Overexpression of the cytotoxic T cell (CT) carbohydrate inhibits muscular dystrophy in the dyW mouse model of congenital muscular dystrophy 1A

Abstract

A number of recent studies have demonstrated therapeutic effects of transgenes on the development of muscle pathology in the mdx mouse model for Duchenne muscular dystrophy, but none have been shown also to be effective in mouse models for laminin alpha2-deficient congenital muscular dystrophy (MDC1A). Here, we show that overexpression of the cytotoxic T cell (CT) GalNAc transferase (Galgt2) is effective in inhibiting the development of muscle pathology in the dy(W) mouse model of MDC1A, much as we had previously shown in mdx animals. Embryonic overexpression of Galgt2 in skeletal muscles using transgenic mice or postnatal overexpression using adeno-associated virus both reduced the extent of muscle pathology in dy(W)/dy(W) skeletal muscle. As with mdx mice, embryonic overexpression of the Galgt2 transgene in dy(W)/dy(W) myofibers inhibited muscle growth, whereas postnatal overexpression did not. Both embryonic and postnatal overexpression of Galgt2 in dy(W)/dy(W) muscle increased the expression of agrin, a protein that, in recombinant form, has been shown to ameliorate disease, whereas laminin alpha1, another disease modifier, was not expressed. Galgt2 over-expression also stimulated the glycosylation of a gly-colipid with the CT carbohydrate, and glycolipids accounted for most of the CT-reactive material in postnatal overexpression experiments. These experiments demonstrate that Galgt2 overexpression is effective in altering disease progression in skeletal muscles of dy(W) mice and should be considered as a therapeutic target in MDC1A.

Figures

Figure 1

Galgt2 transgenic dyW/dyW skeletal muscles have reduced muscle size and reduced muscle pathology. Cross sections of gastrocnemius and tibialis anterior muscles from age-matched dyW/+, Galgt2 transgenic (CT) dyW/+, dyW/dyW, and dyW/dyW/CT mice were stained with H&E. Nuclei (darker spots, arrows) remain centrally localized in dystrophic dyW/dyW muscles that have undergone a cycle of degeneration and regeneration but are primarily absent from dyW/dyW/CT muscle. Scale bar = 50 μm.

Figure 2

Quantitation of muscle growth and muscular dystrophy in Galgt2 transgenic dyW/dyW skeletal muscles. A: Galgt2 transgenic mice (CT) had reduced myofiber diameters in both the dyW/+ and dyW/dyW background at 5 weeks of age. P values are for dyW/+/CT versus dyW/+ or for dyW/dyW/CT versus dyW/dyW. B: Galgt2 transgenic dyW/dyW myofibers had fewer myofibers with central nuclei at 5 weeks of age. P values are for dyW/dyW/CT versus dyW/dyW. TA, tibialis anterior. C: dyW/dyW mice had elevated creatine activity in their serum at 5 weeks of age, whereas Galgt2 transgenic dyW/dyW mice had significantly reduced levels compared with dyW/dyW littermates. P values are for dyW/dyW/CT versus dyW/dyW. Errors are SEM for 3 to 6 animals in which data were collected from 250 myofibers per measurement in A and B and SEM for n = 4 to 13 animals per genotype in C.

Figure 3

Time course of Galgt2 overexpression and CT carbohydrate expression in AAV-Galgt2-infected dyW/dyW skeletal muscles. A: Real-time PCR measurements were made at 1, 2, 3, 4, and 8 weeks after AAV-Galgt2 infection of dyW/dyW skeletal muscle (using AAV1 serotype). Galgt2 overexpression was significant at 1 week after infection and peaked by 4 weeks after infection, remaining high thereafter. P values are all compared with the 0 time point. B: CT carbohydrate overexpression, identified by staining with the CT2 antibody, paralleled Galgt2 gene overexpression, being evident by 1 week and maximal by 3 to 4 weeks of age. Note that although CT carbohydrate is expressed at 0 weeks at the neuromuscular junction and in capillaries, it was not evident at the time exposures used to observe overexpression at this low-level magnification. Secondary only mAb control is shown for 4 weeks. C: Longitudinal section of skeletal muscle (tibialis anterior) at 8 weeks after infection demonstrates that CT carbohydrate overexpression was maintained in most myofibers along their length. Scale bars = 100 μm.

