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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 rescues GFP-expPABPN (13Ala and 17Ala)-associated cell death. (a) Cell survival determined by live-stage microscopy. C2C12 cells were transfected with a GFP vector, GFP-wtPABPN1-10Ala, GFP-expPABPN1-13Ala, and GFP-expPABPN1-17Ala, treated or not with 2.5 mM LiCl. The cells were counted every 24 h post transfection for 6 days consecutively using the live-stage microscope. The percentage of transfected living cells represents the variation of the amount of transfected living cells at different time points compared with the number of transfected cells obtained on day 1. LiCl rescues GFP-expPABPN1-13Ala and 17Ala-associated cell death (*P<0.001 versus non-treated samples). Mean±S.E., *P<0.05 compared with any other groups (ANOVA analysis). The experiment was repeated 3 times. (b) Cell death measured by fluorescent flow cytometry (FFC). Percentage of cell death observed by two-color FFC analysis with 7AAD on day 6 post-treatment with 2.5 mM LiCl. GFP-wtPABPN1-10Ala and GFP were used as controls. Cell death was calculated by dividing the number of 7AAD-stained transfected C2C12 cells over the total number of transfected cells. The experiment was repeated four times. (*P<0.001 versus non-treated samples). (c) A two-color FACS dotplot from a representative experiment in which C2C12 cells were transfected with GFP-expPABPN1-17Ala and treated with 2.5 mM LiCl (bottom) compared with non-treated cells (top), stained with 7AAD to label dead cells, and co-sorted for GFP (green) and 7AAD (red) fluorescence. Grid lines were positioned after calibrating the flow cytometry. Upper right quadrants signify 7AAD-labeled dead or dying transfected cells (GFP+7AAD+), i.e., Q2 (underlined values)
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fig2: LiCl rescues GFP-expPABPN (13Ala and 17Ala)-associated cell death. (a) Cell survival determined by live-stage microscopy. C2C12 cells were transfected with a GFP vector, GFP-wtPABPN1-10Ala, GFP-expPABPN1-13Ala, and GFP-expPABPN1-17Ala, treated or not with 2.5 mM LiCl. The cells were counted every 24 h post transfection for 6 days consecutively using the live-stage microscope. The percentage of transfected living cells represents the variation of the amount of transfected living cells at different time points compared with the number of transfected cells obtained on day 1. LiCl rescues GFP-expPABPN1-13Ala and 17Ala-associated cell death (*P<0.001 versus non-treated samples). Mean±S.E., *P<0.05 compared with any other groups (ANOVA analysis). The experiment was repeated 3 times. (b) Cell death measured by fluorescent flow cytometry (FFC). Percentage of cell death observed by two-color FFC analysis with 7AAD on day 6 post-treatment with 2.5 mM LiCl. GFP-wtPABPN1-10Ala and GFP were used as controls. Cell death was calculated by dividing the number of 7AAD-stained transfected C2C12 cells over the total number of transfected cells. The experiment was repeated four times. (*P<0.001 versus non-treated samples). (c) A two-color FACS dotplot from a representative experiment in which C2C12 cells were transfected with GFP-expPABPN1-17Ala and treated with 2.5 mM LiCl (bottom) compared with non-treated cells (top), stained with 7AAD to label dead cells, and co-sorted for GFP (green) and 7AAD (red) fluorescence. Grid lines were positioned after calibrating the flow cytometry. Upper right quadrants signify 7AAD-labeled dead or dying transfected cells (GFP+7AAD+), i.e., Q2 (underlined values)

Mentions: We previously used transfection assays to establish that the transient expression of a GFP-tagged full-length cDNA of expanded PABPN1 (GFP-expPABPN1-13Ala and 17Ala) in various cell lines leads to significantly more cell death than what can be seen in transfections made with a corresponding vector expressing wild-type PABPN1 (GFP-wtPABPN1-10Ala).6, 40 In order to test the effect of LiCl on cell death associated with expPABPN1 expression, C2C12 cells were pretreated with 2.5 mM LiCl for 3 days before transfection and this treatment was maintained after transfection was made. Cells were transiently transfected with GFP-expPABPN1-13Ala, GFP-expPABPN1-17Ala, and the controls GFP-wtPABPN1-10Ala, as well as with GFP alone. Control samples, where no drug was applied on corresponding transfected cells, were also monitored. We first followed cell viability using automated live-stage fluorescent microscopy to monitor the number of viable green fluorescent cells over 6 days post transfection. We looked at nuclear morphology, and GFP-expressing cells with fragmented or condensed nuclei were counted as dead. The automated microscope precisely returned back to same cell population field. LiCl treatment showed a consistent and significant protective effect against expPABPN1-induced cell death, compared with non-treated counterparts. As shown in Figure 2a, LiCl rescues cells expressing GFP-expPABPN1-13Ala and GFP-expPABPN1-17Ala-associated cell death (*P<0.001 versus non-treated samples). LiCl treatment did not show a reduction of protein aggregation (data not shown). In order to validate this live imaging cell survival assay, we used a fluorescence-based flow cytometry (FFC) assay to analyze the ability of cells to exclude the red-fluorescent 7-aminoactinomycin D (7AAD) biomarker for dead cells. The presence of the GFP-tag in the expression vectors used enabled us to establish the fraction of C2C12 cells that were actually transfected. The use of GFP has been described as a means to assess gene expression and transfection efficiency.41, 42


