bluuebunny/crossref_metadata_2025 huggingface dataset:
Examination of the involvement of protein kinase A in D2 dopamine receptor antagonist‐induced immediate early gene expression
Authors: Adams Amy C. , Keefe Kristen A.
Published: March 2003

Immediate early genes (IEGs) are induced by different signaling pathways. It has been proposed that D2 dopamine receptor blockade induces IEG expression through activation of protein kinase A (PKA), although few studies have examined this issue in vivo. We infused the PKA inhibitor H‐89 into the striatum of male rats, followed 30 min later by systemic administration of eticlopride. Eticlopride‐induced c‐fos and zif268 mRNA expression in striatum was not blocked by H‐89. In addition, eticlopride did not produce measurable levels of PKA activity in striatum, whereas the cAMP activator Sp‐8‐Br‐cAMPs increased levels of activated PKA. Neither the adenosine A2a receptor agonist CGS 21680 nor the phosphodiesterase‐4 inhibitor rolipram, each of which should increase PKA activation, potentiated eticlopride‐induced IEG expression. To test whether other signaling pathways are involved in eticlopride‐mediated gene induction, we also infused inhibitors of the mitogen‐activated and calcium/calmodulin‐dependent protein kinases into animals and then treated them with eticlopride. The data suggest that eticlopride‐induced IEG expression is not solely dependent on these kinases either. These data suggest that PKA activation may not be necessary for induction of IEGs by D2 dopamine receptor antagonists and that other intracellular signaling pathways may be involved.
The heat shock response reduces myelin oligodendrocyte glycoprotein‐induced experimental autoimmune encephalomyelitis in mice
Authors: Heneka Michael T. , Sharp Anthony , Murphy Patricia , Lyons Jeri‐Anne , Dumitrescu Lucia , Feinstein Douglas L.
Published: March 2003

The stress response (SR) can block inflammatory gene expression by preventing activation of transcription factor nuclear factor‐kappa B (NF‐κB). As inflammatory gene expression contributes to the pathogenesis of demyelinating diseases, we tested the effects of the SR on the progression of the demyelinating disease experimental autoimmune encephalomyelitis (EAE). EAE was actively induced in C57BL/6 mice using an encephalitogenic myelin oligodendrocyte glycoprotein (MOG35−55) peptide. Whole body hyperthermia was used to induce a heat shock response (HSR) in immunized mice 2 days after the booster MOG35−55 peptide injection. The HSR reduced the incidence of EAE by 70%, delayed disease onset by 6 days, and attenuated disease severity. The HSR attenuated leukocyte infiltration into CNS assessed by quantitation of perivascular infiltrates, and by reduced staining for CD4 and CD25 immunopositive T‐cells. T‐cell activation, assessed by the production of interferon γ (IFNγ) in response to MOG35−55, was also decreased by the HSR. The HSR reduced inflammatory gene expression in the brain that normally occurs during EAE, including the early increase in RANTES (regulated on activation of normal T‐cell expressed and secreted) expression, and the later expression of the inducible form of nitric oxide synthase. The early activation of transcription factor NF‐κB was also blocked by the HSR. The finding that the SR reduces inflammation in the brain and the clinical severity of EAE opens a novel therapeutic approach for prevention of autoimmune diseases.
Walker‐256 tumor growth causes oxidative stress in rat brain
Authors: Freitas Jofre J. S. , Pompéia Celine , Miyasaka Célio K. , Curi Rui
Published: March 2003

The elevated rate of oxygen consumption and high amount of polyunsaturated fatty acids make the central nervous system vulnerable to oxidative stress. The effect of Walker‐256 tumor growth on oxi–reduction indexes in the hypothalamus (HT), cortex (CT), hippocampus (HC) and cerebellum (CB) of male Wistar rats was investigated. The presence of the tumor caused an increase in thiobarbituric acid reactant substances (TBARs) in the HT, CB and HC. Due to tumor growth, the activity of glucose‐6‐phosphate dehydrogenase increased in the HT and CB, whereas citrate synthase activity was reduced in the HT, CT and CB. Therefore, the potential for generation of reducing power is increased in the cytosol and decreased in the mitochondria of various brain regions of Walker‐256 tumor‐bearing rats. These changes occurred concomitantly with an unbalance in the brain enzymatic antioxidant system. The tumor decreased the activities of catalase in the HT and CB and of glutathione peroxidase in the HT, CB and HC, and raised the CuZn‐superoxide dismutase activity in the HT, CB and HC. These combined findings indicate that Walker‐256 tumor growth causes oxidative stress in the brain.
N‐Acetylaspartate, a marker of both cellular dysfunction and neuronal loss: its relevance to studies of acute brain injury
Authors: Demougeot Céline , Garnier Philippe , Mossiat Claude , Bertrand Nathalie , Giroud Maurice , Beley Alain , Marie Christine
Published: March 2003

To evaluate the contribution of cellular dysfunction and neuronal loss to brain N‐acetylaspartate (NAA) depletion, NAA was measured in brain tissue by HPLC and UV detection in rats subjected to cerebral injury, associated or not with cell death. When lesion was induced by intracarotid injection of microspheres, the fall in NAA was related to the degree of embolization and to the severity of brain oedema. When striatal lesion was induced by local injection of malonate, the larger the lesion volume, the higher the NAA depletion. However, reduction of brain oedema and striatal lesion by treatment with the lipophilic iron chelator dipyridyl (20 mg/kg, 1 h before and every 8 h after embolization) and the inducible nitric oxide synthase inhibitor aminoguanidine (100 mg/kg given 1 h before malonate and then every 9 h), respectively, failed to ameliorate the fall in NAA. Moreover, after systemic administration of 3‐nitropropionic acid, a marked reversible fall in NAA striatal content was observed despite the lack of tissue necrosis. Overall results show that cellular dysfunction can cause higher reductions in NAA level than neuronal loss, thus making of NAA quantification a potential tool for visualizing the penumbra area in stroke patients.
Prostaglandin release by spinal cord injury mediates production of hydroxyl radical, malondialdehyde and cell death: a site of the neuroprotective action of methylprednisolone
Authors: Liu Danxia , Li Liping , Augustus Lezel
Published: March 2003

