bluuebunny/crossref_metadata_2025 huggingface dataset:
Transport Is the Primary Determinant of Glycine Content in Retinal Neurons
Authors: Pow David V.
Published: July 2010

Abstract: This study demonstrates that in mammalian and nonmammalian species it is possible to deplete selectively and reversibly retinal glycinergic neurons of their content of glycine by exposure to sarcosine, a competitive inhibitor of glycine transporter 1 (glyt‐1). This observation was used as a tool to test the hypothesis that uptake of glycine rather than de novo synthesis is the main determinant of glycine content in retinal neurons. Isolated retinae were depleted of immunocytochemically detectable pools of glycine. Thereafter retinae were exposed either to physiological medium containing glycine or to medium lacking glycine but containing precursors for the synthesis of glycine. Retinae exposed to glycine‐containing medium rapidly recovered their content of glycine, whereas retinae exposed to medium lacking glycine but containing serine, a substrate for synthesis of glycine, showed only a slow recovery of immunoreactivity for glycine in a few amacrine cells. These data indicate that uptake of glycine is the primary determinant of glycine content in most retinal glycinergic neurons. The origins of the extracellular pools of glycine remain to be identified; however, it is suggested that such glycine may be derived from the vitreous humor and that in turn this glycine may be derived from the peripheral circulation.
Nuclear Factor of Activated T Cells (NFAT) as a New Component of the Signal Transduction Pathway in Glioma Cells
Authors: Mosieniak Grazyna , Pyrzynska Beata , Kaminska Bozena
Published: July 2010

Abstract: Cyclosporin A (CsA) exerts its immunosuppressive effect by inhibiting the activity of nuclear factor of activated T cells (NFAT), thus preventing transcriptional induction of several cytokine genes. This effect is mediated through inactivation of the phosphatase calcineurin, which inhibits translocation of an NFAT component to the nucleus. We have previously reported that CsA inhibits the growth of rat C6 glioma cells in a dose‐dependent manner and induces apoptotic cell death. Here, we report that NFAT DNA‐binding activity is present in the nuclear extracts from C6 glioma cells and that CsA treatment inhibits the formation of a functional NFAT complex. We provide evidence for the presence of a group of NFATc proteins in proliferating glioma cells. Immunoblot analyses show that stimulation of C6 glioma cells with a calcium‐inducing agent, ionomycin, alters NFATc migration on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. This alteration is inhibited by simultaneous treatment with CsA, suggesting a calcineurin involvement in the regulation of glioma NFATc proteins. Direct immunofluorescence reveals the presence of NFATc proteins in nuclei of proliferating glioma cells and their disappearance in CsA‐treated cells. These data point to a new mechanism of transcription regulation in glioma cells and provide an explanation for the observed sensitivity of glioma cells to CsA.
Src Homology Domains of Phospholipase C γ1 Inhibit Nerve Growth Factor‐Induced Differentiation of PC12 Cells
Authors: Bae Sun Sik , Lee Young Han , Chang Jong‐Soo , Galadari Sehamuddin H. , Kim Yong Sik , Ryu Sung Ho , Suh Pann‐Ghill
Published: July 2010

Abstract: Phospholipase C γ1 (PLC‐γ1) is phosphorylated on treatment of cells with nerve growth factor (NGF). To assess the role of PLC‐γ1 in mediating the neuronal differentiation induced by NGF treatment, we established PC12 cells that overexpress whole PLC‐γ1 (PLC‐γ1PC12), the SH2‐SH2‐SH3 domain (PLC‐γ1SH223PC12), SH2‐SH2‐deleted mutants (PLC‐γ1ΔSH22PC12), and SH3‐deleted mutants (PLC‐γ1ΔSH3PC12). Overexpressed whole PLC‐γ1 or the SH2‐SH2‐SH3 domain of PLC‐γ1 stimulated cell growth and inhibited NGF‐induced neurite outgrowth of PC12 cells. However, cells expressing PLC‐γ1 lacking the SH2‐SH2 domain or the SH3 domain had no effect on NGF‐induced neuronal differentiation. Overexpression of intact PLC‐γ1 resulted in a threefold increase in total inositol phosphate accumulation on treatment with NGF. However, overexpression of the SH2‐SH2‐SH3 domain of PLC‐γ1 did not alter total inositol phosphate accumulation. To investigate whether the SH2‐SH2‐SH3 domain of PLC‐γ1 can mediate the NGF‐induced signal, tyrosine phosphorylation of the SH2‐SH2‐SH3 domain of PLC‐γ1 on NGF treatment was examined. The SH2‐SH2‐SH3 domain of PLC‐γ1 as well as intact PLC‐γ1 could be tyrosine‐phosphorylated on NGF treatment. These results indicate that the overexpressed SH2‐SH2‐SH3 domain of PLC‐γ1 can block the differentiation of PC12 cells induced by NGF and that the inhibition appears not to be related to the lipase activity of PLC‐γ1 but to the SH2‐SH2‐SH3 domain of PLC‐γ1.
Evidence for Activation of Caspase‐3‐Like Protease in Excitotoxin‐ and Hypoxia/Hypoglycemia‐Injured Neurons
Authors: Nath Rathna , Probert Albert , McGinnis Kim M. , Wang Kevin K. W.
Published: July 2010

