bluuebunny/crossref_metadata_2025 huggingface dataset:
Squid visual arrestin: cDNA cloning and calcium‐dependent phosphorylation by rhodopsin kinase (SQRK)
Authors: Mayeenuddin Linnia H. , Mitchell Jane
Published: July 2010

Arrestin binding to rhodopsin is one of the major mechanisms of termination of photoresponses in both vertebrates and invertebrates. Here we report the cDNA cloning and characterization of a 48‐kDa visual arrestin from squid (Loligo pealei). The cDNA encoded a protein that had 56–64% amino acid sequence similarity to reported arrestin sequences. This protein does not encode any distinct modular domains but contains five fingerprint regions that have been identified within arrestins. Antibodies raised to the recombinant arrestin protein detected arrestin expression only in the eye and recognized a doublet in photoreceptor membranes, representing unphosphorylated and phosphorylated arrestin. In squid eye membranes, arrestin was phosphorylated in a Ca2+‐dependent manner and this phosphorylation was inhibited by antibodies raised against squid rhodopsin kinase, but not by inhibitors of protein kinase C or calmodulin kinase. Addition of purified squid rhodopsin kinase to washed rhabdomeric membranes resulted in phosphorylation of rhodopsin, and arrestin was also phosphorylated when calcium was present. This is the first report of a rhodopsin kinase phosphorylating an arrestin substrate, and suggests a dual role for this kinase in the inactivation of the squid visual system.
Hypoxia induces de‐stabilization of the LAT1 large neutral amino acid transporter mRNA in brain capillary endothelial cells
Authors: Boado Ruben J. , Li Jian Yi , Tsukamoto Haruhisa , Pardridge William M.
Published: July 2010

Blood–brain barrier (BBB) transport of large neutral amino acids is mediated by the large neutral amino acid transporter type 1 (LAT1 transporter). Although the gene encoding the Glut1 glucose transporter is up‐regulated in hypoxia, the response of the LAT1 gene to hypoxia is not known. The present study investigates the changes in the LAT1 mRNA in cultured bovine brain capillary endothelial cells exposed to 1% O2 for 24–48 h. The LAT1 mRNA was initially down‐regulated in hypoxia with reciprocal changes in the Glut1 mRNA. No changes in the 4F2hc mRNA in hypoxia were observed. Hypoxia caused an initial de‐stabilization of the LAT1 mRNA, and the t1/2 of the LAT1 mRNA in control and hypoxic cells was 6.4 ± 0.5 and 2.4 ± 0.1 h, respectively. To further explore post‐transcriptional regulation of LAT1 gene expression, the polysome and cytosol fractions of the control and hypoxic endothelial cells were isolated, and LAT1 mRNA binding proteins were detected by ultraviolet light cross‐linking. Whereas the cytosol contained no LAT1 mRNA binding proteins, the cell polysome fraction expressed several LAT1 mRNA binding proteins, including principal 40‐, 70‐ and 80‐kDa proteins. These studies are consistent with post‐transcriptional de‐stabilization of the LAT1 large neutral amino acid transporter in hypoxia.
Antipsychotic drugs differentially modulate apolipoprotein D in rat brain
Authors: Khan M. M. , Parikh V. V. , Mahadik S. P.
Published: July 2010

Apolipoprotein‐D (apoD), a member of the lipocalin family of proteins, binds to arachidonic acid and cholesterol among other hydrophobic molecules. Recently, elevated apoD levels have been reported in the post‐mortem brains, as well as plasma, of schizophrenic patients and in rodent brains after chronic treatment with clozapine (CLOZ). These findings and the evidence for altered membrane lipid metabolism in schizophrenia suggest that apoD may have a role in the pathophysiology of illness, and also in the differential clinical outcome following treatment with typical and atypical antipsychotic drugs. Here, we compared the effects of these antipsychotics on the expression of apoD in rat brain. Chronic treatment with typical antipsychotic, haloperidol (HAL) reduced apoD expression in hippocampus, piriform cortex and caudate‐putamen (p = 0.027–0.002), whereas atypical antipsychotics, risperidone (RISP) and olanzapine (OLZ) increased (p = 0.051 to < 0.001 and p = 0.048 to < 0.001, respectively) apoD expression. In hippocampus, HAL‐induced changes were present in CA1, CA3 and dentate gyrus, however, apoD levels in motor cortex were unchanged. There were also very dramatic effects of HAL on the neuronal morphology, particularly, cellular shrinkage and disorganization with the loss of neuropil. Post‐treatment, either with RISP or OLZ, was very effective in restoring the HAL‐induced reduction of apoD, as well as cellular morphology. Similarly, pre‐treatments were also effective, but slightly less than post‐treatment, in preventing HAL‐induced reduction of apoD. The increased expression of apoD by atypical antipsychotics may reflect a novel molecular mechanism underlying their favorable effects compared with HAL on cognition, negative symptoms and extra‐pyramidal symptoms in schizophrenia.
Conditions associated with ER dysfunction activate homer 1a expression
Authors: Paschen Wulf , Mengesdorf Thorsten
Published: July 2010