The present study explores in vivo whether and how prostaglandin F2α (PGF2α), a membrane phospholipid hydrolysis product, causes neuronal death. The concentration of PGF2α measured by microdialysis sampling increased threefold immediately following impact injury to the rat spinal cord. Administration of PGF2α into the cord through a dialysis fiber caused significant cell loss, increased extracellular levels of hydroxyl radicals and malondialdehyde – an end product of membrane lipid peroxidation – to 3.3 and 2.3 times basal levels, respectively. This suggests that PGF2α‐induced cell death is partly due to hydroxyl radical‐triggered peroxidation. Generating hydroxyl radical by administering Fenton's reagents into the cord through the fibers significantly increased malondialdehyde production – the first direct in vivo evidence that hydroxyl radical triggers membrane lipid peroxidation. Methylprednisolone significantly reduced the release of PGF2α upon spinal cord injury and blocked PGF2α‐induced hydroxyl radical and malondialdehyde production, but did not significantly reduce Fenton's reagent‐induced malondialdehyde production, despite the production of more malondialdehyde by PGF2α. This suggests that methylprednisolone may not directly scavenge hydroxyl radical, and that its ‘antioxidant’ effect is a consequence of blocking the pathways for producing toxic PGF2α and for PGF2α‐induced hydroxyl radical formation, thereby reducing membrane lipid peroxidation.
Cerebroside synthesis as a measure of the rate of remyelination following cuprizone‐induced demyelination in brain
Authors: Jurevics Helga , Hostettler Janell , Muse Evan D. , Sammond Deanne W. , Matsushima Glenn K. , Toews Arrel D. , Morell Pierre
Published: March 2003

We studied markers of myelin content and of the rate of myelination in brains of mice between 8 and 20 weeks of age. During the 12‐week time‐course, control animals showed slight increases in the content of oligodendroglial‐specific cerebroside, as well as cholesterol (enriched in, but not specific to, myelin). In contrast, synthesis of these lipids, as assayed by in vivo incorporation of 3H2O, was substantial, indicating turnover of 0.4% and 0.7% of total brain cerebroside and cholesterol, respectively, each day. We also studied mice exposed to a diet containing 0.2% of the copper chelator, cuprizone. After 6 weeks 20%, and by 12 weeks, over 30% of brain cerebroside was gone. Demyelination was accompanied by down‐regulation of mRNA expression for enzymes controlling myelin lipid synthesis (ceramide galactosyl transferase for cerebroside; hydroxymethylglutaryl‐CoA reductase for cholesterol), and for myelin basic protein. Synthesis of myelin lipids was also greatly depressed. The 20% cerebroside deficit consequent to 6 weeks of cuprizone exposure was restored 6 weeks after return to a control diet. During remyelination, expression of myelin‐related mRNA species, as well as cerebroside and cholesterol synthesis were restored to normal. However, in contrast to the steady state metabolic turnover in the control situation, all the cerebroside and cholesterol made were accumulated. To the extent that accumulating cerebroside is targeted for eventual inclusion in myelin (discussed) the rate of its synthesis is proportional to remyelination. With our assay, in vivo rates of cerebroside synthesis can be determined for a time window of the order of hours. This offers greater temporal resolution and accuracy relative to classical methods assaying accumulation of myelin components at time intervals of several days. We propose this experimental design, and the reproducible cuprizone model, as appropriate for studies of how to promote remyelination.
Parkin is associated with cellular vesicles
Authors: Kubo Shin‐ichiro , Kitami Toshiaki , Noda Setsuko , Shimura Hideki , Uchiyama Yasuo , Asakawa Shuichi , Minoshima Shinsei , Shimizu Nobuyoshi , Mizuno Yoshikuni , Hattori Nobutaka
Published: March 2003

We recently identified a novel gene, parkin, as a pathogenic gene for autosomal recessive juvenile parkinsonism. Parkin encodes a 52‐kDa protein with a ubiquitin‐like domain and two RING‐finger motifs. To provide a insight into the function of parkin, we have examined its intracellular distribution in cultured cells. We found that parkin was localized in the trans‐Golgi network and the secretory vesicles in U‐373MG or SH‐SY5Y cells by immunocytochemical analyses. In the subsequent subcellular fractionation studies of rat brain, we showed that parkin was copurified with the synaptic vesicles (SVs) when we used low ionic conditions throughout the procedure. An immunoelectromicroscopic analysis indicated that parkin was present on the SV membrane. Parkin was readily released from SVs into the soluble phase by increasing ionic strength at neutral pH, but not by a non‐ionic detergent. To elucidate its responsible region for membrane association, we transfected with green fluorescent protein‐tagged deletion mutants of parkin into COS‐1 cells followed by subcellular fractionation. We demonstrated the ability of parkin to bind to the membranes through a broad region except for the ubiquitin‐like domain. The significance of SV localization of parkin is discussed.
The specificity of monoclonal antibody A2B5 to c‐series gangliosides
Authors: Saito Megumi , Kitamura Hisayo , Sugiyama Kiyoshi
Published: March 2003

