Multiplex array proteomics detects increased MMP-8 in CSF after spinal cord injury.

INTRODUCTION: A variety of methods have been used to study inflammatory changes in the acutely injured spinal cord. Recently novel multiplex assays have been used in an attempt to overcome limitations in numbers of available targets studied in a single experiment. Other technical challenges in developing pre-clinical rodent models to investigate biomarkers in cerebrospinal fluid (CSF) include relatively small volumes of sample and low concentrations of target proteins. The primary objective of this study was to characterize the inflammatory profile present in CSF at a subacute time point in a clinically relevant rodent model of traumatic spinal cord injury (SCI). Our other aim was to test a microarray proteomics platform specifically for this application. METHODS: A 34 cytokine sandwich ELISA microarray was used to study inflammatory changes in CSF samples taken 12 days post-cervical SCI in adult rats. The difference between the median foreground signal and the median background signal was measured. Bonferroni and Benjamini-Hochburg multiple testing corrections were applied to limit the False Discovery Rate (FDR), and a linear mixed model was used to account for repeated measures in the array. RESULTS: We report a novel subacute SCI biomarker, elevated levels of matrix metalloproteinase-8 protein in CSF, and discuss application of statistical models designed for multiplex testing. CONCLUSIONS: Major advantages of this assay over conventional methods include high-throughput format, good sensitivity, and reduced sample consumption. This method can be useful for creating comprehensive inflammatory profiles, and biomarkers can be used in the clinic to assess injury severity and to objectively grade response to therapy.

Plasminogen activator promotes recovery following spinal cord injury.

Plasminogen activators play an important role in synaptic plasticity associated with the crossed phrenic phenomenon (CPP) and recovery of respiratory function after spinal cord injury. A genetic approach using knockout mice lacking various genes in the plasminogen activator/plasmin system has shown that induction of urokinase plasminogen activator (uPA) is required during the first hour after a C2-hemisection for the acquisition of the CPP response. The uPA knockout mice do not show the structural remodeling of phrenic motor neuron synapses characteristic of the CPP response. As shown here uPA acts in a cell signaling manner via binding to its receptor uPAR rather than as a protease, since uPAR knockout mice or knock-in mice possessing a modified uPA that is unable to bind to uPAR both fail to generate a CPP and recover respiratory function. Microarray data and real-time PCR analysis of mRNAs induced in the phrenic motor nucleus after C2-hemisection in C57Bl/6 mice as compared to uPA knockout mice indicate a potential cell signaling cascade downstream possibly involving ß-integrin and Src, and other pathways. Identification of these uPA-mediated signaling pathways may provide the opportunity to pharmacologically upregulate the synaptic plasticity necessary for recovery of phrenic motoneuron activity following cervical spinal cord injury.

Decorin, erythroblastic leukaemia viral oncogene homologue B4 and signal transducer and activator of transcription 3 regulation of semaphorin 3A in central nervous system scar tissue.

Scar tissue at sites of traumatic injury in the adult central nervous system presents a combined physical and molecular impediment to axon regeneration. Of multiple known central nervous system scar associated axon growth inhibitors, semaphorin 3A has been shown to be strongly expressed by invading leptomeningeal fibroblasts. We have previously demonstrated that infusion of the small leucine-rich proteoglycan decorin results in major suppression of several growth inhibitory chondroitin sulphate proteoglycans and growth of adult sensory axons across acute spinal cord injuries. Furthermore, decorin treatment of leptomeningeal fibroblasts significantly increases their ability to support neurite growth of co-cultured adult dorsal root ganglion neurons. In the present study we show that decorin has the ability to suppress semaphorin 3A expression within adult rat cerebral cortex scar tissue and in primary leptomeningeal fibroblasts in vitro. Infusion of decorin core protein for eight days resulted in a significant reduction of semaphorin 3A messenger RNA expression within injury sites compared with saline-treated control animals. Both in situ hybridization and immunostaining confirmed that semaphorin 3A messenger RNA expression and protein levels are significantly reduced in decorin-treated animals. Similarly, decorin treatment decreased the expression of semaphorin 3A messenger RNA in cultured rat leptomeningeal fibroblasts compared with untreated cells. Mechanistic studies revealed that decorin-mediated suppression of semaphorin 3A critically depends on erythroblastic leukaemia viral oncogene homologue B4 and signal transducer and activator of transcription 3 function. Collectively, our studies show that in addition to suppressing the levels of inhibitory chondroitin sulphate proteoglycans, decorin has the ability to suppress semaphorin 3A in the injured central nervous system. Our findings provide further evidence for the use of decorin as a potential therapy for promoting axonal growth and repair in the injured adult mammalian brain and spinal cord.

