Ryk ICD binds to β-catenin.

<p>(A) β-catenin binds to Ryk. Constructs of Myc-tagged Ryk or uncleavable Ryk (Ryk:EGFR Rc) were transfected into 293T cells. Ryk proteins were immunoprecipitated with anti-Myc antibody, and beta-catenin associated with Ryk was determined by immunoblotting. Ryk ICD can be detected in the cells expressing wild-type Ryk. (B) The ICD of Ryk binds to β-catenin. Cells expressing Flag-tagged Ryk-ICD were used for anti-Flag immunoprecipitation. The ICD and associated β-catenin were determined by Western blot. NE, nuclear extract; FUIGW-FLAG, vector FUIGW plus a Flag sequence.</p

Reducing presenilin 1 expression counteracts the cytotoxicity of full-length Ryk overexpression in mutant htt striatal cells.

<p>Assays were performed using caspase 3/7 activity in cells subjected to serum deprivation. (A) PS1 and PS2 siRNA treatment enhances the viability of mutant htt striatal cells. Data are mean ± SD (<i>n</i> = 3), *<i>p</i><0.01 compared to scramble RNA. Significance was tested using one-way ANOVA, with correction for multiple testing by Tukey's Multiple Comparison Test. (B) Representative Western blots showing decreased levels of PS1/PS2. (C) Knockdown of PS1 reduces the cytotoxic effects of overexpressing full-length Ryk in mutant htt striatal cells, with no effect detected on the cytotoxic effects of overexpressing Ryk-ICD. Data are mean ± SD (<i>n</i> = 4), *<i>p</i><0.05 and **<i>p</i><0.01 compared to scramble RNA. Significance was tested using one-way ANOVA, with correction for multiple testing by Tukey's Multiple Comparison Test. EV, empty vector; ns, not significant. (D) Representative Western blots showing decreased levels of PS1 and expression of Myc-tagged Ryk species and Myc-tagged Ryk-ICD. *Nonspecific signal.</p

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

<p>(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 (<i>n</i> = 4). *<i>p</i><0.001 compared to scramble; **<i>p</i><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 <i>Renilla</i> luciferase reporter construct. Luciferase and <i>Renilla</i> luciferase activities were measured and the ratio (luciferase/<i>Renilla</i> 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. *<i>p</i><0.001 compared to FHRE-luc, **<i>p</i><0.001 compared to scramble RNA and ***<i>p</i><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).</p

Analysis of Ryk in polyQ nematodes and striatal cells derived from HdhQ111 mice.

<p>(A) Modulation of touch response of polyQ nematodes by LOF of <i>lin-18</i>/RYK. Shown are the results in <i>C. elegans</i> transgenics expressing expanded (128Q) or normal (19Q) exon-1–like htt transgenes in touch receptor neurons. The 128Q-mediated loss of touch response is ameliorated by LOF of <i>lin-18</i>/RYK. No effects are detected in 19Q nematodes. Data are mean ± SEM with more than 200 animals tested per genotype. *<i>p</i><0.001 versus 128Q controls. LOF of <i>lin-18</i>/RYK does not modify 128Q transgene expression levels as tested by qRT-PCR (right panel, data are mean ± SEM with <i>n</i> = 5). Significance was tested using one-way ANOVA, with correction for multiple testing by Tukey's Multiple Comparison Test. (B) Mutant htt (109Q/109Q) striatal cells have increased Ryk levels. Data for qRT-PCR are mean ± SEM (<i>n</i> = 7). **<i>p</i><0.01 versus normal htt (7Q/7Q) cells. Data for Western blotting are mean ± SD (<i>n</i> = 3). **<i>p</i><0.01 versus normal htt (7Q/7Q) cells. Significance was tested using paired <i>t</i> tests. (C) Reducing Ryk levels decreased the mortality of 109Q/109Q striatal cells induced by serum deprivation compared to scramble situation, with no effect detected in 7Q/7Q cells. Western blotting was used to test expression levels as 109Q/109Q cells do not display HTT aggregation. Mutant htt levels were unchanged by knockdown of Ryk. Data are mean ± SD (<i>n</i> = 4). *<i>p</i><0.01 versus scramble. The RNA sequences shown are indicated in the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001895#s4" target="_blank">Materials and Methods</a> section. Significance was tested using paired <i>t</i> tests.</p

The Ryk ICD is cytotoxic in <i>C. elegans</i> neurons and mouse striatal cells expressing expanded polyQs.