To examine the specificity of monoclonal antibody A2B5, four A2B5‐reactive gangliosides (designated as G‐1, G‐2, G‐3 and G‐4) were purified from bonito fish brain. Ganglioside‐1, ‐2, and ‐3 migrated above GD1b, below GQ1b, and far below GQ1b on thin‐layer chromatography. Ganglioside‐4 had the slowest chromatographic mobility and migrated below G‐3. The structures of these gangliosides were characterized by overlay analysis with glycolipid‐specific ligands, product analysis after sialidase or mild acid treatment, and electrospray ionization‐mass spectrometry (ESI‐MS). Accordingly, G‐1, G‐2 and G‐3 were identified to be GT3, GQ1c and GP1c, respectively. The ganglioside G‐4 was shown to have the following structure: NeuAc‐NeuAc‐NeuAc‐Galβ1‐3Gal NAcβ1‐4(NeuAc‐NeuAc‐NeuAcα2‐3)Galβ1‐4Glcβ1‐1′Cer. The antibody A2B5 reacted with these c‐series gangliosides, but not with GD3 and other gangliosides and neutral glycosphingolipids. The antigenic epitope for A2B5 was assumed to include the trisialosyl residue connected to the inner galactose of the hemato‐ or ganglio‐type oligosaccharide structure of gangliosides. Phylogenetic analysis of brain gangliosides using the A2B5 preparation demonstrated that c‐series gangliosides are enriched in lower animals, especially bony fish of different species. The monoclonal antibody A2B5 would be a useful tool for examining the distribution and function of c‐series gangliosides.
Cleavage of Bax is mediated by caspase‐dependent or ‐independent calpain activation in dopaminergic neuronal cells: protective role of Bcl‐2
Authors: Choi Won‐Seok , Lee Eun‐Hee , Chung Chul‐Woong , Jung Yong‐Keun , Jin Byung K. , Kim Seung U. , Oh Tae H. , Saido Takaomi C. , Oh Young J.
Published: March 2003

Two cysteine protease families, caspase and calpain, are known to participate in cell death. We investigated whether a stress‐specific protease activation pathway exists, and to what extent Bcl‐2 plays a role in preventing drug‐induced protease activity and cell death in a dopaminergic neuronal cell line, MN9D. Staurosporine (STS) induced caspase‐dependent apoptosis while a dopaminergic neurotoxin, MPP+ largely induced caspase‐independent necrotic cell death as determined by morphological and biochemical criteria including cytochrome c release and fluorogenic caspase cleavage assay. At the late stage of both STS‐ and MPP+‐induced cell death, Bax was cleaved into an 18‐kDa fragment. This 18‐kDa fragment appeared only in the mitochondria‐enriched heavy membrane fraction of STS‐treated cells, whereas it was detected exclusively in the cytosolic fraction of MPP+‐treated cells. This proteolytic cleavage of Bax appeared to be mediated by calpain as determined by incubation with [35S]methionine‐labelled Bax. Thus, cotreatment of cells with calpain inhibitor blocked both MPP+‐ and STS‐induced Bax cleavage. Intriguingly, overexpression of baculovirus‐derived inhibiting protein of caspase, p35 or cotreatment of cells with caspase inhibitor blocked STS‐ but not MPP+‐induced Bax cleavage. This appears to indicate that calpain activation may be either dependent or independent of caspase activation within the same cells. However, cotreatment with calpain inhibitor rescued cells from MPP+‐induced but not from STS‐induced neuronal cell death. In these paradigms of dopaminergic cell death, overexpression of Bcl‐2 prevented both STS‐ and MPP+‐induced cell death and its associated cleavage of Bax. Thus, our results suggest that Bcl‐2 may play a protective role by primarily blocking drug‐induced caspase or calpain activity in dopaminergic neuronal cells.
Role of DNA‐dependent protein kinase in neuronal survival
Authors: Chechlacz Magdalena , Vemuri Mohan C. , Naegele Janice R.
Published: March 2003

DNA‐dependent protein kinase (DNA‐PK) is a DNA repair enzyme composed of a DNA‐binding component called Ku70/80 and a catalytic subunit called DNA‐PKcs. Many investigators have utilized DNA‐PKcs‐deficient cells and cell lines derived from severe combined immunodeficiency (scid) mice to study DNA repair and apoptosis. However, little is known about the CNS of these mice. This study was carried out using primary neuronal cultures derived from the cerebral hemispheres of new‐born wild‐type and scid mice to investigate the effects of loss of DNA‐PK function on neuronal maturation and survival. Purified neuronal cultures developed comparably in terms of neurite formation and expression of neuronal markers, but scid cultures showed a significant increase in the percentage of dying cells. Furthermore, when apoptosis was induced by staurosporine, scid neurons died more rapidly and in higher numbers. Apoptotic scid neurons exhibited nuclear condensation, DNA fragmentation and caspase‐3 activation, but treatment with the general caspase inhibitor, N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐(O‐methyl) fluoromethyl ketone did not prevent staurosporine‐induced apoptosis. We conclude that a DNA‐PK deficiency in cultured scid neurons may cause an accumulation of DNA damage and increased susceptibility to caspase‐independent forms of programmed cell death.
Pentyl‐4‐yn‐valproic acid enhances both spatial and avoidance learning and attenuates age‐related NCAM‐mediated neuroplastic decline within the rat medial temporal lobe
Authors: Murphy Keith J. , Fox Gerard B. , Foley Andrew G. , Gallagher Helen C. , O'Connell Alan , Griffin Anne‐Marie , Nau Heinz , Regan Ciaran M.
Published: March 2003

