0.001). Error bars denote mean ± SD. (c) Reporter gene assay of RKO B/R cells treated for 6 h (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.001). Error bars denote mean ± SD. (d) Reporter gene assay of RKO B/R cells treated for 12 h (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.001). Error bars denote mean ± SD. (e) Immunoblot assay of RKO cells lysate after 4 h treatment. RKO cells were treated with L-cell CM (lane 1) or Wnt3a CM (lanes 2–4). (f) Densitometric analysis of (e) showing that quercitrin increases β-catenin protein levels (n = 4, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.001). Error bars denote mean ± SD. (g) Immunoblotting of RKO cells treated for 24 h. RKO cells were treated with L-cell CM (lane 1) or Wnt3a CM (lanes 2–4). (h) Densitometric analysis of (g) pLRP6/LRP6 protein levels revealing that quercitrin increases pLRP6 S1490 and decreases pβ-catenin S33/S37 protein levels (n = 4, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05, * p < 0.001). Error bars denote mean ± SD. (i) Densitometric analysis of (g) pβ-catenin/β-catenin protein levels revealing that quercitrin decreases pβ-catenin S33/S37 protein levels (n = 4, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.001). Error bars denote mean ± SD.
	Figure 2. Quercitrin potentiates the Wnt/β-catenin signaling pathway in Xenopus laevis. (a–f) Representative embryos injected with increasing concentrations of xWnt8 mRNA into the ventral blastomere, co-injected with DMSO or 1.6 pmol quercitrin. The injection induces the formation of an incomplete (arrow head) or a complete secondary axis (arrow). (g) Phenotype quantification of (a–f). The coinjection with quercitrin increases the complete secondary axis phenotype occurrence. The number of embryos in each condition is shown above the graph bars. (h) S01234 reporter gene assay. Quercitrin coinjection potentiates the S01234 reporter gene activation induced by Xwnt8 mRNA (n = 2, performed in triplicate, Unpaired t-test comparing the DMSO and quercitrin injected groups. ** p < 0.01). Error bars represent mean ± SD. See Figure S2 for the injection scheme.
	Figure 3. Quercitrin facilitates GSK3 S9 phosphorylation and hinders GSK3 activity. (a–c) TOPFLASH reporter gene assay of HEK293T cells transfected with (a) β-catenin S33A, (b) β-catenin WT, or (c) LRP6 (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test, ns = not significant). (d) Reporter gene assay of HEK293T cells treated with 0.3, 0.5, 0.7, and 1.0 μM of BIO, a GSK3 inhibitor. (n = 3, performed in triplicate, Two-way ANOVA followed by Bonferroni’s multiple comparisons test, * p < 0.05). Error bars denote mean ± SD. (e) GSK3 biosensor scheme. The biosensor contains the GFP protein at the N-terminus and a FLAG-tag at the C-terminus. The phosphorylation of the biosensor by GSK3 triggers its degradation. (f) Immunoblotting assay of HEK293T transfected with the GSK3 biosensor after 6 h treatment. Cells were treated with L-cell CM (lane 1) or Wnt3a CM (lanes 2–5). (g) Densitometric analysis of β-catenin/α-tubulin protein levels (n = 4, Unpaired t-test, two-tailed, * p < 0.001). Error bars denote mean ± SD. (h) Densitometric analysis of GSK3 biosensor/α-tubulin protein levels showing that quercitrin treatment hinders GSK3 activity (n = 4, Unpaired t-test, two-tailed, * p < 0.001). Error bars denote mean ± SD. (i) Immunoblotting of RKO cells lysate after 24 h treatment. RKO cells were treated with L-cell CM (lane 1) or Wnt3a CM (lanes 2–4). (j) Densitometry analysis of pGSK3βS9/GSK3β protein levels demonstrating that quercitrin increases the pGSK3βS9/GSK3β ratio (n = 4, one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05). Error bars denote mean ± SD. (k) Reporter gene assay of RKO B/R cells treated with L-cell CM or Wnt3a CM, quercitrin, and 5 μM XAV939 for 24 h. The treatment reveals that XAV939 impairs the quercitrin potentiation effect (n = 3, performed in triplicate, one-way ANOVA followed by Dunnett’s multiple comparisons test considering Wnt3a CM + DMSO condition as control, * p < 0.05, *** p < 0.001, or considering Wnt3a CM + DMSO + XAV939 as the control condition, ‡‡‡ p < 0.001. An Unpaired t-test of DMSO and DMSO + XAV939 conditions was performed to assess the XAV939 effect on the reporter gene activity, †† p < 0.01). Error bars denote mean ± SD. (l) Scheme of quercitrin mechanism of action. Quercitrin hinders GSK3 activity, thus impairing β-catenin degradation and increasing its stabilization. The red arrow indicates β-catenin degradation. The black arrow denotes β-catenin translocation to the nucleus and Wnt target genes transcription.