Abstract: Caspase activation has been shown to be a critical step in several models of neuronal apoptosis such as staurosporine treatment of human neuroblastoma SH‐SY5Y cells and potassium deprivation of rat cerebellar granule neurons. One common event is the appearance of caspase‐mediated 120‐kDa nonerythroid α‐spectrin breakdown product (SBDP120). Second, inhibitors of the caspase family are effective blockers of such neuronal death. In this study, we report the appearance of caspase‐mediated SBDP120 in excitotoxin‐challenged fetal rat cerebrocortical neurons [N‐methyl‐d‐aspartate (NMDA), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, and kainate] and rat cerebellar granule neurons (NMDA and kainate). A general caspase inhibitor, carbobenzoxy‐Asp‐CH2OC(O)‐2,6‐dichlorobenzene (Z‐D‐DCB), blocked the formation of SBDP120 under these conditions and attenuated the observed NMDA‐induced lactate dehydrogenase (LDH) release in both cell types. Furthermore, hydrolytic activity toward a caspase‐3‐preferred synthetic peptide substrate, acetyl‐DEVD‐7‐amido‐4‐methylcoumarin, was significantly elevated in NMDA‐treated granule neurons. Lastly, oxygen‐glucose deprivation (OGD)‐challenged cerebrocortical cultures also showed the appearance of SBDP120. Again, Z‐D‐DCB blocked the SBDP120 formation as well as attenuated the LDH release from the OGD‐challenged neurons. Taken together, the presence of caspase‐specific SBDP120 and the neuroprotective effects of Z‐D‐DCB strongly suggest that caspase activation contributes at least in part to excitotoxin‐ and OGD‐induced neuronal death.
Ornithine Decarboxylase in Human Brain: Influence of Aging, Regional Distribution, and Alzheimer's Disease
Authors: Morrison Lesley D. , Cao Xiao‐Chun , Kish Stephen J.
Published: July 2010

Abstract: Although experimental animal data have implicated ornithine decarboxylase, a key regulatory enzyme of polyamine biosynthesis, in brain development and function, little information is available on this enzyme in normal or abnormal human brain. We examined the influence, in autopsied human brain, of postnatal development and aging, regional distribution, and Alzheimer's disease on the activity of ornithine decarboxylase. Consistent with animal data, human brain ornithine decarboxylase activity was highest in the perinatal period, declining sharply (by ∼60%) during the first year of life to values that remained generally unchanged up to senescence. In adult brain, a moderately heterogeneous regional distribution of enzyme activity was observed, with high levels in the thalamus and occipital cortex and low levels in cerebellar cortex and putamen. In the Alzheimer's disease group, mean ornithine decarboxylase activity was significantly increased in the temporal cortex (+76%), reduced in occipital cortex (−70%), and unchanged in hippocampus and putamen. In contrast, brain enzyme activity was normal in patients with the neurodegenerative disorder spinocerebellar ataxia type I. Our demonstration of ornithine decarboxylase activity in neonatal and adult human brain suggests roles for ornithine decarboxylase in both developing and mature brain function, and we provide further evidence for the involvement of abnormal polyamine system activity in Alzheimer's disease.
Pramipexole Reduces Reactive Oxygen Species Production In Vivo and In Vitro and Inhibits the Mitochondrial Permeability Transition Produced by the Parkinsonian Neurotoxin Methylpyridinium Ion
Authors: Cassarino David S. , Fall Christopher P. , Smith Trisha S. , Bennett James P.
Published: July 2010

Abstract: Sporadic Parkinson's disease is associated with a defect in the activity of complex I of the mitochondrial electron transport chain. This electron transport chain defect is transmitted through mitochondrial DNA, and when expressed in host cells leads to increased oxygen free radical production, increased antioxidant enzyme activities, and increased susceptibility to programmed cell death. Pramipexole, a chemically novel dopamine agonist used for the treatment of Parkinson's disease symptoms, possesses antioxidant activity and is neuroprotective toward substantia nigral dopamine neurons in hypoxic‐ischemic and methamphetamine models. We found that pramipexole reduced the levels of oxygen radicals produced by methylpyridinium ion (MPP+) both when incubated with SH‐SY5Y cells and when perfused into rat striatum. Pramipexole also exhibited a concentration‐dependent inhibition of opening of the mitochondrial transition pore induced by calcium and phosphate or MPP+. These results suggest that pramipexole may be neuroprotective in Parkinson's disease by attenuating intracellular processes such as oxygen radical generation and the mitochondrial transition pore opening, which are associated with programmed cell death.
Regulation of Drosophila Ca2+/Calmodulin‐Dependent Protein Kinase II by Autophosphorylation Analyzed by Site‐Directed Mutagenesis
Authors: Wang Zheng , Palmer Glenn , Griffith Leslie C.
Published: July 2010

Abstract: In this study we demonstrate that Drosophila calcium/calmodulin‐dependent protein kinase II (CaMKII) is capable of complex regulation by autophosphorylation of the three threonines within its regulatory domain. Specifically, we show that autophosphorylation of threonine‐287 in Drosophila CaMKII is equivalent to phosphorylation of threonine‐286 in rat α CaMKII both in its ability to confer calcium independence on the enzyme and in the mechanistic details of how it becomes phosphorylated. Autophosphorylation of this residue occurs only within the holoenzyme structure and requires calmodulin (CaM) to be bound to the substrate subunit. Phosphorylation of threonine‐306 and threonine‐307 in the CaM binding domain of the Drosophila kinase occurs only in the absence of CaM, and this phosphorylation is capable of inhibiting further CaM binding. Additionally, our findings suggest that phosphorylation of threonine‐306 and threonine‐307 does not mimic bound CaM to alleviate the requirement for CaM binding to the substrate subunit for intermolecular threonine‐287 phosphorylation. These results demonstrate that the mechanism of regulatory autophosphorylation of this kinase predates the split between invertebrates and vertebrates.
Differential Effects of Increasing Gestational Age and Placental Restriction on Tyrosine Hydroxylase, Phenylethanolamine N‐Methyltransferase, and Proenkephalin A mRNA Levels in the Fetal Sheep Adrenal
Authors: Adams Michael B. , Phillips Ian D. , Simonetta Giuseppe , McMillen I. Caroline
Published: July 2010