2‐N‐Pentyl‐4‐pentynoic acid [pentyl‐4‐yn‐valproic acid (VPA)] is an analogue of valproic acid that induces neuritogenesis and increases neural cell adhesion molecule (NCAM) prevalence in cultured neural cells. As memory consolidation involves synapse growth, aided by cell adhesion molecule function, we determined whether or not pentyl‐4‐yn‐VPA had cognition‐enhancing properties. Pentyl‐4‐yn‐VPA (16–85 mg/kg) significantly improved water maze learning and task retention when given prior to each training session. Acute administration of pentyl‐4‐yn‐VPA also influenced memory consolidation processes as, when given at 3 h post‐passive avoidance training, the amnesia induced by scopolamine given 6 h post‐training was prevented in a dose‐dependent manner. Chronic administration of pentyl‐4‐yn‐VPA (16.8 or 50.4 mg/kg) also significantly reduced escape latencies in the water maze task, 24 h following the last drug administration. This improved spatial learning was accompanied by enhanced neuroplasticity as the expression of NCAM polysialylated neurons in the infragranular zone of the dentate gyrus and in layer II of the perirhinal and piriform cortex was increased significantly following chronic drug treatment. The cognition‐enhancing qualities of pentyl‐4‐yn‐VPA, combined with its ability to attenuate the age‐related loss of the NCAM polysialylation state, suggest that it may effectively slow the onset of cognitive decline.
Role of α2‐macroglobulin in regulating amyloid β‐protein neurotoxicity: protective or detrimental factor?
Authors: Fabrizi Cinzia , Businaro Rita , Lauro Giuliana M. , Fumagalli Lorenzo
Published: March 2003

α2‐Macroglobulin (α2M) has been identified as a carrier protein for β‐amyloid (Aβ) decreasing fibril formation and affecting the neurotoxicity of this peptide. The α2‐macroglobulin receptor/low density lipoprotein receptor related protein (LRP) is involved in the internalization and degradation of the α2M/Aβ complexes and its impairment has been reported to occur in Alzheimer's disease. Previous studies have shown α2M to determine an enhancement or a reduction of Aβ toxicity in different culture systems. In order to clarify the role of α2M in Aβ neurotoxicity, we challenged human neuroblastoma cell lines with activated α2M in combination with Aβ. Our results show that in neuroblastoma cells expressing high levels of LRP, the administration of activated α2M protects the cells from Aβ neurotoxicity. Conversely, when this receptor is not present α2M determines an increase in Aβ toxicity as evaluated by MTT and TUNEL assays. In LRP‐negative cells transfected with the full‐length human LRP, the addition of activated α2M resulted to be protective against Aβ‐induced neurotoxicity. By means of recombinant proteins we ascribed the neurotoxic activity of α2M to its FP3 fragment which has been previously shown to bind and neutralize transforming growth factor‐β. These studies provide evidence for both a neuroprotective and neurotoxic role of α2M regulated by the expression of its receptor LRP.
Regulation of functional nitric oxide synthase‐1 expression in cerebellar granule neurons by heregulin is post‐transcriptional, and involves mitogen‐activated protein kinase
Authors: Krainock Randy , Murphy Sean
Published: March 2003

The neuregulins, and the ErbB family of tyrosine kinase receptors, play important roles in the development of the nervous system. Recently, the C‐terminal region of the ErbB4 receptor has been reported to associate with domains of post‐synaptic density proteins. The latter are, in turn, known to assemble nitric oxide synthase (NOS)‐1 at cell junctions. Previously, we showed that heregulin can up‐regulate the expression of NOS‐1 via the ErbB4 receptor in cerebellar granule cell cultures. We have now determined that this up‐regulation is post‐transcriptional, and results in an associated increase in NOS activity in these neurons. Furthermore, we find that heregulin activates both MAP kinase and phosphoinositide 3 kinase (PI 3‐K) in granule cells. While inhibition of MAP kinase reduces the ability of heregulin to up‐regulate NOS‐1 expression, a specific inhibitor of PI 3‐K was without effect. Our results suggest that NO could mediate some of the downstream effects of heregulin in the nervous system.
Cellular resistance to Evans blue toxicity involves an up‐regulation of a phosphate transporter implicated in vesicular glutamate storage
Authors: Israël M. , Tomasi M. , Bostel S. , Meunier F.‐M.
Published: March 2003

It has recently been suggested that the brain‐specific Na+‐dependent phosphate inorganic co‐transporter (BNPI) is able to support glutamate transport and storage in synaptic vesicles. A procedure for measuring the vesicular pool of glutamate is described and was used to select cell lines according to their glutamate storage capacity. Two cell lines were selected: C6BU‐1, with a large intracellular glutamate storage capacity, and NG108‐15, devoid of it. Their contents in BNPI mRNA were compared by RT‐PCR. We found that both cell lines had BNPI, but in addition C6BU‐1 alone expresses the other isoform, DNPI. We also carried out a clonal selection of NG108‐15 cells in the presence of the dye Evans blue, a competitive inhibitor of vesicular glutamate transport, very toxic for cells in culture. It was assumed that only those that sequester and eliminate the drug by overexpressing a vesicular glutamate transporter would survive. We found that the NG108‐15 clones resistant to Evans blue had an increased storage capacity for glutamate. These cells also up‐regulated the BNPI isoform of the phosphate transporter as shown by RT‐PCR and northern blot.
Cyclic AMP‐dependent protein kinase phosphorylates group III metabotropic glutamate receptors and inhibits their function as presynaptic receptors
Authors: Cai Zhaohui , Saugstad Julie A. , Sorensen Scott D. , Ciombor Kelly J. , Zhang Congxiao , Schaffhauser Hervé , Hubalek Frantisek , Pohl Jan , Duvoisin Robert M. , Conn P. Jeffrey
Published: March 2003

