non-invasive-imaging-analysis-cerebral-ischemia-living-rats-using

Non-invasive Imaging and Analysis of Cerebral Ischemia in Living Rats Using Positron Emission Tomography with <sup>18</sup>F-FDG

Stroke is the third leading cause of death among Americans 65 years of age or older1. The quality of life for patients who suffer from a stroke fails to ...

University of Notre Dame

We have identified a novel DEAD box RNA helicase (97 kDa, DP97) from a breast cancer cDNA library that interacts in a hormone-dependent manner with nuclear receptors and represses their transcriptional activity. DP97 has RNA-dependent ATPase activity, and mapping studies localize the interacting regions of DP97 and nuclear receptors to the C-terminal region of DP97 and the hormone binding/activation function-2 region of estrogen receptors (ER), as well as several other nuclear receptors. Repression by DP97 maps to a small region (amino acids 589-631) that has homology to a repression domain in the corepressor protein NCoR2/SMRTe. This region of DP97 is necessary and sufficient for its intrinsic repression activity. The N-terminal helicase region of DP97 is, however, dispensable for its transcriptional repressor activity. The knockdown of endogenous cellular DP97 by antisense DP97 or RNA interference (siRNA for DP97) results in significant enhancement of the expression of estradiol-ER-stimulated genes and attenuation of the repression of genes inhibited by the estradiol-ER. This implies that endogenous DP97 normally dampens stimulation and intensifies repression of estradiol-ER-regulated genes. Our findings add to the growing evidence that RNA helicases can associate with nuclear receptors and function as coregulators to modulate receptor transcriptional activity.

Regulation of nuclear receptor transcriptional activity by a novel DEAD box RNA helicase (DP97).

Plasminogen activator inhibitor-1 is the main physiological regulator of tissue-type plasminogen activator in normal plasma. In addition to its critical function in fibrinolysis, plasminogen activator inhibitor-1 has been implicated in roles in other physiological and pathophysiological processes. To investigate structure-function aspects of mouse plasminogen activator inhibitor-1, the recombinant protein was expressed in Escherichia coli and purified. Five variant recombinant murine proteins (R76E, Q123K, R346A, R101A, and Q123K/R101A) were also generated using site-directed mutagenesis. The variant (R346A) was found to be defective in its inhibitory activity against tissue plasminogen activator relative to its wild-type counterpart. Enzyme-linked immunosorbent assay and surface plasmon resonance experiments demonstrated reduced vitronectin-binding affinity of the (Q123K) variant (K(D) = 1800 nm) relative to the wild-type protein (K(D) = 5.4 nm). Kinetic analyses indicated that the (Q123K) variant had a slower association (k(on) = 2.92 x 10(4) m(-1) s(-1)) to, and a faster dissociation from, vitronectin (k(off) = 5.3 x 10(-2) s(-1)), (wild-type k(on) = 1.03 x 10(6) m(-1) s(-1) and k(off) = 5.27 x 10(-3) s(-1)). The Q123K/R101A variant demonstrated an even lower vitronectin-binding ability. Low density lipoprotein receptor-related protein binding was decreased for the (R76E) variant. It was also demonstrated that the plasminogen activator inhibitor-1/vitronectin complex decreased the interaction of plasminogen activator inhibitor-1 with low density lipoprotein receptor-related protein. These results indicate that the complex interactions traditionally associated with different plasminogen activator inhibitor-1 functions apply to the murine system, thus showing a commonality of subtle functions among different species and evolutionary conservation of this protein. Further, this study provides additional evidence that the human hemostasis system can be studied effectively in the mouse, which is a great asset for investigations with gene-altered mice.

Conservation of critical functional domains in murine plasminogen activator inhibitor-1.

Cell proliferation, an event associated with angiogenesis, involves coordinated activities of a number of proteins. The role of plasminogen activator inhibitor-1 (PAI-1) in angiogenesis remains controversial. Utilizing proliferating PAI-1-/- endothelial cells (EC), the impact of a host PAI-1 deficiency on Akt activation was evaluated. Hyperactivation of Akt(Ser(P)473) was observed in PAI-1-/- EC, and this was probably due to enhanced inactivation of tumor suppressor PTEN, thus rendering the cells resistant to apoptotic signals. Higher levels of inactivated caspase-9 in PAI-1-/- EC led to lower levels of procaspase-3 and cleaved caspase-3, thereby promoting survival. These effects were reversed when recombinant PAI-1 was added to PAI-1-/- EC. Additional studies demonstrated that regulation of proliferation is dependent on its interaction with low density lipoprotein receptor-related protein. Thus, PAI-1 is a negative regulator of cell growth, exerting its effect on the phosphatidylinositol 3-kinase/Akt pathway and allowing controlled cell proliferation.