Role of plasminogen activator in spinal cord remodeling after spinal cord injury.

Plasminogen activators play an active role in synaptic plasticity associated with the crossed phrenic phenomenon (CPP) and recovery of respiratory function following spinal cord injury. A genetic approach has been used to identify molecular mechanisms underlying this synaptic plasticity. Knockout mice lacking different genes in the plasminogen activator/plasmin system demonstrate that expression of urokinase plasminogen activator (uPA) is required during the critical 1-2h delay period following C2-hemisection for the acquisition of a good CPP response. uPA knockout mice fail to show the structural remodeling of phrenic motorneuron synapses that underlie the CPP response. Potential mechanisms by which uPA may promote phrenic motorneuron synaptic plasticity have been explored. Expression of uPA receptors, uPAR and LRP-1, are both up-regulated in the ipsilateral phrenic motor nucleus (PMN) following C2-hemisection. A comparison of microarray data and real-time PCR analysis of mRNAs induced in the PMN after hemisection indicate potential cell signaling pathways downstream of uPA's interaction with these cell surface receptors in the PMN. Knowledge of these uPA-mediated signaling pathways may identify potential means for the pharmacological activation of the synaptic plasticity required for recovery of phrenic motorneuron activity.

Tissue plasminogen activator promotes axonal outgrowth on CNS myelin after conditioned injury.

Following CNS injury, myelin-associated inhibitors represent major obstacles to axonal regeneration and functional recovery. The following study suggests that the proteolytic enzyme tissue plasminogen activator (tPA) plays a major function in 'conditioning-injury induced' axon regeneration. In this paradigm, prior peripheral nerve injury leads to an enhanced ability of sensory neurons to regenerate their central axons in the presence of the CNS inhibitory microenvironment. tPA is widely expressed by CNS and PNS neurons and plays major roles in synaptic reorganization and plasticity. This study shows that cultured neurons from mice deficient in tPA, in contrast to wild-type mice, fail to undergo conditioning-injury induced axonal regeneration in the presence of purified myelin membranes. Interestingly, neurons from mice deficient in plasminogen, the best known substrate for tPA, showed active axon regeneration. These results suggest a novel plasminogen-independent role for tPA in promoting axonal regeneration on CNS myelin.

Interactions of plasminogen activator inhibitor-1 with vitronectin involve an extensive binding surface and induce mutual conformational rearrangements.

In order to explore early events during the association of plasminogen activator inhibitor-1 (PAI-1) with its cofactor vitronectin, we have applied a robust strategy that combines protein engineering, fluorescence spectroscopy, and rapid reaction kinetics. Fluorescence stopped-flow experiments designed to monitor the rapid association of PAI-1 with vitronectin indicate a fast, concentration-dependent, biphasic binding of PAI-1 to native vitronectin but only a monophasic association with the somatomedin B (SMB) domain, suggesting that multiple phases of the binding interaction occur only when full-length vitronectin is present. Nonetheless, in all cases, the initial fast interaction is followed by slower fluorescence changes attributed to a conformational change in PAI-1. Complementary experiments using an engineered, fluorescently silent PAI-1 with non-natural amino acids showed that concomitant structural changes occur as well in native vitronectin. Furthermore, we have measured the effect of vitronectin on the rate of insertion of the reactive center loop into beta-sheet A of PAI-1 during reaction with target proteases. With a variety of PAI-1 variants, we observe that both full-length vitronectin and the SMB domain have protease-specific effects on the rate of loop insertion but that the two exhibit clearly different effects. These results support a model for PAI-1 binding to vitronectin in which the interaction surface extends beyond the region of PAI-1 occupied by the SMB domain. In support of this model are recent results that define a PAI-1-binding site on vitronectin that lies outside the somatomedin B domain (Schar, C. R., Blouse, G. E., Minor, K. H., and Peterson, C. B. (2008) J. Biol. Chem. 283, 10297-10309) and the complementary site on PAI-1 (Schar, C. R., Jensen, J. K., Christensen, A., Blouse, G. E., Andreasen, P. A., and Peterson, C. B. (2008) J. Biol. Chem. 283, 28487-28496).