Recent evidence suggests that the functions of presynaptic metabotropic glutamate receptors (mGluRs) are tightly regulated by protein kinases. We previously reported that cAMP‐dependent protein kinase (PKA) directly phosphorylates mGluR2 at a single serine residue (Ser843) on the C‐terminal tail region of the receptor, and that phosphorylation of this site inhibits coupling of mGluR2 to GTP‐binding proteins. This may be the mechanism by which the adenylyl cyclase activator forskolin inhibits presynaptic mGluR2 function at the medial perforant path‐dentate gyrus synapse. We now report that PKA also directly phosphorylates several group III mGluRs (mGluR4a, mGluR7a, and mGluR8a), as well as mGluR3 at single conserved serine residues on their C‐terminal tails. Furthermore, activation of PKA by forskolin inhibits group III mGluR‐mediated responses at glutamatergic synapses in the hippocampus. Interestingly, β‐adrenergic receptor activation was found to mimic the inhibitory effect of forskolin on both group II and III mGluRs. These data suggest that a common PKA‐dependent mechanism may be involved in regulating the function of multiple presynaptic group II and group III mGluRs. Such regulation is not limited to the pharmacological activation of adenylyl cyclase but can also be elicited by the stimulation of endogenous Gs‐coupled receptors, such as β‐adrenergic receptors.
Stimulated D1 dopamine receptors couple to multiple Gα proteins in different brain regions
Authors: Jin Li‐Qing , Wang Hoau‐Yan , Friedman Eitan
Published: March 2003

Previous studies have revealed that activation of rat striatal D1 dopamine receptors stimulates both adenylyl cyclase and phospholipase C via Gs and Gq, respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D1 dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5‐biphosphate. In these brain regions, D1 dopamine receptors couple differentially to multiple Gα protein subunits. Antisera against Gαq blocks dopamine‐stimulated PIP2 hydrolysis in hippocampal and in striatal membranes. The binding of [35S]GTPγS or [α‐32P]GTP to Gαi was enhanced in all brain regions. Dopamine also increased the binding of [35S]GTPγS or [α‐32P]GTP to Gαq in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine‐stimulated binding of [35S]GTPγS to Gαs only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Gα proteins. Dopamine induced increases in GTPγS binding to Gαs and Gαq was blocked by the D1 antagonist SCH23390 but not by D2 receptor antagonist l‐sulpiride, suggesting that D1 dopamine receptors couple to both Gαs and Gαq proteins. Co‐immunoprecipitation of Gα proteins with receptor‐binding sites indicate that in the frontal cortex, D1 dopamine‐binding sites are associated with both Gαs and Gαq and, in hippocampus or amygdala, D1 dopamine receptors couple solely to Gαq. The results indicate that in addition to the D1/Gs/adenylyl cyclase system, brain D1‐like dopamine receptor sites activate phospholipase C through Gαq protein.
Differential capacities of the RGS1, RGS16 and RGS–GAIP regulators of G protein signaling to enhance α2A‐adrenoreceptor agonist‐stimulated GTPase activity of Go1α
Authors: Hoffmann Marcel , Ward Richard J. , Cavalli Antonella , Carr I. Craig , Milligan Graeme
Published: March 2003

Recombinant RGS1, RGS16 and RGS–GAIP, but not RGS2, were able to substantially further stimulate the maximal GTPase activity of Go1α promoted by agonists at the α2A‐adrenoreceptor in a concentration‐dependent manner. Kinetic analysis of the regulation of an α2A‐adrenoreceptor–Go1α fusion protein by all three RGS proteins revealed that they had similar affinities for the receptor–G protein fusion. However, their maximal effects on GTP hydrolysis varied over threefold with RGS16 > RGS1 > RGS–GAIP. Both RGS1 and RGS16 reduced the potency of the α2A‐adrenoreceptor agonist adrenaline by some 10‐fold. A lower potency shift was observed for the partial agonist UK14304 and the effect was absent for the weak partial agonist oxymetazoline. Each of these RGS proteins altered the intrinsic activity of both UK14304 and oxymetazoline relative to adrenaline. Such results require the RGS interaction with Go1α to alter the conformation of the α2A‐adrenoreceptor and are thus consistent with models invoking direct interactions between RGS proteins and receptors. These studies demonstrate that RGS1, RGS16 and RGS–GAIP show a high degree of selectivity to regulate α2A‐adrenoreceptor‐activated Go1α rather than Gi1α, Gi2α or Gi3α and different capacities to inactivate this G protein.
Autoreceptors do not regulate routinely neurotransmitter release: focus on adrenergic systems
Authors: Kalsner Stanley
Published: March 2003

The theory that neurotransmitter release is regulated locally at the individual terminals of neurons has achieved a rapid and seemingly secure status in our understanding of neuronal function both in the periphery and in the central nervous system. This concept of negative feedback control through the monitoring of the perineuronal concentration of previously released transmitter has been extended to a multiplicity of transmitters and utilized to explain the mechanisms of action of diverse classes of drugs, ranging from antihypertensives to antidepressants. It is my view that negative feedback by terminal and by somadendritic receptors cannot account for the existing body of experimental work. Analyses of the profiles of action of agonists and antagonists, and of the per pulse release of transmitter in the absence of drugs in a variety if peripheral organ systems, as well as in superfused brain slices, demonstrates the need for alternate interpretations of the available data. Evidence is provided that the actions of agonists to inhibit transmitter release and that of antagonists to enhance release occur at different cellular loci and that the purported unitary action of these two classes that is so central to the validity of presynaptic theory is unsupportable.
The amyloid precursor protein (APP)‐cytoplasmic fragment generated by γ‐secretase is rapidly degraded but distributes partially in a nuclear fraction of neurones in culture
Authors: Cupers Philippe , Orlans Isabelle , Craessaerts Katleen , Annaert Wim , De Strooper Bart
Published: March 2003