A novel function of plasminogen activator inhibitor-1 in modulation of the AKT pathway in wild-type and plasminogen activator inhibitor-1-deficient endothelial cells.

Wnt signaling.

Plasminogen activator inhibitor type-1 (PAI-1) is a multi-functional protein. It is a fast-acting inhibitor of plasminogen activators; urokinase-plasminogen activator and tissue type plasminogen activator, and also plays an important role in regulating cell proliferation, adhesion, migration, and signal transduction pathways. These biological events are important processes during angiogenesis and restenosis. PAI-1 has been shown to regulate proliferation, migration, and apoptosis of vascular smooth muscle cells and endothelial cells. The ability of PAI-1 to regulate cellular proliferation and migration has been attributed to its ability to control plasmin production, modify signaling pathways, and its inherent multifactorial ability to bind to vitronectin and lipoprotein receptor-related protein. However, the mechanism by which PAI-1 regulates the apoptotic pathway is not well understood. Evidence from the literature suggests that PAI-1 or its deficiency alters key signalling pathways, such as the PI3-k/Akt and the Jak/STAT pathways, and is involved in maintaining endothelial cell integrity thereby regulating cell death. Other investigators have demonstrated that PAI-1 directly binds to caspases as a mechanism of PAI-1-mediated cellular apoptosis. Moreover, results from studies assessing the role of PAI-1 in apoptosis have suggested that PAI-1 can exert pathogenic or protective effects, which may be related to the disease model or type of injury employed.

Plasminogen activator inhibitor-1: the double-edged sword in apoptosis.

The effects of conantokin (con)-G, con-R[1-17], and con-T on ion flow through N-methyl-D-aspartate receptor (NMDAR) ion channels were determined in cultured primary rat hippocampal neurons. The potency of con-G diminished, whereas inhibition by con-R[1-17] and con-T did not change, as the neurons matured. Con-G, con-R[1-17], and con-T effectively diminished NMDA-induced Ca(2+) influx into the cells. A similar age-dependent decrease in con-G-mediated inhibition of the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) was observed, compared to con-R[1-17] and con-T. The effects of the conantokins on NMDA-induced cAMP response element-binding protein (CREB) phosphorylation in immature (DIV 9) and mature (DIV 16) neurons showed that, at DIV 9, con-G, con-R[1-17], and con-T inhibited NMDA-mediated P-CREB levels, whereas in DIV 16 neurons the conantokins did not inhibit overall levels of NMDA-induced P-CREB. In contrast, P-CREB levels were enhanced through inhibition of the protein phosphatases, PP1 and PP2B (calcineurin). This ability of conantokins to sustain CREB phosphorylation can thus enhance neuronal survival and plasticity.

Conantokins inhibit NMDAR-dependent calcium influx in developing rat hippocampal neurons in primary culture with resulting effects on CREB phosphorylation.

The angiogenic potential of a cell requires dynamic reorganization of the cytoskeletal architecture that involves the interaction of urokinase-type plasminogen activator receptor (uPAR) with the extracellular matrix. This study focuses on the effect of uPAR deficiency (uPAR-/-) on angiogenic function and associated cytoskeletal organization. Utilizing murine endothelial cells, it was observed that adhesion, migration, proliferation, and capillary tube formation were altered in uPAR-/- cells compared to wild-type (WT) cells. On a vitronectin (Vn) matrix, uPAR-/- cells acquired a "fried egg" morphology characterized by circular actin organization and lack of lamellipodia formation. The up-regulation of β1 integrin, FAK(P-Tyr925), and paxillin (P-Tyr118), and decreased Rac1 activation, suggested increased focal adhesions, but delayed focal adhesion turnover in uPAR-/- cells. This accounted for the enhanced adhesion, but attenuated migration, on Vn. VEGF-enriched Matrigel implants from uPAR-/- mice demonstrated a lack of mature vessel formation compared to WT mice. Collectively, these results indicate that a uPAR deficiency leads to decreased angiogenic functions of endothelial cells.

