bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of brain prostanoid EP3 receptors via arachidonic acid cascade during behavioral suppression induced by Δ8‐tetrahydrocannabinol
Authors: Yamaguchi Taku , Kubota Takashi , Watanabe Shigenori , Yamamoto Tsuneyuki
Published: July 2010

We have previously shown that behavioral changes induced by cannabinoid were due to an elevation of prostaglandin E2(PGE2) via the arachidonic acid cascade in the brain. In the present study, we investigated the participation of the prostanoid EP3 receptor, the target of PGE2in the brain, in behavioral suppression induced by Δ8‐tetrahydrocannabinol (Δ8‐THC), an isomer of the naturally occurring Δ9‐THC, using a one‐lever operant task in rats. Intraperitoneal administration of Δ8‐THC inhibited the lever‐pressing behavior, which was significantly antagonized by both the selective cannabinoid CB1 receptor antagonist SR141716A and the cyclooxygenase inhibitor diclofenac. Furthermore, intracerebroventricular (i.c.v.) administration of PGE2significantly inhibited the lever‐pressing performance similar to Δ8‐THC. Prostanoid EP3 receptor antisense‐oligodeoxynucleotide (AS‐ODN; twice a day for 3 days, i.c.v.) significantly decreased prostanoid EP3 receptor mRNA levels as determined by the RT‐PCR analysis in the cerebral cortex, hippocampus and midbrain. AS‐ODN also antagonized the PGE2‐induced suppression of the lever pressing. In the same way, the suppression of lever‐pressing behavior by Δ8‐THC was significantly improved by AS‐ODN. It is concluded that the suppression of lever‐pressing behavior by cannabinoid is due to activation of the prostanoid EP3 receptor through an elevation of PGE2in the brain.
CEP11004, a novel inhibitor of the mixed lineage kinases, suppresses apoptotic death in dopamine neurons of the substantia nigra induced by 6‐hydroxydopamine
Authors: Ganguly Anindita , Oo Tinmarla Frances , Rzhetskaya Margarita , Pratt Robert , Yarygina Olga , Momoi Takashi , Kholodilov Nikolai , Burke Robert E.
Published: July 2010

There is much evidence that the kinase cascade which leads to the phosphorylation of c‐jun plays an important signaling role in the mediation of programmed cell death. We have previously shown that c‐jun is phosphorylated in a model of induced apoptotic death in dopamine neurons of the substantia nigrain vivo. To determine the generality and functional significance of this response, we have examined c‐jun phosphorylation and the effect on cell death of a novel mixed lineage kinase inhibitor, CEP11004, in the 6‐hydroxydopamine model of induced apoptotic death in dopamine neurons. We found that expression of total c‐jun and Ser73‐phosphorylated c‐jun is increased in this model and both colocalize with apoptotic morphology. CEP11004 suppresses apoptotic death to levels of 44 and 58% of control values at doses of 1.0 and 3.0 mg/kg, respectively. It also suppresses, to approximately equal levels, the number of profiles positive for the activated form of capase 9. CEP11004 markedly suppresses striatal dopaminergic fiber loss in these models, to only 22% of control levels. We conclude that c‐jun phosphorylation is a general feature of apoptosis in living dopamine neurons and that the mixed lineage kinases play a functional role as up‐stream mediators of cell death in these neurons.
Membrane properties of rat embryonic multipotent neural stem cells
Authors: Cai Jingli , Cheng Aiwu , Luo Yongquan , Lu Chengbiao , Mattson Mark P. , Rao Mahendra S. , Furukawa Katsutoshi
Published: July 2010

We have characterized several potential stem cell markers and defined the membrane properties of rat fetal (E10.5) neural stem cells (NSC) by immunocytochemistry, electrophysiology and microarray analysis. Immunocytochemical analysis demonstrates specificity of expression of Sox1, ABCG2/Bcrp1, and shows that nucleostemin labels both progenitor and stem cell populations. NSCs, like hematopoietic stem cells, express high levels of aldehyde dehydrogenase (ALDH) as assessed by Aldefluor labeling. Microarray analysis of 96 transporters and channels showed that Glucose transporter 1 (Glut1/Slc2a1) expression is unique to fetal NSCs or other differentiated cells. Electrophysiological examination showed that fetal NSCs respond to acetylcholine and its agonists, such as nicotine and muscarine. NSCs express low levels of tetrodotoxin (TTX) sensitive and insensitive sodium channels and calcium channels while expressing at least three kinds of potassium channels. We find that gap junction communication is mediated by connexin (Cx)43 and Cx45, and is essential for NSC survival and proliferation. Overall, our results show that fetal NSCs exhibit a unique signature that can be used to determine their location and assess their ability to respond to their environment.
Calpain‐mediated proteolytic cleavage of the neuronal glycine transporter, GlyT2
Authors: Baliova Martina , Betz Heinrich , Jursky Frantisek
Published: July 2010

The glycine transporter 2 (GlyT2) belongs to the family of Na+/CL–‐dependent plasma membrane transporters and is localized on the presynaptic terminals of glycinergic neurons. GlyT2 differs from other family members by its extended N‐terminal cytoplasmic region. We report that activation of a Ca2+‐dependent protease, most likely calpain, in spinal cord synaptosomes or cultured spinal cord neurons, results in partial proteolysis of GlyT2. Regions sensitive to calpain cleavagein vivoare located in the N‐terminal and, to a lesser extent, C‐terminal regions of the transporter protein. Incubation of a GlyT2 N‐terminal fusion protein with spinal cord extract in the presence of calcium followed by protein sequence analysis localized the major N‐terminal cleavage site after methionine 156, with a second cleavage site being situated after glycine 164. Interestingly, the size of the N‐terminally truncated GlyT2 protein (70 kDa) is similar to that of most other transporter family members, and truncated GlyT2 displayed full transport activity upon expression in HEK293 cells. Our data suggest that Ca2+‐triggered proteolysis may contribute to the regulation of GlyT2 trafficking and/or function in the neuronal plasma membrane.
Hypoxic remodelling of Ca2+ mobilization in type I cortical astrocytes: involvement of ROS and pro‐amyloidogenic APP processing
Authors: Smith Ian F. , Boyle John P. , Green Kim N. , Pearson Hugh A. , Peers Chris
Published: July 2010

