bluuebunny/crossref_metadata_2025 huggingface dataset:
Alcohol Exposure Alters the Expression Pattern of Neural Cell Adhesion Molecules During Brain Development
Authors: Miñana R. , Climent E. , Barettino D. , Segui J. M. , Renau‐Piqueras J. , Guerri C.
Published: March 2003

Abstract: Neural cell adhesion molecules (NCAMs) play critical roles during development of the nervous system. The aim of this study is to investigate the possible effect of ethanol exposure on the pattern of expression and sialylation of NCAM isoforms during postnatal rat brain development because alterations in NCAM content and distribution have been associated with defects in cell migration, synapse formation, and memory consolidation, and deficits in these processes have been observed after in utero alcohol exposure. The expression of NCAM isoforms in the developing cerebral cortex of pups from control and alcohol‐fed mothers was assessed by western blotting, ribonuclease protection assay, and immunocytochemistry. The highly sialylated form of NCAM [polysialic acid (PSA)‐NCAM] is mainly expressed during the neonatal period and then is down‐regulated in parallel with the appearance of NCAM 180 and NCAM 140. Ethanol exposure increases PSA‐NCAM levels during the neonatal period, delays the loss of PSA‐NCAM, decreases the amount of NCAM 180 and NCAM 140 isoforms, and reduces sialyltransferase activity during postnatal brain development. Neuraminidase treatment of ethanol‐exposed neonatal brains leads to more intense band degradation products, suggesting a higher content of NCAM polypeptides carrying PSA in these samples. However, NCAM mRNA levels are not changed by ethanol. Immunocytochemical analysis demonstrates that ethanol triggers an increase in PSA‐NCAM immunolabeling in the cytoplasm of astroglial cells, accompanied by a decrease in immunogold particles over the plasma membrane. These findings indicate that ethanol exposure during brain development alters the pattern of NCAM expression and suggest that modification of NCAM could affect neuronal‐glial interactions that might contribute to the brain defects observed after in utero alcohol exposure.
Nitric‐Oxide‐Induced Necrosis and Apoptosis in PC12 Cells Mediated by Mitochondria
Authors: Bal‐Price Anna , Brown Guy C.
Published: March 2003

Abstract:Nitric oxide (NO) can trigger either necrotic or apoptotic cell death. We have used PC12 cells to investigate the extent to which NO‐induced cell death is mediated by mitochondria. Addition of NO donors, 1 mMS‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) or 1 mMdiethylenetriamine‐NO adduct (NOC‐18), to PC12 cells resulted in a steady‐state level of 1‐3 μMNO, rapid and almost complete inhibition of cellular respiration (within 1 min), and a rapid decrease in mitochondrial membrane potential within the cells. A 24‐h incubation of PC12 cells with NO donors (SNAP or NOC‐18) or specific inhibitors of mitochondrial respiration (myxothiazol, rotenone, or azide), in the absence of glucose, caused total ATP depletion and resulted in 80‐100% necrosis. The presence of glucose almost completely prevented the decrease in ATP level and the increase in necrosis induced by the NO donors or mitochondrial inhibitors, suggesting that the NO‐induced necrosis in the absence of glucose was due to the inhibition of mitochondrial respiration and subsequent ATP depletion. However, in the presence of glucose, NO donors and mitochondrial inhibitors induced apoptosis of PC12 cells as determined by nuclear morphology. The presence of apoptotic cells was prevented completely by benzyloxycarbonyl‐Val‐Ala‐fluoromethyl ketone (a nonspecific caspase inhibitor), indicating that apoptosis was mediated by caspase activation. Indeed, both NO donors and mitochondrial inhibitors in PC12 cells caused the activation of caspase‐3‐ and caspase‐3‐processing‐like proteases. Caspase‐1 activity was not activated. Cyclosporin A (an inhibitor of the mitochondrial permeability transition pore) decreased the activity of caspase‐3‐ and caspase‐3‐processing‐like proteases after treatment with NO donors, but was not effective in the case of the mitochondrial inhibitors. The activation of caspases was accompanied by the release of cytochromecfrom mitochondria into the cytosol, which was partially prevented by cyclosporin A in the case of NO donors. These results indicate that NO donors (SNAP or NOC‐18) may trigger apoptosis in PC12 cells partially mediated by opening the mitochondrial permeability transition pores, release of cytochromec, and subsequent caspase activation. NO‐induced apoptosis is blocked completely in the absence of glucose, probably due to the lack of ATP. Our findings suggest that mitochondria may be involved in both types of cell death induced by NO donors: necrosis by respiratory inhibition and apoptosis by opening the permeability transition pore. Further, our results indicate that the mode of cell death (necrosis versus apoptosis) induced by either NO or mitochondrial inhibitors depends critically on the glycolytic capacity of the cell.
Characterization of Binding Sites for Chemokines MCP‐1 and MIP‐1α on Human Brain Microvessels
Authors: Andjelkovic Anuska V. , Pachter Joel S.
Published: March 2003