A deficiency of uPAR alters endothelial angiogenic function and cell morphology.

To delineate the critical features of platelets required for formation and stability of thrombi, thromboelastography and platelet aggregation measurements were employed on whole blood of normal patients and of those with Bernard-Soulier Syndrome (BSS) and Glanzmann's Thrombasthenia (GT). We found that separation of platelet activation, as assessed by platelet aggregation, from that needed to form viscoelastic stable whole blood thrombi, occurred. In normal human blood, ristocetin and collagen aggregated platelets, but did not induce strong viscoelastic thrombi. However, ADP, arachidonic acid, thrombin, and protease-activated-receptor-1 and -4 agonists, stimulated both processes. During this study, we identified the genetic basis of a very rare double heterozygous GP1b deficiency in a BSS patient, along with a new homozygous GP1b inactivating mutation in another BSS patient. In BSS whole blood, ADP responsiveness, as measured by thrombus strength, was diminished, while ADP-induced platelet aggregation was normal. Further, the platelets of 3 additional GT patients showed very weak whole blood platelet aggregation toward the above agonists and provided whole blood thrombi of very low viscoelastic strength. These results indicate that measurements of platelet counts and platelet aggregability do not necessarily correlate with generation of stable thrombi, a potentially significant feature in patient clinical outcomes.

Abnormal whole blood thrombi in humans with inherited platelet receptor defects.

A skin-tropic invasive group A Streptococcus pyogenes (GAS) strain, AP53, contains a natural inactivating mutation in the covS gene (covS(M)) of the two-component responder (CovR)/sensor (CovS) gene regulatory system. The effects of this mutation on specific GAS virulence determinants have been assessed, with emphasis on expression of the extracellular protease, streptococcal pyrogenic exotoxin B (SpeB), capsular hyaluronic acid, and proteins that allow host plasmin assembly on the bacterial surface, viz. a high affinity plasminogen (Pg)/plasmin receptor, Pg-binding group A streptococcal M protein (PAM), and the human Pg activator streptokinase. To further illuminate mechanisms of the functioning of CovRS in the virulence of AP53, two AP53 isogenic strains were generated, one in which the natural covS(M) gene was mutated to WT-covS (AP53/covS(WT)) and a strain that contained an inactivated covR gene (AP53/ΔcovR). Two additional strains that do not contain PAM, viz. WT-NS931 and NS931/covS(M), were also employed. SpeB was not measurably expressed in strains containing covR(WT)/covS(M), whereas in strains with natural or engineered covR(WT)/covS(WT), SpeB expression was highly up-regulated. Alternatively, capsule synthesis via the hasABC operon was enhanced in strain AP53/covS(M), whereas streptokinase expression was only slightly affected by the covS inactivation. PAM expression was not substantially influenced by the covS mutation, suggesting that covRS had minimal effects on the mga regulon that controls PAM expression. These results demonstrate that a covS inactivation results in virulence gene alterations and also suggest that the CovR phosphorylation needed for gene up- or down-regulation can occur by alternative pathways to CovS kinase.

A natural inactivating mutation in the CovS component of the CovRS regulatory operon in a pattern D Streptococcal pyogenes strain influences virulence-associated genes.

Group A Streptococcus pyogenes (GAS) strain AP53 is a primary isolate from a patient with necrotizing fasciitis. These AP53 cells contain an inactivating mutation in the sensor component of the cluster of virulence (cov) responder (R)/sensor (S) two-component gene regulatory system (covRS), which enhances the virulence of the primary strain, AP53/covR(+)S(-). However, specific mechanisms by which the covRS system regulates the survival of GAS in humans are incomplete. Here, we show a key role for covRS in the regulation of opsonophagocytosis of AP53 by human neutrophils. AP53/covR(+)S(-) cells displayed potent binding of host complement inhibitors of C3 convertase, viz. Factor H (FH) and C4-binding protein (C4BP), which concomitantly led to minimal C3b deposition on AP53 cells, further showing that these plasma protein inhibitors are active on GAS cells. This resulted in weak killing of the bacteria by human neutrophils and a corresponding high death rate of mice after injection of these cells. After targeted allelic alteration of covS(-) to wild-type covS (covS(+)), a dramatic loss of FH and C4BP binding to the AP53/covR(+)S(+) cells was observed. This resulted in elevated C3b deposition on AP53/covR(+)S(+) cells, a high level of opsonophagocytosis by human neutrophils, and a very low death rate of mice infected with AP53/covR(+)S(+). We show that covRS is a critical transcriptional regulator of genes directing AP53 killing by neutrophils and regulates the levels of the receptors for FH and C4BP, which we identify as the products of the fba and enn genes, respectively.