Chronic hypoxia (CH) alters Ca2+ homeostasis in various cells and may contribute to disturbed Ca2+ homeostasis of Alzheimer's disease. Here, we have employed microfluorimetric measurements of [Ca2+]i to investigate the mechanism underlying augmentation of Ca2+ signalling by chronic hypoxia in type I cortical astrocytes. Application of bradykinin evoked significantly larger rises of [Ca2+]i in hypoxic cells as compared with control cells. This augmentation was prevented fully by either melatonin (150 µm) or ascorbic acid (200 µm), indicating the involvement of reactive oxygen species. Given the association between hypoxia and increased production of amyloid β peptides (AβPs) of Alzheimer's disease, we performed immunofluorescence studies to show that hypoxia caused a marked and consistent increased staining for AβPs and presenilin‐1 (PS‐1). Western blot experiments also confirmed that hypoxia increased PS‐1 protein levels. Hypoxic increases of AβP production was prevented with inhibitors of either γ‐ or β‐secretase. These inhibitors also partially prevented the augmentation of Ca2+ signalling in astrocytes. Our results indicate that chronic hypoxia enhances agonist‐evoked rises of [Ca2+]i in cortical astrocytes, and that this can be prevented by antioxidants and appears to be associated with increased AβP formation.
Brain trauma induces X‐box protein 1 processing indicative of activation of the endoplasmic reticulum unfolded protein response
Authors: Paschen Wulf , Yatsiv Ido , Shoham Shai , Shohami Esther
Published: July 2010

Brain trauma was induced in mice using a closed head injury (CHI) model. At 1, 6 or 24 h after trauma, brains were dissected into the cortex, striatum and hippocampus. Changes in levels of processed X‐box protein 1 (xbp1), glucose‐regulated protein 78 (grp78), growth arrest and DNA damage‐inducible gene 153 (gadd153) and heat‐shock protein 70 (hsp70) mRNA, indicating impaired endoplasmic reticulum (ER) and cytoplasmic functioning, were evaluated by quantitative PCR. In the cortex, processed xbp1 mRNA levels rose to 2000% of control 1 h after CHI, and stayed high throughout the experiments. In the hippocampus and striatum, processed xbp1 mRNA levels rose in a delayed fashion, peaking at 6 h (1000% of control) and 24 h after CHI (1500% of control) respectively. Levels of grp78 mRNA were only slightly increased in the cortex 24 h after CHI (150% of control), and were unchanged or transiently decreased in the hippocampus and striatum. Levels of gadd153 mRNA did not change significantly after trauma. A transient rise in hsp70 mRNA levels was observed only in the cortex, peaking at 1 h after CHI (600% of control). Processing of xbp1 mRNA is a sign of activation of the unfolded protein response indicative of ER dysfunction. The results suggest that brain trauma induces ER dysfunction, which spreads from the ipsilateral cortex to the hippocampus and striatum. These observations may have clinical implications and should therefore be considered for future investigations on therapeutic intervention of brain injury caused by contusion‐induced neurotrauma.
Neuregulin‐1‐β1 enters brain and spinal cord by receptor‐mediated transport
Authors: Kastin Abba J. , Akerstrom Victoria , Pan Weihong
Published: July 2010

Proteins of the neuregulin (NRG) family play important regulatory roles in neuronal survival and synaptic activity. NRG‐1‐β1 has particular potential as a therapeutic agent because it enhances myelination of neurites in spinal cord explants. In this study, we determined the permeation of NRG‐1‐β1 across the blood‐brain and blood–spinal cord barriers (BBB and BSCB respectively). Intact radioactively labeled NRG‐1‐β1 had a saturable and relatively rapid influx rate from blood to the CNS in mice. Capillary depletion studies showed that NRG‐1‐β1 entered the parenchyma of the brain and spinal cord rather than being trapped in the capillaries that compose the BBB. The possible mechanism of receptor‐mediated transport was shown by the ability of antibodies to erbB3 and erbB4 receptors to inhibit the influx. Lipophilicity, less important for such saturable transport mechanisms, was measured by the octanol : buffer partition coefficient and found to be low. The results indicate that NRG‐1‐β1 enters spinal cord and brain by a saturable receptor‐mediated mechanism, which provides the opportunity for possible therapeutic manipulation at the BBB level.
Mice deficient in dihydrolipoamide dehydrogenase show increased vulnerability to MPTP, malonate and 3‐nitropropionic acid neurotoxicity
Authors: Klivenyi Peter , Starkov Anatoly A. , Calingasan Noel Y. , Gardian Gabrielle , Browne Susan E. , Yang Lichuan , Bubber Parvesh , Gibson Gary E. , Patel Mulchand S. , Beal M. Flint
Published: July 2010

