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Fukutin is prerequisite to ameliorate muscular dystrophic phenotype by myofiber-selective LARGE expression.

Ohtsuka Y, Kanagawa M, Yu CC, Ito C, Chiyo T, Kobayashi K, Okada T, Takeda S, Toda T - Sci Rep (2015)

Bottom Line: However, the in vivo therapeutic benefit of using LARGE activity is controversial.Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation.Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan.

ABSTRACT
α-Dystroglycanopathy (α-DGP) is a group of muscular dystrophy characterized by abnormal glycosylation of α-dystroglycan (α-DG), including Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain disease, Walker-Warburg syndrome, and congenital muscular dystrophy type 1D (MDC1D), etc. LARGE, the causative gene for MDC1D, encodes a glycosyltransferase to form [-3Xyl-α1,3GlcAβ1-] polymer in the terminal end of the post-phosphoryl moiety, which is essential for α-DG function. It has been proposed that LARGE possesses the great potential to rescue glycosylation defects in α-DGPs regardless of causative genes. However, the in vivo therapeutic benefit of using LARGE activity is controversial. To explore the conditions needed for successful LARGE gene therapy, here we used Large-deficient and fukutin-deficient mouse models for MDC1D and FCMD, respectively. Myofibre-selective LARGE expression via systemic adeno-associated viral gene transfer ameliorated dystrophic pathology of Large-deficient mice even when intervention occurred after disease manifestation. However, the same strategy failed to ameliorate the dystrophic phenotype of fukutin-conditional knockout mice. Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation. Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.

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Systemic gene transfer of Large into Myf5-fukutin cKO mice after onset.AAV9-MCK-Large was administered to 4-week-old Myf5-fukutin cKO mice via tail vein injection; after 2 months, the skeletal muscles were harvested and analysed for α-DG glycosylation (a, b) and histology (c). Although LARGE was expressed (a), the levels of α-DG glycosylation were unchanged in AAV-treated Myf5-fukutin-cKO mice (a, b). H&E staining for the tibialis anterior muscle did not show improvement of the muscular dystrophic phenotype of Myf5-fukutin-cKO mice (c). WT, litter control mice (fukutinlox/lox without cre-transgene); fukutin-cKO, untreated Myf5-fukutin-cKO mice; and fukutin cKO + Large, Myf5-fukutin-cKO mice with AAV9-MCK-Large treatment. Bar = 50 μm. The full-length blots with α-DG (IIH6), α-DG (core), LARGE, and β-DG are presented in Supplementary Figure S2e-h, respectively.
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f3: Systemic gene transfer of Large into Myf5-fukutin cKO mice after onset.AAV9-MCK-Large was administered to 4-week-old Myf5-fukutin cKO mice via tail vein injection; after 2 months, the skeletal muscles were harvested and analysed for α-DG glycosylation (a, b) and histology (c). Although LARGE was expressed (a), the levels of α-DG glycosylation were unchanged in AAV-treated Myf5-fukutin-cKO mice (a, b). H&E staining for the tibialis anterior muscle did not show improvement of the muscular dystrophic phenotype of Myf5-fukutin-cKO mice (c). WT, litter control mice (fukutinlox/lox without cre-transgene); fukutin-cKO, untreated Myf5-fukutin-cKO mice; and fukutin cKO + Large, Myf5-fukutin-cKO mice with AAV9-MCK-Large treatment. Bar = 50 μm. The full-length blots with α-DG (IIH6), α-DG (core), LARGE, and β-DG are presented in Supplementary Figure S2e-h, respectively.

Mentions: LARGE overexpression increases glycosylation and ligand-binding activity of α-DG in fukutin-deficient cells from FCMD patients37. We examined whether the muscular dystrophic phenotype of fukutin-deficient mice can be improved by LARGE overexpression in vivo. We used muscle precursor cell (MPC)-selective fukutin-deficient conditional knock-out (cKO) mice as a fukutin-deficient model (Myf5-fukutin-cKO mice)27. Myf5-fukutin-cKO mice showed loss of IIH6-positive glycosylation of α-DG in the skeletal muscles at birth27. The dystrophic pathology begins around 4 weeks of age and becomes severe at 12 weeks27. We administered intravenous AAV9-MCK-Large into 4-week-old Myf5-fukutin-cKO mice via the tail vein, and then analysed the glycosylation status of α-DG and therapeutic efficacy after 2 months. Interestingly, although we observed expression of LARGE protein in the AAV-treated Myf5-fukutin-cKO skeletal muscles, IIH6-positive α-DG was hardly produced in AAV-treated Myf5-fukutin cKO mice (Fig. 3a). Immunofluorescence staining also confirmed failure to restore IIH6-positive glycosylation of α-DG by AAV-treatment in Myf5-fukutin-cKO mice (Fig. 3b). H&E staining of skeletal muscles and quantitative muscle pathology showed no significant improvement with AAV treatment (Fig. 3c and Fig. S1a–c). In addition, we found no evidence to support improvements in grip strength, body weight, and serum CK activity after AAV treatment (Fig. S1d–f). These data indicate that the failure to restore α-DG glycosylation in Myf5-fukutin-cKO mice is associated with failure of LARGE therapeutic efficacy.


Fukutin is prerequisite to ameliorate muscular dystrophic phenotype by myofiber-selective LARGE expression.

