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Lithium chloride attenuates cell death in oculopharyngeal muscular dystrophy by perturbing Wnt/β-catenin pathway.

Abu-Baker A, Laganiere J, Gaudet R, Rochefort D, Brais B, Neri C, Dion PA, Rouleau GA - Cell Death Dis (2013)

Bottom Line: Proteins that belong to the Wnt family are known for their role in both human development and adult tissue homeostasis.A hallmark of the Wnt signaling pathway is the increased expression of its central effector, beta-catenin (β-catenin) by inhibiting one of its upstream effector, glycogen synthase kinase (GSK)3β.Furthermore, this effect was also observed in primary cultures of mouse myoblasts expressing expPABPN1.

View Article: PubMed Central - PubMed

Affiliation: The Montreal Neurological Institute and Hospital, Department of Medicine, McGill University, Montréal, Québec H3A2B4, Canada.

ABSTRACT
Expansion of polyalanine tracts causes at least nine inherited human diseases. Among these, a polyalanine tract expansion in the poly (A)-binding protein nuclear 1 (expPABPN1) causes oculopharyngeal muscular dystrophy (OPMD). So far, there is no treatment for OPMD patients. Developing drugs that efficiently sustain muscle protection by activating key cell survival mechanisms is a major challenge in OPMD research. Proteins that belong to the Wnt family are known for their role in both human development and adult tissue homeostasis. A hallmark of the Wnt signaling pathway is the increased expression of its central effector, beta-catenin (β-catenin) by inhibiting one of its upstream effector, glycogen synthase kinase (GSK)3β. Here, we explored a pharmacological manipulation of a Wnt signaling pathway using lithium chloride (LiCl), a GSK-3β inhibitor, and observed the enhanced expression of β-catenin protein as well as the decreased cell death normally observed in an OPMD cell model of murine myoblast (C2C12) expressing the expanded and pathogenic form of the expPABPN1. Furthermore, this effect was also observed in primary cultures of mouse myoblasts expressing expPABPN1. A similar effect on β-catenin was also observed when lymphoblastoid cells lines (LCLs) derived from OPMD patients were treated with LiCl. We believe manipulation of the Wnt/β-catenin signaling pathway may represent an effective route for the development of future therapy for patients with OPMD.

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LiCl increases cell survival and differentiation of primary cultures of mouse myoblasts expressing GFP-expPABPN1 (13Ala and 17Ala). (a) Cell survival determined by live-stage microscopy. Confocal images showing the presence of an increased number of green cells (GFP-expPABPN1-17Ala) following a treatment (2.5 mM LiCl, bottom) support their increased survival by comparison with what is observed when the same cells were not treated with LiCl (top). Immnocytochemistry images were taken at day 6 post-transfection. GFP-expPABPN1-17Ala (green), desmin (red), and DAPI (blue). (b) LiCl increases the number of viable cells. Primary cultures mouse myoblasts expressing GFP-expPABPN1-13Ala, and GF-expPABPN1-17Ala, treated or not with 2.5 mM LiCl were counted at day 6 post-transfection using the live-stage microscope. Three different fields were chosen and cells were counted. Mean±S.E., *P<0.05 compared with any other groups (ANOVA analysis). The experiment was repeated three times. (c) LiCl enhances muscle differentiation of primary mouse myoblasts expressing GFP-expPABPN1-13Ala. Representative images showing the effect of LiCl on cell differentiation. Phase contrast images of myotubes treated with 2.5 mM LiCl (right) show an increased number of many elongated myotubes compared with non-treated cells (left) on day 6 after differentiation. (d) LiCl increases the differentiation index of primary mouse myoblast cells expressing GFP-expPABPN1 (13Ala and 17Ala). The histogram represents the fusion index calculated at the indicated times after DM addition for cells expressing GFP-expPABPN1-13Ala and GFP-expPABPN1-17Ala treated or not with 2.5 mM LiCl. At least three different fields containing 100 cells were counted. Data are the mean±S.E. of three independent experiments
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fig5: LiCl increases cell survival and differentiation of primary cultures of mouse myoblasts expressing GFP-expPABPN1 (13Ala and 17Ala). (a) Cell survival determined by live-stage microscopy. Confocal images showing the presence of an increased number of green cells (GFP-expPABPN1-17Ala) following a treatment (2.5 mM LiCl, bottom) support their increased survival by comparison with what is observed when the same cells were not treated with LiCl (top). Immnocytochemistry images were taken at day 6 post-transfection. GFP-expPABPN1-17Ala (green), desmin (red), and DAPI (blue). (b) LiCl increases the number of viable cells. Primary cultures mouse myoblasts expressing GFP-expPABPN1-13Ala, and GF-expPABPN1-17Ala, treated or not with 2.5 mM LiCl were counted at day 6 post-transfection using the live-stage microscope. Three different fields were chosen and cells were counted. Mean±S.E., *P<0.05 compared with any other groups (ANOVA analysis). The experiment was repeated three times. (c) LiCl enhances muscle differentiation of primary mouse myoblasts expressing GFP-expPABPN1-13Ala. Representative images showing the effect of LiCl on cell differentiation. Phase contrast images of myotubes treated with 2.5 mM LiCl (right) show an increased number of many elongated myotubes compared with non-treated cells (left) on day 6 after differentiation. (d) LiCl increases the differentiation index of primary mouse myoblast cells expressing GFP-expPABPN1 (13Ala and 17Ala). The histogram represents the fusion index calculated at the indicated times after DM addition for cells expressing GFP-expPABPN1-13Ala and GFP-expPABPN1-17Ala treated or not with 2.5 mM LiCl. At least three different fields containing 100 cells were counted. Data are the mean±S.E. of three independent experiments