Altered energy metabolism, including reductions in activities of the key mitochondrial enzymes α‐ketoglutarate dehydrogenase complex (KGDHC) and pyruvate dehydrogenase complex (PDHC), are characteristic of many neurodegenerative disorders including Alzheimer's Disease (AD), Parkinson's disease (PD) and Huntington's disease (HD). Dihydrolipoamide dehydrogenase is a critical subunit of KGDHC and PDHC. We tested whether mice that are deficient in dihydrolipoamide dehydrogenase (Dld+/–) show increased vulnerability to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), malonate and 3‐nitropropionic acid (3‐NP), which have been proposed for use in models of PD and HD. Administration of MPTP resulted in significantly greater depletion of tyrosine hydroxylase‐positive neurons in the substantia nigra of Dld+/– mice than that seen in wild‐type littermate controls. Striatal lesion volumes produced by malonate and 3‐NP were significantly increased in Dld+/– mice. Studies of isolated brain mitochondria treated with 3‐NP showed that both succinate‐supported respiration and membrane potential were suppressed to a greater extent in Dld+/– mice. KGDHC activity was also found to be reduced in putamen from patients with HD. These findings provide further evidence that mitochondrial defects may contribute to the pathogenesis of neurodegenerative diseases.
Identification of a residue in the γ‐aminobutyric acid type A receptor α subunit that differentially affects diazepam‐sensitive and ‐insensitive benzodiazepine site binding
Authors: Derry Jason M. C. , Dunn Susan M. J. , Davies Martin
Published: July 2010

GABAAreceptors that contain either the α4‐ or α6‐subunit isoform do not recognize classical 1,4‐benzodiazepines (BZDs). However, other classes of BZD site ligands, including β‐carbolines, bind to these diazepam‐insensitive receptor subtypes. Some β‐carbolines [e.g. ethyl β‐carboline‐3‐carboxylate (β‐CCE) and methyl 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM)] display a higher affinity for α4‐ compared to α6‐containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric α6/α4 subunits and co‐expressed these with wild‐type rat β2 and γ2Lsubunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site‐directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (α6N204/α4I203). Substitutions at α6N204 did not alter the affinity of the imidazobenzodiazepine Ro15‐4513. A homologous mutation in the diazepam‐sensitive α1 subunit (S205N) resulted in a 7–8‐fold reduction in affinity for the β‐carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the α1 subunit, the affinity for Ro15‐1788 and Ro15‐4513 was decreased by ∼19‐fold and ∼38‐fold respectively. The importance of this residue, located in the Loop C region of the extracellular N‐terminus of the subunit protein, emphasizes the differential interaction of ligands with the α subunit in diazepam‐sensitive and ‐insensitive receptors.
Prothrombin overexpressed in post‐natal neurones requires blood factors for activation in the mouse brain
Authors: Sinnreich Michael , Meins Marita , Niclou Simone P. , Suidan Hana S. , Monard Denis
Published: July 2010

Thrombin is thought to mediate, through protease‐activated receptors, both protective as well as cytotoxic effects. As thrombin receptors are expressed in the CNS, an important question arises as to whether the intact nervous system is able to generate thrombin by activation of its precursor prothrombin, derived endogenously or only upon extravasation following brain injury. To address this question, transgenic mice that express C‐terminally haemagglutinin tagged human prothrombin in post‐mitotic neurones were generated.In situhybridization and immunohistochemical analysis showed abundant and widespread cerebral expression of the transgene. Amidolytic assays of brain homogenates and hippocampal slice cultures demonstrated that activation of transgenic prothrombin required added factors, such as snake venom or blood components. This strongly suggests that any possible action of thrombin in the adult CNS depends on blood‐derived factors that activate prothrombin. Furthermore, the results are consistent with the idea that in the non‐pathological situation an as yet unidentified ligand activates thrombin receptors in the nervous system.
Cell signaling pathways in the neuroprotective actions of the green tea polyphenol (‐)‐epigallocatechin‐3‐gallate: implications for neurodegenerative diseases
Authors: Mandel Silvia , Weinreb Orly , Amit Tamar , Youdim Moussa B. H.
Published: July 2010

Accumulating evidence supports the hypothesis that brain iron misregulation and oxidative stress (OS), resulting in reactive oxygen species (ROS) generation from H2O2and inflammatory processes, trigger a cascade of events leading to apoptotic/necrotic cell death in neurodegenerative disorders, such as Parkinson's (PD), Alzheimer's (AD) and Huntington's diseases, and amyotrophic lateral sclerosis (ALS). Thus, novel therapeutic approaches aimed at neutralization of OS‐induced neurotoxicity, support the application of ROS scavengers, transition metals (e.g. iron and copper) chelators and non‐vitamin natural antioxidant polyphenols, in monotherapy, or as part of antioxidant cocktail formulation for these diseases. Both experimental and epidemiological evidence demonstrate that flavonoid polyphenols, particularly from green tea and blueberries, improve age‐related cognitive decline and are neuroprotective in models of PD, AD and cerebral ischemia/reperfusion injuries. However, recent studies indicate that the radical scavenger property of green tea polyphenols is unlikely to be the sole explanation for their neuroprotective capacity and in fact, a wide spectrum of cellular signaling events may well account for their biological actions. In this article, the currently established mechanisms involved in the beneficial health action and emerging studies concerning the putative novel molecular neuroprotective activity of green tea and its major polyphenol (‐)‐epigallocatechin‐3‐gallate (EGCG), will be reviewed and discussed.
Chronic ethanol consumption impairs the circadian rhythm of pro‐opiomelanocortin and period genes mRNA expression in the hypothalamus of the male rat
Authors: Chen Cui Ping , Kuhn Peter , Advis Juan P. , Sarkar Dipak K.
Published: July 2010