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 rescues GFP-expPABPN (13Ala and 17Ala)-associated cell death. (a) Cell survival determined by live-stage microscopy. C2C12 cells were transfected with a GFP vector, GFP-wtPABPN1-10Ala, GFP-expPABPN1-13Ala, and GFP-expPABPN1-17Ala, treated or not with 2.5 mM LiCl. The cells were counted every 24 h post transfection for 6 days consecutively using the live-stage microscope. The percentage of transfected living cells represents the variation of the amount of transfected living cells at different time points compared with the number of transfected cells obtained on day 1. LiCl rescues GFP-expPABPN1-13Ala and 17Ala-associated cell death (*P<0.001 versus non-treated samples). Mean±S.E., *P<0.05 compared with any other groups (ANOVA analysis). The experiment was repeated 3 times. (b) Cell death measured by fluorescent flow cytometry (FFC). Percentage of cell death observed by two-color FFC analysis with 7AAD on day 6 post-treatment with 2.5 mM LiCl. GFP-wtPABPN1-10Ala and GFP were used as controls. Cell death was calculated by dividing the number of 7AAD-stained transfected C2C12 cells over the total number of transfected cells. The experiment was repeated four times. (*P<0.001 versus non-treated samples). (c) A two-color FACS dotplot from a representative experiment in which C2C12 cells were transfected with GFP-expPABPN1-17Ala and treated with 2.5 mM LiCl (bottom) compared with non-treated cells (top), stained with 7AAD to label dead cells, and co-sorted for GFP (green) and 7AAD (red) fluorescence. Grid lines were positioned after calibrating the flow cytometry. Upper right quadrants signify 7AAD-labeled dead or dying transfected cells (GFP+7AAD+), i.e., Q2 (underlined values)
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fig2: LiCl rescues GFP-expPABPN (13Ala and 17Ala)-associated cell death. (a) Cell survival determined by live-stage microscopy. C2C12 cells were transfected with a GFP vector, GFP-wtPABPN1-10Ala, GFP-expPABPN1-13Ala, and GFP-expPABPN1-17Ala, treated or not with 2.5 mM LiCl. The cells were counted every 24 h post transfection for 6 days consecutively using the live-stage microscope. The percentage of transfected living cells represents the variation of the amount of transfected living cells at different time points compared with the number of transfected cells obtained on day 1. LiCl rescues GFP-expPABPN1-13Ala and 17Ala-associated cell death (*P<0.001 versus non-treated samples). Mean±S.E., *P<0.05 compared with any other groups (ANOVA analysis). The experiment was repeated 3 times. (b) Cell death measured by fluorescent flow cytometry (FFC). Percentage of cell death observed by two-color FFC analysis with 7AAD on day 6 post-treatment with 2.5 mM LiCl. GFP-wtPABPN1-10Ala and GFP were used as controls. Cell death was calculated by dividing the number of 7AAD-stained transfected C2C12 cells over the total number of transfected cells. The experiment was repeated four times. (*P<0.001 versus non-treated samples). (c) A two-color FACS dotplot from a representative experiment in which C2C12 cells were transfected with GFP-expPABPN1-17Ala and treated with 2.5 mM LiCl (bottom) compared with non-treated cells (top), stained with 7AAD to label dead cells, and co-sorted for GFP (green) and 7AAD (red) fluorescence. Grid lines were positioned after calibrating the flow cytometry. Upper right quadrants signify 7AAD-labeled dead or dying transfected cells (GFP+7AAD+), i.e., Q2 (underlined values)
Mentions: We previously used transfection assays to establish that the transient expression of a GFP-tagged full-length cDNA of expanded PABPN1 (GFP-expPABPN1-13Ala and 17Ala) in various cell lines leads to significantly more cell death than what can be seen in transfections made with a corresponding vector expressing wild-type PABPN1 (GFP-wtPABPN1-10Ala).6, 40 In order to test the effect of LiCl on cell death associated with expPABPN1 expression, C2C12 cells were pretreated with 2.5 mM LiCl for 3 days before transfection and this treatment was maintained after transfection was made. Cells were transiently transfected with GFP-expPABPN1-13Ala, GFP-expPABPN1-17Ala, and the controls GFP-wtPABPN1-10Ala, as well as with GFP alone. Control samples, where no drug was applied on corresponding transfected cells, were also monitored. We first followed cell viability using automated live-stage fluorescent microscopy to monitor the number of viable green fluorescent cells over 6 days post transfection. We looked at nuclear morphology, and GFP-expressing cells with fragmented or condensed nuclei were counted as dead. The automated microscope precisely returned back to same cell population field. LiCl treatment showed a consistent and significant protective effect against expPABPN1-induced cell death, compared with non-treated counterparts. As shown in Figure 2a, LiCl rescues cells expressing GFP-expPABPN1-13Ala and GFP-expPABPN1-17Ala-associated cell death (*P<0.001 versus non-treated samples). LiCl treatment did not show a reduction of protein aggregation (data not shown). In order to validate this live imaging cell survival assay, we used a fluorescence-based flow cytometry (FFC) assay to analyze the ability of cells to exclude the red-fluorescent 7-aminoactinomycin D (7AAD) biomarker for dead cells. The presence of the GFP-tag in the expression vectors used enabled us to establish the fraction of C2C12 cells that were actually transfected. The use of GFP has been described as a means to assess gene expression and transfection efficiency.41, 42

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