Complement-mediated opsonization of invasive group A Streptococcus pyogenes strain AP53 is regulated by the bacterial two-component cluster of virulence responder/sensor (CovRS) system.

Three members of a family of small neurotoxic peptides from the venom of Conus parius, conantokins (Con) Pr1, Pr2, and Pr3, function as antagonists of N-methyl-D-aspartate receptors (NMDAR). We report structural characterizations of these synthetic peptides, and also demonstrate their antagonistic properties toward ion flow through NMDAR ion channels in primary neurons. ConPr1 and ConPr2 displayed moderate increases in α-helicity after addition of Mg(2+). Native apo-ConPr3 possessed an α-helical conformation, and the helicity increased only slightly on addition of Mg(2+). Additionally, these peptides diminished NMDA/Gly-mediated currents and intracellular Ca(2+) (iCa(2+)) influx in mature rat primary hippocampal neurons. Electrophysiological data showed that these peptides displayed slower antagonistic properties toward the NMDAR than conantokins from other species of cone snails, e.g., ConT and ConG. Furthermore, to demonstrate selectivity of the C. parius-derived conantokins towards specific NMDAR subunits, cortical neurons from GluN2A(-/-) and GluN2B(-/-) mice were utilized. Robust inhibition of NMDAR-mediated stimulation in GluN2A(-/-)-derived mouse neurons, as compared to those isolated from GluN2B(-/-)-mouse brains, was observed, suggesting a greater selectivity of these antagonists towards the GluN2B subunit. These C. parius conantokins mildly inhibited NMDAR-induced phosphorylation of CREB at Ser(133), suggesting that the peptides modulated iCa(2+) entry and, thereby, activation of CREB, a transcription factor that is required for maintaining long-term synaptic activity. Our data mechanistically show that while these peptides effectively antagonize NMDAR-directed current and iCa(2+) influx, receptor-coupled CREB signaling is maintained. The consequence of sustained CREB signaling is improved neuronal plasticity and survival during neuropathologies.

Antagonist properties of Conus parius peptides on N-methyl-D-aspartate receptors and their effects on CREB signaling.

The spatial distribution of N-methyl-d-aspartate receptor (NMDAR) subunits in layer 5 (L5) neurons of the medial prefrontal cortex (mPFC) is important for integrating input-output signals involved in cognitive functions and motor behavior. In this study, focal laser scanning photostimulation of caged glutamate, slice electrophysiology, and small peptide pharmacology, were used to map the distribution of functional GluN2A and GluN2B subunits of the NMDAR from L5 neurons of wild-type (WT) and GluN2A(-/-) mice. Focal uncaging of glutamate evoked spatially-restricted glutamatergic responses on various dendritic locations of pyramidal neurons in the mPFC. Analyses of the spatial arrangements of the GluN2A and GluN2B subunits were performed by comparing inhibition of glutamatergic responses in the presence of the GluN2A-selective pharmacological antagonist, NVP-AAM077 (NVP), and the GluN2B-selective peptidic antagonist, conantokin-G (con-G). We found that apical and basal expression and distribution of GluN2A and GluN2B were similar in L5 mPFC neurons of WT mice. However, the inhibition of glutamatergic responses by NVP in brain slices of GluN2A(-/-) mice were dramatically decreased, while con-G inhibition remained similar to that observed in WT brain slices. The data obtained show that expression and spatial arrangement of GluN2B subunits is independent of GluN2A in L5 neurons of the mPFC. These findings have important ramifications for NMDAR organization and function in L5 pyramidal neurons of the mPFC, and show that specific populations of NMDARs can be antagonized, while sparing other subgroups of NMDARs, thus preserving selective NMDAR functions, an important therapeutic advantage.

