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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 enhances cell proliferating and differentiation of C2C12 cells transfected with GFP-expPABPN1-17Ala. (a and b) The effect of LiCl treatment on OPMD C2C12 cell model controls. LiCl maintains the ability of cells expressing GFP-wtPABPN1-10Ala (a) and GFP (b) to proliferate and differentiate over the time course. Cells were transiently transfected with GFP-wtPABPN1-10Ala or GFP and treated with 2.5 mM LiCl for 6 days in DM. (c) LiCl protects against cell death and enhances the number of multinucleated differentiated myotubes as well as increasing their diameter's size (bottom panels). Cells were transiently transfected with GFP-expPABPN1-17Ala and treated with 2.5 mM LiCl for 6 days in DM. Cells were visualized daily under the live-stage microscope for morphology and viability. Representative images were captured on day 2 and day 6 post-treatment with the drug. (d) LiCl enhances muscle differentiation of C2C12 cells expressing GFP-expPABPN1-13Ala. Representative images showing the effect of LiCl on cell differentiation. LiCl increases the total number of multinucleated myotubes in cells expressing GFP-expPABPN1-13Ala (bottom left) compared with non-treated GFP-expPABPN1-13Ala cells (top left). Phase contrast image of myotubes treated with 2.5 mM LiCl (bottom right) shows the increased diameter size when compared with non-treated cells (top right) on day 6 after differentiation. White arrows point towards the myotube's diameter. Red arrowheads point towards dead cells or debris of dead cells
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fig3: LiCl enhances cell proliferating and differentiation of C2C12 cells transfected with GFP-expPABPN1-17Ala. (a and b) The effect of LiCl treatment on OPMD C2C12 cell model controls. LiCl maintains the ability of cells expressing GFP-wtPABPN1-10Ala (a) and GFP (b) to proliferate and differentiate over the time course. Cells were transiently transfected with GFP-wtPABPN1-10Ala or GFP and treated with 2.5 mM LiCl for 6 days in DM. (c) LiCl protects against cell death and enhances the number of multinucleated differentiated myotubes as well as increasing their diameter's size (bottom panels). Cells were transiently transfected with GFP-expPABPN1-17Ala and treated with 2.5 mM LiCl for 6 days in DM. Cells were visualized daily under the live-stage microscope for morphology and viability. Representative images were captured on day 2 and day 6 post-treatment with the drug. (d) LiCl enhances muscle differentiation of C2C12 cells expressing GFP-expPABPN1-13Ala. Representative images showing the effect of LiCl on cell differentiation. LiCl increases the total number of multinucleated myotubes in cells expressing GFP-expPABPN1-13Ala (bottom left) compared with non-treated GFP-expPABPN1-13Ala cells (top left). Phase contrast image of myotubes treated with 2.5 mM LiCl (bottom right) shows the increased diameter size when compared with non-treated cells (top right) on day 6 after differentiation. White arrows point towards the myotube's diameter. Red arrowheads point towards dead cells or debris of dead cells

Mentions: In order to investigate how myogenic cells morphologically respond to LiCl treatment, transected C2C12 cells treated with 2.5 mM LiCl were cultivated in growth medium (GM) for 24 h, which was then replaced by differentiation medium (DM) for a further period of 6 days. To evaluate the effect of LiCl on C2C12 differentiation, we followed the morphology of non-transfected C2C12 cells in parallel to the C2C12 cells expressing either GFP-wtPABPN1-10Ala, or GFP-expPABPN1-13Ala or GFP-expPABPN1-17Ala or GFP in the presence of 2.5 mM LiCl. In control samples, with cells expressing GFP-wtPABPN1-10Ala or GFP, LiCl maintains the ability of cells to proliferate and differentiate over the time course (Figures 3a and b). Our results show that LiCl enhances the ability of C2C12 expressing GFP-expPABPN1-17Ala to differentiate from single-cell myoblasts to multinucleated myotubes (Figure 3c). In addition to enhancing the multinucleated differentiation myotubes, the drug enhances the proliferation of viable cells expressing GFP-expPABPN1-17Ala, as detected under the live-stage fluorescent microscope (Figure 3c). Similar findings were also obtained from cells expressing GFP-expPABPN1-13Ala (Figure 3d). LiCl treatment was found to increase myotube formation as a majority of nuclei were present in large multinucleated myotubes after 6 days in DM (Figure 3d). Phase microscopy was used to visualize myotube formation and the morphology of cells. In order to quantitatively evaluate the effect of LiCl on the differentiation grade of C2C12 cells expressing GFP-expPABPN1 (13Al and 17Ala), we calculated the fusion index of the cultures. The results presented in Figure 4 demonstrate that LiCl increases the fusion index of cells expressing GFP-expPABPN1-(13Ala and 17Ala).