	Figure 4. Quercitrin potentiates the canonical Wnt synaptogenic effect and rescues AβO-induced memory impairment in mice. (a–d) Synaptophysin and PSD-95 immunocytochemistry of E16 13DIV cultured hippocampal neurons treated with XAV939 and/or quercitrin for 24 h. Scale bars: 20 μm. (e) Synaptophysin/PSD-95 colocalized puncta quantification of (a–d), showing that quercitrin potentiates the recombinant Wnt-mediated synaptogenesis in vitro, which is impaired by XAV939 (n = 3, performed in duplicate, one-way ANOVA statistical analysis followed by Newman–Keuls multiple comparisons test. ** p < 0.01, comparing hWnt3a + DMSO, and hWnt3a + quercitrin conditions. †† p < 0.01, comparing hWnt3a + quercitrin and hWnt3a + quercitrin + XAV939 conditions). Scale bars denote 20 μm. (f) Immunoblotting analysis of injected mice hippocampus lysate staining for β-catenin, synaptophysin, and PSD-95. β-actin was used as a cytoskeleton loading control. GAPDH was used as a metabolic loading control. Each lane represents a different animal. The animals were injected with vehicle (lanes 1–4), vehicle + AβO (lanes 5–8), or quercitrin + AβO (lanes 9–12). (g–l) Densitometric quantification of (f) showing that quercitrin rescues the decreased β-catenin, synaptophysin, and PSD-95 protein levels caused by AβO ICV injection (n = 4, Unpaired t-test, two-tailed, considering the AβO + vehicle as the control condition, * p < 0.05, p < 0.01, * p < 0.001). Error bars denote mean ± SEM. Each white circle denotes one animal. (m) Workflow scheme of the i.c.v. injections, object recognition training, and testing. Red square represents a familiar object, and blue object represents a novel object. (n) Novel object recognition (NOR) test, 24 h after i.c.v. injection. Quercitrin reverses the memory impairment caused by AβO injection, which is impaired by XAV939 (n = 10, One sample t-test, theoretical mean value of 50%, * p < 0.05, ** p < 0.01). F: familiar object, N: novel object. Error bar represents mean ± SEM. See also Figure S5 for NOR training and open field arena test data.
	Figure S1. Quercitrin potentiates the signaling after Wnt ligand stimulation. TOPFLASH assay of HEK293T transfected with increasing amounts (1, 10, 30 ng/well) of hWnt3a plasmid. HEK293T cells were treated overnight with quercitrin or quercetin (a Wnt signaling inhibitor). (n=3, performed in triplicate, two-way ANOVA analysis considering the DMSO as the control condition followed by a Dunnett multiple comparison test. p<0.05, *p<0.01). Error bars represent mean ± SD
	Figure S2. Injection scheme of 4-cell Xenopus laevis embryos. (a) For the Axis Duplication Assay, the ventral blastomeres were equatorially injected. (b) For the S01234 reporter gene assay, we injected one ventral and one dorsal blastomeres.
	Figure S3. Quercitrin does not potentiate the Wnt signaling in the SW480 tumoral cell line. (a) RKO B/R cell line was treated with L-cell CM, Wnt3a CM, the vehicle DMSO, or increasing concentrations of quercitrin (n=3, performed in triplicate, one-way ANOVA followed by Dunnett's multiple comparisons test, p < 0.01, p < 0.001). Error bars denote mean ± SD. (b) SW480 B/R reporter gene cell lines were treated with DMSO or increasing concentrations of quercitrin (n=3, performed in triplicate, one-way ANOVA followed by Dunnett's multiple comparisons test, p < 0.05, **p < 0.01). Error bars denote mean ± SD.
	Figure S4. Quercitrin per se does not induce synaptogenesis in vitro. (a) Protocol illustration of in vitro hippocampus neuronal culture and treatment. (b-c) Synaptophysin and PSD-95 immunostaining of hippocampal neuronal culture. (d) Colocalized puncta quantification shows that 10 μM quercitrin per se does not induce synaptogenesis (n=3, performed in duplicate, Welch t-test statistical analysis). Error bars denote mean ± SEM. Scale bars denote 10 μm
	Figure S5. ICV injection does not alter the experimental exploration time of injected mice. (a) Total exploration time (s) of the left and right objects in the novel object recognition (NOR) training. (b) Exploration time (% of total time) in both objects under training and testing conditions. (c) Distance travelled (m) in the Open Field analysis. (d) Time (s) in center during the Open Field analysis.