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Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5'-monophospho-(CMP)-N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome-linked muscular dystrophy (mdx) model for DMD. Cmah-/-mdx mice can be induced to develop anti-Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah-/-mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.

Humans and chimpanzees are more sensitive to endotoxin than are mice or monkeys, but any underlying differences in inflammatory physiology have not been fully described or understood. We studied innate immune responses in Cmah-/- mice, emulating human loss of the gene encoding production of Neu5Gc, a major cell surface sialic acid. CMP-N-acetylneuraminic acid hydroxylase (CMAH) loss occurred ∼2-3 million years ago, after the common ancestor of humans and chimpanzees, perhaps contributing to speciation of the genus HomoCmah-/- mice manifested a decreased survival in endotoxemia following bacterial LPS injection. Macrophages from Cmah-/- mice secreted more inflammatory cytokines with LPS stimulation and showed more phagocytic activity. Macrophages and whole blood from Cmah-/- mice also killed bacteria more effectively. Metabolic reintroduction of Neu5Gc into Cmah-/- macrophages suppressed these differences. Cmah-/- mice also showed enhanced bacterial clearance during sublethal lung infection. Although monocytes and monocyte-derived macrophages from humans and chimpanzees exhibited marginal differences in LPS responses, human monocyte-derived macrophages killed Escherichia coli and ingested E. coli BioParticles better. Metabolic reintroduction of Neu5Gc into human macrophages suppressed these differences. Although multiple mechanisms are likely involved, one cause is altered expression of C/EBPβ, a transcription factor affecting macrophage function. Loss of Neu5Gc in Homo likely had complex effects on immunity, providing greater capabilities to clear sublethal bacterial challenges, possibly at the cost of endotoxic shock risk. This trade-off may have provided a selective advantage when Homo transitioned to butchery using stone tools. The findings may also explain why the Cmah-/- state alters severity in mouse models of human disease.

The cytotoxic T cell (CT) GalNAc transferase, or Galgt2, is a UDP-GalNAc:[beta]1,4-N-acetylgalactosaminyltransferase that is localized to the neuromuscular synapse in adult skeletal muscle, where it creates the synaptic CT carbohydrate antigen {GalNAc[beta]1,4[NeuAc(orGc)[alpha]2, 3]Gal[beta]1,4GlcNAc[beta]-}. Overexpression of Galgt2 in the skeletal muscles of transgenic mice inhibits the development of muscular dystrophy in mdx mice, a model for Duchenne muscular dystrophy. Here, we provide physiological evidence as to how Galgt2 may inhibit the development of muscle pathology in mdx animals. Both Galgt2 transgenic wild-type and mdx skeletal muscles showed a marked improvement in normalized isometric force during repetitive eccentric contractions relative to nontransgenic littermates, even using a paradigm where nontransgenic muscles had force reductions of 95% or more. Muscles from Galgt2 transgenic mice, however, showed a significant decrement in normalized specific force and in hindlimb and forelimb grip strength at some ages. Overexpression of Galgt2 in muscles of young adult mdx mice, where Galgt2 has no effect on muscle size, also caused a significant decrease in force drop during eccentric contractions and increased normalized specific force. A comparison of Galgt2 and microdystrophin overexpression using a therapeutically relevant intravascular gene delivery protocol showed Galgt2 was as effective as microdystrophin at preventing loss of force during eccentric contractions. These experiments provide a mechanism to explain why Galgt2 overexpression inhibits muscular dystrophy in mdx muscles. That overexpression also prevents loss of force in nondystrophic muscles suggests that Galgt2 is a therapeutic target with broad potential applications. contraction; extensor digitorum longus; gene therapy; muscular dystrophy