Homer proteins physically link metabotropic glutamate receptors with IP3 receptors located at the endoplasmic reticulum (ER) and thereby modulate receptor‐activated calcium signaling. Homer 1a, the short form of constitutively expressed homer 1 proteins, exerts dominant negative activity with respect to homer 1 proteins by interfering with the formation of multiprotein complexes. Homer 1a is an immediate early gene, the expression of which is activated by various stimuli including glutamate receptor activation. The mechanisms underlying activation of homer 1a expression are however, not fully understood. Here, we show that homer 1a expression is induced in neuronal cell cultures under experimental conditions associated with ER dysfunction. Increased homer 1a mRNA levels were found in 2 sets of cultures: in those exposed to thapsigargin, a specific inhibitor of ER Ca2+‐ATPase, after a transient depletion of ER calcium stores through exposure to calcium‐free medium supplemented with EGTA, and in those exposed to a proteasome inhibitor known to induce ER dysfunction. Thus, homer 1a expression may be activated by impairment of ER functioning just as it is by glutamate receptor activation.
Neuronal‐NOS adaptor protein expression after spreading depression: implications for NO production and ischemic tolerance
Authors: Wiggins Amanda K. , Shen Pei‐Juan , Gundlach Andrew L.
Published: July 2010

Cortical spreading depression (CSD) is characterized by slowly propagating waves of neuronal/astrocytic depolarization and metabolic changes, followed by a period of quiescent neuronal and electroencephalographic activity. CSD acts as a preconditioning stimulus in brain, reducing cell death when elicited up to several days prior to an ischemic insult. Precise mechanisms associated with this neuroprotection are not known, although CSD increases the expression of a number of potentially neuroprotective genes/proteins. The nitric oxide (NO) system may be of particular importance, as it is acutely activated and chronically up‐regulated in cerebral cortex by CSD, and NO can ameliorate and exacerbate cell death under different conditions. Several molecules have recently been identified that modulate the production and/or cellular actions of NO, but it is not known whether their expression is altered by CSD. Therefore, the present study examined the effect of CSD on the spatiotemporal expression of PIN, CAPON, PSD‐95, Mn‐SOD and Cu/Zn‐SOD mRNA in the rat brain. In situ hybridization using specific [35S]‐labelled oligonucleotides revealed that levels of PIN mRNA were significantly increased in the cortex and claustrum (∼30–180%; p ≤ 0.01) after 6 h and 1 and 2 days, but were again equivalent to contralateral (control) cortical values at 7, 14 and 28 days. CAPON mRNA levels were increased (∼30–180%; p ≤ 0.05) in the ipsilateral cortical hemisphere at 6 h and 2 days post treatment, but not at the other times examined. In contrast, levels of PSD‐95, Mn‐ and Cu/Zn‐SOD mRNA were not altered at any time after CSD. These results suggest that following CSD, nNOS activity and NO levels may be tightly regulated by both transcriptional and translational alterations in a range of nNOS adaptor proteins, which may contribute to CSD‐induced neuroprotection against subsequent ischemia.
RNA editing and alternative splicing of human serotonin 2C receptor in schizophrenia
Authors: Dracheva Stella , Elhakem Sharif L. , Marcus Sue M. , Siever Larry J. , McGurk Susan R. , Haroutunian Vahram
Published: July 2010

Serotonin 2C receptor (5‐HT2CR) heterogeneity in the brain occurs mostly from two different sources: (i) 5‐HT2CR mRNA undergoes adenosine‐to‐inosine editing events at five positions, which leads to amino acid substitutions that produce receptor variants with different pharmacological properties; (ii) 5‐HT2CR mRNA is alternatively spliced, resulting in a truncated mRNA isoform (5‐HT2CR‐tr) which encodes a non‐functional serotonin receptor. 5‐HT2CR mRNA editing efficiencies and the expression of the full‐length and the truncated 5‐HT2CR mRNA splice isoforms were analyzed in the prefrontal cortex of elderly subjects with schizophrenia vs. matched controls (ns = 15). No significant differences were found, indicating that there are no alterations in editing or alternative splicing of 5‐HT2CRs that are associated with schizophrenia in persons treated with antipsychotic medications. Quantitation of 5‐HT2CR and 5‐HT2CR‐tr mRNA variants revealed that the expression of 5‐HT2CR‐tr was ∼ 50% of that observed for the full‐length isoform.
Inducible cAMP early repressor (ICER) in the nervous system – a transcriptional regulator of neuronal plasticity and programmed cell death
Authors: Mioduszewska Barbara , Jaworski Jacek , Kaczmarek Leszek
Published: July 2010

The acronym ICER (inducible cAMP early repressor) refers to a group of four proteins produced from the CREM/ICER gene due to use of an internal promoter (P2) placed in an intron of the CREM (cAMP responsive element modulator) gene. The ICER proteins contain DNA binding/leucine zipper domains that make them endogenous inhibitors of transcription driven by CREB (cAMP responsive element binding protein) and its cognates, CREM and ATF‐1 (activating transcription factor‐1). ICER expression is inducible in the brain and in neuronal culture by a variety of stimuli. As a CREB antagonist, ICER appears to be of pivotal importance in neuronal plasticity and programmed cell death.
Cyclin‐dependent kinase 11p110 and casein kinase 2 (CK2) inhibit the interaction between tyrosine hydroxylase and 14‐3‐3
Authors: Sachs Nancy A. , Vaillancourt Richard R.
Published: July 2010