Probing NMDA receptor GluN2A and GluN2B subunit expression and distribution in cortical neurons.

Retinal ganglion cells (RGCs), which are the sole output neurons of the retina, express N-methyl-D-aspartate receptors (NMDARs), rendering these cells susceptible to glutamate excitotoxicity, with implications for loss of normal RGC excitatory responses in disorders such as glaucoma and diabetic retinopathy. Therefore, antagonists that inhibit NMDAR-mediated currents specifically by targeting the GluN2B component of the ion channel have the potential to serve as a basis for developing potential therapeutics. The roles of peptidic conantokins, which are potent brain neuronal NMDAR inhibitors, were studied. By using patch-clamp whole-cell analyses in dissociated RGCs and retinal whole-mount RGCs, we evaluated the effects of synthetic conantokin-G (conG) and conantokin-T (conT), which are small γ-carboxyglutamate-containing peptides, on NMDA-mediated excitatory responses in mouse RGCs. Both conG and conT inhibited the NMDA-mediated currents of dark-adapted dissociated and whole-mount RGCs in a dose-dependent, reversible, noncompetitive manner. Inhibition of NMDA-mediated steady-state currents by NMDAR nonsubunit-selective conT was approximately threefold greater than GluN2B-selective conG or ifenprodil, demonstrating its potential ability to inhibit both GluN2A- and GluN2B-containing ion channels in RGCs. Because the extent of inhibition of NMDA-evoked currents by conG and the pharmacologic GluN2B-selective inhibitor ifenprodil were similar (40-45%) to that of the GluN2A-selective antagonist NVP-AAM0077, we conclude that the levels of GluN2A and GluN2B subunits are similar in RGCs. These results provide a novel basis for developing effective neuroprotective agents to aid in the prevention of undesired glutamatergic excitotoxicity in neurodegenerative diseases of the retina and demonstrate functional assembly of NMDARs in RGCs.

Article	Year
Regulation of nuclear receptor transcriptional activity by a novel DEAD box RNA helicase (DP97). The Journal of biological chemistry\| PubMed ID: 12466272	2003
Conservation of critical functional domains in murine plasminogen activator inhibitor-1. The Journal of biological chemistry\| PubMed ID: 14963029	2004
A novel function of plasminogen activator inhibitor-1 in modulation of the AKT pathway in wild-type and plasminogen activator inhibitor-1-deficient endothelial cells. The Journal of biological chemistry\| PubMed ID: 16785241	2006
Wnt signaling. Current drug targets\| PubMed ID: 18673237	2008
Plasminogen activator inhibitor-1: the double-edged sword in apoptosis. Thrombosis and haemostasis\| PubMed ID: 19132226	2008
Conantokins inhibit NMDAR-dependent calcium influx in developing rat hippocampal neurons in primary culture with resulting effects on CREB phosphorylation. Molecular and cellular neurosciences\| PubMed ID: 20600930	2010
A deficiency of uPAR alters endothelial angiogenic function and cell morphology. Vascular cell\| PubMed ID: 21535874	2011
Abnormal whole blood thrombi in humans with inherited platelet receptor defects. PloS one\| PubMed ID: 23300803	2012
A natural inactivating mutation in the CovS component of the CovRS regulatory operon in a pattern D Streptococcal pyogenes strain influences virulence-associated genes. The Journal of biological chemistry\| PubMed ID: 23316057	2013
Complement-mediated opsonization of invasive group A Streptococcus pyogenes strain AP53 is regulated by the bacterial two-component cluster of virulence responder/sensor (CovRS) system. The Journal of biological chemistry\| PubMed ID: 23928307	2013
Antagonist properties of Conus parius peptides on N-methyl-D-aspartate receptors and their effects on CREB signaling. PloS one\| PubMed ID: 24260577	2013
Probing NMDA receptor GluN2A and GluN2B subunit expression and distribution in cortical neurons. Neuropharmacology\| PubMed ID: 24440368	2014
Synthetic conantokin peptides potently inhibit N-methyl-D-aspartate receptor-mediated currents of retinal ganglion cells. Journal of neuroscience research\| PubMed ID: 25043917	2014

Rashna D. Balsara

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