Abstract: The presence of binding sites for the β chemokines monocyte chemoattractant protein‐1 (MCP‐1) and macrophage inflammatory protein‐1α (MIP‐1α) has recently been identified on human brain microvessels. We extend these findings in this report to reveal that such sites exemplify characteristics of the recognized major receptors for MCP‐1 and MIP‐1α: CCR2, and CCR1 and CCR5, respectively. Specifically, labeled MCP‐1 binding to isolated brain microvessels was inhibited by unlabeled MCP‐1 and MCP‐3, the latter another CCR2 ligand, but not by MIP‐1α. Inhibition of labeled MIP‐1α binding was achieved with unlabeled MIP‐1α and RANTES, the latter a β chemokine that binds to both CCR1 and CCR5, but not by MCP‐1. Labeled MIP‐1α binding was also antagonized by unlabeled MCP‐3, which is also recognized by CCR1, and MIP‐1β, which is a ligand for CCR5. Labeled MCP‐1 and MIP‐1α were further observed to be internalized within the endothelial cells of brain microvessels, following their binding to the microvascular surface at 37°C. Additionally, exposure of microvessels to unlabeled MCP‐1 or MIP‐1α was accompanied by the initial loss and subsequent recovery of surface binding sites for these chemokines, which occurred on a time scale consistent with ligand‐induced endocytosis and recycling. These collective features bear striking similarity to those that characterize interactions of MCP‐1 and MIP‐1α with their receptors on leukocytes and underscore the concept of cognate chemokine receptors on brain microvascular endothelium.
Reduced Binding of Protein Phosphatase 2A to Tau Protein with Frontotemporal Dementia and Parkinsonism Linked to Chromosome 17 Mutations
Authors: Goedert Michel , Satumtira Sivapong , Jakes Ross , Smith Michael J. , Kamibayashi Craig , White Charles L. , Sontag Estelle
Published: March 2003

Abstract: Coding region and intronic mutations in the tau gene cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17). We have previously reported that ABαC, a major form of protein phosphatase 2A (PP2A) in brain, binds tightly to tau protein in vitro and is a major tau phosphatase in vivo. Using in vitro assays, we show here that the FTDP‐17 mutations G272V, ΔK280, P301L, P301S, S305N, V337M, G389R, and R406W inhibit by ∼20‐95% the binding of recombinant three‐repeat and four‐repeat tau isoforms to the ABαC holoenzyme and the AC core enzyme of PP2A. Reduction in binding was maximal for tau proteins with the G272V, ΔK280, and V337M mutations. We also show that tau protein can be specifically coimmunoprecipitated with endogenous PP2A from both rat brain and transfected cell extracts. It is significant that, by using similar coimmunoprecipitation assays, we show that all FTDP‐17 mutations tested, including the N279K mutation, alter the ability of tau to associate with cellular PP2A. Taken together, these results indicate that FTDP‐17 mutations induce a significant decrease in the binding affinity of tau for PP2A in vivo. We propose that altered protein‐protein interactions between PP2A and tau may contribute to FTDP‐17 pathogenesis.
Nuclear Factor κB/p49 Is a Negative Regulatory Factor in Nerve Growth Factor‐Induced Choline Acetyltransferase Promoter Activity in PC12 Cells
Authors: Toliver‐Kinsky T. , Wood T. , Perez‐Polo J. R.
Published: March 2003

Abstract: A novel nuclear factor κB (NF‐κB) binding sitehas been identified within the promoter region of the mouse gene encodingcholine acetyltransferase (ChAT), the enzyme that synthesizes acetylcholineand has been implicated in the cognitive deficits associated with aging andAlzheimer's disease. This binding site, which is located within the nervegrowth factor (NGF)‐responsive enhancer element, was recognized by theNF‐κB protein p49 but not p65 or p50. p49 from both basal forebrain andPC12 nuclear extracts interacted with this specific sequence inelectrophoretic mobility shift assays. Mutation of the NF‐κB site causedan increase in NGF‐induced promoter activation, whereas overexpression of p49in NGF‐differentiated PC12 cells caused a decrease in endogenous ChAT enzymeactivity and a decrease in promoter activity that was specifically mediatedthrough this NF‐κB binding site. Treatment of PC12 cells with NGFresulted in a drastic reduction in nuclear p49 binding to the ChAT NF‐κBsite after 24 h, but nuclear p49 levels were not altered, suggesting that lateNGF‐mediated events prevent binding of p49 to the ChAT promoter by an unknownmechanism other than nuclear translocation. Decreased ChAT expression andincreased NF‐κB activity in the brain are associated with aging andAlzheimer's disease. These data indicate that p49 is a negative regulator ofChAT expression and suggest a possible mechanism for aging‐associated declinesin cholinergic function.
Membrane‐Bound Form of ADP‐Ribosyl Cyclase in Rat Cortical Astrocytes in Culture
Authors: Hotta Taeko , Asai Kiyofumi , Fujita Kaori , Kato Taiji , Higashida Haruhiro
Published: March 2003

Abstract: ADP‐ribosyl cyclase activities in cultured rat astrocytes were examined by using TLC for separation of enzymatic products. A relatively high rate of [3H]cyclic ADP‐ribose production converted from [3H]NAD+ by ADP‐ribosyl cyclase (2.015 ± 0.554 nmol/min/mg of protein) was detected in the crude membrane fraction of astrocytes, which contained ∼50% of the total cyclase activity in astrocytes. The formation rate of [3H]ADP‐ribose from cyclic ADP‐ribose by cyclic ADP‐ribose hydrolase and/or from NAD+ by NAD glycohydrolase was low and enriched in the cytosolic fraction. Although NAD+ in the extracellular medium was metabolized to cyclic ADP‐ribose by incubating cultures of intact astrocytes, the presence of Triton X‐100 in the medium for permeabilizing cells increased cyclic ADP‐ribose production three times as much. Isoproterenol and GTP increased [3H]cyclic ADP‐ribose formation in crude membrane‐associated cyclase activity. This isoproterenol‐induced stimulation of membrane‐associated ADP‐ribosyl cyclase activity was confirmed by cyclic GDP‐ribose formation fluorometrically. This stimulatory action was blocked by prior treatment of cells with cholera toxin but not with pertussis toxin. These results suggest that ADP‐ribosyl cyclase in astrocytes has both extracellular and intracellular actions and that signals of β‐adrenergic stimulation are transduced to membrane‐bound ADP‐ribosyl cyclase via G proteins within cell surface membranes of astrocytes.
The regulation of presenilin‐1 by nerve growth factor
Authors: Counts Scott E. , Lah James J. , Levey Allan I.
Published: March 2003

