Therapeutic potential of proteasome inhibition in Duchenne and Becker muscular dystrophies

Abstract

Duchenne muscular dystrophy (DMD) and its milder allelic variant, Becker muscular dystrophy (BMD), result from mutations of the dystrophin gene and lead to progressive muscle deterioration. Enhanced activation of proteasomal degradation underlies critical steps in the pathogenesis of the DMD/BMD dystrophic process. Previously, we demonstrated that treatment with the proteasome inhibitor MG-132 rescues the cell membrane localization of dystrophin and the dystrophin glycoprotein complex in mdx mice, a natural genetic mouse model of DMD. The current work aims to thoroughly define the therapeutic potential in dystrophinopathies of Velcade, a drug that selectively blocks the ubiquitin-proteasome pathway. Velcade is particularly intriguing since it has been approved for the treatment of multiple myeloma. Therefore, its side effects in humans have been explored. Velcade effects were analyzed through two independent methodological approaches. First, we administered the drug systemically in mdx mice over a 2-week period. In this system, Velcade restores the membrane expression of dystrophin and dystrophin glycoprotein complex members and improves the dystrophic phenotype. In a second approach, we treated with the compound explants from muscle biopsies of DMD or BMD patients. We show that the inhibition of the proteasome pathway up-regulates dystrophin, alpha-sarcoglycan, and beta-dystroglycan protein levels in explants from BMD patients, whereas it increases the proteins of the dystrophin glycoprotein complex in DMD cases.

Figure 1

Inhibition of proteasome activation in muscles from 7-week-old mdx mice treated systemically with the proteasome inhibitor Velcade for 2 weeks. Proteasome activity was evaluated in gastrocnemius muscle lysates by means of specific fluorogenic substrates. Activity was assayed in age-matched wild-type C57BL/10 Sc/Sn (gray bar), untreated (white bar), and Velcade-treated (black bar) mdx mice. Notably, when compared with wild-type animals, proteasome degradation is markedly induced in untreated mdx mice. This effect is decreased by approximately 50% following Velcade exposure. Each column represents the mean ± SE from four to seven animals; *P < 0.05 Wild-type (Wt) versus Velcade-treated mdx; **P < 0.01 wild-type versus untreated mdx; ^§P < 0.05 untreated versus Velcade-treated mdx mice.

Figure 2

Inhibition of the proteasome pathway up-regulates the membrane localization of dystrophin and the dystrophin-glycoprotein complex in 7-week-old mdx mice. Frozen gastrocnemius muscle sections from untreated (saline only) and Velcade-treated mdx mice were immunostained with specific antibodies directed against dystrophin (DYS), β-dystroglycan (β-DG), α-sarcoglycan (α-SG), and spectrin α II (Spectrin). Note that DYS, β-DG, and α-SG, were expressed at low levels in the skeletal muscle fibers from untreated mdx muscles, whereas they were up-regulated on the muscle plasma membrane of the mice treated with the proteasome inhibitor. Spectrin α II was used as a membrane protein control. Final magnification, ×25. Representative images are shown.

Figure 3

Inhibition of the proteasome pathway on caveolin 3 and α-1syntrophin expression in 7-week-old mdx mice. Frozen gastrocnemius muscle sections from untreated (saline only) and Velcade-treated mdx mice were immunostained with specific antibodies directed against caveolin 3 (Cav-3), α−1 synthophin (α−1 Syn) and spectrin α II (Spectrin). Note that Cav-3 is highly expressed in the untreated mdx group, while α−1 Syn staining is absent or just weakly positive. The proteasome inhibitor induced only a slight reduction of Cav-3 expression, while it enhanced α−1 Syn. Spectrin α II was used as a membrane protein control. Final magnification, ×25. Representative images are shown.

