Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. Astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout (Timp1KO) mice do not efficiently remyelinate following a demyelinating injury. Here, we performed an unbiased proteomic analysis and identified a fibronectin-derived peptide called Anastellin (Ana) that was unique to the Timp1KO astrocyte secretome. Ana was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Ana is known to act upon the sphingosine-1-phosphate receptor 1, and we determined that Ana also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro. Administration of FTY720 to wild-type C57BL/6 mice during MOG35-55-experimental autoimmune encephalomyelitis ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 (Timp1KO) had no effect. Analysis of Timp1 and fibronectin (FN1) transcripts from primary human astrocytes from healthy and multiple sclerosis (MS) donors revealed lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Last, analyses of proteomic databases of MS samples identified Ana peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high disease activity. A role for Ana in MS as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and innate remyelination potential in the MS brain.

Binding and Dynamics Demonstrate the Destabilization of Ligand Binding for the S688Y Mutation in the NMDA Receptor GluN1 Subunit.

Encephalopathies are brain dysfunctions that lead to cognitive, sensory, and motor development impairments. Recently, the identification of several mutations within the N-methyl-D-aspartate receptor (NMDAR) have been identified as significant in the etiology of this group of conditions. However, a complete understanding of the underlying molecular mechanism and changes to the receptor due to these mutations has been elusive. We studied the molecular mechanisms by which one of the first mutations within the NMDAR GluN1 ligand binding domain, Ser688Tyr, causes encephalopathies. We performed molecular docking, randomly seeded molecular dynamics simulations, and binding free energy calculations to determine the behavior of the two major co-agonists: glycine and D-serine, in both the wild-type and S688Y receptors. We observed that the Ser688Tyr mutation leads to the instability of both ligands within the ligand binding site due to structural changes associated with the mutation. The binding free energy for both ligands was significantly more unfavorable in the mutated receptor. These results explain previously observed in vitro electrophysiological data and provide detailed aspects of ligand association and its effects on receptor activity. Our study provides valuable insight into the consequences of mutations within the NMDAR GluN1 ligand binding domain.

Astrocytic TIMP-1 regulates production of Anastellin, a novel inhibitor of oligodendrocyte differentiation and FTY720 responses.

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. We have previously demonstrated that murine astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout ( Timp1 KO ) mice do not efficiently remyelinate following a demyelinating injury. To better understand the basis of this, we performed unbiased proteomic analyses and identified a fibronectin-derived peptide called anastellin that is unique to the murine Timp1 KO astrocyte secretome. Anastellin was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Anastellin is known to act upon the sphingosine-1-phosphate receptor 1 (S1PR1), and we determined that anastellin also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro . Further, administration of FTY720 to wild-type C57BL/6 mice during MOG 35-55 -EAE ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 in astrocytes ( Timp1 cKO ) had no effect. Analysis of human TIMP1 and fibronectin ( FN1 ) transcripts from healthy and multiple sclerosis (MS) patient brain samples revealed an inverse relationship where lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Lastly, we analyzed proteomic databases of MS samples and identified anastellin peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high versus low disease activity. The prospective role for anastellin generation in association with myelin lesions as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and the innate remyelination potential of the the MS brain. Significance Statement: Astrocytic production of TIMP-1 prevents the protein catabolism of fibronectin. In the absence of TIMP-1, fibronectin is further digested leading to a higher abundance of anastellin peptides that can bind to sphingosine-1-phosphate receptor 1. The binding of anastellin with the sphingosine-1-phosphate receptor 1 impairs the differentiation of oligodendrocytes progenitor cells into myelinating oligodendrocytes in vitro , and negates the astrocyte-mediated therapeutic effects of FTY720 in the EAE model of chronic CNS inflammation. These data indicate that TIMP-1 production by astrocytes is important in coordinating astrocytic functions during inflammation. In the absence of astrocyte produced TIMP-1, elevated expression of anastellin may represent a prospective biomarker for FTY720 therapeutic responsiveness.

Multimodal imaging of bacterial-host interface in mice and piglets with Staphylococcus aureus endocarditis.