Presenilin‐1 (PS1) protein concentration is linked to neuronal development and to the pathogenesis of Alzheimer's disease, yet little is known about the biological factors and mechanisms that control cellular levels of PS1 protein. As PS1 levels are highest in the developing brain, we tested whether neurotrophin‐induced differentiation influences PS1 expression using neuronotypic pheochromocytoma (PC12) cells. Treatment of PC12 cells with nerve growth factor (NGF) caused ∼ 60–75% increases in the steady‐state levels of endogenous PS1 N‐ and C‐terminal fragments. PS1 protein accumulation was dose‐responsive to NGF and required the presence of the TrkA NGF receptor tyrosine kinase. NGF also induced PS1 fragment accumulation in cultured explants of rat dorsal root ganglia. Quantitative northern blot analysis using PC12 cultures indicated that NGF did not increase steady‐state PS1 mRNA levels. However, pulse‐chase experiments indicated that NGF slowed the degradation rate of endogenous PS1 fragments, increasing the half‐life from t1/2 @22.5 to @25.0 h. This increase in half‐life was insufficient to account for the ∼ 60–75% increase in PS1 fragment levels measured in NGF‐treated cells. Thus, NGF may regulate PS1 protein concentration in NGF‐responsive cells by a complex mechanism that increases PS1 fragment production independent of holoprotein synthesis.
Aging alters regional multichemical profile of the human brain: an in vivo1H‐MRS study of young versus middle‐aged subjects
Authors: Grachev Igor D. , Apkarian A. Vania
Published: March 2003

Age‐related differences in the multichemical proton magnetic resonance spectroscopy (1H‐MRS) profile of the human brain have been reported for several age groups, and most consistently for ages from neonates to 16‐year‐olds. Our recent 1H‐MRS study demonstrated a significant age‐related increase of total chemical concentration (relative to creatine) in the prefrontal and sensorimotor cortices within young adulthood (19–31‐year‐olds). In the present study we test the hypothesis that the level of brain chemicals in the same cortices, which show increased chemical levels during normal development, are reduced with normal aging after young adulthood. The multichemical 1H‐MRS profile of the brain was compared between 19 young and 16 middle‐aged normal subjects across multiple brain regions for all chemicals of 1H‐MRS spectra. Chemical concentrations were measured relative to creatine. Over all age groups the total relative chemical concentration was highest in the prefrontal cortex. Middle‐aged subjects demonstrated a significant decrease of total relative chemical concentration in the dorsolateral prefrontal (F = 54.8, p < 10−7, anova), orbital frontal (F = 3.7, p < 0.05) and sensorimotor (F = 15.1, p < 0.0001) cortices, as compared with younger age. Other brain regions showed no age‐dependent differences. The results indicate that normal aging alters multichemical 1H‐MRS profile of the human brain and that these changes are region‐specific, with the largest changes occuring in the dorsolateral prefrontal cortex. These findings provide evidence that the processes of neuronal maturation of the human brain, and neurotransmitters and other chemical changes as the marker of these neuronal changes are almost finished by young adulthood and then reduced during normal aging toward middle age period of life. The present data also support the notion of heterochronic regressive changes of the aging human brain, where the multichemical brain regional profile seems to inversely recapitulate cortical chemical maturation within normal development.
Contrasting responses by basal ganglia met‐enkephalin systems to low and high doses of methamphetamine in a rat model
Authors: Alburges Mario E. , Keefe Kristin A. , Hanson Glen R.
Published: March 2003

The influence of methamphetamine (METH) on basal ganglia met‐enkephalin (Menk) was studied by determining levels of this peptide in striatal, pallidal and nigral regions after administering a single low (0.5 mg/kg) or high (10 mg/kg) dose of this stimulant. The Menk levels in the striatal and pallidal areas were reduced and increased after the low‐ and high‐dose METH treatments, respectively, 12 h after drug administration in all striatal and pallidal regions examined. The low‐dose effect appeared to be principally influenced by increased activation of the dopamine D2‐like receptor, while the high‐dose effect seemed to result from dominance of D1‐like receptor activation. However, both effects required coactivation of D1‐ and D2‐like receptors. For the most part, both low‐ and high‐dose METH‐induced changes in Menk tissue content were fully recovered by 24 h. The Menk levels were not significantly altered in the substantia nigra 3–24 h after either METH treatment. Results reported herein indicated that striatal and pallidal Menk pathways respond differently after acute treatment with low or high doses of METH.
Autonomous activity and autophosphorylation of CAMPK‐II in rat hippocampal slices: effects of tissue preparation
Authors: Lengyel I. , Cammarota M. , Brent V. A. , Rostas J. A. P.
Published: March 2003

Measurement of the proportion of calcium/calmodulin‐stimulated protein kinase II (CaMPK‐II) that is autonomously active or phosphorylated on Thr286is thought to provide an index of the degree to which CaMPK‐II in a tissue has been activated. We have examined how various ways of handling hippocampal tissue can alter these properties. Both autonomous activity and phospho‐Thr286content was high in freshly dissected hippocampus or freshly cut hippocampal slices. After incubation of hippocampal slices in artificial cerebrospinal fluid for 120 min, both properties of CaMPK‐II decreased to a steady state level. Freeze–thaw or cutting the equilibrated slices could rapidly increase both autonomous activity and phospho‐Thr286immunoreactivity of CaMPK‐II. These increases were comparable to changes induced by experimental treatment. Therefore, our results suggest that considerable care needs to be taken over the way in which hippocampal slices are handled.
Phosphorylation of thr668in the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein by stress‐activated protein kinase 1b (Jun N‐terminal kinase‐3)
Authors: Standen Claire L. , Brownlees Janet , Grierson Andrew J. , Kesavapany Sashi , Lau Kwok‐Fai , McLoughlin Declan M. , Miller Christopher C. J.
Published: March 2003

