Artificial restoration of the linkage between laminin and dystroglycan ameliorates the disease progression of MDC1A muscular dystrophy at all stages

Laminin-α2 deficient congenital muscular dystrophy, classified as MDC1A, is a severe progressive muscle-wasting disease that leads to death in early childhood. MDC1A is caused by mutations in lama2, the gene encoding the laminin-α2 chain being part of laminin-2, the main laminin isoform present in the extracellular matrix of muscles and peripheral nerves. Via selfpolymerization, laminin-2 forms the primary laminin scaffold and binds with high affinity to α- dystroglycan on the cell surface, providing a connection to the cytoskeleton via the transmembranous protein β-dystroglycan. Deficiency in laminin-α2 leads to absence of laminin-2 and to upregulation of laminin-8, a laminin isoform that cannot self-polymerize and does not bind to α-dystroglycan. Therefore, in laminin α2-deficient muscle the chain of proteins linking the intracellular contractile apparatus via the plasma membrane to the extracellular matrix is interrupted. Consequently, muscle fibers loose their stability and degenerate what finally leads to a progressive muscle wasting. In previous studies, we have shown that a miniaturized form of the extracellular matrix protein agrin, which is not related to the disease-causing lama2 gene and was designed to contain highaffinity binding sites for the laminins and for α-dystroglycan, was sufficient to markedly improve muscle function and overall health in the dyW-/- mouse model of MDC1A. In a follow-up study we provided additional evidence that mini-agrin, both increases the tolerance to mechanical load but also improves the regeneration capacity of the dystrophic muscle. We now report on our progress towards further testing the use of this approach for the treatment of MDC1A. To test whether mini-agrin application after onset of the disease would still ameliorate the dystrophic symptoms, we have established the inducible tetracycline-regulated “tet-off” expression system in dyW-/- mice to temporally control mini-agrin expression in skeletal muscles. We show that mini-agrin slows down the progression of the dystrophy when applied at birth or in advanced stages of the disease. However, the extent of the amelioration depends on the dystrophic condition of the muscle at the time of mini-agrin application. Thus, the earlier miniagrin is applied, the higher is the profit of its beneficial properties. In addition to gene therapeutical approaches, the increase of endogenous agrin expression levels in skeletal muscles by pharmacologically active compounds would be a safe and promising strategy for the treatment of MDC1A. To evaluate the potential and pave the way to further expand on the development of such a treatment, we determined whether full-length agrin ameliorates the dystrophic phenotype to a comparable extent as it was observed by application of mini-agrin. We provide evidence that constitutive overexpression of chick full-length agrin in dyW-/- muscle ameliorates the dystrophic phenotype, although not as pronounced as mini-agrin does. In conclusion, our results are conceptual proof that linkage of laminin to the muscle fiber membrane is a means to treat MDC1A at any stage of the disease. Our findings definitely encourage to further expanding on this therapeutic concept, especially in combination with treatment using functionally different approaches. Moreover, these experiments set the basis for further developing clinically feasible and relevant application methods such as gene therapy4 and/or the screening of small molecules able to upregulate production of agrin in muscle

Angiotensin II type 1 receptor antagonists alleviate muscle pathology in the mouse model for laminin-alpha2-deficient congenital muscular dystrophy (MDC1A)

BACKGROUND: Laminin-alpha2-deficient congenital muscular dystrophy (MDC1A) is a severe muscle-wasting disease for which no curative treatment is available. Antagonists of the angiotensin II receptor type 1 (AT1), including the anti-hypertensive drug losartan, have been shown to block also the profibrotic action of transforming growth factor (TGF)-beta and thereby ameliorate disease progression in mouse models of Marfan syndrome. Because fibrosis and failure of muscle regeneration are the main reasons for the severe disease course of MDC1A, we tested whether L-158809, an analog derivative of losartan, could ameliorate the dystrophy in dyW/dyW mice, the best-characterized model of MDC1A. METHODS: L-158809 was given in food to dyW/dyW mice at the age of 3 weeks, and the mice were analyzed at the age of 6 to 7 weeks. We examined the effect of L-158809 on muscle histology and on muscle regeneration after injury as well as the locomotor activity and muscle strength of the mice. RESULTS: We found that TGF-beta signaling in the muscles of the dyW/dyW mice was strongly increased, and that L-158809 treatment suppressed this signaling. Consequently, L-158809 reduced fibrosis and inflammation in skeletal muscle of dyW/dyW mice, and largely restored muscle regeneration after toxin-induced injury. Mice showed improvement in their locomotor activity and grip strength, and their body weight was significantly increased. CONCLUSION: These data provide evidence that AT1 antagonists ameliorate several hallmarks of MDC1A in dyW/dyW mice, the best-characterized mouse model for this disease. Because AT1 antagonists are well tolerated in humans and widely used in clinical practice, these results suggest that losartan may offer a potential future treatment of patients with MDC1A