Certain psychiatric disorders are known to alter the body's biological rhythms. However, currently, very little information is known about the effect of chronic ethanol administration on the circadian clock or the rhythm of β‐endorphin‐containing neurons that participate in the control of the reward and reinforcement of alcohol drinking. Here, we report that administration of ethanol, via a liquid diet paradigm for a period of 2 weeks, abolishes the circadian rhythm of pro‐opiomelanocortin mRNA expression of β‐endorphin neurons in the arcuate nucleus of the hypothalamus. The circadian expression of the clock governing rat period genes (rPeriod1mRNA andrPeriod2mRNA) in the arcuate nucleus was significantly altered, suggesting that ethanol administration disrupted the internal clock. Moreover, ethanol consumption altered the circadian rhythms ofrPeriod2andrPeriod3mRNA levels in the suprachiasmatic nucleus, suggesting that ethanol also affected the function of the central pacemaker. Our findings identified the vulnerability of the body's clock machinery and its opioidergic system to chronic alcohol drinking.
Differentiation‐induced alterations in cyclic AMP signaling in the Cath.a differentiated (CAD) neuronal cell line
Authors: Johnston Christopher A. , Beazely Michael A. , Bilodeau Matthew L. , Andrisani Ourania , Watts Val J.
Published: July 2010

Regulation of intracellular cyclic AMP is critical to the modulation of many cellular activities, including cellular differentiation. Moreover, morphological differentiation has been linked to subsequent alterations in the cAMP signaling pathway in various cellular models. The current study was designed to explore the mechanism for the previously reported enhancement of adenylate cyclase activity in Cath.a differentiated cells following differentiation. Differentiation of Cath.a differentiated cells stably expressing the D2Ldopamine receptor markedly potentiated both forskolin‐ and A2‐adenosine receptor‐stimulated cAMP accumulation. This enhancement was accompanied by a twofold increase in adenylate cyclase 6 (AC6) expression and a dramatic loss in the expression of AC9. The ability of Ca2+to inhibit drug‐stimulated cAMP accumulation was enhanced following differentiation, as was D2Ldopamine receptor‐mediated inhibition of Gαs‐stimulated cAMP accumulation. Differentiation altered basal and drug‐stimulated phosphorylation of the cAMP‐response element‐binding protein, which was independent of changes in protein kinase A expression. The current data suggest that differentiation of the neuronal cell model, Cath.a differentiated cells induces significant alterations in the expression and function of both the proximal and distal portions of the cAMP signaling pathway and may impact cellular operations dependent upon this pathway.
Transcriptional mechanisms of neuroprotection by iron chelators
Authors: Ratan R. R. , Semenza G. , Ryu H. , Zaman K. , Aminova L. , Lee J. , Kung A. , Mwidau A.
Published: July 2010

Iron chelators are pluripotent neuronal antiapoptotic agents that have been shown to enhance metabolic recovery in cerebral ischemia models. The precise mechanisms by which these agents exert their effects remains unclear. Recent studies have demonstrated that iron chelators activate a hypoxia signal transduction pathway that culminates in the stabilization of the transcriptional activator hypoxia‐inducible factor‐1 (HIF‐1) and increased expression of genes that mediate hypoxic adaptation. Previous studies from our lab have shown that structurally distinct iron chelators deferoxamine mesylate and mimosine prevent apoptosis induced by glutathione depletion and oxidative stress in embryonic neuronal cultures. The protective effects are correlated with their ability to enhance DNA binding of HIF‐1 and activating transcription factor (ATF‐1)/cAMP response element‐binding protein (CREB) to the hypoxia response element in cortical cultures and the H19‐7 hippocampal neuronal cell line. mRNA, protein and/or activity levels for genes known to be regulated by HIF‐1, including glycolytic enzymes, p21 waf1/cip1, and erythropoetin, are increased in neuronal cultures in response to iron chelator treatment. Cobalt chloride, which also activates HIF‐1 and ATF1/CREB in cortical cultures, also prevents oxidative stress‐induced death in these cells. Together, these results suggest that iron chelators exert their neuroprotective effects, in part, by activating a signal transduction pathway leading to increased expression of genes known to compensate for hypoxic or oxidative stress. Molecular manipulations are underway to determine whether HIF‐1 is necessary and/or sufficient for cell survival in response to stress in neurons.
Colloquium 15: Role of the Protease‐Activated Receptors in Neural Development, Degeneration and Trauma. Expression and regulation of PARs in brain and spinal cord
Authors: Citron B. A. , Suo Z. , Ameenuddin S. , Davis J. T. , Festoff B. W.
Published: July 2010

Signalling through protease activated receptors (PARs) is important during neural development and reappears during certain CNS disorders resulting in loss of neurons. These heptohelical G‐protein coupled receptors (GPCRs) are activated by specific proteolysis of N‐terminal fragments. How they are regulated at transcriptional and post‐transcriptional levels is of paramount importance in developing neuroprotective strategies. We and others showed in vitro that serine protease signalled apoptosis involves activation of PAR‐1 and a pathway involving coupled Gs, intracellular calcium elevation, activation of caspases, and apoptotic cell death. In vivo, after spinal cord injury in rats and during motor neuron degeneration in wobbler mice, PAR‐1 is up‐regulated in the regions most affected even prior to the onset of phenotypic motor loss in the homozygous recessive wobbler mouse. Information is beginning to appear on transcriptional regulation of PAR expression and involves several transcription factors (TFs) such as AP‐1, SP‐1, NFκB and, more recently, JAK/STAT. The regulation of PARs in the nervous system will be reviewed with an emphasis on those PARs unregulated during neurodegeneration and in response to CNS injury. In summary, immunohistochemistry, quantitative mRNA, gene array analyses of the receptor sequences present and their modulation by exogenous factors indicate that they are potential targets for therapeutic intervention to help preserve neuronal health.

