bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel transverse push–pull microprobe: in vitro characterization and in vivo demonstration of the enzymatic production of adenosine in the spinal cord dorsal horn
Authors: Patterson Shawnna L. , Sluka Kathleen A. , Arnold Mark A.
Published: March 2003

Adenosine produces analgesia in the spinal cord and can be formed extracellularly through enzymatic conversion of adenine nucleotides. A transverse push–pull microprobe was developed and characterized to sample extracellular adenosine concentrations of the dorsal horn of the rat spinal cord. Samples collected via this sampling technique reveal that AMP is converted to adenosine in the dorsal horn. This conversion is decreased by the ecto‐5′‐nucleotidase inhibitor, α,β‐methylene ADP. Related behavioral studies demonstrate that AMP administered directly to the spinal cord can reverse the secondary mechanical hyperalgesia characteristic of the intradermal capsaicin model of inflammatory pain. The specific adenosine A1 receptor antagonist 8‐cyclopentyl‐1,3‐dimethylxanthine (CPT) inhibits the antihyperalgesia produced by AMP. This research introduces a novel microprobe that can be used as an adjunct sampling technique to microdialysis and push–pull cannulas. Furthermore, we conclude that AMP is converted to adenosine in the dorsal horn of the spinal cord by ecto‐5′‐nucleotidase and subsequently may be one source of adenosine, acting through adenosine A1 receptors in the dorsal horn of the spinal cord, which produce antihyperalgesia.
An antisense oligodeoxynucleotide targeted to chromaffin cell scinderin gene decreased scinderin levels and inhibited depolarization‐induced cortical F‐actin disassembly and exocytosis
Authors: Lejen Tatiana , Skolnik Kathy , Rosé Sergio D. , Marcu Monica G. , Elzagallaai Abdelbaset , Trifaró José‐María
Published: March 2003

Chromaffin cell secretion requires cortical F‐actin disassembly and it has been suggested that scinderin, a Ca2+‐dependent F‐actin severing protein, controls cortical actin dynamics. An antisense oligodeoxynucleotide targeting the scinderin gene was used to decrease the expression of the protein and access its role in secretion. Treatment with 2 µm scinderin antisense oligodeoxynucleotide for 4 days produced a significant decrease in scinderin expression and its mRNA levels. The expression of gelsolin, another F‐actin severing protein, was not affected. Scinderin decrease was accompanied by concomitant and parallel decreases in depolarization‐evoked cortical F‐actin disassembly and exocytosis. Similar treatment with a mismatched oligodeoxynucleotide produced no effects. Scinderin antisense oligodeoxynucleotide treatment was also a very effective inhibitor of exocytosis in digitonin‐permeabilized cells stimulated with increasing concentrations of Ca2+. This ruled out scinderin antisense interference with stimulation‐induced depolarization or Ca2+ channel activation. Scinderin antisense treatment decreased the maximum (Bmax) secretory response to Ca2+ without modifying the affinity (Km) of the cation for the exocytotic machinery. Moreover, the antisense treatment did not affect norepinephrine uptake or the expression of dopamine β‐hydroxylase, suggesting that the number and function of chromaffin vesicles was not modified. In addition, scinderin antisense treatment did not alter the expression of proteins involved in vesicle–plasma membrane fusion, such as synaptophysin, synaptotagmin or syntaxin, indicating a lack of effects on the fusion machinery components. These observations strongly suggest that scinderin is a key player in the events involved in the secretory process.
Cyclic AMP‐dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of α4 neuronal nicotinic receptor subunits
Authors: Wecker Lynn , Guo Xiang , Rycerz Anthony M. , Edwards Samuel C.
Published: March 2003

To determine whether α4 subunits of α4β2 neuronal nicotinic receptors are phosphorylated within the M3/M4 intracellular region by cyclic AMP‐dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecipitated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of α4 (α4336−597) were incubated with ATP and either PKA or PKC. Both α4 and α4336−597 were phosphorylated by PKA and PKC, providing the first direct biochemical evidence that the M3/M4 cytoplasmic domain of neuronal nicotinic receptor α4 subunits is phosphorylated by both kinases. When the immunoprecipitated receptors and the α4336−597 fusion protein were phosphorylated and the labeled proteins subjected to phosphoamino acid analysis, results indicated that α4 and α4336−597 were phosphorylated on the same amino acid residues by each kinase. Furthermore, PKA phosphorylated serines exclusively, whereas PKC phosphorylated both serines and threonines. To determine whether Ser368 was a substrate for both kinases, a peptide corresponding to amino acids 356–371 was synthesized (α4356−371) and incubated with ATP and the kinases. The phosphorylation of α4356−371 by both PKA and PKC was saturable with Kms of 15.3 ± 3.3 µm and 160.8 ± 26.8 µm, respectively, suggesting that Ser368 was a better substrate for PKA than PKC.
Regulation of the tyrosine hydroxylase and dopamine β‐hydroxylase genes by the transcription factor AP‐2
Authors: Kim Hee‐Sun , Hong Seok Jong , LeDoux Mark S. , Kim Kwang‐Soo
Published: March 2003