Figure 4

Inhibition of the proteasome pathway increases the protein levels of dystrophin and the dystrophin-glycoprotein complex in 7-week-old mdx mice. A: Gastrocnemius muscle lysates form untreated (saline only) and Velcade-treated mdx mice were separated by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and subjected to WB analysis with specific antibodies against dystrophin (DYS), α-sarcoglycan (α-SG), β-dystroglycan (β-DG), caveolin 3 (Cav-3), α−1 synthophin (α−1-Syn), and Spectrin α II (Spectrin). Note that proteasome inhibition induced an increase of DYS and its associated proteins. Consistently with DYS up-regulation, Cav-3 protein levels were reduced in muscles from Velcade-treated mice, as compared with untreated controls. Notably, in the Velcade-treated mice, DYS migrates at molecular weight equal to 120 kDa. This is in accordance with the predicted size of the protein on the basis of the mdx mutation (premature stop codon in exon 23). Spectrin α II was used as a loading control. B: Bands quantification was performed by densitometric analysis. Data (mean ± SE from four to seven animals) are expressed as percentage of Velcade-treated versus untreated mdx mice. *P < 0.05 untreated (black bar) versus Velcade-treated (white bar) mdx mice.

Figure 5

Inhibition of the proteasome pathway reduces muscle degeneration and necrotic features in skeletal muscle fibers of 7-week-old mdx mice. A: To evaluate muscle fiber damage, Evans blue dye (EBD) agent was injected intraperitoneally in untreated (saline only) and Velcade-treated mdx mice 20 hours before the sacrifice. Frozen gastrocnemius muscle sections were examined by fluorescence microscopy. Sections from untreated mice displayed a strong red autofluorescent signal, whereas muscle fibers from the Velcade-treated group were negative. Representative images are shown. Final magnification, ×10. B: EBD-positive muscle fibers in the gastrocnemius from mdx mice, both untreated (white bar) and Velcade-treated (black bar), were evaluated and expressed as percentage of total fibers (two sections were counted for each muscle; n = 4 mice for each experimental group; n myofibers = 400 to 600). ^§§P < 0.01 untreated versus Velcade-treated mdx mice.

Figure 6

Inhibition of the proteasome pathway improves histopathological features in skeletal muscle fibers of 7-week-old mdx mice. A: Frozen gastrocnemius muscle tissue sections from untreated (saline only) and Velcade-treated mdx mice were stained with standard H&E technique. Skeletal muscle from untreated mdx mice showed clear signs of myopathic changes, including muscle fiber degeneration characterized by central nuclei, variability in fibers size, lymphocytic, and connective tissue infiltration. Note that proteasome inhibition increased the diameter of the myofibers and decreased the signs of the inflammatory and fibrogenic response compared with the untreated mdx mice. Representative images are shown. Final magnification, ×10. B: Cross-sectional area (CSA) in age-matched wild-type C57BL/10 Sc/Sn (gray bar), untreated mdx (white bar), and Velcade-treated (black bar) mdx mice was determined by the Image J software (two sections were counted for each muscle (n = four to seven mice for each experimental group; n myofibers = 400 to 600). CSA data (mean ± SE from four to seven animals) are expressed as percentage of untreated and Velcade-treated mdx versus wild-type mice column. Both central nucleated and necrotic fibers are expressed as percentage of total fibers; **P < 0.001 wild-type versus untreated mdx; ^§P < 0.05 untreated versus Velcade-treated mdx mice; ^§§P < 0.01 untreated versus Velcade-treated mdx mice.

Figure 7

Inhibition of the proteasome pathway up-regulates dystrophin membrane levels and improves histopathological features in diaphragm muscle fibers of 7-week-old mdx mice. A: Paraffin diaphragm muscle sections from untreated (saline only) and Velcade-treated mdx mice were immunostained with a specific antibody directed against dystrophin (DYS) and hence counterstained with hematoxylin. Note that DYS was almost not detectable on the sarcolemma of diaphragm muscle fibers from untreated mdx muscles, whereas it was up-regulated on the muscle plasma membrane of the mice treated with the proteasome inhibitor (white arrows). Final magnification, ×25. Representative images are shown. B: Paraffin-embedded diaphragm muscle tissue sections from untreated (saline only) and Velcade-treated mdx mice were stained with standard H&E technique. Skeletal muscle from untreated mdx mice showed clear signs of myopathic changes, including muscle fiber degeneration characterized by central nuclei, variability in fiber size, and lymphocytic and connective tissue infiltration. Note that proteasome inhibition decreased the signs of the inflammatory and degenerative response. Representative images are shown. Final magnification, ×10. C: Cross-sectional area (CSA) in both untreated (white bar) and Velcade-treated (black bar) mdx mice was determined by the Image J software (two sections were counted for each muscle; n = four to seven mice for each experimental group; n myofibers = 400 to 600). CSA data (mean ± SE from four to seven animals) are expressed as percentage of Velcade-treated versus untreated mdx mice column, while both central nucleated and necrotic fibers are expressed as percentage of total fibers; ^§P < 0.05 untreated versus Velcade-treated mdx mice; ^§§P < 0.001 untreated versus Velcade-treated mdx mice.