Acute bacterial endocarditis is a rapid, difficult to manage, and frequently lethal disease. Potent antibiotics often cannot efficiently kill Staphylococcus aureus that colonizes the heart's valves. S. aureus relies on virulence factors to evade therapeutics and the host's immune response, usurping the host's clotting system by activating circulating prothrombin with staphylocoagulase and von Willebrand factor-binding protein. An insoluble fibrin barrier then forms around the bacterial colony, shielding the pathogen from immune cell clearance. Targeting virulence factors may provide previously unidentified avenues to better diagnose and treat endocarditis. To tap into this unused therapeutic opportunity, we codeveloped therapeutics and multimodal molecular imaging to probe the host-pathogen interface. We introduced and validated a family of small-molecule optical and positron emission tomography (PET) reporters targeting active thrombin in the fibrin-rich environment of bacterial colonies. The imaging agents, based on the clinical thrombin inhibitor dabigatran, are bound to heart valve vegetations in mice. Using optical imaging, we monitored therapy with antibodies neutralizing staphylocoagulase and von Willebrand factor-binding protein in mice with S. aureus endocarditis. This treatment deactivated bacterial defenses against innate immune cells, decreased in vivo imaging signal, and improved survival. Aortic or tricuspid S. aureus endocarditis in piglets was also successfully imaged with clinical PET/magnetic resonance imaging. Our data map a route toward adjuvant immunotherapy for endocarditis and provide efficient tools to monitor this drug class for infectious diseases.

Improvement of kynurenine aminotransferase-II inhibitors guided by mimicking sulfate esters.

The mammalian kynurenine aminotransferase (KAT) enzymes are a family of related isoforms that are pyridoxal 5'-phosphate-dependent, responsible for the irreversible transamination of kynurenine to kynurenic acid. Kynurenic acid is implicated in human diseases such as schizophrenia where it is found in elevated levels and consequently KAT-II, as the isoform predominantly responsible for kynurenic acid production in the brain, has been targeted for the development of specific inhibitors. One class of compounds that have also shown inhibitory activity towards the KAT enzymes are estrogens and their sulfate esters. Estradiol disulfate in particular is very strongly inhibitory and it appears that the 17-sulfate makes a significant contribution to its potency. The work here demonstrates that the effect of this moiety can be mirrored in existing KAT-II inhibitors, from the development of two novel inhibitors, JN-01 and JN-02. Both inhibitors were based on NS-1502 (IC50: 315 µM), but the deliberate placement of a sulfonamide group significantly improved the potency of JN-01 (IC50: 73.8 µM) and JN-02 (IC50: 112.8 µM) in comparison to the parent compound. This 3-4 fold increase in potency shows the potential of these moieties to be accommodated in the KAT-II active site and the effect they can have on improving inhibitors, and the environments in the KAT-II have been suitably modelled using docking calculations.

High resolution crystal structures of human kynurenine aminotransferase-I bound to PLP cofactor, and in complex with aminooxyacetate.

In this study, we report two high-resolution structures of the pyridoxal 5' phosphate (PLP)-dependent enzyme kynurenine aminotransferase-I (KAT-I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT-I at 1.28 Å resolution, and the other with the general PLP-dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP-bound structures. We also report the inhibition of KAT-1 by AOAA and aminooxy-phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 µM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT-I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.

Inhibiting GPIbα Shedding Preserves Post-Transfusion Recovery and Hemostatic Function of Platelets After Prolonged Storage.

OBJECTIVE: The platelet storage lesion accelerates platelet clearance after transfusion, but the underlying molecular mechanism remains elusive. Although inhibiting sheddase activity hampers clearance of platelets with storage lesion, the target platelet protein responsible for ectodomain shedding-induced clearance is not definitively identified. Monoclonal antibody 5G6 was developed recently to bind specifically human platelet receptor glycoprotein (GP)Ibα and inhibit its shedding but not shedding of other receptors. Here, the role of GPIbα shedding in platelet clearance after transfusion was addressed. APPROACH AND RESULTS: Both human leukoreduced apheresis-derived platelets and transgenic mouse platelets expressing human GPIbα were stored at room temperature in the presence and absence of 5G6 Fab fragment. At various time points, aliquots of stored platelets were analyzed and compared. 5G6 Fab inhibited GPIbα shedding in both platelets during storage and preserved higher level of GPIbα on the platelet surface. Compared with age-matched control platelets, 5G6 Fab-stored platelets exhibited similar levels of platelet activation, degranulation, and agonist-induced aggregation. 5G6 Fab-stored human GPIbα platelets exhibited significantly higher post-transfusion recovery and in vivo hemostatic function in recipient mice than control platelets. Consistently, 5G6 Fab-stored, 8-day-old human platelets produced similar improvement in post-transfusion recovery in immunodeficient mice and in ex vivo thrombus formation over collagen under shear flow. CONCLUSIONS: Specific inhibition of GPIbα shedding in the stored platelets improves post-transfusion platelet recovery and hemostatic function, providing clear evidence for GPIbα shedding as a cause of platelet clearance. These results suggest that specific inhibition of GPIbα shedding may be used to optimize platelet storage conditions.

