Oncogenic mutations of the Wnt (wingless)/β-catenin pathway are frequently observed in major cancer types. Thus far, however, no therapeutic agent targeting Wnt/β-catenin signaling is available for clinical use. Here we demonstrate that axitinib, a clinically approved drug, strikingly blocks Wnt/β-catenin signaling in cancer cells, zebrafish, and Apc min/+ mice. Notably, axitinib dramatically induces Wnt asymmetry and nonrandom DNA segregation in cancer cells by promoting nuclear β-catenin degradation independent of the GSK3β (glycogen synthase kinase3β)/APC (adenomatous polyposis coli) complex. Using a DARTS (drug affinity-responsive target stability) assay coupled to 2D-DIGE (2D difference in gel electrophoresis) and mass spectrometry, we have identified the E3 ubiquitin ligase SHPRH (SNF2, histone-linker, PHD and RING finger domain-containing helicase) as the direct target of axitinib in blocking Wnt/β-catenin signaling. Treatment with axitinib stabilizes SHPRH and thereby increases the ubiquitination and degradation of β-catenin. Our findings suggest a previously unreported mechanism of nuclear β-catenin regulation and indicate that axitinib, a clinically approved drug, would provide therapeutic benefits for cancer patients with aberrant nuclear β-catenin activation.axitinib | β-catenin | asymmetric cell division | SHPRH

DJ-1, a Parkinson's disease-associated protein, is strongly up-regulated in reactive astrocytes in Parkinson's disease. This is proposed to represent a neuronal protective response, although the mechanism has not yet been identified. We have generated a transgenic zebrafish line with increased astroglial DJ-1 expression driven by regulatory elements from the zebrafish GFAP gene. Larvae from this transgenic line are protected from oxidative stress-induced injuries as caused by MPP+, a mitochondrial complex I inhibitor shown to induce dopaminergic cells death. In a global label-free proteomics analysis of wild type and transgenic larvae exposed to MPP+, 3418 proteins were identified, in which 366 proteins were differentially regulated. In particular, we identified enzymes belonging to primary metabolism to be among proteins affected by MPP+ in wild type animals, but not affected in the transgenic line. Moreover, by performing protein profiling on isolated astrocytes we showed that an increase in astrocytic DJ-1 expression up-regulated a large group of proteins associated with redox regulation, inflammation and mitochondrial respiration. The majority of these proteins have also been shown to be regulated by Nrf2. These findings provide a mechanistic insight into the protective role of astroglial up-regulation of DJ-1 and show that our transgenic zebrafish line with astrocytic DJ-1 over-expression can serve as a useful animal model to understand astrocyte-regulated neuroprotection associated with oxidative stress-related neurodegenerative disease.

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Potassium channel tetramerization domain containing proteins (KCTDs), which share a conserved BTB (Brica-brac, Tramtrack, Broad complex) domain at their N-terminus, are known to be involved in both developmental and neural processes. However, the developmental expression patterns and functional roles of most vertebrate KCTDs remain unknown. Using enhancer-trapping technology, we have identified a transgenic zebrafish line (ub49) where the vector insertion is in close proximity to kctd15a, and where transgenic marker (eGFP) expression closely reflects endogenous kctd15a expression. Both ub49 and kctd15a show strong maternal expression that suggests a functional role during epiboly and gastrulation. At later developmental stages, expression of eGFP in ub49 also shares the same spatiotemporal features as kctd15a in several neural tissues, including cranial placode precursors, retina, and different areas of the developing brain. In the retina, we observed eGFP labeling of the inner nuclear layer (INL), including a heterogenous population of amacrine cells, and both laminae of the inner plexiform layer (IPL). This expression pattern suggests that Kctd15a proteins have several contextdependent functional roles in both developmental and neural processes. The enhancer trap line, which is the first transgenic reporter of Kctd gene expression in vertebrates, also provides a novel tool to study kctd15a function in vivo.

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