Figure 4

Postnatal CT carbohydrate overexpression after AAV-Galgt2 infection inhibits muscle pathology but not muscle growth. AAV-Galgt2-infected dyW/dyW myofibers were analyzed for CT carbohydrate overexpression (using CT2 immunostaining, green) and for the presence of central nuclei (red). Several central localized myofiber nuclei are indicated with white arrows (F). Most CT-overexpressing myofibers did not have central nuclei. Scale bars: 100 μm (A–E); 50 μm (F).

Figure 5

Quantitation of myofiber diameters and central nuclei in AAV-Galgt2-infected dyW/dyW skeletal muscles. dyW/dyW skeletal muscles were infected with AAV-Galgt2 at 2 weeks of age and analyzed at 10 weeks of age. A: The percentage of myofibers with central nuclei was significantly reduced in both the gastrocnemius and tibialis anterior (TA) muscles in CT-overexpressing myofibers. B: CT-overexpressing myofibers did not have reduced myofiber diameters. Errors are SEM measured from three to six animals per genotype in which data were collected from 250 myofibers per measurement. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 6

Endogenous Galgt2 gene expression is increased in mdx and dyW/dyW muscle and the Galgt2 transgene is differentially overexpressed in the two disease models. A: Galgt2 gene expression was measured by TaqMan RT-PCR in mdx and dyW/dyW skeletal muscle compared with strain-specific control littermates. Endogenous Galgt2 expression is increased in both muscular dystrophy models. Errors are SEM for n = 12 to 18 animals per condition. B: Galgt2 transgenic dyW/dyW skeletal muscle has 60-fold less Galgt2 gene expression than Galgt2 transgenic mdx skeletal muscle, and AAV-Galgt2-infected muscles are also reduced (4.7-fold). Errors are SEM for n = 6 to 18 animals per condition. All data are from gastrocnemius muscles of 6- to 8-week-old animals. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 7

α-Dystroglycan glycosylation with the CT carbohydate is increased in Galgt2 transgenic dyW/dyW muscles but not after postnatal infection with AAV-Galgt2. Identical amounts of skeletal muscle Nonidet P-40 protein homogenates were precipitated by WGA agarose, a lectin that binds the GlcNAc/sialic acid, or by Wisteria floribunda agglutinin (WFA) agarose, a GalNAc-binding lectin that recognizes the CT carbohydrate. Precipitates were blotted for α-dystroglycan (αDG), β-dystroglycan (βDG), or CT2, an anti-CT carbohydrate antibody. α-Dystroglycan glycosylation with the CT carbohydrate was increased in Galgt2 transgenic muscles but not in AAV-Galgt2-infected muscles. α-Dystroglycan appears broader than CT carbohydrate because it was separated on a 6% SDS-PAGE gel, to allow greater resolution of its glycosylation, whereas β-dystroglycan and CT2 blots were separated on a 12% SDS-PAGE gel, to allow visualization of greater numbers of proteins.

Figure 8

Ionic and nonionic detergent extraction yield similar results with respect to protein glycosylation in Galgt2 transgenic and AAV-Galgt2-infected dyW/dyW skeletal muscles. A and B: Enzyme-linked immunosorbent assay assays were done for CT carbohydrate (A, using CT2) or α-dystroglycan (B, using IIH6) using 20 μg of skeletal muscle cell lysate made by extraction with nonionic detergent (1% Nonidet P-40) or with ionic denaturing detergent (2% SDS with 4 mol/L urea). Errors are SD for n = 3 to 6 animals. C: SDS-urea-extracted glycoproteins were dialyzed against 1% Nonidet P-40 and compared with Nonidet P-40-extracted samples (Figure 7) for the ability of the GalNAc-binding lectin WFA to precipitate CT-glycosylated glycoproteins. No significant change was observed compared with extraction in Nonidet P-40 alone.

