Biglycan is an extracellular MuSK binding protein important for synapse stability.

The receptor tyrosine kinase MuSK is indispensable for nerve-muscle synapse formation and maintenance. MuSK is necessary for prepatterning of the endplate zone anlage and as a signaling receptor for agrin-mediated postsynaptic differentiation. MuSK-associated proteins such as Dok7, LRP4, and Wnt11r are involved in these early events in neuromuscular junction formation. However, the mechanisms regulating synapse stability are poorly understood. Here we examine a novel role for the extracellular matrix protein biglycan in synapse stability. Synaptic development in fetal and early postnatal biglycan null (bgn(-/o)) muscle is indistinguishable from wild-type controls. However, by 5 weeks after birth, nerve-muscle synapses in bgn(-/o) mice are abnormal as judged by the presence of perijunctional folds, increased segmentation, and focal misalignment of acetylcholinesterase and AChRs. These observations indicate that previously occupied presynaptic and postsynaptic territory has been vacated. Biglycan binds MuSK and the levels of this receptor tyrosine kinase are selectively reduced at bgn(-/o) synapses. In bgn(-/o) myotubes, the initial stages of agrin-induced MuSK phosphorylation and AChR clustering are normal, but the AChR clusters are unstable. This stability defect can be substantially rescued by the addition of purified biglycan. Together, these results indicate that biglycan is an extracellular ligand for MuSK that is important for synapse stability.

Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities.

Rapamycin is an immunosuppressive immunophilin ligand reported as having neurotrophic activity. We show that modification of rapamycin at the mammalian target of rapamycin (mTOR) binding region yields immunophilin ligands, WYE-592 and ILS-920, with potent neurotrophic activities in cortical neuronal cultures, efficacy in a rodent model for ischemic stroke, and significantly reduced immunosuppressive activity. Surprisingly, both compounds showed higher binding selectivity for FKBP52 versus FKBP12, in contrast to previously reported immunophilin ligands. Affinity purification revealed two key binding proteins, the immunophilin FKBP52 and the beta1-subunit of L-type voltage-dependent Ca(2+) channels (CACNB1). Electrophysiological analysis indicated that both compounds can inhibit L-type Ca(2+) channels in rat hippocampal neurons and F-11 dorsal root ganglia (DRG)/neuroblastoma cells. We propose that these immunophilin ligands can protect neurons from Ca(2+)-induced cell death by modulating Ca(2+) channels and promote neurite outgrowth via FKBP52 binding.

The development of stroke therapeutics: promising mechanisms and translational challenges.

Ischemic stroke is the second most common cause of death worldwide and a major cause of disability. Intravenous thrombolysis with rt-PA remains the only available acute therapy in patients who present within 3h of stroke onset other than the recently approved mechanical MERCI device, substantiating the high unmet need in available stroke therapeutics. The development of successful therapeutic strategies remains challenging, as evidenced by the continued failures of new therapies in clinical trials. However, significant lessons have been learned and this knowledge is currently being incorporated into improved pre-clinical and clinical design. Furthermore, advancements in imaging technologies and continued progress in understanding biological pathways have established a prolonged presence of salvageable penumbral brain tissue and have begun to elucidate the natural repair response initiated by ischemic insult. We review important past and current approaches to drug development with an emphasis on implementing principles of translational research to achieve a rigorous conversion of knowledge from bench to bedside. We highlight current strategies to protect and repair brain tissue with the promise to provide longer therapeutic windows, preservation of multiple tissue compartments and improved clinical success.

Biglycan regulates the expression and sarcolemmal localization of dystrobrevin, syntrophin, and nNOS.

The dystrophin-associated protein complex (DAPC) provides a linkage between the cytoskeleton and the extracellular matrix (ECM) and is also a scaffold for a host of signaling molecules. The constituents of the DAPC must be targeted to the sarcolemma in order to properly function. Biglycan is an ECM molecule that associates with the DAPC. Here, we show that biglycan null mice exhibit a mild dystrophic phenotype and display a selective reduction in the localization of alpha-dystrobrevin-1 and -2, alpha- and beta1-syntrophin, and nNOS at the sarcolemma. Purified biglycan induces nNOS redistribution to the plasma membrane in cultured muscle cells. Biglycan protein injected into muscle becomes stably associated with the sarcolemma and ECM for at least 2 wk. This injected biglycan restores the sarcolemmal expression of alpha-dystrobrevin-1 and -2, and beta1- and beta2-syntrophin in biglycan null mice. We conclude that biglycan is important for the maintenance of muscle cell integrity and plays a direct role in regulating the expression and sarcolemmal localization of the intracellular signaling proteins dystrobrevin-1 and -2, alpha- and beta1-syntrophin and nNOS.

