Eva Engvall

Canine and feline models of human inherited muscle diseases

Animal models are of immense importance for studying mechanisms of disease and testing new therapies, and rodents have been used extensively in the field of neuromuscular disorders. Mice and rats can be genetically manipulated to over-express or not express genes that are important to muscle function, and these animals can be available in large numbers for analysis. Other species, such as cats and dogs, cannot be manipulated in the same ways or be used in large numbers, but they have spontaneously occurring muscle diseases with clinical presentations more closely resembling those of the human disorders. Therefore, cats and dogs may become valuable as intermediate disease models. This review focuses on canine and feline models of human inherited muscle diseases with comparisons to rodent models and an emphasis on the muscular dystrophies.

The new frontier in muscular dystrophy research: booster genes

More than 30 different forms of muscular dystrophy (MD) have been molecularly characterized and can be diagnosed, but progress toward treatment has been slow. Gene replacement therapy has met with great difficulty because of the large size of the defective genes and because of difficulties in delivering a gene to all muscle groups. Cell replacement therapy has also been difficult to realize. Will it even be possible to design specific therapy protocols for all MDs? Or is a more realistic goal to treat some of the secondary manifestations that are common to several forms of MD, such as membrane instability, necrosis, and inflammation, and to promote regeneration? As reviewed here, enhanced expression of a range of proteins provides a boost for degenerating dystrophic muscle in mouse models. Expression of a mini‐agrin promotes basement membrane formation instead of laminin α2; integrin α7, GalNac transferase, and ADAM12 promote cell adhesion and muscle stability in the absence of dystrophin; calpastatin prevents muscle necrosis; and nitric oxide synthase prevents inflammation. ADAM12, IGF‐I, and myostatin blockade promote regeneration and reduce fibrosis. One can envision numerous other candidate booster genes which encode proteins that promote survival and/or regeneration of the compromised muscle or proteins that affect post‐translational modifications of critical proteins. Finally, fibrosis, which is the curse of many human diseases, may also be attacked. Once the mechanisms of the boosters are better understood, drugs may be developed to provide the boost to muscle. Some of the experiences in models of muscular dystrophy may inspire new approaches in other genetic degenerative diseases as well.—Engvall, E., Wewer, U. M. The new frontier in muscular dystrophy research: booster genes. FASEB J. 17, 1579–1584 (2003)

Laminins of the adult mammalian CNS; laminin-α2 (merosin M-) chain immunoreactivity is associated with neuronal processes

Laminins are glycoproteins with three subunits, i.e. a longer alpha chain, a shorter beta chain and a shorter gamma chain. Well-characterized laminins are laminin-1 (EHS laminin; alpha1-beta1-gamma1), laminin-2 (merosin; alpha2-beta1-gamma1), laminin-3 (alpha1-beta2-gamma1) and laminin-4 (alpha2-beta2-gamma1). The present study shows that in the adult mammalian CNS (rat, rabbit, pig and monkey) alpha2 chain immunoreactivity is associated most evidently with neuronal fibers and punctate, potentially synaptic, structures of limbic brain regions. Third ventricle tanycytes and ensheathing cells of the olfactory nerve also express intense alpha2 chain immunoreactivity. Immunostaining for gamma1 chain is present throughout the central nervous system (CNS) in essentially all neuronal cell bodies and their most proximal processes. Immunoreactivity for all chains investigated (alpha1, alpha2, beta1, beta2 and gamma1) were present around blood vessels, especially evident in lightly fixed tissues. The finding that, other than blood vessels, neurons and other structures exhibited immunoreactivity for only one or two (and not three) chains, suggests that variant forms of laminin with yet undiscovered chains or other configurations than the heterotrimeric form are present in the CNS. The association of alpha2-like immunoreactivity with neuronal fibers and synaptic structures is of great interest in light of the known neurite-promoting and cell attachment activities of laminin-2.