Threonine668(thr668) within the carboxy‐terminus of the Alzheimer's disease amyloid precursor protein (APP) is a knownin vivophosphorylation site. Phosphorylation of APPthr668is believed to regulate APP function and metabolism. Thr668precedes a proline, which suggests that it is targeted for phosphorylation by proline‐directed kinase(s). We have investigated the ability of four major neuronally active proline‐directed kinases, cyclin dependent protein kinase‐5, glycogen synthase kinase‐3β, p42 mitogen‐activated protein kinase and stress‐activated protein kinase‐1b, to phosphorylate APPthr668and report here that SAPK1b induces robust phosphorylation of this site bothin vitroandin vivo. This finding provides a molecular framework to link cellular stresses with APP metabolism in both normal and disease states.
IPF, a vesicular uptake inhibitory protein factor, can reduce the Ca2+‐dependent, evoked release of glutamate, GABA and serotonin
Authors: Tamura Yutaka , Özkan Eric D. , Bole David G. , Ueda Tetsufumi
Published: March 2003

Synaptic vesicles in the nerve terminal play a pivotal role in neurotransmission. Neurotransmitter accumulation into synaptic vesicles is catalyzed by distinct vesicular transporters, harnessing an electrochemical proton gradient generated by V‐type proton‐pump ATPase. However, little is known about regulation of the transmitter pool size, particularly in regard to amino acid neurotransmitters. We previously provided evidence for the existence of a potent endogenous inhibitory protein factor (IPF), which causes reduction of glutamate and GABA accumulation into isolated, purified synaptic vesicles. In this study we demonstrate that IPF is concentrated most in the synaptosomal cytosol fraction and that, when introduced into the synaptosome, it leads to a decrease in calcium‐dependent exocytotic (but not calcium‐independent) release of glutamate in a concentration‐dependent manner. In contrast, α‐fodrin (non‐erythroid spectrin), which is structurally related to IPF and thought to serve as the precursor for IPF, is devoid of such inhibitory activity. Intrasynaptosomal IPF also caused reduction in exocytotic release of GABA and the monoamine neurotransmitter serotonin. Whether IPF affects vesicular storage of multiple neurotransmitters in vivo would depend upon the localization of IPF. These results raise the possibility that IPF may modulate synaptic transmission by acting as a quantal size regulator of one or more neurotransmitters.
Auto‐inhibition of Ca2+/calmodulin‐dependent protein kinase II by its ATP‐binding domain
Authors: Lengyel I. , Nairn A. C. , McCluskey A. , Tóth G. , Penke B. , Rostas J. A. P.
Published: March 2003

Ca2+/calmodulin dependent protein kinase (CaMPK) II is a key enzyme in many physiological processes. The enzyme is inactive unless Ca2+/CaM binds to it. In this inactive form CaMPK‐II does not bind ATP suggesting that the ATP‐binding domain is involved in an intramolecular interaction. We show here that F12, a 12 amino acid long peptide fragment of the ATP‐binding domain (CaMPK‐II23–34, GAFSVVRRCVKV) can inhibit the Ca2+/CaM‐dependent activity (IC50 of 3 µm) but has no effect on the Ca2+/CaM‐independent activity of CaMPK‐II. Kinetic analysis exhibited mixed inhibition with respect to autocamtide‐2 and ATP. The inhibition by F12 showed specificity towards CaMPK‐II, but also inhibited CaMPK‐I (IC50 = 12.5 µm), while CaMPK‐IV (IC50 = 85 µm) was inhibited poorly and cAMP‐dependent protein kinase (PKA) was not inhibited. Substitution of phenylalanine at position 25 to alanine (A12), had little effect on the inhibition of different Ca2+/CaM‐dependent protein kinases, suggesting that phenylalanine 25 does not play a crucial role in the interactions involving F12. Thus the molecular interactions involving the ATP‐binding domain appears to play a role in the regulation of nonphosphorylated CaMPK‐II activity.
Brain succinic semialdehyde dehydrogenase: identification of reactive lysyl residues labeled with pyridoxal‐5′‐phosphate
Authors: Choi Soo Young , Bahn Jae Hoon , Lee Byung Ryong , Jeon Seong Gyu , Jang Joong Sik , Kim Choong Kwon , Jin Li Hua , Kim Kyung Hee , Park Jin Sun , Park Jinseu , Cho Sung‐Woo
Published: March 2003

An NAD+‐dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by pyridoxal‐5′‐ phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH4reduction of the pyridoxal‐5′‐phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by pyridoxal‐5′‐phosphate. The absorption spectrum of the reduced and dialyzed pyridoxal‐5′‐phosphate‐inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with pyridoxal‐5′‐phosphate followed by [3H]NaBH4reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse‐phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of pyridoxal‐5′‐phosphate to specific Lys347residue at or near the coenzyme‐binding site of the protein.
Preferential expression of antioxidant response element mediated gene expression in astrocytes
Authors: Murphy T. H. , Yu J. , Ng R. , Johnson D. A. , Shen H. , Honey C. R. , Johnson J. A.
Published: March 2003