<p>(A) Expression of LIN-18 ICD cDNA (4 ng/µl) in touch receptor neurons using the <i>mec-3</i> promoter is sufficient to abolish the neuroprotective activity of <i>lin-18</i> LOF in 128Q nematodes with no effect detected in 19Q nematodes as tested in two independent extrachromosomal arrays (128Q: A1 is ID1333, A2 is ID1334; 19Q: A1 is ID1331, A2 is ID1332; see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001895#pbio.1001895.s018" target="_blank">Table S8</a>) per polyQ genotype. The expression of LIN-18 ICD cDNA was confirmed by RT-PCR for all of the arrays generated. Data are mean ± SEM (more than 200 animals tested). **<i>p</i><0.001 compared to 128Q animals, **<i>p</i><0.001 compared to 128Q;<i>lin-18</i> animals. ns, not significant. Significance was tested using one-way ANOVA, with correction for multiple testing by Tukey's Multiple Comparison Test. (B) Expression of LIN-18 ICD cDNA (4 ng/µl) in touch receptor neurons using the <i>mec-3</i> promoter is also sufficient to abolish the protective activity of <i>lin-18</i> LOF on axonal swelling in the PLM neurons of 128Q nematodes as tested in two independent extrachromosomal arrays (Lin-18 ICD: A1 is ID1333, A2 is ID1334; Lin-18: A1 is ID1325, A2 is ID1326). The expression of LIN-18 ICD cDNA was confirmed by RT-PCR for all of the arrays generated. Expression of empty vector (4 ng/µl) showed no effect as tested in two independent extrachromosomal arrays (A1 is ID1339, A2 is ID1340). Data are mean ± SEM (more than 200 animals tested). *<i>p</i><0.001 compared to 128Q animals, **<i>p</i><0.001 compared to 128Q;<i>lin-18</i> animals. ns, not significant. Significance was tested using one-way ANOVA, with correction for multiple testing by Tukey's Multiple Comparison Test. The lower panel shows a representative image of axonal swelling in the anterior process of posterior touch receptor neurons of 128Q nematodes co-expressing HTT1-57::CFP and YFP <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001895#pbio.1001895-Parker1" target="_blank">[7]</a>. Swelling (white arrows, YFP signals are pseudocolored in green) and HTT::CFP aggregation (yellow arrows, CFP signals are pseudocolored in red) are shown. Magnification is 100× and scale bar is 5 µM. (C) Overexpressing either V5-tagged β-catenin or Myc-tagged Ryk-ICD or both has no effect on the mortality induced by serum deprivation in normal htt striatal cells. Overexpressing β-catenin reduces the mortality induced by serum deprivation in mutant htt striatal cells, whereas overexpressing the Ryk-ICD aggravates cell mortality. Co-expressing Ryk-ICD and β-catenin resulted in cell mortality levels that are similar to those induced by empty vector overexpression. Data are mean ± SD (<i>n</i> = 4). *<i>p</i><0.01 and **<i>p</i><0.05 compared to empty vector. ns, not significant. Significance was tested using paired <i>t</i> tests. (D) Representative Western blot showing increased V5-tagged β-catenin and Myc-tagged Ryk-ICD levels after transfection of 7Q/7Q and 109Q/109Q striatal cells.</p

Neuroprotection by <i>lin-18</i>/Ryk LOF is a cell-autonomous process that requires <i>daf-16</i>/FoxO activity.