Chimeric protein repair of laminin polymerization ameliorates muscular dystrophy phenotype

Mutations in laminin α2-subunit (Lmα2, encoded by LAMA2) are linked to approximately 30% of congenital muscular dystrophy cases. Mice with a homozygous mutation in Lama2 (dy2J mice) express a nonpolymerizing form of laminin-211 (Lm211) and are a model for ambulatory-type Lmα2-deficient muscular dystrophy. Here, we developed transgenic dy2J mice with muscle-specific expression of αLNNd, a laminin/nidogen chimeric protein that provides a missing polymerization domain. Muscle-specific expression of αLNNd in dy2J mice resulted in strong amelioration of the dystrophic phenotype, manifested by the prevention of fibrosis and restoration of forelimb grip strength. αLNNd also restored myofiber shape, size, and numbers to control levels in dy2J mice. Laminin immunostaining and quantitation of tissue extractions revealed increased Lm211 expression in αLNNd-transgenic dy2J mice. In cultured myotubes, we determined that αLNNd expression increased myotube surface accumulation of polymerization-deficient recombinant laminins, with retention of collagen IV, reiterating the basement membrane (BM) changes observed in vivo. Laminin LN domain mutations linked to several of the Lmα2-deficient muscular dystrophies are predicted to compromise polymerization. The data herein support the hypothesis that engineered expression of αLNNd can overcome polymerization deficits to increase laminin, stabilize BM structure, and substantially ameliorate muscular dystrophy

Improving Reproducibility of Phenotypic Assessments in the DyW Mouse Model of Laminin-α2 Related Congenital Muscular Dystrophy.

Laminin-α2 related Congenital Muscular Dystrophy (LAMA2-CMD) is a progressive muscle disease caused by partial or complete deficiency of laminin-211, a skeletal muscle extracellular matrix protein. In the last decade, basic science research has queried underlying disease mechanisms in existing LAMA2-CMD murine models and identified possible clinical targets and pharmacological interventions. Experimental rigor in preclinical studies is critical to efficiently and accurately quantify both negative and positive results, degree of efficiency of potential therapeutics and determine whether to move a compound forward for additional preclinical testing. In this review, we compare published available data measured to assess three common parameters in the widely used mouse model DyW, that mimics LAMA2-CMD, we quantify variability and analyse its possible sources. Finally, on the basis of this analysis, we suggest standard set of assessments and the use of available standardized protocols, to reduce variability of outcomes in the future and to improve the value of preclinical research

Improving Reproducibility of Phenotypic Assessments in the DyW Mouse Model of Laminin-α2 Related Congenital Muscular Dystrophy

Laminin-α2 related Congenital Muscular Dystrophy (LAMA2-CMD) is a progressive muscle disease caused by partial or complete deficiency of laminin-211, a skeletal muscle extracellular matrix protein. In the last decade, basic science research has queried underlying disease mechanisms in existing LAMA2-CMD murine models and identified possible clinical targets and pharmacological interventions. Experimental rigor in preclinical studies is critical to efficiently and accurately quantify both negative and positive results, degree of efficiency of potential therapeutics and determine whether to move a compound forward for additional preclinical testing. In this review, we compare published available data measured to assess three common parameters in the widely used mouse model DyW, that mimics LAMA2-CMD, we quantify variability and analyse its possible sources. Finally, on the basis of this analysis, we suggest standard set of assessments and the use of available standardized protocols, to reduce variability of outcomes in the future and to improve the value of preclinical research

Fatigue and Muscle Atrophy in a Mouse Model of Myasthenia Gravis Is Paralleled by Loss of Sarcolemmal nNOS