Structure and Potential Cellular Targets of HAMLET-like Anti-Cancer Compounds made from Milk Components.

The HAMLET family of compounds (Human Alpha-lactalbumin Made Lethal to Tumours) was discovered during studies on the properties of human milk, and is a class of protein-lipid complexes having broad spectrum anti-cancer, and some specific anti-bacterial properties. The structure of HAMLET-like compounds consists of an aggregation of partially unfolded protein making up the majority of the compound's mass, with fatty acid molecules bound in the hydrophobic core. This is a novel protein-lipid structure and has only recently been derived by small-angle X-ray scattering analysis. The structure is the basis of a novel cytotoxicity mechanism responsible for anti-cancer activity to all of the around 50 different cancer cell types for which the HAMLET family has been trialled. Multiple cytotoxic mechanisms have been hypothesised for the HAMLET-like compounds, but it is not yet clear which of those are the initiating cytotoxic mechanism(s) and which are subsequent activities triggered by the initiating mechanism(s). In addition to the studies into the structure of these compounds, this review presents the state of knowledge of the anti-cancer aspects of HAMLET-like compounds, the HAMLET-induced cytotoxic activities to cancer and non-cancer cells, and the several prospective cell membrane and intracellular targets of the HAMLET family. The emerging picture is that HAMLET-like compounds initiate their cytotoxic effects on what may be a cancer-specific target in the cell membrane that has yet to be identified. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Ketogenic diet alters dopaminergic activity in the mouse cortex.

The present study was conducted to determine if the ketogenic diet altered basal levels of monoamine neurotransmitters in mice. The catecholamines dopamine (DA) and norephinephrine (NE) and the indolamine serotonin (5HT) were quantified postmortem in six different brain regions of adult mice fed a ketogenic diet for 3 weeks. The dopamine metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) and the serotonin metabolite 5-hydroxyindole acetic acid (5HIAA) were also measured. Tissue punches were collected bilaterally from the motor cortex, somatosensory cortex, nucleus accumbens, anterior caudate-putamen, posterior caudate-putamen and the midbrain. Dopaminergic activity, as measured by the dopamine metabolites to dopamine content ratio - ([DOPAC]+[HVA])/[DA] - was significantly increased in the motor and somatosensory cortex regions of mice fed the ketogenic diet when compared to those same areas in brains of mice fed a normal diet. These results indicate that the ketogenic diet alters the activity of the meso-cortical dopaminergic system, which may contribute to the diet's therapeutic effect in reducing epileptic seizure activity.

Homology modeling of human kynurenine aminotransferase III and observations on inhibitor binding using molecular docking.

Kynurenine aminotransferase (KAT) isozymes are responsible for catalyzing the conversion of kynurenine (KYN) to kynurenic acid (KYNA), which is considered to play a key role in central nervous system (CNS) disorders, including schizophrenia. The levels of KYNA in the postmortem prefrontal cortex and in the Cerebrospinal fluid (CSF) of schizophrenics are greater than normal brain. A basic strategy to decrease kynurenic acid levels is to promote the inhibition of the biosynthetic KAT isozymes. As there is no crystallographic model for human kynurenine aminotransferase III (KAT III), therefore, homology modeling has been performed based on the Mus musculus kynurenine aminotransferase III crystal structure (PDB ID: 3E2Y) as a template, and the model of the human KAT III was refined and optimized with molecular dynamics simulations. Further evaluation of the model quality was accomplished by investigating the interaction of KAT III inhibitors with the modeled enzyme. Such interactions were determined employing the AutoDock 4.2 program using the MGLTools 1.5.6 package. The most important interactions for the binding of the inhibitors, which are probably also central components of the active site of KAT III, were identified as Ala134, Tyr135, Lys 280, Lys 288, Thr285 and Arg429, which provide hydrogen bond interactions. Additionally, Tyr135 and Arg429 have good electrostatic interactions with inhibitors consistent with these residues also being essential for inhibition of the enzyme activity. We expect that this model and these docking data will be a useful resource for the rational design of novel drugs for treating neuropathologies.

Essential structural features of novel antischizophrenic drugs: a review.

Due to the wide range of chemical structures and variety of mechanisms of action of antischizophrenic agents, it is difficult to identify and confirm a common pharmacophore. The present review summarizes various pharmacophore models for antischizophrenic activity including those based on the new targets, the kynurenine aminotransferase (KATs), which may facilitate the development of novel drugs. Some models illustrate the structural differences of compounds with mechanisms of action considered similar, and yet others demonstrate pharmacophore models for similar chemical classes of compounds for which the mechanism of antischizophrenic action is still not clear. In this study, we discuss the pharmacophore models for antipsychotics including phenothiazine, butyrophenone, thioxanthene, and atypical agents along with the novel antischizophrenic agents which are inhibitors of KATs isozymes.