Acknowledgements:  Supported by the Dept. of Veterans Affairs, Christopher Reeve Paralysis Foundation, ALS Association, Lied Endowment, KUMCRI and Midwest Biomedical Research Foundation.
Role of proteinases and proteinase‐activated receptors in pain pathways
Authors: Vergnolle N. , Bunnett N. W. , Wallace J. L. , Hollenberg M. D.
Published: July 2010

Proteinase‐activated receptors are G‐protein‐coupled receptors that are activated by the proteolytic cleavage of their N‐terminal domain. PAR‐1, PAR‐3 and PAR‐4 are activated by thrombin, while PAR‐2 is activated by trypsin. Using in vivo models of nociception in response to thermal or mechanical stimuli and Fos immunochemistry, we have shown that PAR‐2 activation by selective peptidic agonists or proteinases (trypsin and tryptase) provoked hyperalgesia and activation of nociceptive neurons. The formalin‐ or compound 48/80‐induced hyperalgesia was significantly decreased in mice deficient for the PAR‐2 gene, compared to wild‐type, showing that PAR‐2 activation contributes to the generation of pain associated with inflammation. In contrast, intraplantar injection of selective PAR‐1 peptidic agonists increased nociceptive threshold and withdrawal latency, leading to mechanical and thermal analgesia, while control peptides had no effect. Intraplantar injection of thrombin also showed analgesic properties in response to mechanical, but not to thermal stimulus. Co‐injection of PAR‐1 selective agonist with carrageenan significantly reduced carrageenan‐induced mechanical and thermal hyperalgesia, while thrombin reduced carrageenan‐induced mechanical but not thermal hyperalgesia. The fact that thrombin is not a selective agonist for PAR‐1 may explain the different effects of thrombin and PAR‐1‐AP. These results identified novel roles for proteinases and their receptors in pain pathways. PAR‐2 antagonists and/or PAR‐1 agonists might then constitute valuable addition to analgesics used in the management of inflammatory pain.
Protease‐activated receptors (PARs), microglial reactivity and neurodegeneration
Authors: Suo Z. , Citron B. A. , Ameenuddin S. , Andrade‐Gordon P. , Festoff B. W.
Published: July 2010

Microglial activity accompanies many types of neurodegeneration and neurotrauma. Thrombin is an early arriving modulator at sites of CNS damage. How it affects cascades leading to neurodegeneration is an area of important study. To investigate the roles of PARs in thrombin‐induced microglial activation, primary microglial cultures from wild type and PAR1 deficient (PAR1 −/−) mice, along with N9 microglial cell line, were treated with thrombin, PAR1 (SFLLRN) and PAR4 (GYPGQV) agonist peptides (AP). Changes in [Ca2+]i, phosphorylation states of mitogen‐activated protein kinases (MAPKs), p38, p44/42, cell proliferation and tumor necrosis factor‐α (TNFα) secretion were examined by calcium imaging, Western blots, MTS assay and ELISA. We found that activation of microglial PAR1 or PAR4 induces a rapid cytosolic free [Ca2+]i increase and transient activation of both p38 and p44/42 MAPKs. Moreover, activation of either PAR1 or PAR4 directly contributes to thrombin‐induced microglial proliferation. In addition, activation of PAR4, but not PAR1, alone mimics the direct effect of thrombin on TNF‐α induction. Nonetheless, activation of PAR1 by PAR1AP potentiates PAR4AP‐induced TNF‐α production. We conclude that activation of PAR1 and PAR4 alone each contributes to different aspects of microglial activation by thrombin. In addition, PAR1 and PAR4 function in concert to mediate thrombin's overall effect on microglia.

Acknowledgements:  Supported by V.A. Medical Research, Missouri Alzheimer's Research Fund, Univ. of Kansas medical Center Research Institute, ALS Association, Chrsitorpher reeve Paralysis Foundation, Lied Endowment and Midwest Biomedical Research Foundation.
Developmental expression of NMDA receptors in primary cortical cell culture: relationship to function and excitotoxicity
Authors: Christmann B. , Uliasz T. F. , Hewett S. J.
Published: July 2010

Over‐activation of the N‐methyl‐d‐aspartate (NMDA) receptor results in a Ca2+‐dependent neurotoxicity termed excitotoxicity. Primary neuronal cell cultures are often used to study the mechanisms of excitotoxicity. While the expression of the NMDA receptor (NR) subunits and their relationship to Ca2+ entry/accumulation and excitotoxicity has been studied extensively, all three parameters have not been examined concurrently. To determine unequivocally whether developmental expression of NR protein and function do indeed coincide with the appearance of excitotoxicity, we examined the temporal relationship between NR subunit expression, NMDA‐induced Ca2+ accumulation, and NMDA‐mediated excitotoxicity simultaneously using sister plates derived from the same mixed cortical cell culture preparations. Western Blot analysis of total protein isolated from cells cultured for 1, 4, 7, 10 and 14 days revealed a time‐dependent increase in NR1, NR2A and NR2B subunit expression, which surprisingly did not correlate with NMDA receptor function, as assessed by measurement of NMDA‐induced 45Ca2+ accumulation. However, when only NR subunit surface expression was quantified, a correlation between expression and 45Ca2+ accumulation did indeed exist. To our surprise, the emergence of excitotoxicity did not show a direct relationship to 45Ca2+ accumulation as has been reported previously. Thus, it appears that other factors besides total Ca2+ accumulation must contribute to the emergence of excitotoxicity in mixed murine cortical cell cultures.