Neurite outgrowth by the alternatively spliced region of human tenascin-C is mediated by neuronal alpha7beta1 integrin.

The region of tenascin-C containing only alternately spliced fibronectin type-III repeat D (fnD) increases neurite outgrowth by itself and also as part of tenascin-C. We previously localized the active site within fnD to an eight amino acid sequence unique to tenascin-C, VFDNFVLK, and showed that the amino acids FD and FV are required for activity. The purpose of this study was to identify the neuronal receptor that interacts with VFDNFVLK and to investigate the hypothesis that FD and FV are important for receptor binding. Function-blocking antibodies against both alpha7 and beta1 integrin subunits were found to abolish VFDNFVLK-mediated process extension from cerebellar granule neurons. VFDNFVLK but not its mutant, VSPNGSLK, induced clustering of neuronal beta1 integrin immunoreactivity. This strongly implicates FD and FV as important structural elements for receptor activation. Moreover, biochemical experiments revealed an association of the alpha7beta1 integrin with tenascin-C peptides containing the VFDNFVLK sequence but not with peptides with alterations in FD and/or FV. These findings are the first to provide evidence that the alpha7beta1 integrin mediates a response to tenascin-C and the first to demonstrate a functional role for the alpha7beta1 integrin receptor in CNS neurons.

Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors: strategies to improve neuronal regeneration.

Peptides derived from extracellular matrix proteins have the potential to function as potent therapeutic reagents to increase neuronal regeneration following central nervous system (CNS) injury, yet their efficacy as pharmaceutical reagents is dependent upon the expression of cognate receptors in the target tissue. This type of codependency is clearly observed in successful models of axonal regeneration in the peripheral nervous system, but not in the normally nonregenerating adult CNS. Successful regeneration is most closely correlated with the induction of integrins on the surface of peripheral neurons. This suggests that in order to achieve optimal neurite regrowth in the injured adult CNS, therapeutic strategies must include approaches that increase the number of integrins and other key receptors in damaged central neurons, as well as provide the appropriate growth-promoting peptides in a "regeneration cocktail." In this review, we describe the ability of peptides derived from tenascin- C, fibronectin, and laminin-1 to influence neuronal growth. In addition, we also discuss the implications of peptide/receptor interactions for strategies to improve neuronal regeneration.

Developmental regulation of biglycan expression in muscle and tendon.

Biglycan is an extracellular ligand for the dystrophin-associated protein complex (DAPC) that is upregulated in both dystrophic and regenerating muscle. Biglycan also binds to collagen VI, mutations of which cause a congenital muscular dystrophy (Ullrich's; UCMD) that is also characterized by connective tissue abnormalities. The expression of biglycan in early development and postnatal ages has not been well characterized. Here we show that biglycan transcript levels peak at approximately 21 weeks' gestation in human fetal muscle. Immunocytochemical analysis of developing mouse muscle shows that biglycan can be detected in muscle as early as embryonic day (E)16 and is most abundant between postnatal day (P)1 and P7. Biglycan is also highly expressed in developing tendon, with maximal levels observed at E16-18. This robust tendon expression is correlated with a sharp peak in biglycan transcript levels in the hindlimb. Finally, at E18 collagen VI colocalizes with biglycan in tendon. These results suggest that biglycan has a particularly important function during muscle and connective tissue development. Moreover, biglycan may play a role in the pathogenesis of collagen VI-associated congenital muscular dystrophies.

Biglycan binds to alpha- and gamma-sarcoglycan and regulates their expression during development.

The dystrophin-associated protein complex (DAPC), which links the cytoskeleton to the extracellular matrix, is essential for muscle cell survival, and is defective in a wide range of muscular dystrophies. The DAPC contains two transmembrane subcomplexes-the dystroglycans and the sarcoglycans. Although several extracellular binding partners have been identified for the dystroglycans, none have been described for the sarcoglycan subcomplex. Here we show that the small leucine-rich repeat (LRR) proteoglycan biglycan binds to alpha- and gamma-sarcoglycan as judged by ligand blot overlay and co-immunoprecipitation assays. Our studies with biglycan-decorin chimeras show that alpha- and gamma-sarcoglycan bind to distinct sites on the polypeptide core of biglycan. Both biglycan proteoglycan as well as biglycan polypeptide lacking glycosaminoglycan (GAG) side chains are components of the dystrophin glycoprotein complex isolated from adult skeletal muscle membranes. Finally, our immunohistochemical and biochemical studies with biglycan null mice show that the expression of alpha- and gamma-sarcoglycan is selectively reduced in muscle from young (P14-P21) animals, while levels in adult muscle (> or = P35) are unchanged. We conclude that biglycan is a ligand for two members of the sarcoglycan complex and regulates their expression at discrete developmental ages.