Blocking extracellular activation of myostatin as a strategy for treating muscle wasting.

Many growth factors are intimately bound to the extracellular matrix, with regulated processing and release leading to cellular stimulation. Myostatin and GDF11 are closely related members of the TGFß family whose activation requires two proteolytic cleavages to release the growth factor from the prodomain. Specific modulation of myostatin and GDF11 activity by targeting growth factor-receptor interactions has traditionally been challenging. Here we demonstrate that a novel strategy for blocking myostatin and GDF11, inhibition of growth factor release, specifically and potently inhibits signaling both in vitro and in vivo. We developed human monoclonal antibodies that selectively bind the myostatin and GDF11 precursor forms, including a subset that inhibit myostatin proteolytic activation and prevent muscle atrophy in vivo. The most potent myostatin activation-blocking antibodies promoted robust muscle growth and resulted in significant gains in muscle performance in healthy mice. Altogether, we show that blocking the extracellular activation of growth factors is a potent method for preventing signaling, serving as proof of concept for a novel therapeutic strategy that can be applied to other members of the TGFß family of growth factors.

Sonic hedgehog promotes the survival of specific CNS neuron populations and protects these cells from toxic insult In vitro.

Sonic hedgehog (Shh), an axis-determining secreted protein, is expressed during early vertebrate embryogenesis in the notochord and ventral neural tube. In this site it plays a role in the phenotypic specification of ventral neurons along the length of the CNS. For example, Shh induces the differentiation of motor neurons in the spinal cord and dopaminergic neurons in the midbrain. Shh expression, however, persists beyond this induction period, and we have asked whether the protein shows novel activities beyond phenotype specification. Using cultures derived from embryonic day 14.5 (E14. 5) rat ventral mesencephalon, we show that Shh is also trophic for dopaminergic neurons. Interestingly, Shh not only promotes dopaminergic neuron survival, but also promotes the survival of midbrain GABA-immunoreactive (GABA-ir) neurons. In cultures derived from the E15-16 striatum, Shh promotes the survival of GABA-ir interneurons to the exclusion of any other cell type. Cultures derived from E15-16 ventral spinal cord reveal that Shh is again trophic for interneurons, many of which are GABA-ir and some of which express the Lim-1/2 nuclear marker, but it does not appear to support motorneuron survival. Shh does not support the survival of sympathetic or dorsal root ganglion neurons. Finally, using the midbrain cultures, we show that in the presence of MPP+, a highly specific neurotoxin, Shh prevents dopaminergic neuron death that normally would have occurred. Thus Shh may have therapeutic value as a protective agent in neurodegenerative disease.

Glial growth factor 2, a soluble neuregulin, directly increases Schwann cell motility and indirectly promotes neurite outgrowth.

Schwann cells proliferate, migrate, and act as sources of neurotrophic support during development and regeneration of peripheral nerves. Recent studies have demonstrated that neuregulins, a family of growth factors secreted by developing motor and peripheral neurons, influence Schwann cell development. In this study, we use three distinct assays to show that glial growth factor 2 (GGF2), a secreted neuregulin, exerts multiple effects on mature Schwann cells in vitro. At doses submaximal for proliferation, GGF2 increases the motility of Schwann cells cultured on peripheral nerve cryosections. Furthermore, in a novel bioassay, focal application of GGF2 causes directed migration in conventional monolayer cultures of directed migration of Schwann cells. At higher doses, GGF2 causes proliferation, as described previously. In a new explant culture system designed to emulate entubulation repair of transected peripheral nerves, GGF2 concentrations greater than necessary to saturate the mitotic response induce the secretion by Schwann cells of activities that promote sympathetic neuron survival and outgrowth. These findings support a model in which neuregulins secreted by peripheral neurons are key components of reciprocal neuron-glia interactions that are important for peripheral nerve development and regeneration.

Schwann cell heparan sulfate proteoglycans play a critical role in glial growth factor/neuregulin signaling.