Abstract: We have demonstrated that there are differential changes in the levels of tyrosine hydroxylase (TH), phenylethanolamine N‐methyltransferase (PNMT), and proenkephalin A (Pro Enk A) mRNA in the fetal sheep adrenal during late gestation. Adrenal TH mRNA:18S rRNA ratios increased between gestational days 100 (0.98 ± 0.13; n = 6) and 125 (1.40 ± 0.15; n = 6) and then decreased, whereas adrenal PNMT mRNA:18S rRNA ratios increased regularly between gestational days 100 (0.08 ± 0.01) and 146 (0.17 ± 0.03). The ratio of adrenal Pro Enk A mRNA to 18S rRNA was higher at gestational day 125 (0.085 ± 0.005) than at either 80–100 days (0.038 ± 0.007) or 140–146 days of gestation (0.055 ± 0.013). In 12 ewes, the growth and development of the placenta were restricted (placental restriction group) from conception. The ratio of adrenal PNMT mRNA to 18S rRNA was significantly reduced in the placental restriction group of fetal sheep (0.003 ± 0.002) compared with controls (0.011 ± 0.002), and there was a significant correlation between the ratio of adrenal PNMT mRNA to 18S rRNA and the mean arterial Po2 (r = 0.88, p < 0.0005). In contrast, TH mRNA and Pro Enk mRNA were unaffected by placental restriction. Adrenaline and nonadrenaline syntheses are therefore differentially regulated in the adrenal during late gestation and in response to chronic intrauterine hypoxemia.
Norepinephrine Increases Cyclic GMP Levels in Cerebellar Cells from Neuronal Nitric Oxide Synthase Knockout Mice
Authors: Morton David B. , Bredt David S.
Published: July 2010

Abstract: Cyclic GMP is an important intracellular messenger in the nervous system that may mediate cellular forms of neuronal plasticity. Previous studies show that most neurotransmitters stimulate cyclic GMP levels by the activation of nitric oxide synthase (NOS). In this study, we report that in primary cell cultures from the cerebellum of neuronal NOS knockout mice, norepinephrine stimulates an increase in cyclic GMP content. This increase is seen in both granule cell and astrocyte cultures and is not blocked by inhibitors of NOS or by inhibition of soluble guanylyl cyclase. These results suggest a novel pathway by which norepinephrine enhances cyclic GMP levels in the nervous system.
Identification of Translin and Trax as Components of the GS1 Strand‐Specific DNA Binding Complex Enriched in Brain
Authors: Taira Eiichi , Finkenstadt Patricia M. , Baraban Jay M.
Published: July 2010

Abstract: In previous gel‐shift assays, we identified a protein complex, referred to as GS1, that binds in a sequence‐specific manner to single‐stranded DNA and is highly enriched in brain. As an initial step in clarifying the function of this complex, we have undertaken studies aimed at defining its protein components. In particular, we focused on identifying two protein bands that were covalently labeled when the GS1‐DNA complex was subjected to UV irradiation to induce cross‐linking between the radiolabeled probe and GS1 components. By following GS1 binding activity through a series of conventional chromatographic steps, as well as an affinity column based on the DNA oligonucleotide used for gel‐shift assays, we were able to achieve ∼500,000‐fold enrichment of GS1 compared with that in crude cerebellar extracts used as starting material. This highly purified fraction contained both protein bands detected by UV cross‐linking in crude extracts. Sequencing of peptides derived from these proteins led to their identification as Translin and Trax, interacting proteins identified in studies of DNA recombination in lymphocytes. A distinct line of research has provided evidence that a complex containing Translin can bind to specific mRNAs and block their translation. Whether one or both of these proposed functions of the Translin/Trax complex explains the high basal level of GS1 binding activity present in the brain remains to be determined.
Human Amylin Induces “Apoptotic” Pattern of Gene Expression Concomitant with Cortical Neuronal Apoptosis
Authors: Tucker H. Michael , Rydel Russell E. , Wright Sarah , Estus Steven
Published: July 2010

Abstract: Amylin forms large β‐pleated neurotoxic oligomers but shows only 38% sequence similarity to Aβ. As patterns of gene expression during neuronal apoptosis appear stimulus and cell type specific, we compared the pattern of amylin‐induced gene expression in rat cortical neurons with that shown previously to be induced by Aβ in order to evaluate whether these two peptides with different primary but similar secondary structure induce apoptosis similarly. Morphologic and quantitative measures of cell death show widespread apoptotic death after amylin treatment. Amylin treatment results in time‐ and concentration‐dependent inductions of oxidative stress genes, such as cox‐2 and IκB‐α. “Apoptotic” genes are also induced in a time‐ and concentration‐dependent manner, including c‐jun, junB, c‐fos, and fosB, followed temporally by a gene known to be modulated by these transcription factors, i.e., transin. In situ hybridization analyses show that c‐fos expression is restricted largely to neurons with condensed chromatin, a hallmark of apoptosis. As these genes are not induced in all models of apoptosis, that amylin‐induced neuronal death is genetically similar to that of Aβ suggests that these peptides may be neurotoxic through a common mechanism.
Selective Activation of Phospholipase Cγ1 and Distinct Protein Kinase C Subspecies in Intracellular Signaling by Hepatocyte Growth Factor/Scatter Factor in Primary Cultured Rat Neocortical Cells
Authors: Machide Mitsuru , Kamitori Kazuyo , Nakamura Yasuko , Kohsaka Shinichi
Published: July 2010