Acknowledgements: Supported by a grant from The Patrick and Catherine Weldon Donaghue Medical Foundation.
NAAG and NAALADase influence NMDA receptor activation and axon‐to‐glia signalling in crayfish medial giant nerve fibers
Authors: Urazaev, A. K. , Gafurov B. S. , Grossfeld R. M. , Lieberman E. M.
Published: July 2010

Action potential generation in crayfish giant nerve fibers causes a transient depolarization followed by a prolonged hyperpolarization in their associated glial cells. The glial potential changes, involving a nicotinic acetylcholine receptor (nAChR)‐induced decrease in Cl– permeability, are caused by axonal release of N‐acetylaspartylglutamate (NAAG) acting specifically on glial group II metabotropic glutamate (mGLURII). In addition, GLU formed by NAAG hydrolysis by NAALADase acts on NMDARs as well as mGLURs. Application of NMDA or l‐aspartate also produces glial cell hyperpolarization that can be inhibited by antagonists of NMDA receptors (MK801) but not by antagonists of AMPA/kainate (AP4) or mGLURs (EGLU). Reducing extracellular Ca2+ depresses the NMDA effect while Na+ depletion, block of voltage‐dependent Ca2+ channels (verapamil) or the nAChR (d‐tubocurarin) completely prevent the response to NMDA. The hyperpolarization caused by high frequency stimulation of the nerve fiber is only partially reduced by NMDAR blockade suggesting these receptors are not solely responsible for initiating the glial hyperpolarization under physiological conditions. These results also indicate that the transient depolarization, induced by physiological activation of glial NMDARs, opens voltage‐sensitive Ca2+ channels leading to Ca2+ influx. An increase in [Ca2+]i by this mechanism potentiates the ACh release due to mGLURII activation and the subsequent hyperpolarization mediated by the nAChR.

Acknowledgements: Supported by NIH grant NS34799 and Guilford Pharmaceuticals, Baltimore, MD, USA.
Poster Sessions AP13: Novel Techniques and Technologies. A simple and sensitive HPLC‐method for simultaneous monitoring of oxidative stress indices and monoamine metabolites
Authors: Yao J. K. , Cheng P.
Published: July 2010

Studies of the antioxidant defense system and the monoamine metabolic pathways are often complicated by cumbersome analytical methods, which require separate and multistep extraction and chemical reaction procedures. Thus, measurements of multiple parameters are limited in relatively small biological samples. High performance liquid chromatography (HPLC) coupled with a Coulometric Multi‐Electrode Array System (CMEAS) provides us a convenient and most sensitive tool to measure low molecular weight, redox‐active compounds in biological sample. The deproteinized sample was analyzed on a HPLC coupled with a 16‐channel CMEAS, which incremented from 60 to 960 mV in 60 mV steps. Each sample was run on a single column (Meta‐250, 4.6 × 250 mm) under a 150‐minute complex gradient that ranged from 0% B (A: 1.1% pentane sulfonic acid) to 20% B (B: 0.1 m lithium acetate in mixture of methanol, acetonenitrile and isopropanol), with a flow rate of 0.5 mL/min. We have developed an automated procedure to simultaneously measure various antioxidant, oxidative stress marker, and monoamine metabolites in a single column with binary gradient. No other chemical reactions are necessary. In order to reduce the running time and yet achieve a reproducible retention time by the autosampler injection, our gradient elution profile was modified to produce a shorter equilibration time and to compensate for the initial contamination of mobile phase B following the first injection. Without the use of two columns in series and peak suppresser/gradient mixer, we have simplified the previously published method to measure over 20 different antioxidants, oxidative stress markers and monoamine metabolites simultaneously in biological samples.
An essential role for P38 MAP kinase signalling in oligodendrocyte differentiation
Authors: Bhat N. R. , Fan F. , Pawate S.
Published: July 2010

Proliferating oligodendrocyte progenitors (OLPs) cease to divide and undergo differentiation to express myelin‐specific markers and genes upon growth factor withdrawal. We report here that signalling via p38 mitogen‐activated protein kinase (p38 MAPK) pathway plays an essential role in this process. SB203580, a specific inhibitor of p38 MAPK, interferes with the characteristic morphological transformation (i.e. from a simple bi‐/tri‐polar shape to a complex multipolar structure) of OLPs and the expression of oligodendrocyte differentiation‐specific antigenic markers, i.e. O4, O1 and myelin basic protein (MBP). RT–PCR analysis reveals a reduced level of mRNA for MBP and myelin associated glycoprotein (MAG) in the inhibitor‐treated cultures. Switching of OLPs from a growth medium to a mitogen‐free differentiation medium leads to an increased phosphorylation of CREB, a transcription factor implicated in myelin gene expression, in a p38 MAPK‐dependent manner. Kinase assays indicate a differential regulation of the p38 MAPK isoforms i.e. a decline, with time in culture, in the activity of the α isoform from an initial high level present in OLPs, in contrast to increased activity levels of the β isoform in differentiating cells. Enforced activation of p38 MAPK by transfection with activated forms of its upstream kinases i.e. MAPK kinase 6 [MKK6b(E)] and [MKK3b(E)], results in a precocious induction of MBP and MAG mRNAs and a marked stimulation of the activity of an MBP promoter‐reporter which can be blocked by the p38 MAPK inhibitor. The results presented strongly support a prominent role for p38 MAPK pathway in oligodendrocyte differentiation and myelin‐specific gene expression.