Tyrosine hydroxylase (TH) is regulated by the reversible phosphorylation of serines 8, 19, 31 and 40. Upon initiation of this study, serine 19 was unique due to its requirement of 14‐3‐3 binding after phosphorylation for optimal enzyme activity, although it has been more recently demonstrated that phosphorylated serine 40 also binds 14‐3‐3. To identify proteins that interact with TH following phosphorylation of serine 19, this amino acid was mutated to alanine and THS19A was used as bait in a yeast two‐hybrid system. From this, mouse‐derived cyclin‐dependent kinase 11 (CDK11)p110 was identified as an interacting partner with THS19A. The interaction was confirmed using human CDK11p110 cDNA in a mammalian system. Previous research has demonstrated that casein kinase 2 (CK2) interacts with CDK11p110, and both were observed to phosphorylate TH in vitro. In addition, CDK11p110 overexpression was observed to inhibit the interaction between TH and 14‐3‐3. A mechanism contributing to disruption of the interaction between TH and 14‐3‐3 may be due to CK2 phosphorylation of specific 14‐3‐3 isoforms, i.e. 14‐3‐3 τ. Collectively, these results imply that CDK11p110 and CK2 negatively regulate TH catecholamine biosynthetic activity since phosphoserine 19 of TH requires 14‐3‐3 binding for optimal enzyme activity and a decreased rate of dephosphorylation.
Over‐expression of antioxidant enzymes protects cultured hippocampal and cortical neurons from necrotic insults
Authors: Wang Hui , Cheng Elise , Brooke Sheila , Chang Pearl , Sapolsky Robert
Published: July 2010

There is now considerable knowledge concerning neuron death following necrotic insults, and it is believed that the generation of reactive oxygen species (ROS) and oxidative damage play a pivotal role in the neuron death. Prompted by this, we have generated herpes simplex virus‐1 amplicon vectors over‐expressing the genes for the antioxidant enzymes catalase (CAT) or glutathione peroxidase (GPX), both of which catalyze the degradation of hydrogen peroxide. Over‐expression of each of these genes in primary hippocampal or cortical cultures resulted in increased enzymatic activity of the cognate protein. Moreover, each enzyme potently decreased the neurotoxicity induced by kainic acid, glutamate, sodium cyanide and oxygen/glucose deprivation. Finally, these protective effects were accompanied by parallel decreases in hydrogen peroxide accumulation and the extent of lipid peroxidation. These studies not only underline the key role played by ROS in the neurotoxicity of necrotic insults, but also suggest potential gene therapy approaches.
Repeated amphetamine treatment induces neurite outgrowth and enhanced amphetamine‐stimulated dopamine release in rat pheochromocytoma cells (PC12 cells) via a protein kinase C‐ and mitogen activated protein kinase‐dependent mechanism
Authors: Park Yang Hae , Kantor Lana , Guptaroy Bipasha , Zhang Minjia , Wang Kevin K. W. , Gnegy Margaret E.
Published: July 2010

Repeated intermittent treatment with amphetamine (AMPH) induces both neurite outgrowth and enhanced AMPH‐stimulated dopamine (DA) release in PC12 cells. We investigated the role of protein kinases in the induction of these AMPH‐mediated events by using inhibitors of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) or protein kinase A (PKA). PKC inhibitors chelerythrine (100 nmand 300 nm), Ro31‐8220 (300 nm) and the MAP kinase kinase inhibitor, PD98059 (30 µm) inhibited the ability of AMPH to elicit both neurite outgrowth and the enhanced AMPH‐stimulated DA release. The direct‐acting PKC activator, 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA, 250 nm) mimicked the ability of AMPH to elicit neurite outgrowth and enhanced DA release. On the contrary, a selective PKA inhibitor, 100 µmRp‐8‐Br‐cAMPS, blocked only the development of AMPH‐stimulated DA release but not the neurite outgrowth. Treatment of the cells with acute AMPH elicited an increase in the activity of PKC and MAP kinase but not PKA. These results demonstrated that AMPH‐induced increases in MAP kinase and PKC are important for induction of both the enhancement in transporter‐mediated DA release and neurite outgrowth but PKA was only required for the enhancement in AMPH‐stimulated DA release. Therefore the mechanisms by which AMPH induces neurite outgrowth and the enhancement in AMPH‐stimulated DA release can be differentiated.
Non‐genomic modulation of dopamine release by bisphenol‐A in PC12 cells
Authors: Yoneda Takashi , Hiroi Toyoko , Osada Mayuko , Asada Akira , Funae Yoshihiko
Published: July 2010

An endocrine disruptor chemical, bisphenol‐A (BPA), is reported to have several short‐term actions in various tissues and/or cells; however, the mechanisms of these actions have not been fully elucidated. We investigated short‐term actions evoked by BPA in pheochromocytoma PC12 cells. BPA elicited dopamine release in PC12 cells in a dose‐dependent manner. A selective N‐type calcium channel antagonist (ω‐conotoxin GVIA) and a ryanodine receptor blocker (ryanodine) inhibited the BPA‐induced dopamine release. The expression of ryanodine receptor mRNA was detected by RT–PCR in PC12 cells. Subsequently, in order to prove whether membrane receptors participate in BPA‐evoked dopamine release, a guanine nucleotide‐binding protein inhibitor [guanosine 5′‐(β‐thio) diphosphate], cyclic AMP antagonist (Rp‐cAMPS) or protein kinase A inhibitor (H7 or H89) was added to PC12 cells prior to BPA‐treatment. All of these agents suppressed BPA‐evoked dopamine release, indicating that multiple signaling pathways may be involved in BPA‐evoked dopamine release in PC12 cells. In conclusion, we demonstrated that BPA induced dopamine release in a non‐genomic manner through guanine nucleotide‐binding protein and N‐type calcium channels. These findings illustrate a novel function of BPA and suggest that exposure to BPA influences the function of dopaminergic neurons.
Glutamate regulates Oct‐2 DNA‐binding activity through α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors in cultured chick Bergmann glia cells
Authors: Méndez J. Alfredo , López‐Bayghen Esther , Rojas Fausto , Hernández María Elena , Ortega Arturo
Published: July 2010

