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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 leads to a significant increase of the β-catenin level in the OPMD C2C12 cell model. (a and b) LiCl leads to a significant increase of β-catenin in the OPMD C2C12 cell model while leaving the GFP level not affected. A western blot showing the effect of LiCl treatment on the β-catenin level at 48 h post transfection. C2C12 myoblast cells were transiently transfected with different PABPN1constructs as well as GFP, and non-transfected cells, and treated or not with two different doses of LiCl as indicated. Actin antibody was used to confirm equal loading. (c and d) LiCl treatment results in re-distribution of β-catenin from a cytoplasmic to a nuclear compartment. Confocal images of immunocytochemistry showing the subcellular distribution of β-catenin. A clear accumulation of β-catenin in the nucleus was observed in the C2C12-transfected cells with GFP-wtPABPN1-10Ala (c) and GFP-expPABPN1-17Ala (d) after LiCl treatment. GFP-PABPN1 constructs (green), β-catenin (red)
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fig6: LiCl leads to a significant increase of the β-catenin level in the OPMD C2C12 cell model. (a and b) LiCl leads to a significant increase of β-catenin in the OPMD C2C12 cell model while leaving the GFP level not affected. A western blot showing the effect of LiCl treatment on the β-catenin level at 48 h post transfection. C2C12 myoblast cells were transiently transfected with different PABPN1constructs as well as GFP, and non-transfected cells, and treated or not with two different doses of LiCl as indicated. Actin antibody was used to confirm equal loading. (c and d) LiCl treatment results in re-distribution of β-catenin from a cytoplasmic to a nuclear compartment. Confocal images of immunocytochemistry showing the subcellular distribution of β-catenin. A clear accumulation of β-catenin in the nucleus was observed in the C2C12-transfected cells with GFP-wtPABPN1-10Ala (c) and GFP-expPABPN1-17Ala (d) after LiCl treatment. GFP-PABPN1 constructs (green), β-catenin (red)

Mentions: The cellular localization of β-catenin is regulated by its phosphorylation by GSK-3β, which lies upstream. In the absence of the Wnt signal, β-catenin is phosphorylated by GSK-3β and targeted for degradation by the ubiquitin–proteasome system. Upon Wnt signaling, it cannot be phosphorylated by GSK-3β resulting in its translocation to the nucleus.43 Li+ ions were in fact shown to inhibit GSK-3β activity, resulting in the elevation of β-catenin protein levels.44, 45, 46 Given the protection effect of LiCl against cell death associated with GFP-expPABPN1 (13Ala and 17Ala) (Figures 2a and b), we investigated whether LiCl protected against expPABPN1-associated cell death by upregulating the level of β-catenin protein. We first analyzed by western blot the transfected cells, which had been treated with 2.5 mM LiCl, and increased expression could be seen in this instance (Figure 6a) at 48 h post transfection; while leaving the level of GFP protein unaffected in cells transfected with GFP-expPABPN1 (13Ala and 17Ala) (Figure 6b). This suggests that the cell survival protection is mediated through induction of the β-catenin protein and/or other proteins downstream from β-catenin rather than affecting the level of PABPN1 protein. The effect of increasing the LiCl concentration on enhancing β-catenin level was also studied in non-transfected C2C12 cells (Supplementary Figure S1). Using immunocytochemistry, we then examined the subcellular localization of β-catenin in cells and compared it before and after a LiCl treatment. In the absence of LiCl treatment, expression of β-catenin appears cytoplasmic (Figures 6c and d, left panels). But following treatment with LiCl, a clear nuclear accumulation of β-catenin can be observed in cells expressing GFP-wtPABPN1-10Ala and GFP-expPABPN1-17Ala, respectively (Figures 6c and d, right panels). Figures 6c and d show that treatment with LiCl leads to a significant nuclear translocation of β-catenin from the cytoplasm to the nuclear compartment in both cells transfected with GFP-wtPABPN1-10Ala and GFP-expPABPN1-17Ala, respectively. Thus, we conclude that LiCl could activate a pro-survival pathway in the C2C12 OPMD model through activation of the β-catenin protein.