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 enhances cell proliferating and differentiation of C2C12 cells transfected with GFP-expPABPN1-17Ala. (a and b) The effect of LiCl treatment on OPMD C2C12 cell model controls. LiCl maintains the ability of cells expressing GFP-wtPABPN1-10Ala (a) and GFP (b) to proliferate and differentiate over the time course. Cells were transiently transfected with GFP-wtPABPN1-10Ala or GFP and treated with 2.5 mM LiCl for 6 days in DM. (c) LiCl protects against cell death and enhances the number of multinucleated differentiated myotubes as well as increasing their diameter's size (bottom panels). Cells were transiently transfected with GFP-expPABPN1-17Ala and treated with 2.5 mM LiCl for 6 days in DM. Cells were visualized daily under the live-stage microscope for morphology and viability. Representative images were captured on day 2 and day 6 post-treatment with the drug. (d) LiCl enhances muscle differentiation of C2C12 cells expressing GFP-expPABPN1-13Ala. Representative images showing the effect of LiCl on cell differentiation. LiCl increases the total number of multinucleated myotubes in cells expressing GFP-expPABPN1-13Ala (bottom left) compared with non-treated GFP-expPABPN1-13Ala cells (top left). Phase contrast image of myotubes treated with 2.5 mM LiCl (bottom right) shows the increased diameter size when compared with non-treated cells (top right) on day 6 after differentiation. White arrows point towards the myotube's diameter. Red arrowheads point towards dead cells or debris of dead cells
© Copyright Policy - open-access
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fig3: LiCl enhances cell proliferating and differentiation of C2C12 cells transfected with GFP-expPABPN1-17Ala. (a and b) The effect of LiCl treatment on OPMD C2C12 cell model controls. LiCl maintains the ability of cells expressing GFP-wtPABPN1-10Ala (a) and GFP (b) to proliferate and differentiate over the time course. Cells were transiently transfected with GFP-wtPABPN1-10Ala or GFP and treated with 2.5 mM LiCl for 6 days in DM. (c) LiCl protects against cell death and enhances the number of multinucleated differentiated myotubes as well as increasing their diameter's size (bottom panels). Cells were transiently transfected with GFP-expPABPN1-17Ala and treated with 2.5 mM LiCl for 6 days in DM. Cells were visualized daily under the live-stage microscope for morphology and viability. Representative images were captured on day 2 and day 6 post-treatment with the drug. (d) LiCl enhances muscle differentiation of C2C12 cells expressing GFP-expPABPN1-13Ala. Representative images showing the effect of LiCl on cell differentiation. LiCl increases the total number of multinucleated myotubes in cells expressing GFP-expPABPN1-13Ala (bottom left) compared with non-treated GFP-expPABPN1-13Ala cells (top left). Phase contrast image of myotubes treated with 2.5 mM LiCl (bottom right) shows the increased diameter size when compared with non-treated cells (top right) on day 6 after differentiation. White arrows point towards the myotube's diameter. Red arrowheads point towards dead cells or debris of dead cells
Mentions: In order to investigate how myogenic cells morphologically respond to LiCl treatment, transected C2C12 cells treated with 2.5 mM LiCl were cultivated in growth medium (GM) for 24 h, which was then replaced by differentiation medium (DM) for a further period of 6 days. To evaluate the effect of LiCl on C2C12 differentiation, we followed the morphology of non-transfected C2C12 cells in parallel to the C2C12 cells expressing either GFP-wtPABPN1-10Ala, or GFP-expPABPN1-13Ala or GFP-expPABPN1-17Ala or GFP in the presence of 2.5 mM LiCl. In control samples, with cells expressing GFP-wtPABPN1-10Ala or GFP, LiCl maintains the ability of cells to proliferate and differentiate over the time course (Figures 3a and b). Our results show that LiCl enhances the ability of C2C12 expressing GFP-expPABPN1-17Ala to differentiate from single-cell myoblasts to multinucleated myotubes (Figure 3c). In addition to enhancing the multinucleated differentiation myotubes, the drug enhances the proliferation of viable cells expressing GFP-expPABPN1-17Ala, as detected under the live-stage fluorescent microscope (Figure 3c). Similar findings were also obtained from cells expressing GFP-expPABPN1-13Ala (Figure 3d). LiCl treatment was found to increase myotube formation as a majority of nuclei were present in large multinucleated myotubes after 6 days in DM (Figure 3d). Phase microscopy was used to visualize myotube formation and the morphology of cells. In order to quantitatively evaluate the effect of LiCl on the differentiation grade of C2C12 cells expressing GFP-expPABPN1 (13Al and 17Ala), we calculated the fusion index of the cultures. The results presented in Figure 4 demonstrate that LiCl increases the fusion index of cells expressing GFP-expPABPN1-(13Ala and 17Ala).

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