Decorin promotes robust axon growth on inhibitory CSPGs and myelin via a direct effect on neurons.

Inhibitory chondroitin sulfate proteoglycans (CSPGs) and myelin-associated molecules are major impediments to axon regeneration within the adult central nervous system (CNS). Decorin infusion can however suppress the levels of multiple inhibitory CSPGs and promote axon growth across spinal cord injuries [Davies, J.E., Tang, X., Denning, J.W., Archibald, S.J., and Davies, S.J., 2004. Decorin suppresses neurocan, brevican, phosphacan and NG2 expression and promotes axon growth across adult rat spinal cord injuries. Eur. J. Neurosci. 19, 1226-1242]. A question remained as to whether decorin can also increase axon growth on inhibitory CSPGs and myelin via a direct effect on neurons. We have therefore conducted an in vitro analysis of neurite extension by decorin-treated adult dorsal root ganglion (DRG) neurons cultured on substrates of inhibitory CSPGs or myelin membranes mixed with laminin. Decorin treatment promoted 14.5 and 5-fold increases in average neurite length/neuron over untreated controls on CSPGs or myelin membranes respectively. In addition to suppressing inhibitory scar formation, our present data shows that decorin can directly boost the ability of neurons to extend axons within CSPG or myelin rich environments.

A deletion mutant of vitronectin lacking the somatomedin B domain exhibits residual plasminogen activator inhibitor-1-binding activity.

Vitronectin and plasminogen activator inhibitor-1 (PAI-1) are important physiological binding partners that work in concert to regulate cellular adhesion, migration, and fibrinolysis. The high affinity binding site for PAI-1 is located within the N-terminal somatomedin B domain of vitronectin; however, several studies have suggested a second PAI-1-binding site within vitronectin. To investigate this secondary site, a vitronectin mutant lacking the somatomedin B domain (rDeltasBVN) was engineered. The short deletion had no effect on heparin-binding, integrin-binding, or cellular adhesion. Binding to the urokinase receptor was completely abolished while PAI-1 binding was still observed, albeit with a lower affinity. Analytical ultracentrifugation on the PAI-1-vitronectin complex demonstrated that increasing NaCl concentration favors 1:1 versus 2:1 PAI-1-vitronectin complexes and hampers formation of higher order complexes, pointing to the contribution of charge-charge interactions for PAI-1 binding to the second site. Furthermore, fluorescence resonance energy transfer between differentially labeled PAI-1 molecules confirmed that two independent molecules of PAI-1 are capable of binding to vitronectin. These results support a model for the assembly of higher order PAI-1-vitronectin complexes via two distinct binding sites in both proteins.

Plasminogen activator induction facilitates recovery of respiratory function following spinal cord injury.

The possibility that plasminogen activator (PA) plays a role in synaptic plasticity was explored in the spinal cord during the crossed phrenic phenomenon (CPP), where respiratory functional plasticity develops following spinal cord injury. Synaptic remodeling on phrenic motorneurons occurs during the characteristic delay period following spinal cord injury before CPP recovery of respiratory function. The molecular mechanisms underlying this plasticity are not well-defined. During the critical 1-2 h delay period required for this synaptic plasticity following a C2 hemisection in mice, uPA and tPA mRNAs are rapidly induced in C4-5 ventral spinal cord neurons in the ipsilateral phrenic motor nucleus (PMN), as are uPA and tPA protein levels. A role for uPA in CPP spinal cord plasticity is confirmed by the impaired ability of uPA knockout mice to acquire a good CPP response by 6 h post-hemisection and their lack of structural remodeling of PMN synapses that underlies development of the CPP response.

Spinal cord injury-induced plasticity in the mouse--the crossed phrenic phenomenon.