Transcriptional control of target genes by antioxidant/electrophile response elements has been well described in peripheral tissues. Genes that are regulated by this mechanism include the antioxidant enzymes NAD(P)H:quinone oxidoreductase, γ‐glutamyl cystine synthetase and glutathione‐S‐transferase. Antioxidant/electrophile response elements within a gene's promoter confer induction by low‐molecular‐weight electrophilic compounds such as tert‐butylhydroquinone and dimethyl fumarate. We have now examined the ability of antioxidant/electrophile response elements to elicit gene expression in neurons and astrocytes in both brain slices and primary cultures using transient transfection of promoter reporter constructs. Our results using a heat‐stable human placental alkaline phosphatase reporter indicate that antioxidant/electrophile response element mediated gene expression is largely restricted to astrocyte cell populations. Placental alkaline phosphatase expression was significantly elevated in astrocytes treated with the antioxidant/electrophile response element inducer dimethyl fumarate. Mutant constructs lacking a functional antioxidant/electrophile response element abolished all placental alkaline phosphatase expression in astrocytes. We suggest that astrocytic metabolic processes that normally aid and/or protect neurons may be controlled via this inducible system.
Neuroprotective effects of non‐steroidal anti‐inflammatory drugs by direct scavenging of nitric oxide radicals
Authors: Asanuma Masato , Nishibayashi‐Asanuma Sakiko , Miyazaki Ikuko , Kohno Masahiro , Ogawa Norio
Published: March 2003

Recently, it has been reported that inflammatory processes are associated with the pathophysiology of Alzheimer's disease and that treatment of non‐steroidal anti‐inflammatory drugs reduce the risk for Alzheimer's disease. In the present study, we examined nitric oxide radical quenching activity of non‐steroidal anti‐inflammatory drugs and steroidal drugs using our established direct in vitro nitric oxide radical detecting system by electron spin resonance spectrometry. The non‐steroidal anti‐inflammatory drugs, aspirin, mefenamic acid, indomethacin and ketoprofen directly and dose‐dependently scavenged generated nitric oxide radicals. In experiments of nitric oxide radical donor, NOC18‐induced neuronal damage, these four non‐steroidal drugs significantly prevented the NOC18‐induced reduction of cell viability and apoptotic nuclear changes in neuronal cells without affecting the induction of inducible nitric oxide synthase‐like immunoreactivity. However, ibuprofen, naproxen or steroidal drugs, which had less or no scavenging effects in vitro, showed almost no protective effects against NOC18‐induced cell toxicity. These results suggest that the protective effects of the former four non‐steroidal anti‐inflammatory drugs against apoptosis might be mainly due to their direct nitric oxide radical scavenging activities in neuronal cells. These direct NO· quenching activities represent novel effects of non‐steroidal anti‐inflammatory drugs. Our findings identified novel pharmacological mechanisms of these drugs to exert not only their anti‐inflammatory, analgesic, antipyretic activities but also neuroprotective activities against neurodegeneration.
A synthetic inhibitor of p53 protects neurons against death induced by ischemic and excitotoxic insults, and amyloid β‐peptide
Authors: Culmsee Carsten , Zhu Xiaoxiang , Yu Qian‐Sheng , Chan Sic L. , Camandola Simonetta , Guo Zhihong , Greig Nigel H. , Mattson Mark P.
Published: March 2003

The tumor suppressor protein p53 is essential for neuronal death in several experimental settings and may participate in human neurodegenerative disorders. Based upon recent studies characterizing chemical inhibitors of p53 in preclinical studies in the cancer therapy field, we synthesized the compound pifithrin‐α and evaluated its potential neuroprotective properties in experimental models relevant to the pathogenesis of stroke and neurodegenerative disorders. Pifithrin‐α protected neurons against apoptosis induced by DNA‐damaging agents, amyloid β‐peptide and glutamate. Protection by pifithrin‐α was correlated with decreased p53 DNA‐binding activity, decreased expression of the p53 target gene Bax and suppression of mitochondrial dysfunction and caspase activation. Mice given pifithrin‐α exhibited increased resistance of cortical and striatal neurons to focal ischemic injury and of hippocampal neurons to excitotoxic damage. These preclinical studies demonstrate the efficacy of a p53 inhibitor in models of stroke and neurodegenerative disorders, and suggest that drugs that inhibit p53 may reduce the extent of brain damage in related human neurodegenerative conditions.
Aging alters the multichemical networking profile of the human brain: an in vivo1H‐MRS study of young versus middle‐aged subjects
Authors: Grachev Igor D. , Swarnkar Amar , Szeverenyi Nikolaus M. , Ramachandran Tarakad S. , Apkarian A. Vania
Published: March 2003

In our most recent study of normal aging, we found decreased concentration of multiple chemicals in the brain of middle‐aged subjects, as compared with younger subjects using in vivo proton magnetic resonance spectroscopy (1H‐MRS). We hypothesized that these age‐dependent differences in brain chemistry changes might be a reflection of the multichemical‐networking‐profile (MCNP) changes during aging. Using 1H‐MRS and correlation analysis, we examined the patterns of regional chemical levels and MCNP within and across multiple brain regions for all nine chemicals of 1H‐MR spectra. The brain chemistry changes and MCNP patterns were compared between 21 young (19–31‐year‐old) and 31 middle‐aged (40–52‐year‐old) normal volunteers. Middle‐aged subjects demonstrated a significant decrease of chemical levels in the prefrontal cortex and sensorimotor cortex (SMC), as compared with the young age group. Of these, neurotransmitters GABA and glutamate in the dorsolateral prefrontal cortex (DLPFC) were altered the most. We also found a significant increase of overall chemical correlation strength in MCNP within and across all studied brain regions with increased age. These changes were caused by alterations in the pattern of negative chemical connectivity across brain regions, which become weaker (less negative) in middle‐aged subjects. The interregional chemical connectivity for the cingulate cortex, SMC and the thalamus was changed the most with increased age. Increased levels of chemical correlation strength across brain regions in aging were found for most chemicals studied (including neurotransmitters GABA and glutamate), and not for N‐acetyl aspartate. These age‐related differences in the connectivity of neurotransmitters were not region dependent. The results suggest that aging is associated with changes of the regional brain chemistry and the brain MCNP. The latter process may reflect an adaptive or compensatory response (possibly related to the elongation of dendrites with aging) to reduced levels of regional brain chemicals. The 1H‐MRS approach proposed here can be used as a valuable tool in the study of the brain chemistry, MCNP and their relationships in normal and abnormal aging.
G protein‐coupled receptor kinase 2 mediates µ‐opioid receptor desensitization in GABAergic neurons of the nucleus raphe magnus
Authors: Li Allen H. , Wang Hung‐Li
Published: March 2003