The γ‐secretase cleavage is the last step in the generation of the β‐amyloid peptide (Aβ) from the amyloid precursor protein (APP). The Aβ precipitates in the amyloid plaques in the brain of Alzheimer's disease patients. The fate of the intracellular APP carboxy‐terminal stub generated together with Aβ has been, in contrast, only poorly documented. The analogies between the processing of APP and other transmembrane proteins like SREBP and Notch suggests that this intracellular fragment could have important signalling functions. We demonstrate here that APP‐C59 is rapidly degraded (half‐life ∼5 min) when overexpressed in baby hamster kidney cells or primary cultures of neurones by a mechanism that is not inhibited by endosomal/lysosomal or proteasome inhibitors. Furthermore, APP‐C59 binds to the DNA binding protein Fe65, although this does not increase the half‐life of APP‐C59. Finally, we demonstrate that a fraction of APP‐C59 becomes redistributed to the nuclear detergent‐insoluble pellet, in which the transcription factor SP1 is also present. Overall our results reinforce the analogy between Notch and APP processing, and suggest that the APP intracellular domain, like the Notch intracellular domain, could have a role in signalling events from the plasma membrane to the nucleus.
Parallel increases in lipid and protein oxidative markers in several mouse brain regions after methamphetamine treatment
Authors: Gluck Martin R. , Moy Lily Y. , Jayatilleke Elizabeth , Hogan Kelly A. , Manzino Lawrence , Sonsalla Patricia K.
Published: March 2003

The neurotoxic actions of methamphetamine (METH) may be mediated in part by reactive oxygen species (ROS). Methamphetamine administration leads to increases in ROS formation and lipid peroxidation in rodent brain; however, the extent to which proteins may be modified or whether affected brain regions exhibit similar elevations of lipid and protein oxidative markers have not been investigated. In this study we measured concentrations of TBARs, protein carbonyls and monoamines in various mouse brain regions at 4 h and 24 h after the last of four injections of METH (10 mg/kg/injection q 2 h). Substantial increases in TBARs and protein carbonyls were observed in the striatum and hippocampus but not the frontal cortex nor the cerebellum of METH‐treated mice. Furthermore, lipid and protein oxidative markers were highly correlated within each brain region. In the hippocampus and striatum elevations in oxidative markers were significantly greater at 24 h than at 4 h. Monoamine levels were maximally reduced within 4 h (striatal dopamine [DA] by 95% and serotonin [5‐HT] in striatum, cortex and hippocampus by 60–90%). These decrements persisted for 7 days after METH, indicating effects reflective of nerve terminal damage. Interestingly, NE was only transiently depleted in the brain regions investigated (hippocampus and cortex), suggesting a pharmacological and non‐toxic action of METH on the noradrenergic nerve terminals. This study provides the first evidence for concurrent formation of lipid and protein markers of oxidative stress in several brain regions of mice that are severely affected by large neurotoxic doses of METH. Moreover, the differential time course for monoamine depletion and the elevations in oxidative markers indicate that the source of oxidative stress is not derived directly from DA or 5HT oxidation.
Vaccination with soluble Aβ oligomers generates toxicity‐neutralizing antibodies
Authors: Lambert Mary P. , Viola Kirsten L. , Chromy Brett A. , Chang Lei , Morgan Todd E. , Yu Jiaxin , Venton Duane L. , Krafft Grant A. , Finch Caleb E. , Klein William L.
Published: March 2003

In recent studies of transgenic models of Alzheimer's disease (AD), it has been reported that antibodies to aged beta amyloid peptide 1–42 (Aβ1−42) solutions (mixtures of Aβ monomers, oligomers and amyloid fibrils) cause conspicuous reduction of amyloid plaques and neurological improvement. In some cases, however, neurological improvement has been independent of obvious plaque reduction, and it has been suggested that immunization might neutralize soluble, non‐fibrillar forms of Aβ. It is now known that Aβ toxicity resides not only in fibrils, but also in soluble protofibrils and oligomers. The current study has investigated the immune response to low doses of Aβ1−42 oligomers and the characteristics of the antibodies they induce. Rabbits that were injected with Aβ1−42 solutions containing only monomers and oligomers produced antibodies that preferentially bound to assembled forms of Aβ in immunoblots and in physiological solutions. The antibodies have proven useful for assays that can detect inhibitors of oligomer formation, for immunofluorescence localization of cell‐attached oligomers to receptor‐like puncta, and for immunoblots that show the presence of SDS‐stable oligomers in Alzheimer's brain tissue. The antibodies, moreover, were found to neutralize the toxicity of soluble oligomers in cell culture. Results support the hypothesis that immunizations of transgenic mice derive therapeutic benefit from the immuno‐neutralization of soluble Aβ‐derived toxins. Analogous immuno‐neutralization of oligomers in humans may be a key in AD vaccines.
Neuroprotective effect of aspirin by inhibition of glutamate release after permanent focal cerebral ischaemia in rats
Authors: De Cristóbal Javier , Moro María A. , Dávalos Antoni , Castillo José , Leza Juan C. , Camarero Jorge , Colado M. Isabel , Lorenzo Pedro , Lizasoain Ignacio
Published: March 2003

Aspirin reduces the size of infarcts after ischaemic stroke. Although this fact has been attributed to its anti‐platelet actions, direct neuroprotective effects have also been reported. We have recently demonstrated that aspirin is neuroprotective by inhibiting glutamate release in ‘in vitro’ models of brain ischaemia, via an increase in ATP production. The present study was designed to determine whether the inhibition of glutamate release induced by aspirin might be protective in a whole‐animal model of permanent focal brain ischaemia. Focal brain ischaemia was produced in male adult Fischer rats by occluding both the common carotid and middle cerebral arteries. Central and serum glutamate levels were determined at fixed intervals after occlusion. The animals were then killed and infarct volume was measured. Aspirin (30 mg/kg i.p. administered 2 h before the occlusion) produced a significant reduction in infarct volume, an effect that correlated with the inhibition caused by aspirin on ischaemia‐induced increase in brain and serum glutamate concentrations after the onset of the ischaemia. Aspirin also inhibited ischaemia‐induced decrease in brain ATP levels. Our present findings show a novel mechanism for the neuroprotective effects of aspirin, which takes place at concentrations in the anti‐aggregant–analgesic range, useful in the management of patients with risk of ischaemic events.
Thrombin and PAR‐1 acitvating peptide increase iNOS expression in cytokine‐stimulated C6 glioma cells
Authors: Meli Rosaria , Mattace Giuseppina ,   Raso,, Cicala Carla , Esposito Emanuela , Fiorino Ferdinando , Cirino Giuseppe
Published: March 2003