Ionotropic glutamate receptors in cerebellar Bergmann glial cells are linked to transcriptional regulation and, by these means, are thought to play an important role in plasticity, learning and memory and in several neuropathologies. Within the CNS, the transcription factors of the POU family bind their target DNA sequences after a growth factor‐dependent phosphorylation–dephosphorylation cascade. Exposure of cultured Bergmann glial cells to glutamate leads to a time‐ and dose‐dependent increase in Oct‐2 DNA‐binding activity. The use of specific pharmacological tools established the involvement of Ca2+‐permeable α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptors. Furthermore, the signaling cascade includes phosphatidyl inositol 3‐kinase as well as protein kinase C activation. Interestingly, transcriptional as well as translational inhibitors abolish the glutamate effect, suggesting a transcriptional up‐regulation of the oct‐2 gene. These data demonstrate that Oct‐2 expression is not restricted to neurons and further strengthen the notion that the glial glutamate receptors participate in the modulation of glutamatergic cerebellar neurotransmission.
Expression and involvement of gicerin, a cell adhesion molecule, in the development of chick optic tectum
Authors: Taira Eiichi , Tsukamoto Yasuhiro , Kohama Keiko , Maeda Mitsuyo , Kiyama Hiroshi , Miki Naomasa
Published: July 2010

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It has both a homophilic binding activity and a heterophilic binding activity to neurite outgrowth factor (NOF) a molecule belonging to the laminin family. We have reported many studies on the heterophilic activity of gicerin and NOF, but the function of its homophilic binding activity in vivo had been unclear. In the retina, gicerin is expressed in retinal ganglion cells only when they extend neurites to the optic tectum. In this report we have found that gicerin is also transiently expressed in the optic tectum during this time. First, cell aggregation assays were used to show that gicerin expressed in the optic tectum displays homophilic binding activity. Then, explant cultures of embryonic day 6 chick optic tectum on gicerin‐Fc chimeric protein‐coated dishes and NOF‐coated dishes were carried out. It was found that gicerin–gicerin homophilic interactions promoted cell migration, whereas heterophilic interactions with NOF induced neurite formation. Furthermore, when anti‐gicerin antibodies were injected in order to examine the effect of gicerin protein in the formation of the tectal layer in ovo, cell migration was strongly inhibited. These data suggest that homophilic interaction of gicerin participates in the migration of neural cells during the layer formation and plays a crucial role in the organization of the optic tectum.
Protection by pioglitazone in the MPTP model of Parkinson's disease correlates with IκBα induction and block of NFκB and iNOS activation
Authors: Dehmer Thomas , Heneka Michael T. , Sastre Magdalena , Dichgans Johannes , Schulz Jörg B.
Published: July 2010

Inflammation has been implicated in the pathogenesis of Parkinson's disease (PD). In the chronic 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) model of PD, inducible NO synthase (iNOS) derived nitric oxide (NO) is an important mediator of dopaminergic cell death. Ligands of the peroxisome proliferator‐activated receptor (PPAR) exert anti‐inflammatory effects. We here investigated whether pioglitazone, a PPARγ agonist, protected mice from MPTP‐induced dopaminergic cell loss, glial activation, and loss of catecholamines in the striatum. As shown by western blot, PPARγ was expressed in the striatum and the substantia nigra of vehicle‐ and MPTP‐treated mice. Oral administration of 20 mg/(kg day) of pioglitazone protected tyrosine hydroxylase (TH)‐positive substantia nigra neurons from death induced by 5 × 30 mg/kg MPTP. However, the decrease of dopamine in the striatum was only partially prevented. In mice treated with pioglitazone, there were a reduced activation of microglia, reduced induction of iNOS‐positive cells and less glial fibrillary acidic protein positive cells in both striatum and substantia nigra pars compacta. In addition, treatment with pioglitazone almost completely blocked staining of TH‐positive neurons for nitrotyrosine, a marker of NO‐mediated cell damage. Because an increase in inhibitory protein‐κ‐Bα (IκBα) expression and inhibition of translocation of the nuclear factor kappaB (NFκB) subunit p65 to the nucleus in dopaminergic neurons, glial cells and astrocytes correlated with the protective effects of pioglitazone, our results suggest that pioglitazone sequentially acts through PPARγ activation, IκBα induction, block of NFκB activation, iNOS induction and NO‐mediated toxicity. In conclusion, treatment with pioglitazone may offer a treatment opportunity in PD to slow the progression of disease that is mediated by inflammation.
Sphingosine‐1‐phosphate induces proliferation and morphological changes of neural progenitor cells
Authors: Harada Jun , Foley Melissa , Moskowitz Michael A. , Waeber Christian
Published: July 2010

Sphingosine‐1‐phosphate (S1P) is a lipid mediator that exerts multiple cellular functions through activation of G‐protein‐coupled receptors. Although the role of S1P on angiogenesis is well established, its role in neurogenesis is unknown. We examined the effects of S1P on G‐protein activation in brain sections of rat embryo and on neural progenitor cells in culture. Intense S1P‐stimulated [35S]GTPγS labeling was observed as early as E15 in the neuroepithelium and differentiating fields throughout the brain, suggesting that functional S1P receptors are expressed in brain areas with active neurogenesis. mRNA transcripts for several S1P receptor subtypes (S1P1, S1P2, S1P3 and S1P5) were expressed in neural progenitor cells prepared from embryonic rat hippocampus. S1P induced phosphorylation of extracellular signal‐regulated kinase (ERK) and proliferation of neural progenitor cells as determined by BrdU incorporation in a pertussis toxin‐sensitive manner. These effects were prevented by the ERK signaling inhibitor U0126. S1P augmented telomerase activity in neural progenitor cells with similar potency as that of FGF‐2. Furthermore, S1P induced cell‐cell aggregation. This morphological change was transient and prevented by Y‐27632, an inhibitor of Rho‐associated kinase. These results suggest that S1P plays a pleiotropic role in neurogenesis via pathways involving S1P receptors, MAP kinases and Rho kinase.
Normal levels of tryptophan hydroxylase immunoreactivity in the dorsal raphe of depressed suicide victims
Authors: Bonkale Willy L. , Murdock Shayna , Janosky Janine E. , Austin Mark C.
Published: July 2010

