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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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Quantitative analysis of the therapeutic effects of AAV9-MCK-Large treatment in Largemyd mice.Amelioration of dystrophic histology after AAV9-MCK-Large treatment was evaluated by quantifying muscle fibres with centrally located nuclei (a; P = 0.007), measuring infiltration of connective tissue by collagen I-immunofluorescence staining (b; P = 0.007) and infiltration of macrophages by F4/80-immunofluorescence staining (c; P = 0.011). Therapeutic efficacy over time was evaluated by grip strength (d; P = 0.007, 0.006, 0.008, and 0.014 for 8, 12, 16, and 24 weeks), body weight (e; P = 0.019, 0.019, 0.024, 0.017, and 0.032 for 6, 8, 10, 12, and 14 weeks), and serum CK activity (f; P = 0.021, 0.008, and 0.011 for 8, 12, and 24 weeks). Data shown are mean ± s.e.m. for each group (n is indicated in the graph). *P ≤ 0.05 vs. non-treated Largemyd homozygous mice (Mann–Whitney U test). Het, Largemyd heterozygous controls; homo, untreated Largemyd homozygous mice; and homo + Large, Largemyd homozygous mice with AAV9-MCK-Large treatment.
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f2: Quantitative analysis of the therapeutic effects of AAV9-MCK-Large treatment in Largemyd mice.Amelioration of dystrophic histology after AAV9-MCK-Large treatment was evaluated by quantifying muscle fibres with centrally located nuclei (a; P = 0.007), measuring infiltration of connective tissue by collagen I-immunofluorescence staining (b; P = 0.007) and infiltration of macrophages by F4/80-immunofluorescence staining (c; P = 0.011). Therapeutic efficacy over time was evaluated by grip strength (d; P = 0.007, 0.006, 0.008, and 0.014 for 8, 12, 16, and 24 weeks), body weight (e; P = 0.019, 0.019, 0.024, 0.017, and 0.032 for 6, 8, 10, 12, and 14 weeks), and serum CK activity (f; P = 0.021, 0.008, and 0.011 for 8, 12, and 24 weeks). Data shown are mean ± s.e.m. for each group (n is indicated in the graph). *P ≤ 0.05 vs. non-treated Largemyd homozygous mice (Mann–Whitney U test). Het, Largemyd heterozygous controls; homo, untreated Largemyd homozygous mice; and homo + Large, Largemyd homozygous mice with AAV9-MCK-Large treatment.

Mentions: Western blot analysis confirmed LARGE was overexpressed in AAV-treated Largemyd mice; consequently, the reactivity of IIH6 antibody exceeded even the baseline levels observed in untreated heterozygous animals (Fig. 1b). Immunofluorescence analysis also confirmed increased IIH6-reactivity in the treated Largemyd skeletal muscles (Fig. 1c). Haematoxylin and eosin (H&E) staining of skeletal muscles indicated decreases in the number of necrotic fibres and recovery of the polygonal contour of myofibres in AAV-treated Largemyd versus untreated Largemyd mice (Fig. 1d). The number of muscle fibres with centrally located nuclei as well as infiltration of connective tissues and macrophages were significantly reduced in comparison to the findings obtained for untreated Largemyd mice (Fig. 2a–c). After the AAV-injection, we tracked changes in grip strength, body weight, and serum creatine kinase (CK). Our results showed significant improvements of these parameters even 4 weeks after the injection (Fig. 2d–f). These results demonstrated that myofibre-selective LARGE expression in Largemyd mice via systemic administration ameliorates the dystrophic pathology even if the initial intervention occurs after onset.


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)

Quantitative analysis of the therapeutic effects of AAV9-MCK-Large treatment in Largemyd mice.Amelioration of dystrophic histology after AAV9-MCK-Large treatment was evaluated by quantifying muscle fibres with centrally located nuclei (a; P = 0.007), measuring infiltration of connective tissue by collagen I-immunofluorescence staining (b; P = 0.007) and infiltration of macrophages by F4/80-immunofluorescence staining (c; P = 0.011). Therapeutic efficacy over time was evaluated by grip strength (d; P = 0.007, 0.006, 0.008, and 0.014 for 8, 12, 16, and 24 weeks), body weight (e; P = 0.019, 0.019, 0.024, 0.017, and 0.032 for 6, 8, 10, 12, and 14 weeks), and serum CK activity (f; P = 0.021, 0.008, and 0.011 for 8, 12, and 24 weeks). Data shown are mean ± s.e.m. for each group (n is indicated in the graph). *P ≤ 0.05 vs. non-treated Largemyd homozygous mice (Mann–Whitney U test). Het, Largemyd heterozygous controls; homo, untreated Largemyd homozygous mice; and homo + Large, Largemyd homozygous mice with AAV9-MCK-Large treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Quantitative analysis of the therapeutic effects of AAV9-MCK-Large treatment in Largemyd mice.Amelioration of dystrophic histology after AAV9-MCK-Large treatment was evaluated by quantifying muscle fibres with centrally located nuclei (a; P = 0.007), measuring infiltration of connective tissue by collagen I-immunofluorescence staining (b; P = 0.007) and infiltration of macrophages by F4/80-immunofluorescence staining (c; P = 0.011). Therapeutic efficacy over time was evaluated by grip strength (d; P = 0.007, 0.006, 0.008, and 0.014 for 8, 12, 16, and 24 weeks), body weight (e; P = 0.019, 0.019, 0.024, 0.017, and 0.032 for 6, 8, 10, 12, and 14 weeks), and serum CK activity (f; P = 0.021, 0.008, and 0.011 for 8, 12, and 24 weeks). Data shown are mean ± s.e.m. for each group (n is indicated in the graph). *P ≤ 0.05 vs. non-treated Largemyd homozygous mice (Mann–Whitney U test). Het, Largemyd heterozygous controls; homo, untreated Largemyd homozygous mice; and homo + Large, Largemyd homozygous mice with AAV9-MCK-Large treatment.
Mentions: Western blot analysis confirmed LARGE was overexpressed in AAV-treated Largemyd mice; consequently, the reactivity of IIH6 antibody exceeded even the baseline levels observed in untreated heterozygous animals (Fig. 1b). Immunofluorescence analysis also confirmed increased IIH6-reactivity in the treated Largemyd skeletal muscles (Fig. 1c). Haematoxylin and eosin (H&E) staining of skeletal muscles indicated decreases in the number of necrotic fibres and recovery of the polygonal contour of myofibres in AAV-treated Largemyd versus untreated Largemyd mice (Fig. 1d). The number of muscle fibres with centrally located nuclei as well as infiltration of connective tissues and macrophages were significantly reduced in comparison to the findings obtained for untreated Largemyd mice (Fig. 2a–c). After the AAV-injection, we tracked changes in grip strength, body weight, and serum creatine kinase (CK). Our results showed significant improvements of these parameters even 4 weeks after the injection (Fig. 2d–f). These results demonstrated that myofibre-selective LARGE expression in Largemyd mice via systemic administration ameliorates the dystrophic pathology even if the initial intervention occurs after onset.

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