Laminin α2 deficiency and muscular dystrophy; genotype-phenotype correlation in mutant mice

Deficiency of laminin alpha2 is the cause of one of the most severe muscular dystrophies in humans and other species. It is not yet clear how particular mutations in the laminin alpha2 chain gene affect protein expression, and how abnormal levels or structure of the protein affect disease. Animal models may be valuable for such genotype-phenotype analysis and for determining mechanism of disease as well as function of laminin. Here, we have analyzed protein expression in three lines of mice with mutations in the laminin alpha2 chain gene and in two lines of transgenic mice overexpressing the human laminin alpha2 chain gene in skeletal muscle. The dy(3K)/dy(3K) experimental mutant mice are completely deficient in laminin alpha2; the dy/dy spontaneous mutant mice have small amounts of apparently normal laminin; and the dy(W)/dy(W) mice express even smaller amounts of a truncated laminin alpha2, lacking domain VI. Interestingly, all mutants lack laminin alpha2 in peripheral nerve. We have demonstrated previously, that overexpression of the human laminin alpha2 in skeletal muscle in dy(2J)/dy(2J) and dy(W)/dy(W) mice under the control of a striated muscle-specific creatine kinase promoter substantially prevented the muscular dystrophy in these mice. However, dy(W)/dy(W) mice, expressing the human laminin alpha2 under the control of the striated muscle-specific portion of the desmin promoter, still developed muscular dystrophy. This failure to rescue is apparently because of insufficient production of laminin alpha2. This study provides additional evidence that the amount of laminin alpha2 is most critical for the prevention of muscular dystrophy. These data may thus be of significance for attempts to treat congenital muscular dystrophy in human patients.

Elimination of myostatin does not combat muscular dystrophy in dy mice but increases postnatal lethality.

Myostatin is a TGF-beta family member and a negative regulator of skeletal muscle growth. It has been proposed that reduction or elimination of myostatin could be a treatment for degenerative muscle diseases such as muscular dystrophy. Laminin-deficient congenital muscular dystrophy is one of the most severe forms of muscular dystrophy. To test the possibility of ameliorating the dystrophic phenotype in laminin deficiency by eliminating myostatin, we crossed dy(W) laminin alpha2-deficient and myostatin null mice. The resulting double-deficient dy(W)/dy(W);Mstn(-/-) mice had a severe clinical phenotype similar to that of dy(W)/dy(W) mice, even though muscle regeneration was increased. Degeneration and inflammation of muscle were not alleviated. The pre-weaning mortality of dy(W)/dy(W);Mstn(-/-) mice was increased compared to dy(W)/dy(W), most likely due to significantly less brown and white fat in the absence of myostatin, and postweaning mortality was not significantly improved. These results show that eliminating myostatin in laminin-deficiency promotes muscle formation, but at the expense of fat formation, and does not reduce muscle pathology. Any future therapy based on myostatin may have undesirable side effects.

ADAM12 Alleviates the Skeletal Muscle Pathology in mdx Dystrophic Mice

Muscular dystrophy is characterized by muscle degeneration and insufficient regeneration and replacement of muscle fibers by connective tissue. New therapeutic strategies directed toward various forms of muscular dystrophy are needed to preserve muscle mass and promote regeneration. In this study we examined the role of the transmembrane ADAM12, a disintegrin and metalloprotease, which is normally associated with development and regeneration of skeletal muscle. We demonstrate that ADAM12 overexpression in the dystrophin-deficient mdx mice alleviated the muscle pathology in these animals, as evidenced by less muscle cell necrosis and inflammation, lower levels of serum creatine kinase, and less uptake of Evans Blue dye into muscle fibers. These studies demonstrate that ADAM12 directly or indirectly contributes to muscle cell regeneration, stability, and survival.

Muscular dystrophies and other inherited myopathies.