The retinoic acid‐inducible and developmentally regulated transcription factor AP‐2 plays an important role during development. In adult mammals, AP‐2 is expressed in both neural and non‐neural tissues. However, the function of AP‐2 in different neuronal phenotypes is poorly understood. In this study, transcriptional regulation of tyrosine hydroxylase (TH) and dopamine β‐hydroxylase (DBH) genes by AP‐2 was investigated. AP‐2 binding sites were identified in the upstream regions of both genes. Electrophoretic mobility shift assays (EMSA) and DNase I footprinting analyses indicate that the AP−2 interaction with these motifs is more prominent in catecholaminergic SK‐N‐BE(2)C and CATH.a than in non‐catecholaminergic HeLa and HepG2 cell lines. Exogenous expression of AP‐2 robustly transactivated TH and DBH promoter activities in non‐catecholaminergic cell lines. While AP‐2 regulates the DBH promoter activity via a single site, transactivation of the TH promoter by AP‐2 appears to require multiple sites. In support of this, mutation of multiple AP‐2 binding sites but not that of single site diminished the basal promoter activity of the TH gene in cell lines that express TH and abolished transactivation by exogenous AP‐2 expression in cell lines that do not express TH. In contrast, mutation of a single AP‐2 binding site of the DBH gene completely abolished transactivation by AP‐2. Double‐label immunohistochemistry showed that AP‐2 is coexpressed with TH in noradrenergic and adrenergic neurons in both the central and peripheral nervous systems of adult rodents. Numerous non‐catecholaminergic cell groups within the spinal cord, medulla, cerebellum, and pons also express AP‐2. The concentration of AP‐2 in dorsomedial locations along the neuraxis suggests a regionally specific role for this transcription factor in the regulation of neuronal function. Based on these findings we propose that AP‐2 may coregulate TH and DBH gene expression and thus participate in expression/maintenance of neurotransmitter phenotypes in (nor)adrenergic neurons and neuroendocrine cells.
ApoE protects cortical neurones against neurotoxicity induced by the non‐fibrillar C‐terminal domain of the amyloid‐β peptide
Authors: Drouet Béatrice , Fifre Alexandre , Pinçon‐Raymond Martine , Vandekerckhove Joël , Rosseneu Maryvonne , Guéant Jean‐Louis , Chambaz Jean , Pillot Thierry
Published: March 2003

Although the genetic link between the ε4 allele of apolipoprotein E (apoE) and Alzheimer's disease (AD) is well established, the apoE isoform‐specific activity underlying this correlation remains unclear. We have recently characterized the interaction of the soluble the amyloid‐β peptide (Aβ) with model membrane and demonstrated that non‐fibrillar Aβ peptide, including N‐terminal truncated forms of Aβ, induced apoptotic cell death in primary rat cortical neurones in vitro. To further investigate the potential interaction between apoE and Aβ in the pathogenesis of AD, we have determined the effect of apoE isoforms on the neurotoxicity of non‐fibrillar Aβ peptides. We demonstrate here that the apoE2 and E3 isoforms protect cortical neurones against apoptotic cell death induced by a non‐fibrillar form of the Aβ1−40, Aβ12−42, Aβ29−40 and Aβ29−42 peptides, whereas apoE4 had no effect. This effect involves the formation of stable complexes between apoE and the C‐terminal domain (e.g. amino acids 29–40) of Aβ1−40. Interestingly, apoE had no effect on the toxicity induced by aggregated Aβ peptides, suggesting a lack of interaction between apoE and amyloid fibrils. Our results provide evidence that interaction with the C‐terminal domain of Aβ, apoE2 and E3, but not apoE4, inhibits the interactions of the non‐fibrillar Aβ peptide with the plasma membrane of neurones, Aβ peptide aggregation and subsequent neurotoxicity.
The octadecaneuropeptide ODN stimulates neurosteroid biosynthesis through activation of central‐type benzodiazepine receptors
Authors: Do‐Rego Jean‐Luc , Mensah‐Nyagan Ayikoe Guy , Beaujean Delphine , Leprince Jérôme , Tonon Marie‐Christine , Luu‐The Van , Pelletier Georges , Vaudry Hubert
Published: March 2003