Myasthenia Gravis (MG) patients suffer from chronic fatigue of skeletal muscles, even after initiation of proper immunosuppressive medication. Since the localization of neuronal nitric oxide synthase (nNOS) at the muscle membrane is important for sustained muscle contraction, we here study the localization of nNOS in muscles from mice with acetylcholine receptor antibody seropositive (AChR+) experimental autoimmune MG (EAMG). EAMG was induced in 8 week-old male mice by immunization with AChRs purified from torpedo californica. Sham-injected wild type mice and mdx mice, a model for Duchenne muscular dystrophy, were used for comparison. At EAMG disease grade 3 (severe myasthenic weakness), the triceps, sternomastoid and masseter muscles were collected for analysis. Unlike in mdx muscles, total nNOS expression as well as the presence of its binding partner syntrophin ?-1, were not altered in EAMG. Immunohistological and biochemical analysis showed that nNOS was lost from the muscle membrane and accumulated in the cytosol, which is likely the consequence of blocked neuromuscular transmission. Atrophy of all examined EAMG muscles were supported by up-regulated transcript levels of the atrogenes atrogin-1 and MuRF1, as well as MuRF1 protein, in combination with reduced muscle fiber diameters. We propose that loss of sarcolemmal nNOS provides an additional mechanism for the chronic muscle fatigue and secondary muscle atrophy in EAMG and MG

Apoptosis inhibitors and mini-agrin have additive benefits in congenital muscular dystrophy mice

Mutations in LAMA2 cause a severe form of congenital muscular dystrophy, called MDC1A. Studies in mouse models have shown that transgenic expression of a designed, miniaturized form of the extracellular matrix molecule agrin ('mini-agrin') or apoptosis inhibition by either overexpression of Bcl2 or application of the pharmacological substance omigapil can ameliorate the disease. Here, we tested whether mini-agrin and anti-apoptotic agents act on different pathways and thus exert additive benefits in MDC1A mouse models. By combining mini-agrin with either transgenic Bcl2 expression or oral omigapil application, we show that the ameliorating effect of mini-agrin, which acts by restoring the mechanical stability of muscle fibres and, thereby, reduces muscle fibre breakdown and concomitant fibrosis, is complemented by apoptosis inhibitors, which prevent the loss of muscle fibres. Treatment of mice with both agents results in improved muscle regeneration and increased force. Our results show that the combination of mini-agrin and anti-apoptosis treatment has beneficial effects that are significantly bigger than the individual treatments and suggest that such a strategy might also be applicable to MDC1A patients

Total transcript and protein levels of nNOS are normal in muscles of EAMG mice.

<p>(A) Total mRNA amount of nNOS in triceps, masseter and sternomastoid muscles. Negative controls were muscles from healthy CFA/PBS injected control mice (ctrl; n = 3) and as positive control muscles from <i>mdx</i> mice were used (<i>mdx</i>; n = 3). In the AChR+ EAMG muscles (AChR+; n = 6), total nNOS levels were unchanged as compared to controls (triceps p = 0.55; triceps p = 0.32; triceps p = 0.90). nNOS mRNA was clearly reduced in <i>mdx</i> muscles. (B) Western blot analysis of nNOS (160 kD) levels in total protein extracts from triceps (Trc), masseter (Mass) and sternomastoid (STM) muscles. Representative results are shown. No difference was seen between nNOS protein content in AChR+ EAMG (AChR+; n = 3) and control (ctrl; n = 3) muscles, whereas nNOS protein was reduced in muscles of <i>mdx</i> (<i>mdx</i>; n = 3)-mice to approximately 20%. β-actin was used as reference protein.</p

Clinical characteristics of EAMG mice that developed a severe disease phenotype (EAMG grade 3; n = 8).

<p>(A) Loss of body weight in the AChR+ EAMG mice started after day 28. Day of dissection is indicated by a square (35, 42 or 49 days after post-immunization). (B) The time AChR+ EAMG mice could hold on to the upside-down grid is displayed. Mice with a grid time <0 seconds (EAMG grade 3) were sacrificed for analysis. The day of dissection is indicated by a square. (C) ELISA analyzed the immunoreactivity of AChR autoantibodies in sera from immunized mice. The background from negative controls was subtracted and anti-AChR antibody mAb35 was used as positive control. Half maximum titer is indicated. Control mice (ctrl) were immunized with CFA/PBS.</p