Ohtsuka Y, Kanagawa M, Yu CC, Ito C, Chiyo T, Kobayashi K, Okada T, Takeda S, Toda T - Sci Rep (2015)

Systemic gene transfer of Large into Myf5-fukutin cKO mice after onset.AAV9-MCK-Large was administered to 4-week-old Myf5-fukutin cKO mice via tail vein injection; after 2 months, the skeletal muscles were harvested and analysed for α-DG glycosylation (a, b) and histology (c). Although LARGE was expressed (a), the levels of α-DG glycosylation were unchanged in AAV-treated Myf5-fukutin-cKO mice (a, b). H&E staining for the tibialis anterior muscle did not show improvement of the muscular dystrophic phenotype of Myf5-fukutin-cKO mice (c). WT, litter control mice (fukutinlox/lox without cre-transgene); fukutin-cKO, untreated Myf5-fukutin-cKO mice; and fukutin cKO + Large, Myf5-fukutin-cKO mice with AAV9-MCK-Large treatment. Bar = 50 μm. The full-length blots with α-DG (IIH6), α-DG (core), LARGE, and β-DG are presented in Supplementary Figure S2e-h, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4321163&req=5

f3: Systemic gene transfer of Large into Myf5-fukutin cKO mice after onset.AAV9-MCK-Large was administered to 4-week-old Myf5-fukutin cKO mice via tail vein injection; after 2 months, the skeletal muscles were harvested and analysed for α-DG glycosylation (a, b) and histology (c). Although LARGE was expressed (a), the levels of α-DG glycosylation were unchanged in AAV-treated Myf5-fukutin-cKO mice (a, b). H&E staining for the tibialis anterior muscle did not show improvement of the muscular dystrophic phenotype of Myf5-fukutin-cKO mice (c). WT, litter control mice (fukutinlox/lox without cre-transgene); fukutin-cKO, untreated Myf5-fukutin-cKO mice; and fukutin cKO + Large, Myf5-fukutin-cKO mice with AAV9-MCK-Large treatment. Bar = 50 μm. The full-length blots with α-DG (IIH6), α-DG (core), LARGE, and β-DG are presented in Supplementary Figure S2e-h, respectively.
Mentions: LARGE overexpression increases glycosylation and ligand-binding activity of α-DG in fukutin-deficient cells from FCMD patients37. We examined whether the muscular dystrophic phenotype of fukutin-deficient mice can be improved by LARGE overexpression in vivo. We used muscle precursor cell (MPC)-selective fukutin-deficient conditional knock-out (cKO) mice as a fukutin-deficient model (Myf5-fukutin-cKO mice)27. Myf5-fukutin-cKO mice showed loss of IIH6-positive glycosylation of α-DG in the skeletal muscles at birth27. The dystrophic pathology begins around 4 weeks of age and becomes severe at 12 weeks27. We administered intravenous AAV9-MCK-Large into 4-week-old Myf5-fukutin-cKO mice via the tail vein, and then analysed the glycosylation status of α-DG and therapeutic efficacy after 2 months. Interestingly, although we observed expression of LARGE protein in the AAV-treated Myf5-fukutin-cKO skeletal muscles, IIH6-positive α-DG was hardly produced in AAV-treated Myf5-fukutin cKO mice (Fig. 3a). Immunofluorescence staining also confirmed failure to restore IIH6-positive glycosylation of α-DG by AAV-treatment in Myf5-fukutin-cKO mice (Fig. 3b). H&E staining of skeletal muscles and quantitative muscle pathology showed no significant improvement with AAV treatment (Fig. 3c and Fig. S1a–c). In addition, we found no evidence to support improvements in grip strength, body weight, and serum CK activity after AAV treatment (Fig. S1d–f). These data indicate that the failure to restore α-DG glycosylation in Myf5-fukutin-cKO mice is associated with failure of LARGE therapeutic efficacy.

Bottom Line: However, the in vivo therapeutic benefit of using LARGE activity is controversial.Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation.Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.

View Article: PubMed Central - PubMed

Affiliation: Division of Neurology/Molecular Brain Science, Kobe University Graduate School of Medicine, Kobe, 650-0017, Japan.

ABSTRACT
α-Dystroglycanopathy (α-DGP) is a group of muscular dystrophy characterized by abnormal glycosylation of α-dystroglycan (α-DG), including Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain disease, Walker-Warburg syndrome, and congenital muscular dystrophy type 1D (MDC1D), etc. LARGE, the causative gene for MDC1D, encodes a glycosyltransferase to form [-3Xyl-α1,3GlcAβ1-] polymer in the terminal end of the post-phosphoryl moiety, which is essential for α-DG function. It has been proposed that LARGE possesses the great potential to rescue glycosylation defects in α-DGPs regardless of causative genes. However, the in vivo therapeutic benefit of using LARGE activity is controversial. To explore the conditions needed for successful LARGE gene therapy, here we used Large-deficient and fukutin-deficient mouse models for MDC1D and FCMD, respectively. Myofibre-selective LARGE expression via systemic adeno-associated viral gene transfer ameliorated dystrophic pathology of Large-deficient mice even when intervention occurred after disease manifestation. However, the same strategy failed to ameliorate the dystrophic phenotype of fukutin-conditional knockout mice. Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation. Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.

Show MeSH
Related in: MedlinePlus