Neurosteroids may play a major role in the regulation of various neurophysiological and behavioural processes. However, while the biochemical pathways involved in the synthesis of neuroactive steroids in the central nervous system are now elucidated, the mechanisms controlling the activity of neurosteroid‐producing cells remain almost completely unknown. In the present study, we have investigated the effect of the octadecaneuropeptide (ODN), an endogenous ligand of benzodiazepine receptors, in the control of steroid biosynthesis in the frog hypothalamus. Glial cells containing ODN‐like immunoreactivity were found to send their thick processes in the close vicinity of neurones expressing the steroidogenic enzyme 3β‐hydroxysteroid dehydrogenase. Exposure of frog hypothalamic explants to graded concentrations of ODN (10−10−10−5m) produced a dose‐dependent increase in the conversion of tritiated pregnenolone into various radioactive steroids, including 17‐hydroxypregnenolone, progesterone, 17‐hydroxyprogesterone, dehydroepiandrosterone and dihydrotestosterone. The ODN‐induced stimulation of neurosteroid biosynthesis was mimicked by the central‐type benzodiazepine receptor (CBR) inverse agonists methyl β‐carboline‐3‐carboxylate (β‐CCM) and methyl 6,7‐dimethoxy‐4‐ethyl‐β‐carboline‐3‐carboxylate (DMCM). The stimulatory effects of ODN, β‐CCM and DMCM on steroid formation was markedly reduced by the CBR antagonist flumazenil. The ODN‐evoked stimulation of neurosteroid production was also significantly attenuated by GABA. Collectively, these data indicate that the endozepine ODN, released by glial cell processes in the vicinity of 3β‐hydroxysteroid dehydrogenase‐containing neurones, stimulates the biosynthesis of neurosteroids through activation of central‐type benzodiazepines receptors.
5‐HT1B Autoreceptors limit the effects of selective serotonin re‐uptake inhibitors in mouse hippocampus and frontal cortex
Authors: Malagié Isabelle , Trillat Anne‐Cécile , Bourin Michel , Jacquot Christian , Hen René , Gardier Alain M.
Published: March 2003

We used knockout mice and receptor antagonist strategies to investigate the contribution of the serotonin (5‐hydroxytryptamine, 5‐HT) 1B receptor subtype in mediating the effects of selective serotonin re‐uptake inhibitors (SSRIs). Using in vivo intracerebral microdialysis in awake mice, we show that a single systemic administration of paroxetine (1 or 5 mg/kg, i.p.) increased extracellular serotonin levels [5‐HT]ext in the ventral hippocampus and frontal cortex of wild‐type and mutant mice. However, in the ventral hippocampus, paroxetine at the two doses studied induced a larger increase in [5‐HT]ext in knockout than in wild‐type mice. In the frontal cortex, the effect of paroxetine was larger in mutants than in wild‐type mice at the 1 mg/kg, but not at 5 mg/kg. In addition, either the absence of the 5‐HT1B receptor or its blockade with the mixed 5‐HT1B/1D receptor antagonist, GR 127935, potentiated the effect of a single administration of paroxetine on extracellular 5‐HT levels more in the ventral hippocampus than in the frontal cortex. These data suggest that 5‐HT1B autoreceptors limit the effects of SSRIs on dialysate 5‐HT levels at serotonergic nerve terminals.
Heregulin upregulates the expression of nitric oxide synthase (NOS)‐1 in rat cerebellar granule neurons via the ErbB4 receptor
Authors: Krainock Randy , Murphy Sean
Published: March 2003