Mentions: To further confirm the observed protective effects of LiCl on C2C12 cells, we assessed both proliferation and differentiation in primary cultures of mouse myoblasts expressing GFP-expPABPN1 (13Ala and 17Ala) after LiCl treatment. We followed cell viability using automated live-stage fluorescent microscopy to monitor the number of viable green fluorescent cells over 6 days post transfection. LiCl treatment showed a consistent and significant protective effect against expPABPN1-induced cell death, compared with non-treated counterparts. As shown in Figures 5a and b, LiCl increases the survival of cells expressing GFP-expPABPN1-13Ala and GFP-expPABPN1-17Ala (*P<0.001 when either transiently expressing LiCl treated cells are compared with their non-treated counterparts). In addition to enhancing the proliferation of viable cells expressing GFP-expPABPN1-13Ala and GFP-expPABPN1-17Ala, the drug also enhanced the differentiation of primary myoblast cultures into myotubes (Figures 5c and d). We also calculated the fusion index of the cultures. The results presented in Figure 5d demonstrate that LiCl increases the fusion index of primary myoblast cells expressing GFP-expPABPN1 (13Ala and 17Ala). In conclusion, the results we present from primary cultures of mouse myoblasts support the protective effects of LiCl obtained in the OPMD C2C12 cell model.


Lithium chloride attenuates cell death in oculopharyngeal muscular dystrophy by perturbing Wnt/β-catenin pathway.

Abu-Baker A, Laganiere J, Gaudet R, Rochefort D, Brais B, Neri C, Dion PA, Rouleau GA - Cell Death Dis (2013)

LiCl increases cell survival and differentiation of primary cultures of mouse myoblasts expressing GFP-expPABPN1 (13Ala and 17Ala). (a) Cell survival determined by live-stage microscopy. Confocal images showing the presence of an increased number of green cells (GFP-expPABPN1-17Ala) following a treatment (2.5 mM LiCl, bottom) support their increased survival by comparison with what is observed when the same cells were not treated with LiCl (top). Immnocytochemistry images were taken at day 6 post-transfection. GFP-expPABPN1-17Ala (green), desmin (red), and DAPI (blue). (b) LiCl increases the number of viable cells. Primary cultures mouse myoblasts expressing GFP-expPABPN1-13Ala, and GF-expPABPN1-17Ala, treated or not with 2.5 mM LiCl were counted at day 6 post-transfection using the live-stage microscope. Three different fields were chosen and cells were counted. Mean±S.E., *P<0.05 compared with any other groups (ANOVA analysis). The experiment was repeated three times. (c) LiCl enhances muscle differentiation of primary mouse myoblasts expressing GFP-expPABPN1-13Ala. Representative images showing the effect of LiCl on cell differentiation. Phase contrast images of myotubes treated with 2.5 mM LiCl (right) show an increased number of many elongated myotubes compared with non-treated cells (left) on day 6 after differentiation. (d) LiCl increases the differentiation index of primary mouse myoblast cells expressing GFP-expPABPN1 (13Ala and 17Ala). The histogram represents the fusion index calculated at the indicated times after DM addition for cells expressing GFP-expPABPN1-13Ala and GFP-expPABPN1-17Ala treated or not with 2.5 mM LiCl. At least three different fields containing 100 cells were counted. Data are the mean±S.E. of three independent experiments
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Related In: Results  -  Collection