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 leads to a significant increase of the β-catenin level in the OPMD C2C12 cell model. (a and b) LiCl leads to a significant increase of β-catenin in the OPMD C2C12 cell model while leaving the GFP level not affected. A western blot showing the effect of LiCl treatment on the β-catenin level at 48 h post transfection. C2C12 myoblast cells were transiently transfected with different PABPN1constructs as well as GFP, and non-transfected cells, and treated or not with two different doses of LiCl as indicated. Actin antibody was used to confirm equal loading. (c and d) LiCl treatment results in re-distribution of β-catenin from a cytoplasmic to a nuclear compartment. Confocal images of immunocytochemistry showing the subcellular distribution of β-catenin. A clear accumulation of β-catenin in the nucleus was observed in the C2C12-transfected cells with GFP-wtPABPN1-10Ala (c) and GFP-expPABPN1-17Ala (d) after LiCl treatment. GFP-PABPN1 constructs (green), β-catenin (red)
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Related In: Results  -  Collection

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fig6: LiCl leads to a significant increase of the β-catenin level in the OPMD C2C12 cell model. (a and b) LiCl leads to a significant increase of β-catenin in the OPMD C2C12 cell model while leaving the GFP level not affected. A western blot showing the effect of LiCl treatment on the β-catenin level at 48 h post transfection. C2C12 myoblast cells were transiently transfected with different PABPN1constructs as well as GFP, and non-transfected cells, and treated or not with two different doses of LiCl as indicated. Actin antibody was used to confirm equal loading. (c and d) LiCl treatment results in re-distribution of β-catenin from a cytoplasmic to a nuclear compartment. Confocal images of immunocytochemistry showing the subcellular distribution of β-catenin. A clear accumulation of β-catenin in the nucleus was observed in the C2C12-transfected cells with GFP-wtPABPN1-10Ala (c) and GFP-expPABPN1-17Ala (d) after LiCl treatment. GFP-PABPN1 constructs (green), β-catenin (red)
Mentions: The cellular localization of β-catenin is regulated by its phosphorylation by GSK-3β, which lies upstream. In the absence of the Wnt signal, β-catenin is phosphorylated by GSK-3β and targeted for degradation by the ubiquitin–proteasome system. Upon Wnt signaling, it cannot be phosphorylated by GSK-3β resulting in its translocation to the nucleus.43 Li+ ions were in fact shown to inhibit GSK-3β activity, resulting in the elevation of β-catenin protein levels.44, 45, 46 Given the protection effect of LiCl against cell death associated with GFP-expPABPN1 (13Ala and 17Ala) (Figures 2a and b), we investigated whether LiCl protected against expPABPN1-associated cell death by upregulating the level of β-catenin protein. We first analyzed by western blot the transfected cells, which had been treated with 2.5 mM LiCl, and increased expression could be seen in this instance (Figure 6a) at 48 h post transfection; while leaving the level of GFP protein unaffected in cells transfected with GFP-expPABPN1 (13Ala and 17Ala) (Figure 6b). This suggests that the cell survival protection is mediated through induction of the β-catenin protein and/or other proteins downstream from β-catenin rather than affecting the level of PABPN1 protein. The effect of increasing the LiCl concentration on enhancing β-catenin level was also studied in non-transfected C2C12 cells (Supplementary Figure S1). Using immunocytochemistry, we then examined the subcellular localization of β-catenin in cells and compared it before and after a LiCl treatment. In the absence of LiCl treatment, expression of β-catenin appears cytoplasmic (Figures 6c and d, left panels). But following treatment with LiCl, a clear nuclear accumulation of β-catenin can be observed in cells expressing GFP-wtPABPN1-10Ala and GFP-expPABPN1-17Ala, respectively (Figures 6c and d, right panels). Figures 6c and d show that treatment with LiCl leads to a significant nuclear translocation of β-catenin from the cytoplasm to the nuclear compartment in both cells transfected with GFP-wtPABPN1-10Ala and GFP-expPABPN1-17Ala, respectively. Thus, we conclude that LiCl could activate a pro-survival pathway in the C2C12 OPMD model through activation of the β-catenin protein.

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