The Wnt Receptor Ryk Reduces Neuronal and Cell Survival Capacity by Repressing FOXO Activity During the Early Phases of Mutant Huntingtin Pathogenicity

The Wnt receptor Ryk is an evolutionary-conserved protein important during neuronal differentiation through several mechanisms, including γ-secretase cleavage and nuclear translocation of its intracellular domain (Ryk-ICD). Although the Wnt pathway may be neuroprotective, the role of Ryk in neurodegenerative disease remains unknown. We found that Ryk is up-regulated in neurons expressing mutant huntingtin (HTT) in several models of Huntington's disease (HD). Further investigation in Caenorhabditis elegans and mouse striatal cell models of HD provided a model in which the early-stage increase of Ryk promotes neuronal dysfunction by repressing the neuroprotective activity of the longevity-promoting factor FOXO through a noncanonical mechanism that implicates the Ryk-ICD fragment and its binding to the FOXO co-factor β-catenin. The Ryk-ICD fragment suppressed neuroprotection by lin-18/Ryk loss-of-function in expanded-polyQ nematodes, repressed FOXO transcriptional activity, and abolished β-catenin protection of mutant htt striatal cells against cell death vulnerability. Additionally, Ryk-ICD was increased in the nucleus of mutant htt cells, and reducing γ-secretase PS1 levels compensated for the cytotoxicity of full-length Ryk in these cells. These findings reveal that the Ryk-ICD pathway may impair FOXO protective activity in mutant polyglutamine neurons, suggesting that neurons are unable to efficiently maintain function and resist disease from the earliest phases of the pathogenic process in HD. © 2014 Tourette et al

The Sirtuin 2 Inhibitor AK-7 Is Neuroprotective in Huntington’s Disease Mouse Models

Inhibition of sirtuin 2 (SIRT2) deacetylase mediates protective effects in cell and invertebrate models of Parkinson’s disease and Huntington’s disease (HD). Here we report the in vivo efficacy of a brain-permeable SIRT2 inhibitor in two genetic mouse models of HD. Compound treatment resulted in improved motor function, extended survival, and reduced brain atrophy and is associated with marked reduction of aggregated mutant huntingtin, a hallmark of HD pathology. Our results provide preclinical validation of SIRT2 inhibition as a potential therapeutic target for HD and support the further development of SIRT2 inhibitors for testing in humans

Reduced creatine kinase as a central and peripheral biomarker in Huntington's disease

AbstractA major goal of current clinical research in Huntington's disease (HD) has been to identify preclinical and manifest disease biomarkers, as these may improve both diagnosis and the power for therapeutic trials. Although the underlying biochemical alterations and the mechanisms of neuronal degeneration remain unknown, energy metabolism defects in HD have been chronicled for many years. We report that the brain isoenzyme of creatine kinase (CK-BB), an enzyme important in buffering energy stores, was significantly reduced in presymptomatic and manifest disease in brain and blood buffy coat specimens in HD mice and HD patients. Brain CK-BB levels were significantly reduced in R6/2 mice by ∼18% to ∼68% from 21 to 91days of age, while blood CK-BB levels were decreased by ∼14% to ∼44% during the same disease duration. Similar findings in CK-BB levels were observed in the 140 CAG mice from 4 to 12months of age, but not at the earliest time point, 2months of age. Consistent with the HD mice, there was a grade-dependent loss of brain CK-BB that worsened with disease severity in HD patients from ∼28% to ∼63%, as compared to non-diseased control patients. In addition, CK-BB blood buffy coat levels were significantly reduced in both premanifest and symptomatic HD patients by ∼23% and ∼39%, respectively. The correlation of CK-BB as a disease biomarker in both CNS and peripheral tissues from HD mice and HD patients may provide a powerful means to assess disease progression and to predict the potential magnitude of therapeutic benefit in this disorder

Defining the role of syndecan-4 in mechanotransduction using surface-modification approaches

The ability of cells to respond to external mechanical stimulation is a complex and robust process involving a diversity of molecular interactions. Although mechanotransduction has been heavily studied, many questions remain regarding the link between physical stimulation and biochemical response. Of significant interest has been the contribution of the transmembrane proteins involved, and integrins in particular, because of their connectivity to both the extracellular matrix and the cytoskeleton. Here, we demonstrate the existence of a mechanically based initiation molecule, syndecan-4. We first demonstrate the ability of syndecan-4 molecules to support cell attachment and spreading without the direct extracellular binding of integrins. We also examine the distribution of focal adhesion-associated proteins through controlling surface interactions of beads with molecular specificity in binding to living cells. Furthermore, after adhering cells to elastomeric membranes via syndecan-4-specific attachments we mechanically strained the cells via our mechanical stimulation and polymer surface chemical modification approach. We found ERK phosphorylation similar to that shown for mechanotransductive response for integrin-based cell attachments through our elastomeric membrane-based approach and optical magnetic twisting cytometry for syndecan-4. Finally, through the use of cytoskeletal disruption agents, this mechanical signaling was shown to be actin cytoskeleton dependent. We believe that these results will be of interest to a wide range of fields, including mechanotransduction, syndecan biology, and cell–material interactions

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

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

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 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

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