Nucleus raphe magnus (NRM) sends the projection to spinal dorsal horn and inhibits nociceptive transmission. Analgesic effect produced by µ‐opioid receptor agonists including morphine partially results from activating the NRM‐spinal cord pathway. It is generally believed that µ‐opioid receptor agonists disinhibit spinally projecting neurons of the NRM and produce analgesia by hyperpolarizing GABAergic interneurons. In the present study, whole‐cell patch‐clamp recordings combined with single‐cell RT‐PCR analysis were used to test the hypothesis that DAMGO ([D‐Ala2,N‐methyl‐Phe4,Gly‐ol5]enkephalin), a specific µ‐opioid receptor agonist, selectively hyperpolarizes NRM neurons expressing mRNA of glutamate decarboxylase (GAD67). Homologous desensitization of µ‐opioid receptors in NRM neurons could result in the development of morphine‐induced tolerance. G protein‐coupled receptor kinase (GRK) is believed to mediate µ‐opioid receptor desensitization in vivo. Therefore, we also investigated the involvement of GRK in mediating homologous desensitization of DAMΑΜGO‐induced electrophysiological effects on NRM neurons by using two experimental strategies. First, single‐cell RT‐PCR assay was used to study the expression of GRK2 and GRK3 mRNAs in individual DAMGO‐responsive NRM neurons. Whole‐cell recording was also performed with an internal solution containing the synthetic peptide, which corresponds to Gβγ‐binding domain of GRK and inhibits Gβγ activation of GRK. Our results suggest that DAMGO selectively hyperpolarizes NRM GABAergic neurons by opening inwardly rectifying K+ channels and that GRK2 mediates short‐term homologous desensitization of µ‐opioid receptors in NRM GABAergic neurons.
Reduced activation and expression of ERK1/2 MAP kinase in the post‐mortem brain of depressed suicide subjects
Authors: Dwivedi Y. , Rizavi H. S. , Roberts R. C. , Conley R. C. , Tamminga C. A. , Pandey G. N.
Published: March 2003

The extracellular regulated kinases (ERK) 1 and ERK2 are members of mitogen‐activated protein (MAP) kinase family that play an important role in transducing extracellular signals to the nucleus and have been implicated in a broad spectrum of biological responses. To test the hypothesis that MAP kinases may be involved in depression, we examined the activation of p44/42 MAP kinase and expression of ERK1 and ERK2 in the post‐mortem brain tissue obtained from non‐psychiatric control subjects (n = 11) and age‐ and the post‐mortem interval‐matched depressed suicide subjects (n = 11). We observed that p44/42 MAP kinase activity was significantly decreased in the prefrontal cortical areas (Brodmann's areas 8, 9 and 10) and the hippocampus of depressed suicide subjects without any change in the cerebellum. This decrease was associated with a decrease in mRNA and protein levels of ERK1 and ERK2. In addition, the expression of MAP kinase phosphatase (MKP)2, a ‘dual function’ ERK1/2 phosphatase, was increased in the prefrontal cortex and hippocampus. These studies suggest that p44/42 MAP kinases are less activated in the post‐mortem brain of depressed suicide subjects and this may be because of reduced expression of ERK1/2 and increased expression of MKP2. Given the role of MAP kinases in various physiological functions and gene expression, alterations in p44/42 MAP kinase activation and expression of ERK1/2 may contribute significantly to the pathophysiology of depressive disorders.
Constitutive expression of functional GABABreceptors in mIL‐tsA58 cells requires both GABAB(1)and GABAB(2)genes
Authors: Chronwall Bibie M. , Davis Tracy D. , Severidt Mathew W. , Wolfe Shannyn E. , McCarson Kenneth E. , Beatty Diane M. , Low Malcolm J. , Morris Stephen J. , Enna Salvatore J.
Published: March 2003

Studies of γ‐aminobutyric acid (GABA)Breceptor function in heterologous cell systems have suggested that expression of two distinct seven transmembrane G‐protein coupled receptor subunits is necessary for receptor activation and signal transduction. Some results suggest that both receptor proteins must be inserted into the plasma membrane to create heterodimers; however, it is possible that subunit monomers or homodimers are functional in cells which constitutively express GABABreceptors. A new pituitary intermediate lobe melanotrope cell clone (mIL tsA58) has been isolated which constitutively expresses GABAB, D2and corticotrophin releasing factor receptors. Here, we report on characterization of the GABABreceptors. Solution hybridization‐nuclease protection assays reveal the presence of GABAB(1)and GABAB(2)transcripts. Western blots show GABAB(1a)and one of two GABAB(2)proteins. Addition of the GABABagonist baclofen to cultured mIL‐tsA58 (mIL) cells inhibits high voltage activated Ca2+channels, as measured by agonist‐induced inhibition of the K+‐depolarization‐stimulated increase in Ca2+influx. CGP55845, a GABABantagonist, blocks the response to baclofen. Knockdown of either GABAB(1)or GABAB(2)subunits with selective antisense oligodeoxynucleotides reduced GABABprotein levels and completely abolished the GABABreceptor response in the mIL cells. Taken together, these results indicate that functionally active GABABreceptors in mIL cells require the constitutive expression of both GABABgenes. This is a physiologic validation of results from recombinant overexpression in naive cells and shows that the mIL cell line is a useful model for studying GABABreceptor expression, regulation and function.
Cytokine‐induced cell death in immortalized Schwann cells: roles of nitric oxide and cyclic AMP
Authors: Nagano Sukehisa , Takeda Margaret , Ma Lan , Soliven Betty
Published: March 2003