Acknowledgements: Supported in part by NINDS grant # NS41035‐01.
Iron mediates gene expression in activated microglia
Authors: Connor J. R. , Cheepsunthorn P. , Reid J. , Radov L. A.
Published: July 2010

Microglia have diverse functions within the brain. Activation of microglia has been described in many neurological diseases and these activated microglia may contribute to the disease process. Following brain injury, activated microglia increase the expression of macrophage‐associated antigens and release neuroactive substances, including reactive oxygen and nitrogen molecules, inflammatory cytokines, and neurotoxins. Iron accumulation accompanies activation of microglia including those associated with neuritic plaques in Alzheimer Disease, infarcted brain areas in stroke, and within the vicinity of plaques in Multiple Sclerosis. The question is whether the effect of iron acummulation in microglia is enhances activation or limits function. To address this question, we examined the influence of iron on gene expression in the murine microglia (BV2) cell line during activation with lipopolysaccharide (LPS) using microarray analysis. The combined presence of LPS and iron resulted in the increased expression of an additional 48 genes and decreased expression of 76 genes compared to the LPS alone group. Among the genes that increased expression in the iron plus LPS group where CD38, CD83, caspase 11, IL15, Mip 1a, interferon alpha family, and Ras superfamily of regulatory GTPase. These results suggest that iron can influence the inflammatory response in microglia elicited by LPS and the removal of tissue debris during any reparative processes.
Anti‐inflammatory actions of transforming growth factor‐β1 on rat microglia
Authors: Krady J. K. , Basu A. , Enterline J. R. , Levison S. W.
Published: July 2010

Microglia rapidly respond to CNS injury yet the mechanisms leading to their activation and inactivation remain poorly defined. In particular, few studies have established how interactions among inflammatory mediators affect the innate immune response of microglia. To begin to understand the hierarchy of cytokine signaling we examined the effects of interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), tumor necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ) and transforming growth factor‐β1 (TGF‐β1) on both newborn and bulk isolated adult microglia. To assess the functional state of the cells we assayed the expression of cyclooxygenase‐2 (Cox‐2); interleukin‐6 and tumor necrosis factor‐α, and two protein tyrosine kinases implicated in microglial responses to activational stimuli, Hck and Fak. These studies demonstrated that IL‐1β, TNF‐α, IL‐6, but not IFN‐γ increase the expression of Cox‐2, whereas they all increase the expression of hck and fak. TGF‐β1 had either no effect, or it decreased basal levels of these proteins. Significantly, TGFβ1 blocked the activational effects of IL‐1β when given prior to or simultaneously with IL‐1β. TGF‐β1 blocked the induction of the tyrosine kinases, Cox‐2, and the induction of IL‐6 and TNF‐α mRNAs. Thus we conclude that TGF‐β1 exerts a stronger effect on the microglial state than IL‐1β. However, TGF‐β1 was ineffective in antagonizing the induction of Cox‐2 by IL‐6. Therefore, we conclude that the TGF‐β receptor signalling cascades intersect with IL‐1, but they may not interact with IL‐6 signalling. Consequently, IL‐6 exerts stronger effects on the microglial state than either IL‐1 or TGF‐β.

Acknowledgements:  Supported by NMSS award #RG3837.
Involvement of P38MAP kinase in the induction of nitric oxide synthase in human astrocytes
Authors: Anderson J. A. , Jana M. , Dasgupta S. , Liu X. , Pahan K.
Published: July 2010

The present study underlines the importance of p38 mitogen‐activated protein kinase (p38MAPK) in regulating the expression of iNOS in human astroglia. The combination of IL‐1β and IFN‐γ activated p38MAPK and induced the production of NO and the expression of iNOS in human U373MG astroglial cells. SB203580, a specific inhibitor of p38MAPK, and Dp38, a dominant‐negative mutant of p38MAPK, markedly inhibited cytokine‐induced production of NO and expression of iNOS. Next we investigated the role of NF‐κB in the expression of iNOS. Inhibition of cytokine‐induced NF‐κB activation and iNOS expression by SN50, a specific inhibitor of p50 NF‐κB, but not by SN50M, a nonfunctional peptide mutant, does suggest that activation of NF‐κB is necessary for the expression of iNOS. However, SB203580 and Dp38 had no effect on the activation of NF‐κB suggesting that SB203580 and Dp38 inhibit the expression of iNOS without inhibiting the activation of NF‐κB. Next we examined the role of C/EBP‐b in the expression of iNOS. In contrast to the effect of SB203580 and Dp38 on the activation of NF‐κB, both SB203580 and Dp38 markedly inhibited the activation of C/EBP‐b in cytokine‐stimulated cells. Similar to U373MG astrocytoma cells, Dp38 also inhibited cytokine‐induced expression of iNOS and activation of C/EBP‐b but not that of NF‐κB in human primary astroglia. Taken together, these studies suggest that p38MAPK regulates the expression of iNOS in human astroglia by regulating the activation of C/EBP‐b but not that of NF‐κB.