Figure 8

Inhibition of the proteasome pathway enhances muscle cell regeneration in 7-week-old mdx mice. A: Frozen skeletal muscle sections from untreated (saline only) and Velcade-treated mdx mice were immunostained with a primary antibody directed toward the native form of E-MHC. Note that proteasome inhibition increases the number of E-MHC positive muscle fibers. Representative images are shown. Final magnification, ×10. B: E-MHC positive fibers in the gastrocnemius from mdx mice, both untreated (white bar) and treated (black bar), were quantified (two sections were counted for each muscle; n = four to seven mice for each experimental group; n myofibers = 400 to 600). Each column represents the mean ± SE from four to seven animals; ^§P < 0.05 untreated versus Velcade-treated mdx mice. C: Skeletal muscle nuclear lysates form untreated (saline only) and Velcade-treated mdx mice were subjected to immunoblot analysis with specific antibodies against MyoD and Myf-5, two common markers of regeneration. GAPDH was used to normalize sample loading. Note that Velcade administration to mdx mice increases both MyoD and Myf-5 levels, suggesting the hypothesis that muscle regeneration in treated animals is enhanced. D: Band quantification was performed by densitometric analysis. Data (mean ± SE from four to seven animals) are expressed as percentage of Velcade-treated versus untreated mdx mice. ^§P < 0.05, ^§§P < 0.01 untreated (white bar) versus Velcade-treated (black bar) mdx mice.

Figure 9

Inhibition of the proteasome pathway reduces signs of inflammation in the skeletal muscle of 7-week-old mdx mice. A: Frozen skeletal muscle sections from untreated (saline only) and Velcade-treated mdx mice were immunostained with a primary antibody directed toward the macrophage marker F4/80. Representative images are shown. Final magnification, ×10. B: In untreated (white bar) and Velcade-treated (black bar) mdx mice, the number of labeled cells (brown colored) was counted within two entire sections for each muscle (n = four to seven mice for each experimental group), and the total area of each section was assessed using a calibrated square grid. The volume of muscle sample was calculated as the product of the section area and thickness (5 μm). Concentrations of macrophages were expressed as number of cells per cubic millimeter (mm³). Each column represents the mean ± SE; ^§§P < 0.01 untreated versus Velcade-treated mdx mice. C: Skeletal muscle lysates form untreated (saline only) and Velcade-treated mdx mice were subjected to Immunoblot analysis with specific antibodies against IL-6. Determination of GAPDH expression was used to normalize sample loading. Please note that Velcade administration to mdx mice reduces the level of IL-6, thus further suggesting that proteasome inhibition is associated with a reduction of the inflammatory state. D: Quantification of IL-6 levels in untreated (white bar) and Velcade-treated (black bar) mdx mice by densitometric analysis. Data (mean ± SE from four to seven animals) are expressed as percentage of Velcade-treated versus untreated mdx mice. ^§P < 0.05 untreated versus Velcade-treated mdx mice.

Figure 10

Inhibition of the proteasome pathway dramatically decreases serum CK levels in 7-week-old mdx mice. Blood samples from untreated (saline only) and Velcade-treated mdx mice were obtained by intracardiac punctures at 2 weeks of treatment. Each column represents the mean ± SE from four to seven animals. *P < 0.05 untreated (white bar) versus Velcade-treated (black bar) mdx mice.