A variety of evidence suggests that serotonin neurotransmission is altered in the brain of suicide victims and depressed patients. While numerous post‐mortem studies have investigated serotonin transporters and receptors, few studies have examined the biosynthetic integrity of the rate‐limiting enzyme, tryptophan hydroxylase (TPH), in post‐mortem specimens of depressed suicide subjects. Therefore, the aim of the present study was to test the hypothesis that the levels of TPH immunoreactivity (IR) are altered in specific subnuclei of the dorsal raphe (DR) in depressed suicide victims. Suicide victims with a confirmed diagnosis of major depression were matched with non‐psychiatric controls based on age, gender and post‐mortem interval. Frozen tissue sections containing the DR were selected from two anatomical levels and processed for TPH radioimmunocytochemistry. The optical density corresponding to the regional levels of TPH‐IR was quantified in specific subnuclei of the DR from the film autoradiographic images. No significant differences in the levels of TPH‐IR were found in any DR subnuclei between depressed suicide victims and control subjects. The lack of change in TPH‐IR levels does not necessarily imply that serotonin synthesis or neurotransmission is not altered in the brain of depressed subjects. Many factors influence and regulate serotonin synthesis, and it is conceivable that alterations exist at other levels of regulation of serotonin biosynthesis in depression. Our findings indicate that TPH biosynthesis, at least at the protein level, is not significantly altered in the DR of depressed suicide victims.
Role of reactive oxygen species in LPS‐induced production of prostaglandin E2in microglia
Authors: Wang Tongguang , Qin Liya , Liu Bin , Liu Yuxin , Wilson Belinda , Eling Thomas E. , Langenbach Robert , Taniura Seijiro , Hong Jau‐Shyong
Published: July 2010

We determined the roles of reactive oxygen species (ROS) in the expression of cyclooxygenase‐2 (COX‐2) and the production of prostaglandin E2(PGE2) in lipopolysaccharide (LPS)‐activated microglia. LPS treatment increased intracellular ROS in rat microglia dose‐dependently. Pre‐treatment with superoxide dismutase (SOD)/catalase, or SOD/catalase mimetics that can scavenge intracellular ROS, significantly attenuated LPS‐induced release in PGE2. Diphenylene iodonium (DPI), a non‐specific NADPH oxidase inhibitor, decreased LPS‐induced PGE2production. In addition, microglia from NADPH oxidase‐deficient mice produced less PGE2than those from wild‐type mice following LPS treatment. Furthermore, LPS‐stimulated expression of COX‐2 (determined by RT‐PCR analysis of COX‐2 mRNA and western blot for its protein) was significantly reduced by pre‐treatment with SOD/catalase or SOD/catalase mimetics. SOD/catalase mimetics were more potent than SOD/catalase in reducing COX‐2 expression and PGE2production. As a comparison, scavenging ROS had no effect on LPS‐induced nitric oxide production in microglia. These results suggest that ROS play a regulatory role in the expression of COX‐2 and the subsequent production of PGE2during the activation process of microglia. Thus, inhibiting NADPH oxidase activity and subsequent ROS generation in microglia can reduce COX‐2 expression and PGE2production. These findings suggest a potential therapeutic intervention strategy for the treatment of inflammation‐mediated neurodegenerative diseases.
Microarray analysis of gene expression on herbal glycoside recipes improving deficient ability of spatial learning memory in ischemic mice
Authors: Wang Zhong , Du Qingyou , Wang Fusheng , Liu Zhongrong , Li Baigang , Wang Anmin , Wang Yongyan
Published: July 2010

In order to reveal the mechanism of herbal glycoside recipes retrieving deficient ability of spatial learning memory in mice suffering from cerebral ischemia/reperfusion, a microarray system was used to analyze gene expression in those groups with increasing ability of spatial learning memory who were different from ischemic mice. In this work, we reported a comprehensive characterization of gene expression profiles of mouse hippocampus by the use of cDNA microarray system containing 1176 known genes in middle cerebral artery occlusion (MCAO) ischemic mice after treating with different dosage recipes of glycoside herbs (30, 90, and 270 mg/kg). The ability of spatial learning memory in ischemic mice was found to be decreased. The pathological process in ischemic mouse brain showed that a complex related to 100 genes' expression yielded 1.8‐fold. Dose‐dependent effects showed an improvement in the deficient ability and reduction in infarct volume when treated with glycoside recipes. Many genes (38–46) in expression were found greater than 1.8‐fold in those effective recipes groups, including genes in cell cycle regulation, signal transduction, nerve system transcription factors, DNA binding protein, etc. Nine genes related to retrieving deficient ability of spatial learning memory treated with glycoside recipes were also found in this study. These results suggest that microarray analysis of gene expression might be useful for elucidating the mechanisms of pharmacological function of recipes.
Nonylphenol induces the death of neural stem cells due to activation of the caspase cascade and regulation of the cell cycle
Authors: Kudo Chiho , Wada Koichiro , Masuda Tomotake , Yonemura Toshimitsu , Shibuya Atsuhito , Fujimoto Yohko , Nakajima Atsushi , Niwa Hitoshi , Kamisaki Yoshinori
Published: July 2010