Abstract: Hepatocyte growth factor/scatter factor (HGF) was recently reported to function as a neurotrophic factor in the CNS. To investigate the intracellular signal pathways after activation of the HGF receptor c‐Met in primary cultured rat neocortical cells, in vitro kinase assays were performed. HGF stimulation enhances the phosphorylation of endogenous 80‐ and 45‐kDa substrates. Studies with protein kinase inhibitors and phorbol 12‐myristate 13‐acetate showed that protein kinase C (PKC) is activated intracellularly. The 80‐kDa protein was identified to be the major PKC substrate MARCKS. Although four PKC subspecies, PKCα, PKCε, PKCγ, and PKCλ, were expressed in the cells, only PKCα, PKCε, and PKCγ were selectively translocated in the plasma membrane after HGF stimulation. As expected from these three PKC subspecies, phosphorylation of phospholipase Cγ1 (PLCγ1) but not phosphatidylinositol 3‐kinase was enhanced, although the stimulation of brain‐derived neurotrophic factor induced phosphorylation of phosphatidylinositol 3‐kinase. In contrast to the neocortical cells, HGF did not enhance phosphorylation of PLCγ1 in primary astrocytes. We also found that activated PKC(s) served as a major mitogen‐activated protein kinase activator in this pathway. These findings suggest that HGF exerts neurotrophic effects through selective phosphorylation of PLCγ1 and activation of distinct PKC subspecies in neocortical cells, most likely neurons.
Phosphorylation of Mitogen‐Activated Protein Kinase in Cultured Rat Cortical Glia by Stimulation of Metabotropic Glutamate Receptors
Authors: Peavy Richard D. , Conn P. Jeffrey
Published: July 2010

Abstract: Activation of metabotropic glutamate receptors (mGluRs) in glia results in significant physiological effects for both the glia and the neighboring neurons; but in many cases, the mGluR subtypes and signal transduction mechanisms mediating these effects have not been determined. In this study, we report that mGluR activation in primary cultures of rat cortical glia results in tyrosine phosphorylation of several proteins, including p44/p42 mitogen‐activated protein kinases, also referred to as extracellular signal‐regulated kinases (ERK1/2). Incubation of glial cultures with the general mGluR agonist 1‐aminocyclopentane‐1S,3R‐dicarboxylate and the mGluR group I‐selective agonists (RS)‐3,5‐dihydroxyphenylglycine (DHPG) and l‐quisqualate resulted in increased tyrosine phosphorylation of ERK1/2. The group II‐selective agonist (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine and group III‐selective agonist l(+)‐2‐amino‐4‐phosphonobutyric acid had no effect on tyrosine phosphorylation. DHPG‐induced ERK1/2 phosphorylation could be inhibited by an antagonist that acts at group I or group II mGluRs but not by antagonists for group II and group III mGluRs. Protein kinase C (PKC) activators also induced ERK1/2 phosphorylation, but the PKC inhibitor bisindolylmaleimide I did not inhibit DHPG‐induced ERK1/2 phosphorylation at a concentration that inhibited the response to phorbol 12,13‐dibutyrate. These data suggest that mGluR activation of ERK1/2 in cultured glia is mediated by group I mGluRs and that this effect is independent of PKC activation. Furthermore, immunoblots with antibodies against various mGluR subtypes show expression of mGluR5, but no other mGluRs in our cultures. Taken together, these results suggest that mGluR5 stimulation results in tyrosine phosphorylation of ERK1/2 and other glial proteins.
Enhanced Acetylcholine Release from Cells that Have More 15‐kDa Proteolipid in Their Membrane, a Constituent V‐ATPase, and Mediatophore
Authors: Israël M. , Lesbats B. , Tomasi M. , Ohkuma S.
Published: July 2010

Abstract: Mediatophore is a protein that translocates acetylcholine (ACh) on calcium action. It is a homopolymer of a 15‐kDa proteolipid that is also a constituent of the membrane sector of vacuolar H+‐adenosine trisphosphatase (V‐ATPase; vacuolar proton pump). Experiments on neuroblastoma cell lines (N18TG‐2) that are deficient for ACh release and on cells that are competent for release, such as the glioma C6BU‐1 or the N18TG‐2/C6BU‐1 fusion product NG108‐15, show that there is a correlation between ACh release and the 15‐kDa proteolipid content of the cell membrane. In another cell line, L‐M(TK−), it has been possible to up‐regulate ACh release and the membrane proteolipid content after treating the cells with dibutyryl‐cyclic AMP or dexamethasone. As mediatophore translocates ACh and as V‐ATPase may help vesicular ACh storage, it was interesting to determine the respective role of the two proteins in the observed correlation between release and proteolipid content. After blocking vesicular loading with vesamicol, we did not affect release from these cells, suggesting that the observed correlation may be attributed to mediatophore. The acquisition of an ACh release mechanism would then depend on the process that guides the proteolipid to the plasma membrane of the cell.
Enhanced Oxidative Stress and Altered Antioxidants in Brains of Bcl‐2‐Deficient Mice
Authors: Hochman Ayala , Sternin Hagit , Gorodin Svetlana , Korsmeyer Stanley , Ziv Ilan , Melamed Eldad , Offen Daniel
Published: July 2010

Abstract: Bcl‐2 is an antiapoptotic protein located in the outer mitochondrial membrane. Cellular perturbations associated with programmed cell death may be the consequence of disrupted mitochondrial function as well as excessive production of reactive oxygen species (ROS). Numerous studies indicate that Bcl‐2 is involved in opposing cell death induced by oxidative stimuli, but its mode of action is uncertain. We reexamined the role of Bcl‐2 by using a loss‐of‐function model, Bcl‐2 knockout mice. Brains from Bcl‐2‐deficient mice had a 43% higher content of oxidized proteins and 27% lower number of cells in the cerebellum relative to wild‐type mice. Incubation of cerebellar neurons from Bcl‐2 +/+ brains with 0.5 mM dopamine caused 25% cell death, whereas in Bcl‐2‐deficient cells, it resulted in 52% death; glial cells provided protection in both cultures. Splenocytes from Bcl‐2‐deficient mice were also killed more effectively by dopamine as well as paraquat. Bcl‐2‐deficient mice did not survive intraperitoneal injection of MPTP, which caused a decrease in dopamine level in the striatum of Bcl‐2 +/− brains, which was more significant than in wild‐type mice. When compared with Bcl‐2 +/+ brains, brains of 8‐day‐old Bcl‐2‐deficient mice had higher activities of the antioxidant enzymes GSH reductase (192%) and GSH transferase (142%), whereas at the age of 30 days, GSH peroxidase was significantly lower (66%). Activities of GSH transferase and GSH reductase increased significantly (158 and 262%, respectively) from day 8 to day 30 in Bcl‐2 +/+ mice, whereas GSH peroxidase decreased (31%) significantly in Bcl‐2−/− animals. In summary, our results demonstrated enhanced oxidative stress and susceptibility to oxidants as well as altered levels of antioxidant enzymes in brains of Bcl‐2‐deficient mice. It is concluded that Bcl‐2 affects cellular levels of ROS, which may be due to an effect either on their production or on antioxidant pathways.
Striatal Expression of Glutamic Acid Decarboxylase Gene in Alzheimer's Disease
Authors: Boissière Florence , Faucheux Baptiste , Duyckaerts Charles , Hauw Jean‐Jacques , Agid Yves , Hirsch Etienne C.
Published: July 2010

