Robert Sealock

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.

Wide genetic variation in the native pial collateral circulation is a major determinant of variation in severity of stroke

Severity of stroke varies widely among individuals. Whether differences in the extent of the native (preexisting) pial collateral circulation exist and contribute to this variability is unknown. We addressed these questions and probed for potential genetic contributions using morphometric analysis of the collateral circulation in 15 inbred mouse strains recently shown to exhibit wide differences in infarct volume. Morphometrics were determined in the unligated left hemisphere (for native collaterals) and ligated right hemisphere (for remodeled collaterals) 6 days after permanent middle cerebral artery (MCA) occlusion. Variation among strains in native collateral number, diameter, MCA, anterior cerebral artery (ACA), and posterior cerebral artery (PCA) tree territories were, respectively: 56-fold, 3-fold, 42%, 56%, and 61%. Collateral length (P<0.001) and the number of penetrating arterioles branching from them also varied (P<0.05). Infarct volume correlated inversely with collateral number (P<0.0001), diameter (P<0.0001), and penetrating arteriole number (P<0.05) and directly with MCA territory (P<0.05). Relative collateral conductance and MCA territory, when factored together, strongly predicted infarct volume (P<0.0001). Outward remodeling of collaterals in the ligated hemisphere varied ∼3-fold. These data show that the extent of the native pial collateral circulation and collateral remodeling after obstruction vary widely with genetic background, and suggest that this variability, due to natural polymorphisms, is a major contributor to variability in infarct volume.

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.

Genetic Architecture Underlying Variation in Extent and Remodeling of the Collateral Circulation

Rationale Collaterals are arteriole-to-arteriole anastomoses that connect adjacent arterial trees. They lessen ischemic tissue injury by serving as endogenous bypass vessels when the trunk of 1 tree becomes narrowed by vascular disease. The number and diameter (“extent”) of native (preexisting) collaterals, plus their amount of lumen enlargement (growth/remodeling) in occlusive disease, show remarkably wide variation among inbred mouse strains (eg, C57BL/6 and BALB/c), resulting in large differences in tissue injury in models of occlusive disease. Evidence suggests similar large differences exist among healthy humans. Objective To identify candidate loci responsible for genetic-dependent collateral variation. Methods and Results Cerebral collateral number and diameter were determined in 221 C57BL/6×BALB/c F2 progeny, followed by linkage analysis to identify quantitative trait loci (QTL) for collateral number and diameter. Four QTL were obtained for collateral number, including epistasis between 2 loci. A QTL that was identical to the strongest QTL for collateral number on chromosome 7 (logarithm of the odds [LOD]=29, effect size=37%) was also mapped for collateral diameter (LOD=17, effect size=30%). Chromosome substitution strain analysis confirmed this locus. We also obtained a unique QTL on chromosome 11 for collateral remodeling after middle cerebral artery occlusion. Association mapping within the chromosome 7 QTL interval using collateral traits measured for 15 inbred strains delineated 172-kbp (P =0.00002) and 290-kbp (P=0.0004) regions on chromosome 7 containing 2 and 7 candidate genes, respectively. Conclusions We conclude that collateral extent and remodeling are unique, highly heritable complex traits, with 1 QTL predominantly affecting native collateral number and diameter.

Talin is a post-synaptic component of the rat neuromuscular junction.

Talin is a protein, recently discovered in chicken gizzard, which occurs at sites of actin-plasma membrane interaction in several cell types. Vinculin also occurs at many of these sites, possibly in association with talin. In this study, three antisera against talin were used to probe the neuromuscular junction of rat skeletal muscle, which is also a site of vinculin accumulation. By immunofluorescence, all three sera stained the junction strongly in frozen sections of rat diaphragm. The extrajunctional periphery was lightly and irregularly stained in some muscle cells; others seemed not to be stained outside the junction. Staining remained at junctions and increased in extrajunctional regions of muscle denervated 6 weeks before sacrifice. The staining in all cases was abolished by competition with purified talin. One serum tested by immunoblotting recognized one protein at Mr 215 000 (identical with the value for chicken gizzard talin) and traces of a second at Mr 190 000 (corresponding to a known proteolytic fragment of talin). We conclude that rat muscle talin is similar in its general protein structure to chicken gizzard talin, and is a post-synaptic component of the neuromuscular junction.

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.

Congenic Fine-Mapping Identifies a Major Causal Locus for Variation in the Native Collateral Circulation and Ischemic Injury in Brain and Lower Extremity

Rationale: Severity of tissue injury in occlusive disease is dependent on the extent (number and diameter) of collateral vessels, which varies widely among healthy mice and humans. However, the causative genetic elements are unknown. Recently, much of the variation among different mouse strains, including C57Bl/6J (B6, high extent) and BALB/cByJ (Bc, low extent), was linked to a quantitative trait locus on chromosome 7 (Candq1). Objective: We used congenic mapping to refine Candq1 and its candidate genes to create an isogenic strain set with large differences in collateral extent to assess their impact and the impact of Candq1, alone, on ischemic injury. Methods and Results: Six congenic strains possessing portions of Candq1 introgressed from B6 into Bc were generated and phenotyped. Candq1 was refined from 27 to 0.737 Mb with full retention of effect, that is, return or rescue of phenotypes from the poor values in Bc to nearly those of wild-type B6 in the B6/B6 congenic mice as follows: 83% rescue of low pial collateral extent and 4.5-fold increase in blood flow and 85% reduction of infarct volume after middle cerebral artery occlusion; 54% rescue of low skeletal muscle collaterals and augmented recovery of perfusion (83%) and function after femoral artery ligation. Gene deletion and in silico analysis further delineated the candidate genes. Conclusions: We have significantly refined Candq1 (now designated determinant of collateral extent 1; Dce1), demonstrated that genetic background–dependent variation in collaterals is a major factor underlying differences in ischemic tissue injury, and generated a congenic strain set with wide allele dose–dependent variation in collateral extent for use in investigations of the collateral circulation.

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.

Genetic dissection of the Canq1 locus governing variation in extent of the collateral circulation.

Background Native (pre-existing) collaterals are arteriole-to-arteriole anastomoses that interconnect adjacent arterial trees and serve as endogenous bypass vessels that limit tissue injury in ischemic stroke, myocardial infarction, coronary and peripheral artery disease. Their extent (number and diameter) varies widely among mouse strains and healthy humans. We previously identified a major quantitative trait locus on chromosome 7 (Canq1, LOD = 29) responsible for 37% of the heritable variation in collateral extent between C57BL/6 and BALB/c mice. We sought to identify candidate genes in Canq1 responsible for collateral variation in the cerebral pial circulation, a tissue whose strain-dependent variation is shared by similar variation in other tissues. Methods and Findings Collateral extent was intermediate in a recombinant inbred line that splits Canq1 between the C57BL/6 and BALB/c strains. Phenotyping and SNP-mapping of an expanded panel of twenty-one informative inbred strains narrowed the Canq1 locus, and genome-wide linkage analysis of a SWRxSJL-F2 cross confirmed its haplotype structure. Collateral extent, infarct volume after cerebral artery occlusion, bleeding time, and re-bleeding time did not differ in knockout mice for two vascular-related genes located in Canq1, IL4ra and Itgal. Transcript abundance of 6 out of 116 genes within the 95% confidence interval of Canq1 were differentially expressed >2-fold (p-value<0.05÷150) in the cortical pia mater from C57BL/6 and BALB/c embryos at E14.5, E16.5 and E18.5 time-points that span the period of collateral formation. Conclusions These findings refine the Canq1 locus and identify several genes as high-priority candidates important in specifying native collateral formation and its wide variation.