Endocrine disruptors (EDs) are a great concern throughout the world, because they have adverse effects on human health and wildlife. In the present study, we investigated the effects of EDs on the proliferation and survival of murine neural stem cells (NSCs). In contrast to bisphenol A, phthalic acid benzyl n‐butyl ester, phthalic acid di‐n‐butyl ester and phthalic acid di(2‐ethylhexyl) ester, the treatment of NSCs with 4‐nonylphenol for 24 h inhibited cell growth in a concentration‐dependent manner. In addition, treatment with 4‐nonylphenol resulted in nuclear condensation and DNA fragmentation (morphological changes due to apoptosis) in NSCs after 12 h of exposure, and activated caspase‐3 after 6 h and 9 h of exposure. Furthermore, an exposure to 4‐nonylphenol led to the accumulation of cells at the G2/M phase interface and down‐regulated the protein levels of cyclin A and B1, which are the major regulatory proteins at the G2to M transition of the cell cycle. Together, these results indicate that, in contrast to other EDs, 4‐nonylphenol may exhibit a potent cytotoxicity through apoptosis via the caspase cascade and cell cycle arrest at the G2/M phase, and suggest that 4‐nonylphenol may affect neurogenesis in the CNS.
Microarray analysis of developmental plasticity in monkey primary visual cortex
Authors: Lachance Pascal E. D. , Chaudhuri Avi
Published: July 2010

We performed microarray gene expression analyses on the visual cortex of Old‐World monkeys (Cercopithicus aethiops) in an effort to identify transcripts associated with developmental maturation and activity‐driven changes during the visual critical period. Samples derived from normal animals and those subjected to monocular enucleation (ME) were hybridized to human Affymetrix HG‐U95Av2 oligonucleotide microarrays (N = 12) and the results were independently validated by real‐time quantitative RT‐PCR. To identify genes exhibiting significant expression differences among our samples, the microarray hybridization data were processed with two software packages that use different analytical models (Affymetrix MicroArray Suite 5.0, dChip 1.2). We identified 108 transcripts within diverse functional categories that differed in their visual cortical expression at the height of the critical period when compared to adults. The expression levels of four transcripts were also globally modulated following ME during the critical period. These transcripts are particularly sensitive to ME during the critical period but are not significantly modulated in ME adults. Three of the ME‐driven genes (NGFI‐B, egr3, NARP) are known immediate‐early genes (IEG) while the other (DUSP6) is a phosphatase that can regulate IEG expression. The putative biological significance of the ME‐driven and developmentally regulated genes is discussed with respect to the critical period for activity‐dependent visual cortical neuroplasticity.
Poster Sessions AP10: Learning, Memory and Behavior. S‐adenosylmethionine induced seizures are anesthetic dependant
Authors: Bottiglieri T. , Anderson D. , Diaz‐Arrastia R. , Arning E.
Published: July 2010

S‐adenosylmethionine (SAMe) is present in all tissues and functions as the sole donor of methyl groups in over 100 different methylation reactions. Recent reports suggest that direct intraventricular injection of SAMe induces Parkinsonian like symptoms in rats including seizures, tremors, hyperkinesia and abnormal posture. In order to assess the influence of SAMe on rat behavior we have undertaken a study to examine the effect of 3 different forms of SAMe. Guide cannulae were sterotaxically implanted into the lateral ventricle of male SD rats (n = 5 for each group) using either ketamine or chloral hydrate anesthesia. 48 h post surgery the rats received a 5‐μL injection containing 1 μmol of either SAMe‐toluenedisulfonate, SAMe‐butanedisulfonate, SAMe‐chloride, or vehicle (butanedisulfonate, toluenedisulfonate or saline). Locomotor activity was monitored using the TruScan monitoring system and by videotape recording for 1 h. The videotape was reviewed by one of the authors (RD‐A) who is experienced with animal models of epilepsy. SAMe injected animals had frequent myoclonic and tonic seizures, and occasional generalized clonic seizures. SAMe induced behavioral seizures and tremors occurred only in rats that had previously been anesthetized with chloral hydrate, and not in rats that received ketamine. The number of movements recorded during the 1‐h period were significantly increased in SAMe injected animals compared to control groups in both chloral hydrate and ketamine anethetized rats. Our studies indicate that there is an anesthetic dependency for SAMe induced seizures and tremors.
The type 1 interleukin‐1 receptor is essential for multiple aspects of brain inflammation
Authors: Levison S. W. , Basu A. , Krady J. K. , O'Malley M. O. , Styren S. D. , DeKosky S. T.
Published: July 2010

Interleukin‐1 (IL‐1) is induced immediately after brain imjury and elevated levels of IL‐1 have been strongly implicated in the neurodegeneration that accompanies stroke, Alzheimer's disease and Multiple Sclerosis. Antagonizing IL‐1 reduces cell death; however, the basis for this protection has not been elucidated. Here we analyzed the response to penetrating brain injury in mice lacking the type 1 interleukin receptor (IL‐1R1) to determine which cellular and molecular mediators of tissue damage require IL‐1 signaling. At the cellular level fewer amoeboid microglia/macrophages appeared adjacent to the injured brain tissue in IL‐1R1 null mice, and those microglia present at early postinjury intervals retained their resting morphology. Astrogliosis also was mildly abrogated. At the molecular level, cyclooxygenase 2 and IL‐6 expression were depressed and delayed. Interestingly, basal levels of cyclooxygenase 2, IL‐1 and IL‐6 were significantly lower in the IL‐1R1 null mice. Additionally, stimulation of VCAM‐1 mRNA was depressed in the IL‐1R1 null mice, and correspondingly, there was reduced migration of peripheral macrophages into the IL‐1R1 null brain after injury. This observation correlated with a reduced number of cyclooxygenase 2+ amoeboid phagocytes adjacent to the injury. By contrast, the production of nerve growth factor was only mildly affected. Since antagonizing IL‐1 protects neural cells in experimental models of stroke and multiple sclerosis, our data suggest that cell preservation is achieved by abrogating microglial/macrophage activation and the subsequent self‐propagating cycle of inflammation.