Glial growth factors are proteins encoded by the neuregulin gene and are thought to signal via receptor tyrosine kinases. Many neuregulin gene products bind heparin, and we hypothesize that affinity for heparin may implicate cell surface heparan sulfate proteoglycans (HeSPGs) as co-receptors for the soluble neuregulin gene product, recombinant human glial growth factor 2 (rhGGF2). Using primary rat Schwann cell cultures, we show that exogenous heparin and heparan sulfate block rhGGF2-induced phosphorylation of putative neuregulin receptors, and block subsequent DNA synthesis; other glycosaminoglycans show no such effect. Inhibition of Schwann cell HeSPG biosynthesis by administration of beta-xyloside also blocks responsiveness to rhGGF2. In cell-free binding assays, rhGGF2 binds heparin and heparan sulfate with high affinity, while suramin and suramin-like molecules block this binding. These suramin-like molecules reversibly block Schwann cell responsiveness to rhGGF2 with a rank order of potency identical to that in the cell-free binding assay. Thus we demonstrate high affinity and specificity in the interaction of rhGGF2 with heparin-like molecules, and show that three distinct perturbations of this interaction on Schwann cells (exogenous heparin/ heparan sulfate treatment, inhibition of HeSPG biosynthesis, and treatment with suramin-like molecules) result in a loss of responsiveness to rhGGF2. These results support a model in which HeSPGs are critical components that modulate extracellular rhGGF2 signaling interactions with appropriate receptor tyrosine kinases.

Rat olfactory neurons can utilize the endogenous lectin, L-14, in a novel adhesion mechanism.

L-14 is a divalent, lactosamine-binding lectin expressed in many vertebrate tissues. In the rat nervous system, L-14 expression has been observed previously in restricted neuronal subsets within the dorsal root ganglia and spinal cord. In this study we report that L-14 is expressed by nonneuronal cells in the rat olfactory nerve. We demonstrate that L-14 binds and co-localizes with two ligands in the rat olfactory system: a beta-lactosamine-containing glycolipid, and a putative member of the laminin family. The former is expressed on the surfaces of nascent olfactory axons originating from neuron cell bodies in the olfactory epithelium. The latter is present in the extracellular matrix of the axonal path leading to synaptic targets in the olfactory bulb. In vitro, we find that recombinant L-14 promotes primary olfactory neuron adhesion to two laminin family members, and promotes intercellular adhesion. Both activities are dose-dependent, and are independent of integrin-mediated mechanisms. We have thus found that L-14 can serve two distinct adhesive functions in vitro, and propose that L-14 in vivo can promote olfactory axon fasciculation by crosslinking adjacent axons and promote axonal adhesion to the extracellular matrix.

Peptide growth factor control of olfactory neurogenesis and neuron survival in vitro: roles of EGF and TGF-beta s.

The olfactory epithelium (OE) is unique in the mammalian nervous system as a site of continual neurogenesis. Though many studies have described this process in vivo and olfactory neurogenesis can be demonstrated in vitro, the specific factors that modulate this process have not been defined. Noting the common ectodermal origin and structural similarity between the OE and epidermis, peptide factors known to modulate epidermal differentiation were tested in OE cultures. Our results demonstrate that EGF acts as a mitogen for the basal cells that give rise to olfactory neurons and that transforming growth factor-beta s (TGF-beta s) promote neurogenesis. Using a neutralizing antibody, we show that it is likely that the endogenous neurogenic factor is TGF-beta 2, or a very closely related factor.

Thy-1 involvement in neurite outgrowth: perturbation by antibodies, phospholipase C, and mutation.