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fig5: LiCl increases cell survival and differentiation of primary cultures of mouse myoblasts expressing GFP-expPABPN1 (13Ala and 17Ala). (a) Cell survival determined by live-stage microscopy. Confocal images showing the presence of an increased number of green cells (GFP-expPABPN1-17Ala) following a treatment (2.5 mM LiCl, bottom) support their increased survival by comparison with what is observed when the same cells were not treated with LiCl (top). Immnocytochemistry images were taken at day 6 post-transfection. GFP-expPABPN1-17Ala (green), desmin (red), and DAPI (blue). (b) LiCl increases the number of viable cells. Primary cultures mouse myoblasts expressing GFP-expPABPN1-13Ala, and GF-expPABPN1-17Ala, treated or not with 2.5 mM LiCl were counted at day 6 post-transfection using the live-stage microscope. Three different fields were chosen and cells were counted. Mean±S.E., *P<0.05 compared with any other groups (ANOVA analysis). The experiment was repeated three times. (c) LiCl enhances muscle differentiation of primary mouse myoblasts expressing GFP-expPABPN1-13Ala. Representative images showing the effect of LiCl on cell differentiation. Phase contrast images of myotubes treated with 2.5 mM LiCl (right) show an increased number of many elongated myotubes compared with non-treated cells (left) on day 6 after differentiation. (d) LiCl increases the differentiation index of primary mouse myoblast cells expressing GFP-expPABPN1 (13Ala and 17Ala). The histogram represents the fusion index calculated at the indicated times after DM addition for cells expressing GFP-expPABPN1-13Ala and GFP-expPABPN1-17Ala treated or not with 2.5 mM LiCl. At least three different fields containing 100 cells were counted. Data are the mean±S.E. of three independent experiments
Mentions: To further confirm the observed protective effects of LiCl on C2C12 cells, we assessed both proliferation and differentiation in primary cultures of mouse myoblasts expressing GFP-expPABPN1 (13Ala and 17Ala) after LiCl treatment. We followed cell viability using automated live-stage fluorescent microscopy to monitor the number of viable green fluorescent cells over 6 days post transfection. LiCl treatment showed a consistent and significant protective effect against expPABPN1-induced cell death, compared with non-treated counterparts. As shown in Figures 5a and b, LiCl increases the survival of cells expressing GFP-expPABPN1-13Ala and GFP-expPABPN1-17Ala (*P<0.001 when either transiently expressing LiCl treated cells are compared with their non-treated counterparts). In addition to enhancing the proliferation of viable cells expressing GFP-expPABPN1-13Ala and GFP-expPABPN1-17Ala, the drug also enhanced the differentiation of primary myoblast cultures into myotubes (Figures 5c and d). We also calculated the fusion index of the cultures. The results presented in Figure 5d demonstrate that LiCl increases the fusion index of primary myoblast cells expressing GFP-expPABPN1 (13Ala and 17Ala). In conclusion, the results we present from primary cultures of mouse myoblasts support the protective effects of LiCl obtained in the OPMD C2C12 cell model.

Bottom Line: Proteins that belong to the Wnt family are known for their role in both human development and adult tissue homeostasis.A hallmark of the Wnt signaling pathway is the increased expression of its central effector, beta-catenin (β-catenin) by inhibiting one of its upstream effector, glycogen synthase kinase (GSK)3β.Furthermore, this effect was also observed in primary cultures of mouse myoblasts expressing expPABPN1.

View Article: PubMed Central - PubMed

Affiliation: The Montreal Neurological Institute and Hospital, Department of Medicine, McGill University, Montréal, Québec H3A2B4, Canada.

ABSTRACT
Expansion of polyalanine tracts causes at least nine inherited human diseases. Among these, a polyalanine tract expansion in the poly (A)-binding protein nuclear 1 (expPABPN1) causes oculopharyngeal muscular dystrophy (OPMD). So far, there is no treatment for OPMD patients. Developing drugs that efficiently sustain muscle protection by activating key cell survival mechanisms is a major challenge in OPMD research. Proteins that belong to the Wnt family are known for their role in both human development and adult tissue homeostasis. A hallmark of the Wnt signaling pathway is the increased expression of its central effector, beta-catenin (β-catenin) by inhibiting one of its upstream effector, glycogen synthase kinase (GSK)3β. Here, we explored a pharmacological manipulation of a Wnt signaling pathway using lithium chloride (LiCl), a GSK-3β inhibitor, and observed the enhanced expression of β-catenin protein as well as the decreased cell death normally observed in an OPMD cell model of murine myoblast (C2C12) expressing the expanded and pathogenic form of the expPABPN1. Furthermore, this effect was also observed in primary cultures of mouse myoblasts expressing expPABPN1. A similar effect on β-catenin was also observed when lymphoblastoid cells lines (LCLs) derived from OPMD patients were treated with LiCl. We believe manipulation of the Wnt/β-catenin signaling pathway may represent an effective route for the development of future therapy for patients with OPMD.

Show MeSH
Related in: MedlinePlus