Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3–05) reversed Rho activation and reduced the number of TUNEL-labeled cells by ∼50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3–05 blocked the increase in p75NTR expression. Experiments with p75NTR-null mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.

The activation state of Rho is an important determinant of axon growth and regeneration in neurons. Axons can extend neurites on growth inhibitory substrates when Rho is inactivated by C3-ADP-ribosyltransferase (C3). We found by Rho-GTP pull-down assay that inhibitory substrates activate Rho. To inactivate Rho, scrapeloading of C3 was necessary because it does not freely enter cells. To overcome the poor permeability of C3, we made and characterized five new recombinant C3-like chimeric proteins designed to cross the cell membrane by receptor-independent mechanisms. These proteins were constructed by the addition of short transport peptides to the carboxyl-terminal of C3 and tested using a bioassay measuring neurite outgrowth of PC-12 cells plated on growth inhibitory substrates. All five constructs stimulated neurite outgrowth but with different dose-response profiles. Biochemical properties of the chimeric proteins were examined using C3-05, the most effective construct tested. Gel shift assays showed that C3-05 retained the ability to ADP-ribosylate Rho. Western blots and immunocytochemistry were used to verify the presence of C3 inside treated cells. C3-05 was also effective at promoting neurite outgrowth in primary neuronal cultures, as well as causing the disassembly of actin stress fibers and focal adhesions complexes in fibroblasts. These studies demonstrate that the new C3-like proteins are effective in delivering biologically active C3 into different cell types, thereby, inactivating Rho.

Nerve growth factor (NGF) mediates the survival and differentiation of neurons by stimulating the tyrosine kinase activity of the TrkA/NGF receptor. Here, we identify SHP-1 as a phosphotyrosine phosphatase that negatively regulates TrkA. SHP-1 formed complexes with TrkA at Y490, and dephosphorylated it at Y674/675. Expression of SHP-1 in sympathetic neurons induced apoptosis and TrkA dephosphorylation. Conversely, inhibition of endogenous SHP-1 with a dominant-inhibitory mutant stimulated basal tyrosine phosphorylation of TrkA, thereby promoting NGF-independent survival and causing sustained and elevated TrkA activation in the presence of NGF. Mice lacking SHP-1 had increased numbers of sympathetic neurons during the period of naturally occurring neuronal cell death, and when cultured, these neurons survived better than wild-type neurons in the absence of NGF. These data indicate that SHP-1 can function as a TrkA phosphatase, controlling both the basal and NGF-regulated level of TrkA activity in neurons, and suggest that SHP-1 regulates neuron number during the developmental cell death period by directly regulating TrkA activity.

No abstract

The formation of stable adhesive contacts between pre- and post-synaptic neurons represents the initial step in synapse assembly. The cell adhesion molecule N-cadherin, the receptor tyrosine phosphatase DLAR, and the scaffolding molecule Liprin-α play critical, evolutionarily conserved roles in this process. However, how these proteins signal to the growth cone, and are themselves regulated, remains poorly understood. Using Drosophila photoreceptors (R cells) as a model, we evaluate genetic and physical interactions among these three proteins. We demonstrate that DLAR function in this context is independent of phosphatase activity, but requires interactions mediated by its intracellular domain. Genetic studies reveal both positive and, surprisingly, inhibitory interactions amongst all three genes. These observations are corroborated by biochemical studies demonstrating that DLAR physically associates via its phosphatase domain with N-cadherin in Drosophila embryos. Together, these data demonstrate that N-cadherin, DLAR, and Liprin-α function in a complex to regulate adhesive interactions between pre- and post-synaptic cells, and provide a novel mechanism for controlling the activity of liprin-α in the developing growth cone.

No abstract

Exfoliation of plasma membrane components is a directed process that consumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are released on exfoliated vesicles. We examine here whether the Fas receptor and its cognate ligand (FasL) are present on vesicles shed from high metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free medium conditioned by CX-1 cells are increased by 1.8-fold and 1.6-fold, respectively, relative to that in medium conditioned by MIP-101 cells. Although vesicles shed from both cancer cell lines contain Fas antigen, the amount of Fas per vesicle and the percentage of vesicles containing Fas are increased for vesicles isolated from MIP-101 cells, relative to those from CX-1 cells, as determined by immunogold particle labeling and electron microscopy and by immunofluorescence microscopy and flow cytometry. Results of metabolic labeling with 35S-methionine indicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 cells, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells. Although mRNA for soluble Fas receptor is detectable in both cell lines, depletion of shed vesicles from serum-free medium by ultracentrifugation removes all detectable biological activity. FasL is detected on vesicles exfoliated from HuT 78 cells by immunoelectron microscopy and Western blot analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from FasL-induced and anti-Fas–mediated apoptosis, indicating that Fas present on shed vesicles is biologically active. We conclude that the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mechanism for long-range signal-directed apoptosis while maintaining Fas/FasL on a membrane surface.

As previously reported, our initial investigation was concerned with the bicyclic olefins, ethylidene norbornane, and dihydrodicyclopentadiene. The former produced a skeletally rearranged γ‐sultone and the latter an allylic sulfonic acid. In an attempt to further clarify the mechanisms of the sulfonation of these two models and to extend the study to models of other types of EPDM, we have now examined the sulfonation of several other model olefins. In addition to the bicyclic olefins reported previously, we have studied the sulfonation of monocyclic type II and III olefins and cyclic type I and type II olefins. The results showed that the bicyclic olefins behaved differently from their monocyclic analogs reinforcing earlier speculation as to a synchronous reaction mechanism. Exocyclics gave allylic sulfonic acids while endocyclics produced β‐sultones, which is the exact reverse of what was observed for the bicyclic counterparts. Acyclic type II olefins produced “stable” β‐sultones, and the influence of the sulfonating reagent on the subsequent chemistry is discussed. Type I olefins, curiously, gave a complex mixture of initial products which tended to rearrange to essentially a single thermodynamic product. The results of the investigation suggest that based on these models, each of the three major types of EPDM should have a different sulfonation mechanism.