Thy-1 is a major cell surface protein anchored in the plasma membrane of neurons and lymphocytes by a covalent glyco-phosphatidyl-inositide linkage. Despite thorough characterization of the molecule's physicochemical properties, its biological function remains elusive. In this study we demonstrate that (i) monoclonal antibodies directed against Thy-1 are capable of enhancing neurite outgrowth from sympathetic neurons in culture, as well as stimulating the initiation of neurite sprouting from cultured adrenal chromaffin cells and PC12 cells. This effect is not observed with monovalent, Fab antibody fragments. Treatment with intact antibodies also results in the shedding of Thy-1 into the culture medium. (ii) Treatment of chromaffin cells with phosphatidyl-inositol-specific phospholipase C also results in an induction of neurite sprouting. The lipase effect can be blocked by preincubating the cells with monovalent anti-Thy-1 Fab fragments, indicating that the outgrowth stimulation is specifically due to removal of Thy-1. (iii) An entirely different approach to elucidating the function of Thy-1 involves mutagenesis of PC12 cells. Selection for Thy-1-deficient mutants revealed that cells lacking Thy-1 sprout neurites spontaneously at a very high frequency. A novel role for Thy-1 is proposed wherein the results of the mutant cell studies are compatible with the antibody and lipase data. Each of the perturbations can be viewed as releasing an inhibition that Thy-1 normally exerts on neurite outgrowth. We suggest that Thy-1 normally acts to stabilize neuronal membranes and processes, possibly through homophilic interactions.

Thy-1 multimerization is correlated with neurite outgrowth.

Thy-1 is abundantly expressed in the vertebrate nervous system. Perturbation studies in vitro suggest that Thy-1 inhibits neurite outgrowth and stabilizes neuronal processes (N. K. Mahanthappa and P. H. Patterson. (1992). Thy-1 involvement in neurite outgrowth: Perturbation by antibodies, phospholipase C, and mutation. Dev. Biol. 150,47-59). We here report that Thy-1 participates in several types of homophilic interactions, each with differential sensitivity to reduction and boiling. The relative abundance of the multimeric forms of Thy-1 vary with the cell's ability to sprout neurites. Gel filtration chromatography of sympathetic neuron and PC12 cell lysates reveals that Thy-1 immunoreactivity appears in 25-, 45-, and 150-kDa forms. In neurons, Thy-1 immunoreactivity is distributed equally in all three forms, whereas in PC12 cells, the majority of Thy-1 immunoreactivity is found in the higher molecular weight forms. When PC12 cells are induced to sprout neurites with NGF, the Thy-1 size distribution becomes identical to that of neurons. The three forms of Thy-1 immunoreactivity are likely to be homomultimers of Thy-1 because immunoaffinity-purified, soluble Thy-1 also forms complexes similar in size to those found in neuronal extracts. To test whether Thy-1 multimerization may occur through interactions like those between immunoglobulin heavy and light chains, synthetic peptides corresponding to candidate sites for such associations in Thy-1 were tested for their effects on multimerization and neurite outgrowth. One peptide increases the amount of monomeric Thy-1 relative to total Thy-1, and promotes outgrowth. These results suggest that multimeric forms of Thy-1 inhibit process outgrowth and neurite sprouting by stabilizing the surface membrane and/or underlying cytoskeleton.

Basic FGF induces neuronal differentiation, cell division, and NGF dependence in chromaffin cells: a sequence of events in sympathetic development.

To define further the molecules that control sympathoadrenal differentiation, we have investigated the effects of FGF, NGF, and glucocorticoid on cultured neonatal rat adrenal chromaffin cells. Basic FGF (bFGF), like NGF, induces cell division and neurite outgrowth from these cells. Dexamethasone inhibits neuronal differentiation but not proliferation induced by bFGF. Unlike NGF, bFGF will not support the survival of chromaffin cell-derived sympathetic neurons. However, bFGF induces a dependence on NGF. The overlapping but distinct responses to NGF and bFGF may underlie a sequence of events in sympathetic differentiation. bFGF (or another factor) may act locally in developing ganglia to stimulate mitotic expansion and initial axon outgrowth. Subsequent survival and maturation are then controlled by NGF, which is provided by peripheral targets of innervation. In the adrenal gland, glucocorticoids may permit bFGF to amplify the chromaffin population, while preventing neuronal differentiation.