The crossed phrenic phenomenon (CPP) describes respiratory functional plasticity that arises following spinal cord injury. Cervical spinal cord hemisection rostral to the phrenic nucleus paralyzes the ipsilateral hemidiaphragm by interrupting the descending flow of respiratory impulses from the medulla to phrenic motoneurons in the spinal cord. This loss of activity converts some synapses on phrenic motoneurons from a "functionally ineffective" state pre-hemisection to a "functionally latent" state post-hemisection. If the animal is subjected to respiratory stress by transecting the contralateral phrenic nerve, this latent respiratory pathway is activated and function is restored to the paralyzed hemidiaphragm. The mechanisms underlying this plasticity are not well-defined, particularly at the molecular level. Therefore, we explored whether it was possible to demonstrate the CPP in mice, a species amenable to a molecular genetic approach. We show the CPP qualitatively in mice using electromyographic (EMG) recordings from the diaphragm. Interestingly, our data also suggest that in the mouse latent fibers in the ventral funiculus ipsilateral to an anatomically incomplete hemisection may also play a role in the CPP. In particular, we examined the inter-operative delay time between the spinal cord injury and contralateral phrenicotomy required for a response. As the inter-operative delay was reduced, the proportion of mice displaying the CPP decreased from 95% for overnight animals, 86% in 4-8 h, to 77% for 1-2 h mice, and less than 28% for animals receiving a phrenicotomy under 0.5 h post-spinal cord lesion. This is the first study to demonstrate the CPP in mice.

A mechanism for assembly of complexes of vitronectin and plasminogen activator inhibitor-1 from sedimentation velocity analysis.

Plasminogen activator inhibitor-1 (PAI-1) and vitronectin are cofactors involved in pathological conditions such as injury, inflammation, and cancer, during which local levels of PAI-1 are increased and the active serpin forms complexes with vitronectin. These complexes become deposited into surrounding tissue matrices, where they regulate cell adhesion and pericellular proteolysis. The mechanism for their co-localization has not been elucidated. We hypothesize that PAI-1-vitronectin complexes form in a stepwise and concentration-dependent fashion via 1:1 and 2:1 intermediates, with the 2:1 complex serving a key role in assembly of higher order complexes. To test this hypothesis, sedimentation velocity experiments in the analytical ultracentrifuge were performed to identify different PAI-1-vitronectin complexes. Analysis of sedimentation data invoked a novel multisignal method to discern the stoichiometry of the two proteins in the higher-order complexes formed (Balbo, A., Minor, K. H., Velikovsky, C. A., Mariuzza, R. A., Peterson, C. B., and Schuck, P. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 81-86). Our results demonstrate that PAI-1 and vitronectin assemble into higher order forms via a pathway that is triggered upon saturation of the two PAI-1-binding sites of vitronectin to form the 2:1 complex. This 2:1 PAI-1-vitronectin complex, with a sedimentation coefficient of 6.5 S, is the key intermediate for the assembly of higher order complexes.

A model for the three-dimensional structure of human plasma vitronectin from small-angle scattering measurements.

Small-angle X-ray scattering (SAXS) measurements were used to characterize vitronectin, a circulatory protein found in human plasma that functions in regulating cell adhesion and migration, as well as proteolytic cascades that affect blood coagulation, fibrinolysis, and pericellular proteolysis. SAXS measurements were taken over a 3-fold range of protein concentrations, yielding data that characterize a monodisperse system of particles with an average radius of gyration of 30.3 +/- 0.6 A and a maximum linear dimension of 110 A. Shape restoration was applied to the data to produce two models of the solution structure of the ligand-free protein. A low-resolution model of the protein was generated that indicates the protein to be roughly peanut-shaped. A better understanding of the domain structure of vitronectin resulted from low-resolution models developed from available high-resolution structures of the domains. These domains include the N-terminal domain that was determined experimentally by NMR [Mayasundari, A., Whittemore, N. A., Serpersu, E. H., and Peterson, C. B. (2004) J. Biol. Chem. 279, 29359-29366] and the docked structure of the central and C-terminal domains that were determined by computational threading [Xu, D., Baburaj, K., Peterson, C. B., and Xu, Y. (2001) Proteins: Struct., Funct., Genet. 44, 312-320]. This model provides an indication of the disposition of the central domain and C-terminal heparin-binding domains of vitronectin with respect to the N-terminal somatomedin B (SMB) domain. This model constructed from the available domain structures, which agrees with the low-resolution model produced from the SAXS data, shows the SMB domain well separated from the central and heparin-binding domains by a disordered linker (residues 54-130). Also, binding sites within the SMB domain are predicted to be well exposed to the surrounding solvent for ease of access to its various ligands.

Studying multiprotein complexes by multisignal sedimentation velocity analytical ultracentrifugation.