Figure 11

Inhibition of the proteasome pathway decreases NF-κB activation in 7-week-old mdx mice. A: Skeletal muscle nuclear lysates from untreated (saline only) and Velcade-treated mdx mice were subjected to immunoblot analysis with specific antibodies against total NF-κB (p65). The membrane was then stripped and reprobed with GAPDH antibody to normalize sample loading. Please note that Velcade administration to mdx mice reduces the level of p65 NF-κB allowed to translocate at nuclear level, thus suggesting that NF-κB activation is inhibited. B: Bands quantification was performed by densitometric analysis. Data (mean ± SE from four to seven animals) are expressed as percentage of Velcade-treated versus untreated mdx mice. ^§§P < 0.01 untreated (white bar) versus Velcade-treated (black bar) mdx mice.

Figure 12

Inhibition of the proteasome pathway up-regulates the membrane expression of dystrophin and its associated proteins in muscle explants from BMD patients. Muscle explants were incubated in the absence or presence of Velcade at 1 and 10 μmol/L concentrations for 16 hours. Frozen skeletal muscle sections from untreated and Velcade-treated BMD explants were immunostained with specific antibodies directed against dystrophin (DYS), β-dystroglycan (β-DG), and α-sarcoglycan (α-SG). DYS, α-SG, and β-DG were reduced and showed an irregular membrane staining in the skeletal muscle fibers from untreated muscle explants, whereas they displayed an enhanced and uniform positivity on the muscle plasma membrane following Velcade exposure. Final magnification, ×25. Representative images are shown.

Figure 13

Inhibition of the proteasome pathway up-regulates protein levels of members of the dystrophin-glycoprotein complex in human muscle explants from BMD patients. Muscle explants were incubated in the absence or presence of Velcade at 1 and 10 μmol/L concentrations for 16 hours. Total protein lysates from control, BMD untreated and Velcade-treated muscle explants were separated by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and subjected to WB analysis with specific antibodies against dystrophin (DYS), α-sarcoglycan (α-SG), and β-dystroglycan (β-DG). In the Velcade-group DYS protein levels were significantly increased. Notably, DYS migrated at a molecular weight of 357 kDa that is expected according to the patient’s mutation (black arrow). A lower protein band (approximately 200 kDa) was detected (black arrowhead). This signal could relate to the dystrophin isoform Dp260, migrating at a lower molecular size because of the patient’s mutation. α-SG and β-DG were increased by 30% and 60%. Vinculin (Vinc) was used as a loading control.

Figure 14

Inhibition of the proteasome pathway up-regulates the membrane expression of members of the dystrophin-glycoprotein complex in human muscle explants from DMD patients. Muscle explants were incubated in the absence or presence of Velcade at 1 and 10 μmol/L concentrations for 16 hours. Frozen skeletal muscle sections from untreated and Velcade-treated DMD explants were immunostained with specific antibodies directed against dystrophin (DYS), β-dystroglycan (β-DG), and α-sarcoglycan (α-SG). A-SG, β-DG were reduced in the skeletal muscle fibers from untreated muscle explants, whereas they displayed an enhanced and uniform membrane staining on the muscle plasma membrane following Velcade exposure. As expected in the untreated group, DYS is negative, while, in the Velcade samples, it displays positivity in few fibers. Final magnification, ×25. Representative images are shown.

Figure 15

Inhibition of the proteasome pathway up-regulates the protein levels of members the dystrophin-glycoprotein complex in muscle explants from DMD patients. Muscle explants were incubated in the absence or presence of Velcade at 1 and 10 μmol/L concentrations for 16 hours. Total protein lysates from control, DMD untreated, and Velcade-treated muscle explants were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and subjected to immunoblot analysis with specific antibodies against α-sarcoglycan (α-SG) and β-dystroglycan (β-DG). Note that α-SG and β-DG are severely reduced in the untreated muscle explant from the DMD patient when compared with the control subject (C). Velcade increases α-SG and β-DG protein levels by 60% and 70%. Vinculin (Vinc) was used as a loading control.