Abstract: To examine potential alteration of GABAergic striatal neurons in Alzheimer's disease, we used quantitative in situ hybridization to analyze the messenger RNA coding for Mr 67,000 glutamic acid decarboxylase (GAD67 mRNA) in the striatum of five patients with Alzheimer's disease (AD) and nine matched control subjects. We found a 51–57% increase in the optical density of hybridization signal in the caudate nucleus and putamen, corresponding to a 30–42% increase in the number of neurons expressing a detectable amount of GAD67 mRNA. By contrast, no alteration was observed in the ventral striatum. The expression of GAD67 mRNA per neuron was similar in AD and control subjects both in the dorsal and ventral striatum. Taken together, our data indicate that, in AD, GABAergic neurotransmission is increased in the dorsal striatum but not in the ventral striatum. We suggest that this increased GABAergic neurotransmission may explain extrapyramidal signs often observed in AD.
Guanosine 5′‐(γ‐[35S]Thio)triphosphate Autoradiography Allows Selective Detection of Histamine H3 Receptor‐Dependent G Protein Activation in Rat Brain Tissue Sections
Authors: Laitinen Jarmo T. , Jokinen Marianne
Published: July 2010

Abstract: Histamine elicits its biological effects via three distinct G protein‐coupled receptors, termed H1, H2, and H3. We have used guanosine 5′‐(γ‐[35S]thio)triphosphate (GTPγ[35S]) autoradiography to localize histamine receptor‐dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A1 receptor‐dependent GTPγ[35S] signal in several brain regions. All further incubations therefore contained 8‐cyclopentyl‐1,3‐dipropylxanthine (10 µM), a selective A1 receptor antagonist. Histamine elicited dose‐dependent increments in GTPγ[35S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine‐evoked binding response closely reflects the known distribution of H3 binding sites and was faithfully mimicked by Nα‐methylhistamine, (R)‐α‐methylhistamine, and immepip, three H3‐selective agonists. In all regions examined, the GTPγ[35S] signal was reversed with thioperamide and clobenpropit, two potent H3‐selective antagonists, whereas mepyramine, a specific H1 antagonist, and cimetidine, a prototypic H2 antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPγ[35S] autoradiography selectively detects H3 receptor‐dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPγ[35S] autoradiography preferentially reveals responses to Gi/o‐coupled receptors, our data indicate that most, if not all, central H3 binding sites represent functional receptors coupling to Gi/o, the inhibitory class of G proteins. Besides allowing more detailed studies on H3 receptor signaling within anatomically restricted regions of the CNS, GTPγ[35S] autoradiography offers a novel approach for functional in vitro screening of H3 ligands.
Up‐Regulation of Type 2 Iodothyronine Deiodinase mRNA in Reactive Astrocytes Following Traumatic Brain Injury in the Rat
Authors: Zou Lanling , Burmeister Lynn A. , Styren Scot D. , Kochanek Patrick M. , DeKosky Steven T.
Published: July 2010

Abstract: Type 2 5′‐deiodinase (5′‐D2), which converts thyroxine to the more active thyroid hormone 3,5,3′‐triiodothyronine (T3), is believed to be an important source of intracellular T3 in the brain. The activity of this enzyme is increased in hypothyroidism and decreased in hyperthyroidism, and as such, it serves an important role to protect the brain from wide fluctuations in T3 during changes in thyroidal state. Although it has been hypothesized that T3 may facilitate neuronal regeneration after CNS injury, the 5′‐D2 response to brain injury is unknown. To assess the 5′‐D2 mRNA response to injury, we performed in situ hybridization following traumatic brain injury. In unlesioned animals, 5′‐D2 mRNA was undetectable. At 3 days posttrauma, 5′‐D2 mRNA was detected in ipsilateral cortex near the contusion. A significant further increase of 5′‐D2 mRNA was noted 7 days posttrauma in both hippocampus and cortex. Similar response was also observed on the contralateral side. Colocalization of 5′‐D2 mRNA with glial fibrillary acidic protein indicates that reactive astrocytes were the major cellular source for the trauma‐induced 5′‐D2 expression. These data demonstrate, for the first time, a trauma‐induced, astrocytic up‐regulation of 5′‐D2 mRNA, suggesting a potential role for T3 action in adult brain's response to injury and recovery.
Enhancement of Extracellular Glutamate Scavenge System in Injured Motoneurons
Authors: Toki Hidenobu , Namikawa Kazuhiko , Su Qingning , Kiryu‐Seo Sumiko , Sato Kohji , Kiyama Hiroshi
Published: July 2010