Protein interactions can promote the reversible assembly of multiprotein complexes, which have been identified as critical elements in many regulatory processes in cells. The biophysical characterization of assembly products, their number and stoichiometry, and the dynamics of their interactions in solution can be very difficult. A classical first-principle approach for the study of purified proteins and their interactions is sedimentation velocity analytical ultracentrifugation. This approach allows one to distinguish different protein complexes based on their migration in the centrifugal field without isolating reversibly formed complexes from the individual components. An important existing limitation for systems with multiple components and assembly products is the identification of the species associated with the observed sedimentation rates. We developed a computational approach for integrating multiple optical signals into the sedimentation coefficient distribution analysis of components, which combines the size-dependent hydrodynamic separation with discrimination of the extinction properties of the sedimenting species. This approach allows one to deduce the stoichiometry and to assign the identity of the assembly products without prior assumptions of the number of species and the nature of their interaction. Although chromophoric labels may be used to enhance the spectral resolution, we demonstrate the ability to work label-free for three-component protein mixtures. We observed that the spectral discrimination can synergistically enhance the hydrodynamic resolution. This method can take advantage of differences in the absorbance spectra of interacting solution components, for example, for the study of protein-protein, protein-nucleic acid or protein-small molecule interactions, and can determine the size, hydrodynamic shape, and stoichiometry of multiple complexes in solution.

Role of Lys-32 residues in R67 dihydrofolate reductase probed by asymmetric mutations.

R67 dihydrofolate reductase (R67 DHFR) is a novel protein encoded by an R-plasmid that confers resistance to the antibiotic, trimethoprim. This homotetrameric enzyme possesses 222 symmetry, which imposes numerous constraints on the single active site pore, including a "one-site-fits-both" strategy for binding its ligands, dihydrofolate (DHF) and NADPH. Previous studies uncovered salt effects on binding and catalysis (Hicks, S. N., Smiley, R. D., Hamilton, J. B., and Howell, E. E. (2003) Biochemistry 42, 10569-10578), however the one or more residues that participate in ionic contacts with the negatively charged tail of DHF as well as the phosphate groups in NADPH were not identified. Several studies predict that Lys-32 residues were involved, however mutations at this residue destabilize the R67 DHFR homotetramer. To study the role of Lys-32 in binding and catalysis, asymmetric K32M mutations have been utilized. To create asymmetry, individual mutations were added to a tandem array of four in-frame gene copies. These studies show one K32M mutation is tolerated quite well, whereas addition of two mutations has variable effects. Two double mutants, K32M:1+2 and K32M: 1+4, which place the mutations on opposite sides of the pore, reduce kcat. However a third double mutant, K32M: 1+3, that places two mutations on the same half pore, enhances kcat 4- to 5-fold compared with the parent enzyme, albeit at the expense of weaker binding of ligands. Because the kcat/Km values for this double mutant series are similar, these mutations appear to have uncovered some degree of non-productive binding. This non-productive binding mode likely arises from formation of an ionic interaction that must be broken to allow access to the transition state. The K32M:1+3 mutant data suggest this interaction is an ionic interaction between Lys-32 and the charged tail of dihydrofolate. This unusual catalytic scenario arises from the 222 symmetry imposed on the single active site pore.

Plasminogen activator inhibitor type 1 promotes the self-association of vitronectin into complexes exhibiting altered incorporation into the extracellular matrix.

Serine proteinase inhibitors, including plasminogen activator inhibitor type 1 (PAI-1) and antithrombin, are key regulators of hemostatic processes such as thrombosis and wound healing. Much evidence suggests that PAI-1 can influence such processes, as well as pathological events like tumor metastasis, through its ability to directly regulate binding of blood platelets and cells to extracellular substrata. One way that PAI-1 influences these processes may be mediated through its binding to the plasma protein vitronectin. Binding to PAI-1 results in the incorporation of vitronectin into a higher order complex with a potential for multivalent interactions (Podor, T. J., Shaughnessy, S. G., Blackburn, M. N., and Peterson, C. B. (2000) J. Biol. Chem. 275, 25402-25410). In this study, evidence is provided to support this concept from studies on the effects of PAI-1-induced multimerization on the interactions of vitronectin with matrix components and cell surface receptors. By monitoring complex formation and stability over time using size-exclusion high performance liquid chromatography, a correlation is made between PAI-1-induced multimerization and enhanced cell/matrix binding properties of vitronectin. This evidence indicates that PAI-1 alters the adhesive functions of vitronectin by converting the protein via the higher order complex to a self-associated, multivalent species that is functionally distinct from the abundant monomeric form found in the circulation.