Heregulin plays key roles in regulating cell number, determining fate and establishing pattern in the developing nervous system via specific receptors (ErbBs), including ErbB4. Two recent reports have shown that ErbB4 forms a complex with postsynaptic density proteins, which are, in turn, known to complex with nitric oxide synthase (NOS)‐1. To reveal whether heregulin might regulate the expression of NOS‐1, cultures enriched in cerebellar granule cells were exposed to heregulin for 72 h. This treatment resulted in an increase in NOS‐1 protein (> 70%), an effect mediated by the ErbB4 receptor. While nitric oxide might mediate some of the downstream effects of heregulin in the nervous system, heregulin treatment neither enhanced granule cell survival, nor protected neurons from acute glutamate excitotoxicity.
Parallel modification of adenosine extracellular metabolism and modulatory action in the hippocampus of aged rats
Authors: Cunha Rodrigo A. , Almeida Teresa , Ribeiro J. A.
Published: March 2003

The neuromodulator adenosine can be released as such, mainly activating inhibitory A1 receptors, or formed from released ATP, preferentially activating facilitatory A2A receptors. We tested if changes in extracellular adenosine metabolism paralleled changes in A1/A2A receptor neuromodulation in the aged rat hippocampus. The evoked release and extracellular catabolism of ATP were 49–55% lower in aged rats, but ecto‐5′‐nucleotidase activity, which forms adenosine, was 5‐fold higher whereas adenosine uptake was decreased by 50% in aged rats. The evoked extracellular adenosine accumulation was 30% greater in aged rats and there was a greater contribution of the ecto‐nucleotidase pathway and a lower contribution of adenosine transporters for extracellular adenosine formation in nerve terminals. Interestingly, a supramaximal concentration of an A1 receptor agonist, N6‐cyclopentyladenosine (250 nm) was less efficient in inhibiting (17% in old versus 34% in young) and A2A receptor activation with 30 nm CGS21680 was more efficient in facilitating (63% in old versus no effect in young) acetylcholine release from hippocampal slices of aged compared with young rats. The parallel changes in the metabolic sources of extracellular adenosine and A1/A2A receptor neuromodulation in aged rats further strengthens the idea that different metabolic sources of extracellular adenosine are designed to preferentially activate different adenosine receptor subtypes.
Light scattering and transmission electron microscopy studies reveal a mechanism for calcium/calmodulin‐dependent protein kinase II self‐association
Authors: Hudmon Andy , Kim Sally A. , Kolb Stephen J. , Stoops James K. , Waxham M. Neal
Published: March 2003

Calmodulin (CaM)‐kinase II holoenzymes composed of either α or β subunits were analyzed using light scattering to determine a mechanism for self‐association. Under identical reaction conditions, only αCaM‐kinase II holoenzymes self‐associated. Self‐association was detected at a remarkably low enzyme concentration (0.14 µmor 7 µg/mL). Light scattering revealed two phases of self‐association: a rapid rise that peaked, followed by a slower decrease that stabilized after 2–3 min. Electron microscopy identified that the rapid rise in scattering was due to the formation of loosely packed clusters of holoenzymes that undergo further association into large complexes of several microns in diameter over time. Self‐association required activation by Ca2+/CaM and was strongly dependent on pH. Self‐association was not detected at pH 7.5, however, the extent of this process increased as reaction pH decreased below 7.0. A peptide substrate (autocamtide‐2) and inhibitor (AIP) designed from the autoregulatory domain of CaM‐kinase II potently prevented self‐association, whereas the peptide substrate syntide‐2 did not. Thus, CaM‐kinase II self‐association is isoform specific, regulated by the conditions of activation, and is inhibited by peptides that bind to the catalytic domain likely via their autoregulatory‐like sequence. A model for CaM‐kinase II self‐association is presented whereby catalytic domains in one holoenzyme interact with the regulatory domains in neighboring holoenzymes. These intersubunit–interholoenzyme autoinhibitory interactions could contribute to both the translocation and inactivation of CaM‐kinase II previously reported in models of ischemia.
Paracrine neuroprotective effect of nitric oxide in the developing retina
Authors: Guimarães Cinthya A. , Assreuy Jamil , Linden Rafael
Published: March 2003