It is certain that more inherited neuromuscular disorders of dogs and cats will be identified as the ability of practicing veterinarians to recognize disorders of muscle, nerve, and neuromuscular junction improves and newer diagnostic tests become available. Two specific points are critical. Before DNA-based genetic tests and specific therapies can be developed, an accurate description of the problem, clinically and histopathologically, must be performed. This is particularly important for the accuracy of a pedigree analysis, because inclusion of dogs with unrelated problems would alter the interpretation. Second, animals with inherited breed-associated disease should not be bred for generation of companion animals.

Laminin-α2 chain-like antigens in CNS dendritic spines

Abstract The laminin-α2 chain is a component of brain capillary basement membranes and appears also to be present in neurons of rat, rabbit, pig and non-human primate brain as evidenced by immunohistochemistry. In the present study, we have further characterized this very distinct neuronal laminin-α2 chain-like immunoreactivity in the hippocampus of various species. Immunoelectron microscopy with poly- and monoclonal antibodies to the laminin-α2 chain G-domain localized laminin-α2 chain immunoreactivity in adult rat and rabbit hippocampus to dendritic processes, primarily to dendritic spines. In the developing rat hippocampus, spine-associated laminin-α2 chain-like immunoreactivity first appeared at a time corresponding to that of active synaptogenesis. After an entorhinal cortex lesion in adult rats, the time course of denervation-induced loss and reactive reappearance of spines in the molecular layer of the dentate gyrus was correlated closely to the loss and reappearance of laminin-α2 chain immunoreactivity. Immunoblot analysis of normal adult rat, rabbit and pig brain revealed a protein similar in size to the reported 80-kDa laminin-α2 chain fragment of human placenta as well as 140/160-kDa proteins. These results suggest the presence of proteins with antigenic homology to the laminin-α2 chain and/or laminin-α2 isoforms in dendrites and dendritic spines in selected areas of the brain, predominately in the hippocampus and other limbic structures. Given the adhesion and neurite promoting functions of laminins, it is possible that neuronal laminin-α2 chain-like proteins play a role in synaptic function and plasticity in the CNS.

Dystrophin-Deficient Muscular Dystrophy in a Labrador Retriever

Sex-linked muscular dystrophy associated with dystrophin deficiency has been reported in several breeds of dogs and is best characterized in the golden retriever. In this case report, a young, male Labrador retriever with dystrophin-deficient muscular dystrophy is presented. Clinical signs included generalized weakness, lingual hypertrophy, and dysphagia. Electromyographic abnormalities including complex repetitive discharges were present. Serum creatine kinase concentration was dramatically elevated. Histopathological changes within a muscle biopsy specimen confirmed a dystrophic myopathy, and dystrophin deficiency was demonstrated by immunohistochemical staining. While X-linked muscular dystrophy has not previously been reported in the Labrador retriever, a hereditary myopathy with an autosomal recessive mode of inheritance has been characterized. A correct diagnosis and classification of these two disorders are critical for breeders and owners since both the mode of inheritance and the prognosis differ.

Laminin α2 (merosin)-deficient muscular dystrophy and demyelinating neuropathy in two cats

We report laminin α2 (merosin) deficiency associated with muscular dystrophy and demyelinating neuropathy in two cats. The cats developed progressive muscle weakness, and atrophy. Either hypotonia or contractures resulted in recumbency, necessitating euthanasia. Muscle biopsies showed dystrophic changes including marked endomysial fibrosis, myofiber necrosis, variability of fiber size, and perimysial lipid accumulation. Immunohistochemistry showed that laminin α2 chain was absent or reduced, while dystrophin and all the components of the dystrophin-associated glycoprotein complex were present and normal. One cat was examined in detail. Motor nerve conduction velocity (MNCV) was decreased, and ultrastructurally the peripheral nerves showed Schwann cell degeneration and demyelination. Brain imaging was not performed, but white matter changes were not apparent in the brain at necropsy. The disease in these cats is similar to primary or secondary merosin (laminin α2)-deficient congenital muscular dystrophy (CMD) in humans and to dystrophia muscularis in mice.