Testing Fmr1KO Phenotypes in Response to GSK3 Inhibitors: SB216763 versus AFC03127

Glycogen synthase kinase 3 (GSK3) is a proline-directed serine-threonine kinase that is associated with several neurological disorders, including Alzheimer’s disease and fragile X syndrome (FXS). We tested the efficacy of a novel GSK3 inhibitor AFC03127, which was developed by Angelini Pharma, in comparison to the metabotropic glutamate receptor 5 inhibitor 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and the GSK3 inhibitor SB216763 in in vivo and in vitro assays in Fmr1KO mice, a mouse model useful for the study of FXS. The in vivo assay tested susceptibility to audiogenic-induced seizures (AGS) whereas the in vitro assays assessed biomarker expression and dendritic spine length and density in cultured primary neurons as a function of drug dose. MPEP and SB216763 attenuated AGS in Fmr1KO mice, whereas AFC03127 did not. MPEP and AFC03127 significantly reduced dendritic expression of amyloid-beta protein precursor (APP). All drugs rescued spine length and the ratio of mature dendritic spines. Spine density was not statistically different between vehicle and GSK3 inhibitor-treated cells. The drugs were tested over a wide concentration range in the in vitro assays to determine dose responses. A bell-shaped dose response decrease in APP expression was observed in response to AFC03127, which was more effective than SB216763. These findings confirm previous studies demonstrating differential effects of various GSK3 inhibitors on AGS propensity in Fmr1KO mice and confirm APP as a downstream biomarker that is responsive to GSK3 activity.

Role of Nrf2 Signaling in Development of Hepatocyte-Like Cells

Generation of hepatocytes from human adipose-derived mesenchymal stem cells (hADSCs) could be a promising alternative source of human hepatocytes. However, mechanisms to differentiate hepatocytes from hADSCs are not fully elucidated. In this study, we investigated the role of nuclear factor erythroid-2 related factor 2 (Nrf2) in differentiation of hepatocyte-like cells (HLCs). We used our established three-step differentiation protocol to develop HLCs from hADSCs. Significant nuclear translocation of Nrf2 occurred from day 11 (Step 2) until the end of HLC differentiation. There were no significant differences in Nrf2 translocation rates among the four experimental groups (activin-A, GSK3 inhibitor, Nrf2 siRNA, and control) at day 6 (end of Step 1). Nuclear translocation of Nrf2 in the GSK3 inhibitor-treated group was obviously higher than the other groups at day 11 (Step 2). Moreover, nuclear translocation of Nrf2 in the GSK3 inhibitor-treated group was notably higher than the other groups during Step 3. CYP3A4 activity (Luciferin-IPA assay) of the GSK3 inhibitor-treated group was significantly higher than the other three groups. Nrf2 was activated during differentiation of HLCs, and inhibition of Nrf2 delayed maturation and impaired the function of HLCs. Thus, Nrf2 might be a notable target for developing highly functional human HLCs.

Activation of Wnt/β-catenin signaling to increase BMI1 by Licl attenuates the toxicity of cisplatin in the HEI-OC1 auditory cells

Cisplatin is a very effective anti-tumor drug; nonetheless, it can induce cochlear hair cell apoptosis and ototoxicity in large doses. WNT/β-catenin signaling is also closely related to aging, embryonic development, and apoptosis. We establish a cisplatin-induced HEI-OC1 auditory cells model. WNT/β-catenin was activated by GSK3 inhibitor Licl to detect the expression level of each component of the WNT pathway and BMI1. The expression of BMI1 in the hair cell line model of HEI-OC1 cells induced by cisplatin was significantly reduced, and cell apoptosis was significantly reduced by increasing the expression level of cell line BMI through activating WNT/β-catenin signaling. Activation of WNT/β-catenin signaling to increase BMI1 expression can reduce the apoptosis of cochlear hair cells induced by cisplatin. BMI1 also has a protective effect on the ototoxicity of cisplatin.

A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells

The canonical Wnt pathway transcriptional co-activator β-catenin regulates self-renewal and differentiation of mammalian nephron progenitor cells (NPCs). We modulated β-catenin levels in NPC cultures using the GSK3 inhibitor CHIR9902 (CHIR) to examine opposing developmental actions of β-catenin. Low CHIR-mediated maintenance and expansion of NPCs is independent of direct engagement of TCF/LEF/β-catenin transcriptional complexes at low CHIR-dependent cell-cycle targets. In contrast, in high CHIR, TCF7/LEF1/β-catenin complexes replaced TCF7L1/TCF7L2 binding on enhancers of differentiation-promoting target genes. Chromosome confirmation studies showed pre-established promoter-enhancer connections to these target genes in NPCs. High CHIR-associated de novo looping was observed in positive transcriptional feedback regulation to the canonical Wnt pathway. Thus, β-catenin's direct transcriptional role is restricted to the induction of NPCs where rising β-catenin levels switch inhibitory TCF7L1/TCF7L2 complexes to activating LEF1/TCF7 complexes at primed gene targets poised for rapid initiation of a nephrogenic program.

Cohesin mutations are synthetic lethal with stimulation of WNT signaling

Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21, and STAG2 and screened for synthetic lethality with 3009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top ‘hits’ was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of β-catenin in cohesin-mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunits stag2b and rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin-mutant cancers.

Lithium inhibits tryptophan catabolism via the inflammation-induced kynurenine pathway in human microglia

SummaryActivation of the kynurenine pathway may lead to depletion of the serotonin precursor tryptophan, which has been implicated in the neurobiology of depression. This study describes a mechanism whereby lithium inhibits inflammatory tryptophan breakdown. Upon activation, immortalized human microglia showed a robust increase in indoleamine-2,3-dioxygenase (IDO1) mRNA transcription, IDO1 protein expression, and activity. Further, chromatin immunoprecipitation verified enriched binding of both STAT1 and STAT3 to the IDO1 promoter. Lithium counteracted these effects, increasing inhibitory GSK3βS9 phosphorylation and reducing STAT1S727 and STAT3Y705 phosphorylation levels in activated cells. Experiments in primary human microglia and human induced pluripotent stem cell (hiPSC)-derived microglia corroborated lithium’s effects. Moreover, IDO activity was reduced by GSK3 inhibitor SB-216763 and STAT inhibitor nifuroxazide via downregulation of P-STAT1S727 and P-STAT3Y705. Our study demonstrates that lithium inhibits the inflammatory kynurenine pathway in the microglia compartment of the human brain.