Acknowledgements:  Supported by NMSS Award #RG 3837.
Myelin transcription factor 1 function in oligodendrocyte development
Authors: Nielsen J. A. , Hudson L. D. , Armstrong R. C.
Published: July 2010

Myelin transcription factor 1 (Myt1) is a CCHC zinc‐finger protein that is expressed in oligodendrocyte progenitors (OP) and is down‐regulated as oligodendrocytes differentiate and accumulate proteolipid protein (PLP). Myt1 contains six zinc‐finger DNA‐binding domains, a putative acidic transcriptional activation domain, and an alpha‐helical protein–protein interaction domain. Myt1 has two zinc‐fingers near the N‐terminus and four zinc‐fingers (4F) near the C‐terminus. Each set of zinc‐fingers can bind to the PLP promoter. The objective of this study was to determine the function of Myt1 by expressing the 4F domain of Myt1, which lacks the putative transcriptional activation domain and protein–protein interaction domain. We predicted the 4F domain would compete for Myt1 response elements, and lacking other functional domains would interfere with endogenous Myt1 function. A retroviral expression system that included a FLAG epitope tag was used to ectopically express the 4F domain of Myt1 in cultured rat neonatal OPs. OPs and their progeny exhibited nuclear immunoreactivity for the 4F‐FLAG fusion protein. Expression of 4F in OPs grown with PDGF and FGF mitogens reduced proliferation as compared to controls. In the absence of mitogens, expression of 4F inhibited the differentiation of OPs as assessed by morphological criteria, O1 immunostaining, and PLP mRNA expression. This 4F inhibition of OP differentiation along the oligodendrocyte pathway was not due to alternative differentiation along an astrocytic pathway. These data suggest that Myt1 regulates a critical transition in oligodendrocyte lineage development in modulating the OP proliferative response relative to terminal differentiation and myelin gene expression.

Acknowledgements: Supported by USUHS grant RO70IE.
Signalling pathways linking proliferation and survival in oligodendrocyte progenitors
Authors: Saini H. S. , Chu A. K. , Boxer L. M. , Sato‐Bigbee C.
Published: July 2010

Our previous results support the idea that the transcription factor CREB mediates the action of neuroligand and growth factor signals that coupled to different signalling pathways, play different roles at specific stages of oligodendrocyte (OLG) development. We have found before that the stimulation of OLG progenitor cell proliferation by neurotrophin‐3 (NT‐3) is preceded by a dramatic increase in CREB phosphorylation by both MAPK and PKC signalling pathways. In addition, we showed that inhibition of CREB expression abolished the NT‐3 dependent stimulation of OLG proliferation. We have now found that treatment of OLG progenitors with NT‐3 also stimulates the expression of the antiapoptotic protein bcl‐2. Interestingly, the bcl‐2 gene promoter contains a CREB binding site and thus, this transcription factor may also be involved in modulating the survival of OLG progenitors. In support of this hypothesis, we found that the NT‐3 dependent stimulation of bcl‐2 expression is dramatically decreased when CREB levels in the cells are down regulated by treatment with CREB antisense oligonucleotide. The role of CREB in regulating bcl‐2 expression in the OLG progenitors is further supported by transient transfection experiments using a luciferase reporter gene driven by the promoter region of the bcl‐2 gene. The experiments indicated that the activity of the bcl‐2 promoter was significantly enhanced by incubating the cells with NT‐3. However, this response was abolished by mutations in the promoter at the CREB binding site. Altogether, these results support the idea that signalling pathways targeting CREB may play an important role in linking cell proliferation and survival of OLG progenitors.
CNTF is up‐regulated in astrocytes during spinal cord remyelination and stimulates FGF‐2 production
Authors: Levison S. W. , Albrecht P. J. , Murtie J. C. , Ness J. K. , Redwine J. M. , Armstrong R. C.
Published: July 2010

FGF‐2 is a potent mitogen and survival factor for oligodendrocyte progenitors in vitro, and it is induced in astrocytes in conjunction with successful remyelination during recovery from murine hepatitis virus (MHV) induced spinal cord demyelination. However, the factor(s) responsible for inducing FGF‐2 after demyelination in astrocytes are unknown. Here we used in vivo and in vitro paradigms to assess whether ciliary neurotrophic factor (CNTF) could be responsible for FGF‐2 induction during remyelination. In mice recovering from MHV‐induced demyelination, increased CNTF expression coincided with spinal cord remyelination. Immunohistochemistry demonstrated that CNTF protein was present within astrocytes surrounding and within remyelinating lesions. After directly injecting CNTF into the spinal cord, FGF‐2 mRNA increased in spinal cord astrocytes. Furthermore, CNTF induced FGF‐2 mRNA over 2.5 fold in cultured mouse spinal cord astrocytes as determined by kinetic RT–PCR analysis. Although IL‐1β has been implicated in FGF‐2 production, IL‐1β mRNA was not detected during remyelination, nor did IL‐1β induce FGF‐2 mRNA from mouse spinal cord astrocytes in vitro. Altogether, these results strongly implicate CNTF as an upstream positive regulator of FGF‐2 production in astrocytes during early remyelination.