The retina of newborn rats consists of the ganglion cell layer (GCL), the inner plexiform layer (IPL), the inner nuclear layer (INL) containing amacrine cells and the neuroblastic layer (NBL). In retinal explants, the GCL enters cell death after sectioning of the optic nerve, whereas there is almost no cell death in the NBL. When protein synthesis is inhibited with anisomycin, cell death is blocked in the GCL and induced in the NBL. We tested the roles of nitric oxide (NO) on cell death in the retina in vitro. Either l‐arginine, the substrate for NO synthase or the NO donor S‐nitroso‐acetylpenicillamine (SNAP) blocked cell death induced by anisomycin in the NBL, but had no effect in the GCL. Sepiapterin, a precursor of the nitric oxide synthase (NOS)‐cofactor tetrahydrobiopterin also had a protective effect against anisomycin. The use of 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one, an inhibitor of soluble form of guanylyl cyclase, showed that anti‐apoptotic effect of SNAP is partially mediated by cGMP generated by activation of guanylyl cyclase. NADPH‐diaphorase histochemistry stained cells only in the GCL and INL. Thus, the degenerative effect of anisomycin is observed within the NBL, whereas the localization of NOS is restricted to the GCL and INL. The protective effect of both the NO substrate and cofactor upon cell death induced by anisomycin in the NBL, indicates that NO produced by amacrine and ganglion cells is a paracrine modulator of cell death within the retinal tissue.
Soluble N‐cadherin stimulates fibroblast growth factor receptor dependent neurite outgrowth and N‐cadherin and the fibroblast growth factor receptor co‐cluster in cells
Authors: Utton Michelle A. , Eickholt Britta , Howell Fiona V. , Wallis Julie , Doherty Patrick
Published: March 2003

A chimeric molecule consisting of the extracellular domain of the adhesion molecule, N‐cadherin, fused to the Fc region of human IgG (NCAD‐Fc) supports calcium‐dependent cell adhesion and promotes neurite outgrowth following affinity‐capture to a tissue culture substrate. When presented to cerebellar neurons as a soluble molecule, the NCAD‐Fc stimulated neurite outgrowth in a manner equivalent to that seen for N‐cadherin expressed as a cell surface glycoprotein. Neurons expressing a dominant‐negative version of the fibroblast growth factor (FGF) receptor did not respond to soluble NCAD‐Fc. In cells transfected with full‐length N‐cadherin and the FGF receptor, antibody‐clustering of N‐cadherin resulted in a co‐clustering of the FGF receptor to discrete patches in the cell membrane. The data demonstrate that the ability of N‐cadherin to stimulate neurite outgrowth can be dissociated from its ability to function as a substrate associated adhesion molecule. The N‐cadherin and the FGF receptor co‐clustering in cells provides a basis for the neurite outgrowth response stimulated by N‐cadherin being dependent on FGF receptor function.
Is Bax a mitochondrial mediator in apoptotic death of dopaminergic neurons in Parkinson's disease?
Authors: Hartmann Andreas , Michel Patrick P. , Troadec Jean‐Denis , Mouatt‐Prigent Annick , Faucheux Baptiste A. , Ruberg Merle , Agid Yves , Hirsch Etienne C.
Published: March 2003

Bax is a proapoptotic member of the Bcl‐2 family of proteins. It is believed to exert its action primarily by facilitating the release of cytochrome c from the mitochondrial intermembrane space into the cytosol, leading to caspase activation and cell death. Because alterations in mitochondrial respiratory function, caspase activation and cell death with morphologic features compatible with apoptosis have been observed post mortem in the brain of patients with Parkinson's disease, we tried to clarify the potential role of Bax in this process in an immunohistochemical study on normal and Parkinson's disease post‐mortem brain and primary mesencephalic cell cultures treated with MPP+. We found that Bax is expressed ubiquitously by dopaminergic (DA) neurons in post‐mortem brain of normal and Parkinson's disease subjects as well as in vitro. Using an antibody to Bax inserted into the outer mitochondrial membrane as an index of Bax activation, no significant differences were observed between control and Parkinson's disease subjects, regardless of the mesencephalic subregion analysed. However, in Parkinson's disease subjects, the percentage of Bax‐positive melanized SNpc neurons containing Lewy bodies, suggestive of DA neuronal suffering, was significantly higher than the overall percentage of Bax‐positive neurons among melanized neurons. Furthermore, all melanized SNpc neurons in Parkinson's disease subjects with activated caspase‐3 were also immunoreactive for Bax, suggesting that Bax anchored in the outer mitochondrial membrane of melanized SNpc neurons showing signs of neuronal suffering or apoptosis is increased compared with DA neurons that are apparently unaltered. Surprisingly, MPP+ treatment of tyrosine hydroxylase (TH)‐positive neurons in primary mesencephalic cultures did not cause redistribution of Bax, although cytochrome c was released from the mitochondria and nuclear condensation/fragmentation was induced. Taken together, these findings suggest that in the human pathology, Bax may be a cofactor in caspase activation, but our in vitro data fail to indicate a central role for Bax in apoptotic death of DA neurons in an experimental Parkinson's disease paradigm.
Tissue distribution and processing of proSAAS by proprotein convertases
Authors: Sayah M. , Fortenberry Y. , Cameron A. , Lindberg I.
Published: March 2003

