Neal R. Kramarcy

β2–syntrophin: Localization at the neuromuscular junction in skeletal muscle

The syntrophins are a multigene family of proteins which bind C-terminal domains of dystrophin, utrophin and homologs thereof. We report here that antibodies specific for one isoform, beta 2-syntrophin, labeled only the neuromuscular junction (NMJ) in rat skeletal muscle. Anti-alpha 1-syntrophin antibodies gave strong labeling of the sarcolemma and NMJ in normal rat and mouse muscle, and similar but much weaker labeling in dystrophin-minus mdx muscle. beta 2-Syntrophin therefore appears to be specific to the NMJ in normal muscle, as is utrophin, and may be involved in acetylcholine receptor clustering. alpha 1-Syntrophin appears to be associated mainly with dystrophin, as expected, but a small portion must be associated with another protein, possibly homologs of the electric tissue 87K protein.

Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junctions of skeletal muscle☆

In this report we have demonstrated that paxillin, a cytoskeletal protein which is present in focal adhesions, localizes in vivo to regions of cell-extracellular matrix interaction which are believed to be analogous to focal adhesions. Specifically, it is enriched in the dense plaques of chicken gizzard smooth muscle tissue and in the myotendinous junctions formed in Xenopus laevis tadpole tail skeletal muscle. In addition, paxillin was identified at the rat diaphragm neuromuscular junction. The distribution of paxillin is thus comparable to that of other focal adhesion proteins, for example, talin and vinculin, in these structures.

Syntrophin isoforms at the neuromuscular junction: Developmental time course and differential localization

The syntrophins are a family of cytoplasmic adapter proteins that associate with dystrophin family proteins and have putative signaling and structural roles at the neuromuscular junction. We have localized the syntrophin family members within the rodent junction from birth to adulthood. Alpha-syntrophin is the only isoform on the postsynaptic membrane at birth. In the adult, it occurs on the crests of the junctional folds, with utrophin, and in the troughs, with dystrophin. Surprisingly, neuronal nitric oxide synthase (nNOS) does not accompany alpha-syntrophin onto the crests. Beta2-syntrophin, a junction-specific form, is not present at birth and occurs mainly in the troughs in the adult. Beta1-syntrophin is a sarcolemmal form at birth, not concentrated at the junction, and disappears entirely from most fibers by 6 weeks. In positive fibers, junctional beta1-syntrophin occurs exclusively in the troughs. These results suggest that the syntrophin isoforms have distinct functions at the junction and show that the known protein-protein associations of the syntrophins and nNOS in skeletal muscle are not sufficient to explain their localizations.

Structural Abnormalities at Neuromuscular Synapses Lacking Multiple Syntrophin Isoforms

The syntrophins are modular adapter proteins that function by recruiting signaling molecules to the cytoskeleton via their direct association with proteins of the dystrophin protein family. We investigated the physiological function of β2-syntrophin by generating a line of mice lacking this syntrophin isoform. The β2-syntrophin null mice show no overt phenotype, or muscular dystrophy, and form structurally normal neuromuscular junctions (NMJs). To determine whether physiological consequences caused by the lack of β2-syntrophin were masked by compensation from the α-syntrophin isoform, we crossed these mice with our previously described α-syntrophin null mice to produce mice lacking both isoforms. The α/β2-syntrophin null mice have NMJs that are structurally more aberrant than those lacking only α-syntrophin. The NMJs of the α/β2-syntrophin null mice have fewer junctional folds than either parent strain, and the remaining folds are abnormally shaped with few openings to the synaptic space. The levels of acetylcholine receptors are reduced to 23% of wild type in mice lacking both syntrophin isoforms. Furthermore, the α/β2-syntrophin null mice ran significantly shorter distances on voluntary exercise wheels despite having normal neuromuscular junction transmission as determined by micro-electrode recording of endplate potentials. We conclude that both α-syntrophin and β2-syntrophin play distinct roles in forming and maintaining NMJ structure and that each syntrophin can partially compensate for the loss of the other.

Dystrophin as a focal adhesion protein. Collocalization with talin and the Mr 48,000 sarcolemmal protein in cultured Xenopus muscle.

Monoclonal antibodies against dystrophin and the postsynaptic 58 kDa protein from Torpedo electric organ were used to localize homologs of these proteins in cultured skeletal muscle (Xenopus laevis). The Xenopus homolog is an M r 48000 protein and, like dystrophin, is a sarcolemmal protein. Both proteins localized precisely to talin‐positive sites, hence with each other, on the substrate‐apposed sarcolemma. Therefore, the first sites of appearance of dystrophin on cultured muscle cells are focal adhesions, i.e. specific sites of cytoskeleton/extracellular matrix interaction. These data also add to evidence that dystrophin and the 58 kDa act together.

Effects of drug-induced changes in brain monoamines on aggression and motor behavior in mice

Mice maintained on a basal casein diet supplemented with 4% L-tyrosine potentiated L-DOPA effects on aggression. At low doses (12.5-25 mg/kg) L-DOPA increased aggression whereas at high doses (50-100 mg/kg) it decreased aggression. 5-HTP (50-200 mg/kg) produced a dose-dependent decrease in aggression and motor activity which was antagonized by pretreatment with dietary L-tyrosine (4%) or L-DOPA (50 mg/kg). L-DOPA induced reductions in motor activity were, in turn, antagonized by 5-HTP. Increases in motor activity following d-amphetamine (3 mg/kg) were sharply reduced by 5-HTP (50-100 mg/kg), but 5-HTP potentiated reductions in aggression following d-amphetamine. The concentration in brain of tyrosine, DOPA, dopamine (DA), noradrenaline (NA), DOPAC, HVA, tryptophan, serotonin (5-HT), and 5-HIAA were obtained following drug and diet treatments. The changes observed, particularly in DA and 5-HT metabolites, provide further evidence for an inhibitory role of brain 5-HT systems in the mediation of the behavioral effects of d-amphetamine and the catecholamine precursors, L-tyrosine and L-DOPA.