Abstract: An increase in glutamine synthetase (GS) mRNA expression after peripheral motor nerve injury was demonstrated by differential display PCR using single arbitrary primer coupled with in situ hybridization screening called in situ display. Differential display PCR was carried out to compare differences in mRNA expression between axotomized (6 h after the transection) and normal hypoglossal nuclei in mice. Several gene fragments were increased after nerve injury; one was identified as GS. Subsequent emulsion autoradiography of hybridization tissue sections revealed that the increase in GS mRNA was observed in injured motoneurons. As GS is a key enzyme participating in the metabolism of the major excitatory neurotransmitter glutamate, we examined the significance of increased GS expression on glutamate‐uptake kinetics. GS‐transfected human embryonic kidney cells showed an up‐regulation in glutamate‐uptake kinetics. Therefore, newly expressed GS together with an increased expression of the neuronal glutamate transporter EAAC1 in the injured motoneurons accelerates glutamate uptake. The present results may suggest that the glutamate‐uptake system involving the neuronal glutamate transporter and GS in injured neurons is enhanced so as to provide resistance against neurotoxic glutamate accumulation during the early process of nerve regeneration.
Cloning of a cDNA Coding for Active Tyrosine Hydroxylase in the Rainbow Trout (Oncorhynchus mykiss): Comparison with Other Hydroxylases and Enzymatic Expression
Authors: Linard B. , Pakdel F. , Marmignon M. H. , Saligaut C.
Published: July 2010

Abstract: Although catecholamines are of critical importance for neuroendocrine function in teleost fishes, there has been no tool to give access to pretranslational regulation of their synthesis enzymes. In this study, we undertook cloning of the cDNA coding for the tyrosine hydroxylase (TH) of the rainbow trout (Oncorhynchus mykiss). First, we looked for a tissue sufficiently rich in TH to make an expression library. The cDNA coding for the rainbow trout TH (rtTH) was then cloned and sequenced. The rtTH sequence encodes a protein of 489 amino acids. Several domains and amino acids required for enzyme activity, like cysteines or phosphorylation sites, are highly conserved between species. Northern blot analysis showed a single rtTH messenger RNA of 4.2 kb expressed in the anteroventral brain. The ability of rtTH to hydroxylate l‐tyrosine was analyzed by transient expression of the rtTH cDNA in COS‐1 cells. In vitro TH activity, using COS‐1 cell extracts, demonstrated that TH activity in transfected cells was 40‐fold higher than in untransfected cells. Western blot analysis revealed a single protein of ∼65 kDa in both COS‐1 cells and in trout brain. This rtTH cDNA provides us with a tool for further studies on pretranslational regulation of the enzyme in salmonids.
Induction of Reactive Oxygen Species in Neurons by Haloperidol
Authors: Sagara Yutaka
Published: July 2010

Abstract: Haloperidol (HP) is widely prescribed for schizophrenia and other affective disorders but has severe side effects such as tardive dyskinesia. Because oxidative stress has been implicated in the clinical side effects of HP, rat primary cortical neurons and the mouse hippocampal cell line HT‐22 were used to characterize the generation of reactive oxygen species (ROS) and other cellular alterations caused by HP. Primary neurons and HT‐22 cells are equally sensitive to HP with an IC50 of 35 µM in the primary neurons and 45 µM in HT‐22. HP induces a sixfold increase in levels of ROS, which are generated from mitochondria but not from the metabolism of catecholamines by monoamine oxidases. Glutathione (GSH) is an important antioxidant for the protection of cells against HP toxicity because (1) the intracellular GSH decreases as the ROS production increases, (2) the exogenous addition of antioxidants, such as β‐estradiol and vitamin E, lowers the level of ROS and protects diol and vitamin E, lowers the level of ROS and protects HT‐22 cells from HP, and (3) treatments that result in the reduction of the intracellular GSH potentiate HP toxicity. The GSH decrease is followed by the increase in the intracellular level of Ca2+, which immediately precedes cell death. Therefore, HP causes a sequence of cellular alterations that lead to cell death and the production of ROS is the integral part of this cascade.
Repeated Administration of the Neurotensin Receptor Antagonist SR 48692 Differentially Regulates Mesocortical and Mesolimbic Dopaminergic Systems
Authors: Azzi Mounia , Betancur Catalina , Sillaber Inge , Spanagel Rainer , Rostène William , Bérod Anne
Published: July 2010

Abstract: The purpose of the present study was to investigate the effects of repeated administration of the neurotensin receptor antagonist, SR 48692, on the activity of the mesocortical and mesolimbic dopaminergic (DA) systems. We showed that daily administration of SR 48692 for 15 days (1 mg/kg i.p.) to Wistar rats increased the expression of tyrosine hydroxylase mRNA and protein in the ventral mesencephalon. Simultaneous in vivo microdialysis in the shell part of the nucleus accumbens (AcbSh) and the medial prefrontal cortex (mPFC) revealed that blockade of neurotensin receptors for 15 days decreased basal extracellular levels of DA (∼50%) and its metabolites in the AcbSh, whereas no modification in DA levels was observed in the mPFC. In animals submitted to a forced swimming stress, which preferentially enhanced extracellular DA levels in the mPFC, treatment with SR 48692 failed to affect the stress‐induced increase in DA. Moreover, given that glucocorticoids can modulate the activity of mesencephalic DA neurons, we examined the effect of the same SR 48692 treatment on corticosterone levels in dialysates from the AcbSh. We found that repeated SR 48692 did not affect the basal levels of free corticosterone, but significantly reduced the increase induced by forced swimming stress. The present results demonstrate that repeated treatment with SR 48692 modulates selectively the DA mesolimbic system when compared with the mesocortical pathway. These findings suggest that long‐term treatment with selective neurotensin receptor antagonists could have potential clinical utility in the treatment of neuropsychiatric disorders associated with hyperactivity of the mesolimbic DA systems or the hypothalamic‐pituitary‐adrenal axis.
β‐N‐Acetylhexosaminidase in Peripheral Blood Lymphocytes and Monocytes in the Different Forms and Stages of Multiple Sclerosis
Authors: Orlacchio Antonio , Martino Sabata , Sarchielli Paola , Gallai Virgilio , Emiliani Carla
Published: July 2010