Acknowledgements:  Supported by NMSS Award #RG 3837 (SWL) and NS 33316 (RCA).
Is the interaction of CNP with tubulin and microtubules required for process extension in oligodendrocytes?
Authors: Lee J. , Gravel M. , Braun P. E.
Published: July 2010

Oligodendrocytes (ODC) differentiate and undergo vast membrane synthesis and process extension to form myelin. What are the molecular mechanisms underlying these events? The myelin protein, CNP, appears to play an important role. Recent work from our lab and others provide strong evidence that CNP is associated with the cytoskeleton and may be involved in process extension. Intriguingly, CNP has an enzymatic activity, whose in vivo relevance is unknown, since substrates with 2′,3′‐cyclic termini have not yet been determined for CNP. To elucidate the function of CNP, a 55‐kDa polypeptide, which copurifies with CNP by immunoprecipitation from rat brain, was identified as tubulin by mass spectrometry. Cultured ODC and CNP‐transfected COS‐7 cells showed extensive microtubule colocalization along the main processes and the cell body. COS‐7 cells expressing high levels of CNP exhibited major morphological changes similar to cultured ODC: rounding of the cell body, process extension, and extensive branching of the processes. Deletion mutant studies reveal that tubulin binding and cell transformation are mediated by the C‐terminal catalytic domain. However, the catalytic activity of CNP is not responsible for these morphological changes, since expression of enzymatic inactive mutants in non‐neuronal cells resembled cells expressing wild‐type CNP. Furthermore, RICH (Regeneration Induced CNPase Homologue), a protein that is expressed in fish retinal ganglion cells and shares extensive homology with CNP in the C‐terminal domain, can bind to tubulin and cause similar cytoskeletal transformations in non‐neuronal cells. Our results suggest that binding of CNP to microtubules facilitates cytoskeletal rearrangements necessary for process extension and myelination in ODC.

Acknowledgements:  Supported by the CIHR and FCAR.
Role of melatonin in affecting disturbances of central regulation of reproduction by neurotoxicants
Authors: Arutjunyan A. V. , Stepanov M. G. , Kerkeshko G. O. , Prokopenko V. M. , Korenevsky A. V.
Published: July 2010

An effect of neurotropic xenobiotic 1,2‐dymethylhydrazine (DMH) on hypothalamic regulation of female rat reproduction has been studied. A disturbance of diurnal rhythms of biogenic amines in brain areas responsible for gonadoliberin synthesis and secretion (preoptic area of the hypothalamus – PA, and median eminence – ME), and in suprachiasmatic nuclei of the hypothalamus as a main circadian rhythms pace‐maker, has been found. DMH has been shown to be able to cause various changes in diurnal rhythms of the biogenic amines (dopamine, norepinephrine, serotonin) contents in these brain areas, and in reactive oxygen species (ROS) formation in PA, which can be due to diurnal variations in neurotransmitter levels in this area of the hypothalamus. Taking into account a role of melatonin produced in the pineal gland in circadian rhythms organization, an effect of exogenous melatonin on the above changes caused by DMH has been investigated. The gonadoliberin contents in PA and ME of rats undergone administration of DMH and melatonin have been studied. DMH has been shown not to cause sensible changes in the gonadoliberin contents in PA and ME, while melatonin itself and on the background of DMH administration significantly decreased gonadoliberin levels. Administration of DMH causes less significant changes in biogenic amines compared to melatonin. Also, melatonin in both absence and presence of DMH aggravates ROS formation in PA as well as in the whole hypothalamus. Our data allow supposition that exogenious melatonin is not capable of recovering processes of regulation of reproduction that were disturbed by neurotoxicant xenobiotics.

Acknowledgements:  Supported by RBRF grant #00‐04‐48967.
The glucose paradox of cerebral ischemia – a possible resolution
Authors: Schurr A. , Payne R. S.
Published: July 2010

The glucose paradox of cerebral ischemia, the aggravation of delayed neuronal damage by preischemic hyperglycemia, is a well‐documented phenomenon. Recently, we demonstrated that corticosterone (CT) plasma levels were significantly elevated within 30 min after glucose administration (i.p.) only to return to baseline levels by 120 min. In this study we examined the postulate that preischemic glucose loading aggravates the postischemic outcome via the induction of a short‐lived, massive elevation in CT plasma levels. Hence, we tested the ability of the CT synthesis inhibitor metyrapone (MT) and the glucocorticoid receptor antagonist, mifepristone (RU486), to attenuate the aggravated delayed neuronal damage in a rat model of cardiac‐arrest‐induced transient global cerebral ischemia (TGI). Seven different groups of 24‐h‐fasted rats were used and were administered several treatments prior to TGI that were aimed at either inhibiting CT synthesis (MT, 100 mg/kg, i.p.) or at blocking the brain glucocorticoid receptors (RU486, 40 mg/kg, i.p.). Plasma glucose and CT levels were measured 2 min pre‐TGI. Neuronal damage was assessed 7 days post‐TGI in hippocampal slices using electrophysiological means. Plasma glucose levels were not reduced in MT‐ or RU486‐treated rats. Both MT and RU 486 prevented preischemic hyperglycemia‐aggravated ischemic damage, the former presumably via inhibition of CT synthesis, the latter by glucocorticoid receptor antagonism. The present data strongly suggest that preischemic hyperglycemia‐aggravated neuronal damage, i.e. the glucose paradox, results from glucose‐induced CT release, rather than intensification of lactic acidosis in the ischemic brain.