Figure_5.tif (984.39 kB)

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

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posted on 2014-06-24, 03:20 authored 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) Expression of LIN-18 ICD cDNA (4 ng/µl) in touch receptor neurons using the mec-3 promoter is sufficient to abolish the neuroprotective activity of lin-18 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 Table S8) 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). **p<0.001 compared to 128Q animals, **p<0.001 compared to 128Q;lin-18 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 mec-3 promoter is also sufficient to abolish the protective activity of lin-18 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). *p<0.001 compared to 128Q animals, **p<0.001 compared to 128Q;lin-18 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 [7]. 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 (n = 4). *p<0.01 and **p<0.05 compared to empty vector. ns, not significant. Significance was tested using paired t 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.