Tumor necrosis factor‐α and interferon‐γ are pleiotropic cytokines that regulate Schwann cell responses during injury and inflammatory demyelination. We have previously shown that cyclic AMP (cAMP)‐elevating agents decrease the demyelination and Wallerian degeneration in experimental allergic neuritis. In this study, we examined the role of cAMP in cytokine‐mediated signaling in a spontaneously immortal Schwann cell clone (iSC). We found that tumor necrosis factor‐α and interferon‐γ exert synergistic inhibitory action on Schwann cell viability via the production of nitric oxide (NO) and ceramide (cer). Furthermore, we found that: (i) NO synthase inhibitors attenuate the cytokine‐induced cer accumulation and cell death indicating that NO acts upstream of cer; and (ii) cytokine‐induced cell death is decreased in iSCs pretreated continuously for 48–72 h with forskolin, an activator of adenylate cyclase. Although forskolin modulates the phosphorylation of ERKs and Akt, it decreases the susceptibility of iSC to cytokines via a separate mechanism operating after NO induction and before cer accumulation. We propose that the protective effect of cAMP‐elevating agents in experimental allergic neuritis may be mediated in part via modulation of Schwann cell responses to cytokines.
Differential role of superoxide and glutathione in S‐nitrosoglutathione‐mediated apoptosis: a rationale for mild forms of familial amyotrophic lateral sclerosis associated with less active Cu,Zn superoxide dismutase mutants
Authors: Ciriolo Maria Rosa , Aquilano Katia , De Martino Angelo , Carrì Maria Teresa , Rotilio Giuseppe
Published: March 2003

SH‐SY5Y cells transfected with the enzymatically inactive Cu,Zn superoxide dismutase mutant H46R were more resistant to S‐nitrosoglutathione (GSNO)‐induced apoptosis. Cytochrome c release from mitochondria, caspase 3 activation, p53 up‐regulation, p21 cleavage and Bcl‐2 modulation, all involved in the apoptotic process, were significantly less altered with respect to untransfected cells. The H46R resistance to NO was associated with a higher content of reduced glutathione (GSH) and was abolished by blockage of glutathione synthesis. On the other hand, H46R cells were as sensitive as SH‐SY5Y cells to puromycin‐induced apoptosis; furthermore, they were more susceptible to apoptosis elicited by the superoxide‐generating drug paraquat and to cell necrosis provoked by t‐butyl hydroperoxide. These results confirm that the level of superoxide dismutase activity is fundamental for protecting cells against oxygen free radical challenge. Its impairment is not detrimental to cells exposed to NO, as long as the overall reducing power represented by GSH is assured. These results are relevant to explain a milder progression of the familial amyotrophic lateral sclerosis disease when associated with the H46R mutation.
Serotonin activation of the ERK pathway in Hermissenda: contribution of calcium‐dependent protein kinase C
Authors: Crow Terry , Xue‐Bian Juan‐Juan , Siddiqi Vilma , Neary Joseph T.
Published: March 2003

The mitogen‐activated protein kinase (MAPK) cascade is an important contributor to synaptic plasticity and learning in both vertebrates and invertebrates. In the nudibranch mollusk Hermissenda, phosphorylation and activation of the extracellular signal‐regulated protein kinase (ERK), a key member of a MAPK cascade, is produced by one‐trial and multitrial Pavlovian conditioning. Several signal transduction pathways that are activated by 5‐hydroxytryptamine (5‐HT) and may contribute to conditioning have been identified in type B photoreceptors. However, the regulation of ERK activity by ‘upstream’ signaling molecules has not been previously investigated in Hermissenda. In the present study we examined the role of protein kinase C (PKC) in the serotonin (5‐HT) activation of the ERK pathway. The phorbol ester TPA produced an increase in ERK phosphorylation that was blocked by the PKC inhibitors GF109203X or Gö6976. TPA‐dependent ERK phosphorylation was also blocked by the MEK1 inhibitors PD098059 or U0126. The increased phosphorylation of ERK by 5‐HT was reduced but not blocked by pretreatment with the calcium chelator BAPTA‐AM or pretreatment with Gö6976 or GF109203X. These results indicate that Ca2+‐dependent PKC activation contributes to ERK phosphorylation, although a PKC‐independent pathway is also involved in 5‐HT‐dependent ERK phosphorylation and activation.
Glutamate induces focal adhesion kinase tyrosine phosphorylation and actin rearrangement in heterologous mGluR1‐expressing CHO cells via calcium/calmodulin signaling
Authors: Shinohara Yoshiaki , Nakajima Yoshiaki , Nakanishi Shigetada
Published: March 2003