The comparative immunogenicity of human and porcine factor VIII in haemophilia A mice.

Inhibitory antibodies to factor VIII (FVIII inhibitors) are the most significant complication in the management of haemophilia A. The immunogenicity of FVIII may be driven in part by structural determinants within the FVIII molecule itself. Regions of nonidentity between human and porcine FVIII possibly could drive differential immune responses. The goal of this study was to compare the overall antibody response and levels of antibodies to the individual FVIII domains in naïve haemophilia A mice immunised with human or porcine FVIII. Haemophilia A mice were immunised with human or porcine FVIII using a protocol that mimics human clinical use. Inhibitor and total anti-FVIII antibody titers were measured and the domain-specificity of antibodies from 1,759 anti-FVIII hybridomas was determined. The overall immunogenicity of human and porcine FVIII was similar but significant differences in domain recognition were discovered. Anti-A2 and anti-C2 antibodies constituted the majority of inhibitors in both the human and porcine FVIII groups, similar to inhibitors that develop in humans. The proportions of anti-A2 or anti-C2 antibodies were not significantly different between the two groups. However, the specific inhibitory activity of anti-A2 antibodies was higher in the human FVIII group. Additionally, proportion of anti-C1 antibodies was significantly higher in the human FVIII group. In contrast, anti-A3 antibodies were more common in the porcine FVIII group. The differential immune response to human and porcine FVIII suggests that it may be possible to reduce the immunogenicity of FVIII by mutagenesis of the A2, A3 and C1 domains.

Column chromatography analysis of brain tissue: an advanced laboratory exercise for neuroscience majors.

Neurochemical analysis of discrete brain structures in experimental animals provides important information on synthesis, release, and metabolism changes following behavioral or pharmacological experimental manipulations. Quantitation of neurotransmitters and their metabolites following unilateral drug injections can be carried out using standard chromatographic equipment typically found in most undergraduate analytical laboratories. This article describes an experiment done in a six session (four hours each) component of a neuroscience research methods course. The experiment provides advanced neuroscience students experience in brain structure dissection, sample preparation, and quantitation of catecholamines using high performance liquid chromatography (HPLC) and protein analysis using ultraviolet spectroscopic methods. The students are exposed to useful laboratory techniques such as standard solution preparation and calibration curve construction, centrifugation, quantitative pipetting, and data evaluation and graphical presentation. Typically, only students that participate in independent neuroscience research are familiar with these types of quantitative skills. The usefulness of this type of experimental design for understanding behavioral or pharmacological effects on neurotransmitter systems is emphasized through a final report requiring a comprehensive literature search.

Relationship between NMDA receptor expression and MPP+ toxicity in cultured dopaminergic cells.

It has been suggested that excitotoxicity could be contributing to dopamine cell loss after methylphenylpyridinium ion (MPP+) exposure, although the literature regarding this is contradictory. Given that in cell culture excitotoxicity has been reported to be dependent on culture age, we postulated that these discrepant results might be explained by a difference in developmental expression of N-methyl-D-aspartate (NMDA) receptors. To test this, mesencephalic cells were cultured and the number of dopaminergic neurons (tyrosine hydroxylase-immunoreactive cells [TH-IR] cells) expressing the NMDA R1 subunit (NR1) was determined using double-label immunofluorescence microscopy. An increase in the percentage of TH-IR cells expressing NR1 occurred over time in culture and this correlated with the toxicity of NMDA. At 7 days in vitro (DIV 7), only 17% (n=167 cells/4 experiments) of TH-IR cells expressed NR1 and these cells were insensitive to NMDA toxicity. This increased to 80% (n=254 cells/6 experiments) by DIV 11 and cultures were now susceptible to NMDA-induced injury. Cultures grown for either 7 or 11 days were treated for 48 hr with increasing concentrations of MPP= (0.5-20 microM) and the loss of dopaminergic neurons was determined by cell counting. Cultures at DIV 7 were more sensitive to MPP= than 11-day-old cultures (LD50= approximately 0.75 microM vs. 15 microM, respectively). Co-exposure to MK-801 (5 microM) did not protect against MPP+ toxicity in young cultures, but attenuated MPP+ toxicity in the older cultures, becoming statistically significant at 20 microM MPP+. These data indicate that the activation of NMDA receptors is not required for, but can contribute to, MPP(+)-induced neurodegeneration of dopaminergic cells in culture.