Acknowledgements:  This study was supported by NIH grants (NS39940 and AG19487).
Purification and plasticity of human cord blood mesenchymal stem cells
Authors: Cohen R. I.
Published: July 2010

Human cord blood is well known to contain hemapoetic stem cells capable of differentiating into blood related cell types. However, it is possible to derive another type of stem cell, referred to as a stromal, or in this study, mesenchymal stem cells, which are capable of producing other differentiated cell types which can include fat, muscle, bone and cartilage. It has also become apparent that neural cells can be derived from mesenchymal stem cells taken from bone marrow, another source like cord blood, of both hemapoetic and mesenchymal stem cells. Recently, Sanchez‐Ramos and coworkers published a study demonstrating that mesenchymal stem cells from total nucleated cord blood differentiate into cells expressing neural antigens. This work indicated the potential of this starting material as a valuable therapeutic source of human neural progenitors. Derivation and definition of these cord blood derived neural cells is unclear since the antigenic characterization of the cord blood mesenchymal stem cells still remains unknown. In this study we are using stem cell antigens such as CD34, CD90, CD117, CD133, to identify the cellular pool of cord blood mesenchymal stem cells and purify them using positive magnetic bead selection. Each population will be antigenically tested for their ability to differentiate into neural cells using defined culture conditions previously used to generate neural cells from multipotent starting material such as; ES cells; bone marrow mesenchymal stem cells; and CNS‐derived multipotent neural stem cells.
Analysis of 12360 Poly A+ RNA message levels in Alzheimer disease (AD) hippocampal CA1
Authors: Lukiw W. J. , Rogaev E. I. , Schurr J. , Carver L. A. , LeBlanc H. J. , Howse K. , Ball M. J. , Bazan N. G.
Published: July 2010

Multiple changes in gene expression patterns are consistently observed in AD brain. To extend our initial studies (Alzheimer's Reports 2000; 3, 161–167) hippocampal CA1 poly A+ RNA populations were analyzed for 12360 expression levels using U95Av2 GeneChips (Affymetrix) and Atlas cDNA microarrays (Clontech). CA1 regions were carefully selected from several hundred potential specimens obtained from domestic or international brain banks. To minimize the effects of individual differences in gene expression, poly A+ RNA of high spectral quality (A260/2801.9) were pooled from 12 AD or 12 control brains and compared as a group. All postmortem interval tissues (<2.5 h) were classified as CDR 0‐4 (CERAD). Data were analyzed using GeneSpring (Silicon Genetics) and Microarray/Data Mining Tool 5.0 (Affymetrix). Compared to 18 control markers, of the 36 strongest signal differences detected, AD brain showed significant decreases in RNAs encoding 27 transcription factors and signalling elements involved in synaptic plasticity. These defects correlate well with the observed general down‐regulation of genetic activity in AD neocortex. Increases in nine potentially apoptotic or inflammatory poly A+ RNAs, including DAXX, βAPP, COX‐2, IL‐1 precursor and cPLA2 were strikingly apparent. These data support the hypothesis of gene misregulation, transcription signalling deficits and the progressive activation of apoptotic and neuroinflammatory elements in AD brain.
MPTP‐treated senescence accelerated mice as a novel experimental model for Parkinson's disease
Authors: Boldyrev A. A. , Sorokina E. , Kasey V. , Bastrikiva N. , Fedorova T. , Stvolinsky S.
Published: July 2010

Known experimental models of Parkinson's Disease (PD) are limited by nonsimultaneous expression of physiological and biochemical features. We described a novel PD model consisting of N‐methyl,4‐phenyl‐tetrahydropyridine (MPTP) treatment of Senescence Accelerated Mice (SAM) characteristic of increased level of reactive oxygen species aggravated by deficiency of antioxidant defense system. MPTP treatment was found to suppress locomotion and enhance the nonmotivated behavior (grooming); short‐term tremor and apparent rigidity were also noted. MPTP effect was accompanied with increased level of protein carbonyls and lipid hydroperoxides indicating numerous disorders in antioxidant defense system. The brain of MPTP treated animals demonstrated higher MAO B activity and low level of SOD. Brain of control animals treated with MPTP was demonstrated by unchanged MAO B and two times decreased SOD activity; behavioral alterations in these mice being less manifested than that of SAM animals. The data presented showed that MPTP treated SAM animals are perspective model for Parkinson's disease study.

Acknowledgements:  The work is supported by RFBR (## 99‐04‐49420; 00‐04‐48767).
NAALADase (GCP II) inhibitors protect in models of amyotrophic lateral sclerosis (ALS)
Authors: Thomas A. G. , Corse A. M. , Coccia C. F. , Wozniak K. M. , Hartman T. , Jada P. , Chandran M. , Rothstein J. F. , DalCanto M. , Slusher B. S.
Published: July 2010

Chronic glutamate toxicity is implicated in the pathogenesis of ALS. The neuropeptide N‐acetyl‐aspartyl glutamate (NAAG) appears to function both as a storage form for glutamate and as a neuromodulator at glutamatergic synapses. Catabolism of NAAG by N‐acetylated‐α‐linked acidic dipeptidase (NAALADase; also termed glutamate carboxypeptidase II), yields N‐acetyl aspartate (NAA) and glutamate. Since prior studies demonstrate an up‐regulation of NAALADase in motor cortex and increased levels of NAA and glutamate in the CSF of ALS patients, we hypothesized that inhibition of NAALADase could protect against neuronal degeneration in ALS. Neuroprotective effects of two NAALADase inhibitors were assessed. 2‐(Phosphonomethyl)pentanedioic acid (2‐PMPA) decreased motor neuron loss and prevented loss of choline acetyltransferase (ChAT) activity in an in vitro model of ALS wherein chronic glutamate toxicity was induced by blocking glutamate transport. Gross morphology was preserved in 2‐PMPA‐treated cultures. In a SOD‐1 transgenic mouse model of ALS, oral administration of a structurally different NAALADase inhibitor (GPI 5693) increased survival by 29 days and delayed onset of clinical symptoms by 17 days. Preliminary analysis of spinal cord pathology revealed severe neuronal depletion and astrocytosis with white matter changes in control mice. In mice treated with GPI 5693, normal neuronal populations with modest vacuolar changes were observed. These data suggest that NAALADase inhibition may provide an exciting therapeutic approach to the devastating disease, ALS.