Abstract: The activity of the acidic glycohydrolase β‐N‐acetylhexosaminidase, an enzyme system normally participating in the stepwise degradation of glycoproteins, glycolipids, and proteoglycans, appears to be modulated in lymphocytes and monocytes from peripheral blood of patients affected by multiple sclerosis during different stages of the disease. In particular, a significant decrease in this enzyme activity, compared with healthy subjects, was observed in patients affected by the relapsing‐remitting form both in a stable clinical status and during a relapse as well as in patients with the progressive form. The decrease in total intracellular hexosaminidase activity in lymphomonocytes of multiple sclerosis patients was accompanied by an enrichment of this activity associated with the plasma membrane fraction as demonstrated by experiments of subcellular fractionation. The analysis carried out using two synthetic substrates, 4‐methylumbelliferyl N‐acetyl‐β‐d‐glucosaminide and its sulfate derivative, enables us to demonstrate that this accumulation is mainly due to isoenzymes with a ββ structure, whereas lysosomal fractions confirmed the classical presence of both αβ and ββ forms (hexosaminidases A and B, respectively). This was particularly evident in the plasma membrane fraction from mononuclear cells of patients with a clinical exacerbation of the disease. Considered together, these observations provide additional insight into the abnormality of peripheral blood immune cells in multiple sclerosis and may contribute to the understanding of the basic mechanisms underlying the pathological events resulting in the demyelinating process.
Role of Group III Metabotropic Glutamate Receptors in Excitotoxin‐Induced Cerebellar Granule Cell Death
Authors: Staton Penelope C. , Bristow David R.
Published: July 2010

Abstract: The neuronal effects of the metabotropic glutamate receptor agonist (1S,3R)‐aminocyclopentane‐1,3‐dicarboxylic acid have been studied in cultured rat cerebellar granule cells, and compared with those of the endogenous excitotoxin glutamate, and the dietary excitotoxin β‐N‐methylamino‐l‐alanine. Glutamate, β‐N‐methylamino‐l‐alanine, and (1S,3R)‐aminocyclopentane‐1,3‐dicarboxylic acid all caused concentration‐dependent cerebellar granule cell death over a 24‐h exposure period. The metabotropic antagonist (RS)‐α‐methyl‐4‐carboxyphenylglycine reduced glutamate‐, β‐N‐methylamino‐l‐alanine‐, and (1S,3R)‐aminocyclopentane‐1,3‐dicarboxylic acid‐induced death by 50, 37, and 90%, respectively. (1S,3R)‐Aminocyclopentane‐1,3‐dicarboxylic acid‐induced death was unaffected by the group I antagonist (RS)‐1‐aminoindan‐1,5‐dicarboxylic acid, increased by the group II antagonist ethylglutamic acid, and markedly decreased by the group III antagonist (RS)‐α‐methylserine‐O‐phosphate. Neither (1S,3R)‐aminocyclopentane‐1,3‐dicarboxylic acid nor the group I agonist (RS)‐3,5‐dihydroxyphenylglycine caused an increase in intracellular free calcium levels. The group III agonist l‐(+)‐2‐amino‐4‐phosphonobutyric acid also induced concentration‐dependent cerebellar granule cell death, and so it was suggested that the group III metabotropic glutamate receptors were responsible for (1S,3R)‐aminocyclopentane‐1,3‐dicarboxylic acid‐induced death. Blocking these receptors with (RS)‐α‐methylserine‐O‐phosphate also prevented a proportion of glutamate‐ and β‐N‐methylamino‐l‐alanine‐induced death.
Persistence of the Interaction of Calmodulin with Adenylyl Cyclase: Implications for Integration of Transient Calcium Stimuli
Authors: Onyike Chiadi U. , Lin Allison H. , Abrams Thomas W.
Published: July 2010

Abstract: Ca2+/calmodulin‐sensitive adenylyl cyclase plays a role in several forms of synaptic plasticity and learning. To understand how cellular signals from neuronal activity during behavioral stimuli might be integrated by adenylyl cyclase, we have characterized the response of type I adenylyl cyclase to transient Ca2+ stimuli. Stimulation by a several second Ca2+ stimulus is delayed, rising to a peak after the Ca2+ stimulus has ended. We attempted to identify the site of the persistent Ca2+ signal that enabled adenylyl cyclase stimulation to increase after free Ca2+ had declined. Free calmodulin itself displayed no persistent activation by Ca2+ and was unable to activate adenylyl cyclase if exposed to low Ca2+ solution <1 s before reaching adenylyl cyclase. In contrast, activation of the calmodulin‐adenylyl cyclase complex persisted for seconds after Ca2+ stimulus. Activation decayed with a time constant of 6 or 13 s depending on assay conditions. These results suggest that the calmodulin‐adenylyl cyclase complex can serve as a site of cellular memory for a Ca2+ transient that has ended even before adenylyl cyclase is fully activated.
Ca2+‐Mediated Activation of c‐Jun N‐Terminal Kinase and Nuclear Factor κB by NMDA in Cortical Cell Cultures
Authors: Ko Hyuk Wan , Park Kye‐Yoon , Kim Hansin , Han Pyung‐Lim , Kim Youn Uck , Gwag Byoung Joo , Choi Eui‐Ju
Published: July 2010