The conversion of inactive precursor proteins into bioactive neuropeptides and peptide hormones involves regulated secretory proteins such as prohormone convertases PC1 and PC2. The neuroendocrine protein 7B2 represents a specific binding protein for PC2, and the protein proSAAS, which interacts with PC1, exhibits certain structural and functional homologies with 7B2. With the intention of better understanding the physiological role of proSAAS and its derived peptides, we investigated its tissue localization using a new radioimmunoassay (RIA) to a C‐terminal proSAAS‐derived peptide. Immunoreactivity corresponding to this SAAS‐derived peptide is mostly localized to the brain and gut. Analysis of the brain distribution of the proSAAS‐derived peptides indicates that the hypothalamus and pituitary are the two richest areas, consistent with the previously described high expression of PC1 in these two areas. In order to investigate the cleavage of proSAAS by prohormone convertases, we incubated recombinant His‐tagged proSAAS with recombinant mouse proPC2 or furin, separated the cleavage products using high‐pressure gel permeation chromatography and analyzed the products by RIA. Our results indicate that either PC2 or furin can accomplish in vitro rapid removal and efficient internal processing of the C‐terminal peptide, exposing the inhibitory hexapeptide to possible further digestion by carboxypeptidases. Finally, we also studied proSAAS processing in the brains of wild‐type and PC2 null mice and found that proSAAS is efficiently processed in vivo. Whereas the C‐terminal peptide is mostly internally cleaved in wild‐type mouse brain, it is not processed as efficiently in the brain of PC2 null mice, suggesting that PC2 is partially responsible for this cleavage in vivo.
Adjuvant‐induced joint inflammation causes very rapid transcription of β‐preprotachykinin and α‐CGRP genes in innervating sensory ganglia
Authors: Bulling Duncan G. S. , Kelly David , Bond Susan , McQueen Daniel S. , Seckl Jonathan R.
Published: March 2003

Neuropeptides synthesized in dorsal root ganglia (DRG) have been implicated in neurogenic inflammation and nociception in experimental and clinical inflammatory arthritis. We examined the very early changes in response to adjuvant injection in a rat model of unilateral tibio‐tarsal joint inflammation and subsequent monoarthritis. Within 30 min of adjuvant injection ipsilateral swelling and hyperalgesia were apparent, and marked increases in β‐preprotachykinin‐A (β‐PPT‐A) and α‐calcitonin gene‐related peptide (CGRP)‐encoding mRNAs were observed in small‐diameter L5 DRG neurones innervating the affected joint. This response was augmented by recruitment of additional small‐diameter DRG neurones expressing β‐PPT‐A and CGRP transcripts. The increased mRNA was paralleled by initial increases in L5 DRG content of the protein products, substance P and calcitonin gene‐related peptide. Within 15 min of adjuvant injection there were increases in electrical activity in sensory nerves innervating a joint. Blockade of this activity prevented the rapid induction in β‐PPT‐A and CGRP mRNA expression in DRG neurones. Increased expression of heteronuclear (intron E) β‐PPT‐A RNA suggests that increases in β‐PPT‐A mRNA levels were, at least in part, due to transcription. Pre‐treatment with the protein synthesis inhibitor cycloheximide had no effect upon the early rise in neuropeptide mRNAs. This and the rapid time course of these changes suggest that increased sensory neural discharge and activation of a latent modulator of transcription are involved.
Homocysteine potentiates copper‐ and amyloid beta peptide‐mediated toxicity in primary neuronal cultures: possible risk factors in the Alzheimer's‐type neurodegenerative pathways
Authors: White Anthony R. , Huang Xudong , Jobling Michael F. , Barrow Colin J. , Beyreuther Konrad , Masters Colin L. , Bush Ashley I. , Cappai Roberto
Published: March 2003