Thrombin (THR) plays a key role in the brain under physiological and pathological conditions. Several of the biological activities of thrombin have been shown to be mainly driven through activation of protease‐activated receptor‐1 (PAR‐1)‐type thrombin receptor. Here we have studied the effect of THR and PAR‐1‐activating peptide (PAR1‐AP), SFLLRN, on cytokine‐induced expression of inducible nitric oxide (iNOS), a prominent marker of astroglial activation using the rat C6 glioma cells. In this cell line, THR (1–10 U/mL) and PAR1‐AP (1–100 µm) induced a significant concentration‐dependent increase both of IFN‐γ‐ (250 U/mL) or TNF‐α‐ (500 U/mL) induced NO release. The observed increase of NO production was related to an enhancement of iNOS expression as measured in cell lysates prepared from different treatments by using SDS–PAGE followed by western blot analysis. The effect of THR, but not that of PAR1‐AP, was significantly inhibited by hirulogTM (60 µg/mL), a specific and stochiometric THR inhibitor or by cathepsin‐G (40 mU/mL), an inhibitor of PAR‐1. In conclusion our data suggest a role for THR through activation of PAR‐1 in the induction of astroglial iNOS, and further support the hypothesis that THR may function as an important pathophysiological modulator of the inflammatory response.
Presence of NK2 binding sites in the rat brain
Authors: Saffroy Monique , Torrens Yvette , Glowinski Jacques , Beaujouan Jean‐Claude
Published: March 2003

Attempts were made to label tachykinin NK2 binding sites in the adult rat brain using [125I]neurokinin A (NKA) as ligand in the presence of NK1 and NK3 agonist or antagonist to avoid labelling of NK1 and NK3 binding sites, respectively. A high‐affinity, specifically NK2‐sensitive, [125I]NKA‐binding, temperature‐dependent, reversible, sensitive to GTPγS and correspondence to a single population of binding sites (KD and Bmax values: 2.2 nm and 7.3 fmol/mg protein) was demonstrated on hippocampal membranes. Competition studies performed with tachykinins and tachykinin‐related compounds indicated that the pharmacological properties of these NK2‐sensitive [125I]NKA binding sites were identical to those identified in the rat urinary bladder and duodenum. NKA, neuropeptide K, and neuropeptide γ, as well as the potent and selective NK2 antagonists SR 144190, SR 48968 and MEN 10627, presented a nanomolar affinity for these sites. The regional distribution of these NK2‐sensitive [125I]NKA binding sites differs markedly from those of NK1 and NK3 binding sites, with the largest labeling being found in the hippocampus, the thalamus and the septum. Binding in other brain structures was low or negligible. A preliminary autoradiographic analysis confirmed [125I]NKA selective binding in hippocampal CA1 and CA3 areas, particularly, and in several thalamic nuclei.
Mechanisms of prostaglandin E2‐induced interleukin‐6 release in astrocytes: possible involvement of EP4‐like receptors, p38 mitogen‐activated protein kinase and protein kinase C
Authors: Fiebich Bernd L. , Schleicher Sandra , Spleiss Olivia , Czygan Martin , Hüll Michael
Published: March 2003

The expression of cyclooxygenase‐2 (COX‐2) and the synthesis of prostaglandin E2 (PGE2) as well as of cytokines such as interleukin‐6 (IL‐6) have all been suggested to propagate neuropathology in different brain disorders such as HIV‐dementia, prion diseases, stroke and Alzheimer's disease. In this report, we show that PGE2‐stimulated IL‐6 release in U373 MG human astroglioma cells and primary rat astrocytes. PGE2‐induced intracellular cAMP formation was mediated via prostaglandin E receptor 2 (EP2), but inhibition of cAMP formation and protein kinase A or blockade of EP1/EP2 receptors did not affect PGE2‐induced IL‐6 synthesis. This indicates that the cAMP pathway is not part of PGE2‐induced signal transduction cascade leading to IL‐6 release. The EP3/EP1‐receptor agonist sulprostone failed to induce IL‐6 release, suggesting an involvement of EP4‐like receptors. PGE2‐activated p38 mitogen‐activated kinase (p38 MAPK) and protein kinase C (PKC). PGE2‐induced IL‐6 synthesis was inhibited by specific inhibitors of p38 MAPK (SB202190) and PKC (GF203190X). Although, up to now, EP receptors have only rarely been linked to p38 MAPK or PKC activation, these results suggest that PGE2 induces IL‐6 via an EP4‐like receptor by the activation of PKC and p38 MAPK via an EP4‐like receptor independently of cAMP.
Structural and functional characterization of the 5′ upstream promoter of the humanPhox2agene: possible direct transactivation by transcription factor Phox2b
Authors: Hong Seok Jong , Kim Chun‐Hyung , Kim Kwang‐Soo
Published: March 2003