Abstract: We examined the possibility that c‐Jun N‐terminal kinase (JNK) and nuclear factor κB (NF‐κB) might be involved in intracellular signaling cascades that mediate NMDA‐initiated neuronal events. Exposure of cortical neurons to 100 µM NMDA induced activation of JNK within 1 min. Activity of JNK was further increased over the next 5 min and then declined by 30 min. Similarly, ionomycin, a selective Ca2+ ionophore, induced activation of JNK. The NMDA‐induced activation of JNK was abrogated in the absence of extracellular Ca2+, suggesting that Ca2+ entry is necessary and sufficient for the JNK activation. Immunohistochemistry with anti‐NF‐κB antibody demonstrated nuclear translocation of NF‐κB within 5 min following NMDA treatment. NMDA treatment also enhanced the DNA binding activity of nuclear NF‐κB in a Ca2+‐dependent manner. Treatment with 3 mM aspirin blocked the NMDA‐induced activation of JNK and NF‐κB. Neuronal death following a brief exposure to 100 µM NMDA was Ca2+ dependent and attenuated by addition of aspirin or sodium salicylate. The present study suggests that Ca2+ influx is required for NMDA‐induced activation of JNK and NF‐κB as well as NMDA neurotoxicity. This study also implies that aspirin may exert its neuroprotective action against NMDA through blocking the NMDA‐induced activation of NF‐κB and JNK.
Rilmenidine Elevates Cytosolic Free Calcium Concentration in Suspended Cerebral Astrocytes
Authors: Ozog Mark A. , Wilson John X. , Dixon S. Jeffrey , Cechetto David F.
Published: July 2010

Abstract: Rilmenidine, a ligand for imidazoline and α2‐adrenergic receptors, is neuroprotective following focal cerebral ischemia. We investigated the effects of rilmenidine on cytosolic free Ca2+ concentration ([Ca2+]i) in rat astrocytes. Rilmenidine caused concentration‐dependent elevation of [Ca2+]i, consisting of a transient increase (1–100 µM rilmenidine) or a transient increase followed by sustained elevation above basal levels (1–10 mM rilmenidine). A similar elevation in [Ca2+]i was induced by the imidazoline ligand cirazoline. The transient response to rilmenidine was observed in Ca2+‐free medium, indicating that rilmenidine evokes release of Ca2+ from intracellular stores. However, the sustained elevation of Ca2+ was completely dependent on extracellular Ca2+, consistent with rilmenidine activating Ca2+ influx.Pretreatment with thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+‐ATPase, abolished the response to rilmenidine, confirming the involvement of intracellular stores and suggesting that rilmenidine and thapsigargin activate a common Ca2+ influx pathway. The α2‐adrenergic antagonist rauwolscine attenuated the increase in [Ca2+]i induced by clonidine (a selective α2 agonist), but not the response to rilmenidine. These results indicate that rilmenidine stimulates both Ca2+ release from intracellular stores and Ca2+ influx by a mechanism independent of α2‐adrenergic receptors. In vivo, rilmenidine may enhance uptake of Ca2+ from the extracellular fluid by astrocytes, a process that may contribute to the neuroprotective effects of this agent.
Transcriptional and Translational Regulation of Phosphodiesterase Type IV Isozymes in Rat Brain by Electroconvulsive Seizure and Antidepressant Drug Treatment
Authors: Suda Shiro , Nibuya Masashi , Ishiguro Takeo , Suda Haruhiko
Published: July 2010

Abstract: We examined the influence of electroconvulsive seizure (ECS) and imipramine (IMI) treatment on the transcription and translation of cyclic nucleotide phosphodiesterase type IV (PDE IV) isozymes in the rat brain. Our in situ hybridization studies revealed an increase of PDE IV‐B mRNA level in various brain regions after acute ECS. However, the increase of PDE IV activity was produced not by acute but by chronic ECS treatment in the frontal cortex. Increased PDE IV‐B mRNA expression in frontal but not in hippocampal subfields was induced also after chronic ECS treatment. Although an increase in PDE IV‐A mRNA expression of the dentate gyrus in the hippocampus was observed, no change of PDE IV activity was produced in the hippocampus by acute or chronic ECS treatment. These results suggest that the repeated increases of PDE IV‐B mRNA expression are attributable to the increase of PDE IV translation. Increased PDE IV‐B transcription and PDE IV translation in the frontal cortex were also produced after chronic IMI treatment. This is the first report demonstrating an expressional regulation of Drosophila melanogaster dunce (dnc) gene homologue PDE IV isozymes in the brain. Although no pathophysiological conditions with reduced PDE IV activity in the nervous system are known except for a learning deficit in the mutant fly dnc−, our results suggest possible treatments to cope with reduced PDE IV activity.
Involvement of Bcl‐2 Family and Caspase‐3‐Like Protease in NO‐Mediated Neuronal Apoptosis
Authors: Tamatani Michio , Ogawa Satoshi , Niitsu Youichirou , Tohyama Masaya
Published: July 2010

Abstract: To clarify mechanisms of neuronal death in the postischemic brain, we examined whether astrocytes exposed to hypoxia/reoxygenation exert a neurotoxic effect, using a coculture system. Neurons cocultured with astrocytes subjected to hypoxia/reoxygenation underwent apoptotic cell death, the effect enhanced by a combination of interleukin‐1β with hypoxia. The synergistic neurotoxic activity of hypoxia and interleukin‐1β was dependent on de novo expression of inducible nitric oxide synthase (iNOS) and on nitric oxide (NO) production in astrocytes. Further analysis to determine the neurotoxic mechanism revealed decreased Bcl‐2 and increased Bax expression together with caspase‐3 activation in cortical neurons cocultured with NO‐producing astrocytes. Inhibition of NO production in astrocytes by NG‐monomethyl‐l‐arginine, an inhibitor of NOS, significantly inhibited neuronal death together with changes in Bcl‐2 and Bax protein levels and in caspase‐3‐like activity. Moreover, treatment of neurons with a bax antisense oligonucleotide inhibited the caspase‐3‐like activation and neuronal death induced by an NO donor, sodium nitroprusside. These data suggest that NO produced by astrocytes after hypoxic insult induces apoptotic death of neurons through mechanisms involving the caspase‐3 activation after down‐regulation of BCl‐2 and up‐regulation of Bax protein levels.