Group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) stimulate phospholipase C (PLC) and lead to mobilization of intracellular Ca2+ and activation of protein kinase C (PKC). In this investigation, using heterologous receptor‐expressing Chinese hamster ovary (CHO) cells, we showed that stimulation of mGluR1 or mGluR5 with glutamate rapidly increases tyrosine phosphorylation of focal adhesion kinase (FAK) (maximum at 1–3 min) in a dose‐dependent manner (half‐maximal responses at approximately 2 µm). In mGluR1‐expressing cells, the glutamate‐induced increase of FAK tyrosine phosphorylation was blocked by not only the PLC inhibitor, U73122, but also depletion of intracellular Ca2+ and effectively abrogated by calmodulin (CaM) inhibitors, calmidazolium and fluphenazine. However, neither the PKC inhibitor, GF109203X, nor the CaM kinase II inhibitor, KN‐62, inhibited glutamate‐stimulated FAK tyrosine phosphorylation. Stimulation of mGluR1 caused a marked increase in actin stress fiber formation. Importantly, this actin rearrangement was prevented by the CaM inhibitor, but not by the PKC inhibitor and is thus in a good agreement with the signaling cascade of the mGluR1‐FAK pathway. These results suggest that the Ca2+/CaM signaling and its downstream FAK tyrosine phosphorylation play an important role in cellular function of mGluR1.
Dependence of excitotoxic neurodegeneration on mitochondrial aconitase inactivation
Authors: Li Qing‐You , Pedersen Cynthia , Day Brian J. , Patel Manisha
Published: March 2003

Using the inactivation of mitochondrial and cytosolic aconitases as markers of compartment‐specific superoxide (O2−) production, we show that oxygen‐glucose deprivation (OGD) or excitotoxin exposure produce a time‐dependent inactivation of mitochondrial, but not cytosolic, aconitase in cortical cultures. To determine if mitochondrial O2− production was an important determinant in neuronal death resulting from OGD, metalloporphyrins with varying superoxide dismutase (SOD) activity were tested for their ability to protect against mitochondrial aconitase inactivation and cell death. OGD‐induced mitochondrial aconitase inactivation and cell death was inhibited by manganese tetrakis (4‐benzoic acid) porphyrin (MnTBAP), manganese tetrakis (N‐ethylpyridinium‐2‐yl) porphyrin (MnTE‐2‐PyP) and NMDA receptor antagonists. By contrast, NMDA‐ or kainate (KA)‐induced mitochondrial aconitase inactivation and cell death was inhibited by MnTBAP, but not MnTE‐2‐PyP. Moreover, both MnTBAP and MnTE‐2‐PyP penetrated mitochondrial fractions of cortical cells. These data suggest that mitochondrial aconitase inactivation closely correlates with subsequent neuronal death following excitotoxicity produced by OGD or NMDA/KA exposure. Assessment of biological rather biochemical antioxidant activities better predicted neuroprotection by metalloporphyrins. Moreover, antioxidants that protect oxidant‐sensitive mitochondrial targets such as aconitase may be useful as therapies for disease states involving excitotoxicity.
Modulation of NMDA‐mediated excitotoxicity by protein kinase C
Authors: Wagey R. , Hu J. , Pelech S. L. , Raymond L. A. , Krieger C.
Published: March 2003

Excessive activation ofN‐methyl‐d‐aspartate (NMDA) receptors leads to cell death in human embryonic kidney‐293 (HEK) cells which have been transfected with recombinant NMDA receptors. To evaluate the role of protein kinase C (PKC) activation in NMDA‐mediated toxicity, we have analyzed the survival of transfected HEK cells using trypan blue exclusion. We found that NMDA‐mediated death of HEK cells transfected with NR1/NR2A subunits was increased by exposure to phorbol esters and reduced by inhibitors of PKC activation, or PKC down‐regulation. The region of NR2A that provides the PKC‐induced enhancement of cell death was localized to a discrete segment of the C‐terminus. Use of isoform‐specific PKC inhibitors showed that Ca2+‐ and lipid‐dependent PKC isoforms (cPKCs), specifically PKCβ1, was responsible for the increase in cell death when phorbol esters were applied prior to NMDA in these cells. PKC activity measured by anin vitrokinase assay was also increased in NR1A/NR2A‐transfected HEK cells following NMDA stimulation. These results suggest that PKC acts on the C‐terminus of NR2A to accentuate cell death in NR1/NR2A‐transfected cells and demonstrate that this effect is mediated by cPKC isoforms. These data indicate that elevation of cellular PKC activity can increase neurotoxicity mediated by NMDA receptor activation.
Selective cannabinoid CB1 receptor‐mediated inhibition of inducible nitric oxide synthase protein expression in C6 rat glioma cells
Authors: Esposito Giuseppe , Izzo Angelo A. , Di Rosa Massimo , Iuvone Teresa
Published: March 2003

We have studied the effects of two cannabinoid receptor agonists, WIN 55,212–2 and cannabinol, on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in the C6 glioma cell line. After 24 h of lipopolysaccharide (LPS) (1 µg/mL) and interferon‐γ (IFN‐γ) (300 U/mL) stimulation, a significant increase in NO production, evaluated as nitrite, was observed in the culture medium. WIN 55,212–2 (0.1–10000 nm) and cannabinol (0.3–30000 nm), dose‐dependently inhibited nitrite production showing a different potency (WIN 55,212–2 EC50: 4.2 nm; cannabinol EC50: 700 nm). WIN 55,212–2 (100 nm), given concomitantly to the stimulus also inhibited iNOS expression but had no effect when added to the cells 2 h after LPS/IFN‐γ, indicating a possible interference at the protein synthesis level or at an earlier step, as gene transcription. The cannabinoid CB1 receptor antagonist, SR141716A (0.1–100 nm), but not the cannabinoid CB2 receptor antagonist, SR144528 (0.1–100 nm), reduced in a dose‐related manner WIN 55,212–2‐and cannabinol‐induced inhibition of nitrite production. SR141161A also reversed the WIN 55,212–2‐induced inhibition of iNOS expression. These data suggest that selective cannabinoid CB1 receptor activation, by inhibiting iNOS expression and NO overproduction in glial cells, might be helpful in NO‐mediated inflammation leading to neurodegeneration.