<p>(A) Expression of <i>lin-18</i> cDNA in touch receptor neurons using the <i>mec-3</i> promoter abolishes the neuroprotective activity of <i>lin-18</i> LOF in 128Q nematodes with no effect detected in 19Q nematodes as tested in two independent extrachromosomal arrays (A1, A2) per polyQ genotype. The expression of <i>lin-18</i> cDNA was confirmed by RT-PCR followed with enzymatic restriction for all of the arrays generated (unpublished data). Data are mean ± SEM (more than 200 animals tested). *<i>p</i><0.001 compared to 128Q transgenics; **<i>p</i><0.001 compared to 128Q;<i>lin-18</i> nematodes. (B) Neuron dysfunction is aggravated by <i>bar-1</i>/β-catenin or <i>daf-16</i>/FoxO LOF in 128Q nematodes, and protection from 128Q toxicity by <i>lin-18</i> LOF is reduced by LOF of <i>bar-1</i> and suppressed by LOF of <i>daf-16</i>. Data are mean ± SEM (more than 200 animals tested). **<i>p</i><0.001 compared to 128Q transgenics; ***<i>p</i><0.001 compared to 128Q;<i>lin-18</i> nematodes. (C) <i>lin-18</i>, <i>bar-1</i> and <i>daf-16</i> LOF alone or in combination do not change transgenic protein expression levels in 128Q nematodes. Data are mean ± SD (<i>n</i> = 3). Significance was tested using one-way ANOVA, with correction for multiple testing by Tukey's Multiple Comparison Test.</p

The ICD of Ryk is increased in the nucleus of mutant htt striatal cells.

<p>(A) Representative confocal microscopy images showing the pattern of Ryk-ICD immunoreactivity in normal htt (7Q/7Q) and mutant htt (109Q/109Q) striatal cells under normal culture conditions (no serum starvation) as detected using the rabbit polyclonal antibody anti-Ryk^ICD. (B) Quantification of Ryk-ICD immunoreactivity in mouse striatal cells. 7Q/7Q and 109Q/109Q cells were grown on the same slides. Comparisons were performed for cells with a nucleus size in the range of 150–250 pixels. Ryk-ICD immunoreactivity was increased in the nucleus of 109Q/109Q cells compared to 7Q/7Q cells. Data are mean ± SEM for the ratio Intensity/Area as detected in either nucleus or cytoplasm (<i>n</i> = 3 for a total of at least 100 cells analyzed), ***<i>p</i><0.0001 compared to normal htt cells; n.s., not significant. Significance was tested using one-way ANOVA, with correction for multiple testing by Tukey's Multiple Comparison Test. Ryk siRNA treatments were observed to reduce nuclear Ryk-ICD immunoreactivity in 109Q/109Q cells (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001895#pbio.1001895.s006" target="_blank">Figure S6</a>). (C) Representative confocal microscopy images showing the pattern of Ryk-ICD immunoreactivity in normal htt (7Q/7Q) and mutant htt (109Q/109Q) striatal cells under normal culture conditions (no serum starvation) as detected using the rabbit polyclonal antibody anti-Ryk^ICD and mouse Pol2 antibody 7C2. (D) Quantification of Ryk-ICD and Pol2 immunoreactivity in mouse striatal cells. 7Q/7Q and 109Q/109Q cells were grown on the same slides. Comparisons were performed for cells with a nucleus size in the range of 150–250 pixels. The ratio for Ryk-ICD/Pol2 immunoreactivity was increased in the nucleus of 109Q/109Q cells compared to 7Q/7Q cells. Data are mean ± SEM for the ratio Intensity Ryk-ICD/Intensity Pol2 as detected in the nucleus (<i>n</i> = 4 for a total of at least 100 cells analyzed), **<i>p</i><0.002 compared to normal htt cells. Significance was tested using Welch's <i>t</i> test.</p