Figure 9

Galgt2 overexpression in skeletal muscle increases the glycosylation of a glycolipid with the CT carbohydrate. A: Glycolipids were extracted from skeletal muscles and separated by high-performance thin layer chromatography, followed by CT2 antibody overlay to identify CT-glycosylated glycolipids. Galgt2 transgenic (Tg) skeletal muscles had a large increase in a single glycolipid (arrow) whose migration was distinct from that of control gangliosides. B: Cross sections of skeletal muscle were immunostained with CT carbohydrate antibody (CT2) before or after extraction of lipids from the section. Some CT staining remained in Galgt2 transgenic (Tg) dyW/dyW muscle after lipid extraction, whereas very little staining remained in AAV-Galgt2-infected dyW/dyW muscles. Arrows point to a few positively stained remaining myofibers. A, arteriole; V, vein. C: Similar results were obtained in Galgt2 transgenic and AAV-Galgt2-infected mdx muscle. Scale bars: 50 μm [B (bottom) and C]; 25 μm [B (top)].

Figure 10

Utrophin and agrin are overexpressed in Galgt2 transgenic dyW/dyW myofibers relative to dyW/dyW muscle. A: Cross sections of dyW/dyW skeletal muscle or Galgt2 transgenic (CT) dyW/dyW skeletal muscle were immunostained with antibodies to the indicated proteins. Galgt2 transgenic muscle had increased expression of utrophin and agrin along extrasynpatic regions of the sarcolemma, whereas laminin α1 was not expressed. B: Staining compares dyW/dyW and dyW/+ muscles. Arrows point to aggregates of CT-stained mononuclear cells, which are probably immune cell infiltrates within dystrophic muscle. nmj labels neuromuscular junction, the CT-positive structure just to the left of the label. Scale bars = 50 μm (A, B).

Figure 11

Expression of proteins in Galgt2 transgenic and AAV-Galgt2-infected dyW/dyW muscles by immunoblotting. Whole-cell muscle protein lysates (20 μg) were immunoblotted with the indicated antibodies. Utrophin protein was increased in Galgt2 transgenic (Tg) dyW/dyW skeletal muscle but not in AAV-Galgt2-infected dyW/dyW skeletal muscle, whereas agrin protein was increased in both. Molecular masses are 400 d for utrophin, agrin, laminin α1, and dystrophin, 200 d for laminin α4, 350 d for laminin α5, 43 d for β-dystroglycan, ∼160 to 180 d for α-dystroglycan, 42 kd for actin.

Figure 12

Transcription of laminin α4 and agrin is increased in dyW/dyW skeletal muscle but is not further increased in Galgt2 transgenic or AAV-Galgt2-infected dyW/dyW muscle. RNA was extracted from skeletal muscles and subjected to semiquantitative TaqMan RT-PCR for the indicated genes. No ECM genes or utrophin were increased in Galgt2 transgenic dyW/dyW or AAV-Galgt2-infected dyW/dyW when compared with dyW/dyW skeletal muscle. Laminin α1 transcription was not present in skeletal muscle. Errors are SEM for n = 3 to 6 animals. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 13

Expression of utrophin and extracellular matrix proteins in CT-overexpressing dyW/dyW myofibers after AAV-Galgt2 infection. dyW/dyW muscles were infected with AAV-Galgt2 at 2 weeks of age and analyzed for CT carbohydrate overexpression at 10 weeks of age using CT2 immunostaining at low (×20, left) and high (×40, right) power. Staining of serial sections with antibodies to utrophin, agrin, laminin α2, laminin α4, or laminin α5 (panels below CT2 immunostains) showed high levels of agrin and laminin α4 in regions of AAV-Galgt2 infection. Laminin α1 was not expressed. Scale bars: 50 μm (left); 25 μm (right).