Carbohydrates have been shown to mediate or modulate a number of important events in the development of the nervous system; however, there is little evidence that they participate directly in the development of synapses. One carbohydrate structure that is likely to be important in synaptic development of the neuromuscular junction is the CT carbohydrate antigen {GalNAc [beta]1,4 [NeuAc [alpha]2,3] Gal [beta]1(-3GalNAc or -4GlcNAc)}. The synaptic localization of the CT antigen is due to the presence of the terminal [beta]1,4 GalNAc linkage, and such linkages are localized to the neuromuscular junction in many species. Here we show that an enzyme that can create the synaptic CT structure, the CT GalNAc transferase, is also confined to the neuromuscular junction in mice. Using transgenic mice, we show that overexpression of the CT GalNAc transferase in extrasynaptic regions in skeletal myofibers caused as much as a 60% reduction in the diameter of adult myofibers and an order of magnitude increase in satellite cells. Neuromuscular junctions of transgenic mice had severely reduced numbers of secondary folds, Schwann cell processes were present in the synaptic cleft, and secondary folds were often misaligned with active zones. In addition, multiple presynaptic specializations occurred on individual myofibers. In addition, some normally synaptic proteins, including laminin [alpha]4, laminin [alpha]5, utrophin, and NCAM, were expressed along extrasynaptic regions of myofibers. One of the muscle proteins that displayed increased glycosylation with the CT antigen in the transgenic mice was [alpha]-dystroglycan. These experiments provide the first in vivo evidence that a synaptic carbohydrate antigen has important roles in the development of the neuromuscular synapse and suggest that the CT antigen is involved in controlling the expression of synaptic molecules. Key Words: synapse; GalNAc; neuromuscular junction; laminin; satellite cell; dystroglycan.

Differentiation of both pre- and postsynaptic structures at the skeletal neuromuscular junction is organized by the basal lamina that occupies the synaptic cleft. As [Beta]1 integrins are a major class of receptors for basal lamina components, we stained muscles with antibodies to the 10 integrin [Alpha] subunits known to form dimers with [Beta]1, to determine if any of these molecules were concentrated at synaptic sites on muscle fibers. In both developing and adult muscle, the integrin [Alpha]1 chain was selectively associated with presynaptic cells (Schwann cells and/or nerve terminals), while [Alpha]7 was present on both synaptic and extrasynaptic portions of the muscle fiber surface. Thus [Alpha]1 and [Alpha]7 integrins are present in synaptic membranes. Expression of the [Alpha]7 chain was analyzed further by staining with antibodies specific for three alternatively spliced products of the [Alpha]7 gene (A, B, and C), all of which were expressed in muscle. The [Alpha]7A and [Alpha]7B isoforms were confined to synaptic sites in adult muscle, while [Alpha]7C was present both synaptically and extrasynaptically. In developing muscle, [Alpha]7A appeared postnatally and specifically at the synapse; [Alpha]7B was present throughout the muscle fiber perinatally, becoming confined to the synapse in the second postnatal week; and [Alpha]7C was present extrasynaptically both perinatally and in adulthood. Thus, two of the [Alpha]7 integrins are synapse-specific, and all three show distinct spatiotemporal patterns of expression within a single cell type. Finally, we asked whether perturbation of laminin expression affected the distribution of the [Alpha]7 integrins. In normal mice, laminin [Beta]2 is concentrated in synaptic basal lamina. In [Beta]2-null mutant mice, [Alpha]7A was still present at synaptic sites, but [Alpha]7B was absent. This result provides genetic evidence that basal lamina composition is a determinant of integrin distribution.

The basal lamina that ensheaths skeletal muscle fibers traverses the synaptic cleft at the neuromuscular junction. Synaptic and extrasynaptic portions of the basal lamina contain different laminin β chains: β2 (or s) at synapses and β1 (or B1) extrasynaptically. Laminin 2 is also confined to synapselike patches on myotube surfaces in vitro, whereas β1 is present throughout the extracellular matrix. This differential localization of laminin β chains was analyzed by expression of chimeric β1-β2 molecules in cultured mouse myotubes. A 16-amino acid carboxyl-terminal sequence in β2 was necessary for synaptic localization, and an amino-terminal domain in β1 promoted association with extracellular fibrils. The synaptic targeting sequence of β2 contains a site previously shown t%f be adhesive for motor neurons., The postsynaptic apparatus of the skeletal neuromuscular junction is assembled from numerous extracellular, membrane, and cytoskeletal components (1). To date, most studies of the assembly of the postsynaptic apparatus have [...]