ABSTRACT
The expression of neutral glycosphingolipids (GSLs) and gangliosides was investigated in cryosections of normal mouse skeletal muscle and in muscle of mice with neuromuscular diseases using indirect immunofluorescence microscopy. Transversal and longitudinal sections were immunostained with specific polyclonal antibodies against lactosylceramide, lacto-N-neotetraosylceramide, globoside, GM3(Neu5Ac), GM3(Neu5Gc) and Gm1(Neu5Ac) as well as monoclonal anti-Forssman GSL antibody. In normal CBA/J mouse muscle (control) the main immunohistochemically detected ganglioside was GM3(Neu5Ac) followed by moderately expressed GM3(Neu5Gc) and GM1. The neutral GSLs lactosylceramide and globoside were stained with almost identical, high fluorescence intensity. Low amounts of lacto-N-neotetraosylceramide and trace quantities of Forssman GSL were immunostained. All GSLs were detected in the sarcolemma, but also in considerable amounts at the intracellular level. Mice with neuromuscular diseases were the A2G-adr mouse mutant (a model for human recessive myotonia of Becker type), the BL6-wr mutant (a model for motor neuron disease) and the BL10-mdx mouse mutant (a model for human Duchenne muscular dystrophy). No changes in GSL expression were found in the A2G-adr mouse, while muscle of the BL6-wr mouse showed increased intensity of immunofluorescence in stainings with anti-lactosylceramide and anti-GM3(Neu5Ac) antibodies. Muscle of BL10-mdx mice showed the most prominent changes in GSL expression with reduced fluorescence intensity for all antibodies. Major differences were not observed in the intensities of GSLs, but there were significant differences in the patterns of distribution on plasma membrane and at the subcellular level. The exact nature and pathogenesis of these changes should be elucidated since such investigations could furnish advances in understanding the functional role of neutral GSLs and gangliosides in normal as well as in diseased muscle.
Subject(s)
Gangliosides/analysis , Glycosphingolipids/analysis , Muscle, Skeletal/cytology , Muscle, Skeletal/pathology , Muscular Dystrophy, Animal/pathology , Neuromuscular Diseases/pathology , Animals , Antibodies , Carbohydrate Sequence , Fluorescent Antibody Technique, Indirect , Gangliosides/chemistry , Glycosphingolipids/chemistry , Humans , Immunohistochemistry , Mice , Mice, Inbred mdx , Mice, Mutant Strains , Molecular Sequence Data , Motor Neuron Disease/pathology , Myotonia/pathology , Reference ValuesABSTRACT
The expression of neutral glycosphingolipids and gangliosides was investigated in mouse and rabbit skeletal muscle by means of biochemical and immunochemical techniques. Neutral glycosphingolipids from muscle of the inbred rabbit strain used in this study showed a simple TLC pattern, comprising mainly monohexosylceramide. In addition to this compound, lactosylceramide, lacto-N-neotetraosylceramide, globoside and Forssman GSL were detected in mouse muscle. The major ganglioside in both species was GM3; GM3 (Neu5Ac) and GM3(Neu5Gc) were found in a 3:1 ratio in mouse muscle, whereas the absence of GM3(Neu5Gc) is characteristic of rabbit muscle. As a general structural feature of all muscle GM3 gangliosides investigated, a C18 fatty acid and C18 sphingosine were the major components besides minor C22 and C24:1 fatty acids of the respective ceramide portions, as revealed by positive and negative ion FAB-MS. alpha 2-3 sialylated lacto-N-neotetraosyl-ceramide (sialylparagloboside) was expressed in both species, whereas the alpha 2-6 sialylated isomeric compound was found only in mouse muscle. Minute quantities of ganglio-series GM1, GD1a, GD1b, and GT1b were detected in muscles from both species. Glycosphingolipid expression could be confirmed immunohistochemically by examining transverse and longitudinal cryosections of skeletal muscle samples. The results provide the basis for the investigation of muscle specific glycosphingolipids that might modulate membrane protein functions in muscle.
