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The Ryk ICD represses the transcriptional activity of FOXO3a, a protein that protects from mutant HTT.

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posted on 24.06.2014, 03:20 by Cendrine Tourette, Francesca Farina, Rafael P. Vazquez-Manrique, Anne-Marie Orfila, Jessica Voisin, Sonia Hernandez, Nicolas Offner, J. Alex Parker, Sophie Menet, Jinho Kim, Jungmok Lyu, Si Ho Choi, Kerry Cormier, Christina K. Edgerly, Olivia L. Bordiuk, Karen Smith, Anne Louise, Michael Halford, Steven Stacker, Jean-Philippe Vert, Robert J. Ferrante, Wange Lu, Christian Neri

(A) Foxo3a siRNA treatment enhances the mortality of mutant htt striatal cells subjected to serum deprivation, whereas FOXO3a overexpression (O/E) has the opposite effect. Data are mean ± SD (n = 4). *p<0.001 compared to scramble; **p<0.001 compared to empty vector control. (B) Representative Western blots showing decreased (si-Foxo3a) or increased (FOXO3a O/E) FOXO3a levels and no change in HTT protein levels. (C) FOXO transcriptional activity was measured in normal htt mouse striatal cells. Cells were cultured in normal conditions and co-transfected with a construct encoding FOXO3a together with the reporter FHRE-luciferase, which contains three canonical FOXO binding sites, and an internal Renilla luciferase reporter construct. Luciferase and Renilla luciferase activities were measured and the ratio (luciferase/Renilla luciferase)10,000 calculated. Data are mean ± SD of four independent experiments performed in triplicate. Treatment with β-catenin siRNA, full-length Ryk cDNA, and Ryk-ICD cDNA reduces luciferase activity to similar levels, whereas treatment with uncleavable Ryk showed no effect. *p<0.001 compared to FHRE-luc, **p<0.001 compared to scramble RNA and ***p<0.001 compared to FOXO3a O/E. Significance was tested using one-way ANOVA, with correction for multiple testing by Tukey's Multiple Comparison Test. (D) Representative Western blots showing increased levels of FOXO3a and decreased levels of β-catenin, and expression of Myc-tagged Ryk, Myc-tagged Ryk-ICD, and Myc-tagged γ-secretase–uncleavable Ryk (all proteins with a Myc tag at the C-terminal end). The Myc-tagged Ryk and γ-secretase–uncleavable Ryk proteins were detected as two fragments, one corresponding to the full-length Ryk precursor (Ryk) and one corresponding to a Ryk CTF (Ryk CTF) resulting from proteolytic cleavage in the extracellular domain near the transmembrane domain. The full-length Ryk precursor is less abundant for wild-type Ryk expression compared to mutant Ryk expression (see Results for the discussion of Ryk expression profiles).

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