The specification of neurotransmitter identity is a critical step in neural development. Recent progresses have indicated that the closely related homeodomain factors Phox2a and 2b are essential for development of noradrenergic (NA) neuron differentiation, and may directly determine the neurotransmitter identity. With a long‐term goal of understanding the regulatory cascade of NA phenotype determination, we isolated and characterized ahPhox2agenomic clone encompassing approximately 7.5 kb of the 5′ upstream promoter region, the entire exon–intron structure, and approximately 4 kb of the 3′ flanking region. Using mRNAs isolated from the Phox2a‐expressing human cell line, both primer extension and 5′‐rapid amplification of cDNA ends analyses identified a single transcription start site that resides 172 nucleotides upstream of the start codon. The transcription start site was preceded by a TATA‐like sequence motif and transcripts from this site contained an additional G residue at the 5′ position, supporting the authenticity of this site as the transcriptional start site ofhPhox2a. We assembled hPhox2a–luciferase reporter constructs containing different lengths of the 5′ upstream sequences. Transient transfection assays of these reporter constructs in bothhPhox2a‐positive and ‐negative cell lines show that 1.3‐kb or longer upstream sequences of thehPhox2agene may confer NA cell‐specific reporter gene expression. Furthermore, cotransfection assays in thePhox2a‐negative HeLa cell line show that forced expression of Phox2b, but not that of Phox2a or MASH1, significantly transactivates the transcriptional activity of hPhox2a. This study will provide a frame to further delineate the regulatory cascade of NA neuron differentiation.
Isolation and characterization of VGF peptides in rat brain. Role of PC1/3 and PC2 in the maturation of VGF precursor
Authors: Trani E. , Giorgi A. , Canu N. , Amadoro G. , Rinaldi A. M. , Halban P. A. , Ferri L. , Possenti R. , Schininà† M. E. , Levi A.
Published: March 2003

The neurotrophin responsive gene vgf is widely expressed in central and peripheral neurones, and in certain neuroendocrine cell populations. Its encoded VGF precursor protein (proVGF1: 617 amino acids in rat, 615 in man, > 85% homology) gives rise to several low molecular weight species. We studied a range of neuroendocrine and neuronal cells, in which VGF‐processing products were prominent with an apparent molecular weight of 20 and 10 kDa (VGF20 and VGF10, respectively). Such peptides were recognized by antibodies specific for the C‐terminal rat VGF nonapeptide, thus indicating that they included the C‐terminus of proVGF. Ectopic expression of the neuroendocrine‐specific prohormone convertases PC1/3 or PC2 in GH3 cells showed that both could generate VGF20, while VGF10 was preferentially produced by PC1/3. Site‐directed mutagenesis was used to identify the KRKRKK488 motif as the target within VGF sequence which leads to the production of VGF20. Molecular characterization of rat VGF10, on the other hand, revealed that this peptide is produced by cleavage at the RPR555 site. By the combined use of high‐resolution separation techniques, matrix‐assisted laser desorption/ionization time of flight (MALDI‐ToF) mass spectrometry and manual Edman degradation we identified in rat brain a VGF fragment analogous to bovine peptide V and two novel peptides also derived from the C‐terminal region of proVGF.
β‐Amyloid catabolism: roles for neprilysin (NEP) and other metallopeptidases?
Authors: Carson Julie A. , Turner Anthony J.
Published: March 2003

The steady‐state level of amyloid β‐peptide (Aβ) represents a balance between its biosynthesis from the amyloid precursor protein (APP) through the action of the β‐ and γ‐secretases and its catabolism by a variety of proteolytic enzymes. Recent attention has focused on members of the neprilysin (NEP) family of zinc metalloproteinases in amyloid metabolism. NEP itself degrades both Aβ1−40and Aβ1−42in vitroandin vivo, and this metabolism is prevented by NEP inhibitors. Other NEP family members, for example endothelin‐converting enzyme, may contribute to amyloid catabolism and may also play a role in neuroprotection. Another metalloproteinase, insulysin (insulin‐degrading enzyme) has also been advocated as an amyloid‐degrading enzyme and may contribute more generally to metabolism of amyloid‐forming peptides. Other candidate enzymes proposed include angiotensin‐converting enzyme, some matrix metalloproteinases, plasmin and, indirectly, thimet oligopeptidase (endopeptidase‐24.15). This review critically evaluates the evidence relating to proteinases implicated in amyloid catabolism. Therapeutic strategies aimed at promoting Aβ degradation may provide a novel approach to the therapy of Alzheimer's disease.
Age‐related loss of extrastriatal dopamine D2‐like receptors in women
Authors: Kaasinen Valtteri , Kemppainen Nina , Någren Kjell , Helenius Hans , Kurki Timo , Rinne Juha O.
Published: March 2003

Positron emission tomography (PET) studies have indicated that thein vivoavailability of dopamine D2‐like receptors declines with age in the human brain. Most of the studies have been carried out with healthy male subjects, or with subject groups containing both sexes. The authors have recently demonstrated that the availability of D2‐like receptors in the frontal cortex is higher in women than in men. The present study was aimed to further examine this phenomenon. Thirty‐seven healthy women (age range 22–78 years) were examined with PET and [11C]FLB 457, a high‐affinity tracer for the extrastriatal D2‐like receptors. A negative relationship between age and dopamine D2‐like receptor availability was seen in the frontal cortex (decrease of 12% per decade of life), the temporal cortex (9%) and the thalamus (6%). A non‐linear s‐shape association explained the relationship only in the frontal cortex, while in other regions the association was linear. Neither oestradiol nor progesterone levels had a significant relationship with the [11C]FLB 457 uptake in any of the brain regions studied after the effect of age was partialled out. The results indicate that: (i) the extrastriatal D2‐like receptor availability decreases with age in healthy women with the fastest rate in the frontal cortex and with the overall rate close to the rate reported in healthy men; (ii) around midlife (age 40–60 years) in women, the frontal receptor decline plateaus while the decline continues to be linear in other extrastriatal brain regions; and (iii) serum oestradiol or progesterone levels are not associated with cortical or thalamic D2‐like receptor availability in women. The results may prove to be important in studies where the biochemical basis of clinical sex differences is examined in patients with dopamine‐related neuropsychiatric disorders.