Oxidative stress may have an important role in the progression of neurodegenerative disorders such as Alzheimer's disease (AD) and prion diseases. Oxidative damage could result from interactions between highly reactive transition metals such as copper (Cu) and endogenous reducing and/or oxidizing molecules in the brain. One such molecule, homocysteine, a thiol‐containing amino acid, has previously been shown to modulate Cu toxicity in HeLa and endothelial cells in vitro. Due to a possible link between hyperhomocysteinemia and AD, we examined whether interaction between homocysteine and Cu could potentiate Cu neurotoxicity. Primary mouse neuronal cultures were treated with homocysteine and either Cu (II), Fe (II or III) or Zn (II). Homocysteine was shown to selectively potentiate toxicity from low micromolar concentrations of Cu. The toxicity of homocysteine/Cu coincubation was dependent on the ability of homocysteine to reduce Cu (II) as reflected by the inhibition of toxicity with the Cu (I)‐specific chelator, bathocuproine disulphonate. This was supported by data showing that homocysteine reduced Cu (II) more effectively than cysteine or methionine but did not reduce Fe (III) to Fe (II). Homocysteine also generated high levels of hydrogen peroxide in the presence of Cu (II) and promoted Aβ/Cu‐mediated hydrogen peroxide production and neurotoxicity. The potentiation of metal toxicity did not involve excitotoxicity as ionotropic glutamate receptor antagonists had no effect on neurotoxicity. Homocysteine alone also had no effect on neuronal glutathione levels. These studies suggest that increased copper and/or homocysteine levels in the elderly could promote significant oxidant damage to neurons and may represent additional risk factor pathways which conspire to produce AD or related neurodegenerative conditions.
Activation of 5‐HT1A but not 5‐HT1B receptors attenuates an increase in extracellular dopamine derived from exogenously administered l‐DOPA in the striatum with nigrostriatal denervation
Authors: Kannari Kazuya , Yamato Hiroshi , Shen Huo , Tomiyama Masahiko , Suda Toshihiro , Matsunaga Muneo
Published: March 2003

In order to determine whether l‐DOPA‐derived extracellular dopamine (DA) in the striatum with dopaminergic denervation is affected by activation of serotonin autoreceptors (5‐HT1A and 5‐HT1B receptors), we applied in vivo brain microdialysis technique to 6‐hydroxydopamine‐lesioned rats and examined the effects of the selective 5‐HT1A receptor agonist 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT) and the selective 5‐HT1B receptor agonist CGS‐12066 A on l‐DOPA‐derived extracellular DA levels. Single l‐DOPA injection (50 mg/kg i.p.) caused a rapid increase and a following decrease of extracellular DA, with a peak value at 100 min after l‐DOPA injection. Pretreatment with both 0.3 mg/kg and 1 mg/kg 8‐OH‐DPAT (i.p.) significantly attenuated an increase in l‐DOPA‐derived extracellular DA and the times of peak DA levels were prolonged to 150 min and 225 min after l‐DOPA injection, respectively. These 8‐OH‐DPAT‐induced changes in l‐DOPA‐derived extracellular DA were antagonized by further pretreatment with WAY‐100635, a selective 5‐HT1A antagonist. In contrast, intrastriatal perfusion with the 5‐HT1B agonist CGS‐12066 A (10 nm and 100 nm) did not induce any changes in l‐DOPA‐derived extracellular DA. Thus, stimulation of 5‐HT1A but not 5‐HT1B receptors attenuated an increase in extracellular DA derived from exogenous l‐DOPA. These results support the hypothesis that serotonergic neurons are primarily responsible for the storage and release of DA derived from exogenous l‐DOPA in the absence of dopaminergic neurons.
The human δ opioid receptor activates Gi1α more efficiently than Go1α
Authors: Moon Hyo‐Eun , Cavalli Antonella , Bahia Daljit S. , Hoffmann Marcel , Massotte Dominique , Milligan Graeme
Published: March 2003

To assess the relative capacity of the human δ opioid receptor to activate closely related G proteins, fusion proteins were constructed in which the α‐subunits of either Gi1 or Go1, containing point mutations to render them insensitive to the actions of pertussis toxin, were linked in‐frame with the C‐terminus of the receptor. Following transient and stable expression in HEK 293 cells, both constructs bound the antagonist [3H]naltrindole with high affinity. d‐ala2,d‐leu5 Enkephalin effectively inhibited forskolin‐stimulated adenylyl cyclase activity in intact cells in a concentration‐dependent, but pertussis toxin‐insensitive, manner. The high‐affinity GTPase activity of both constructs was also stimulated by d‐ala2,d‐leu5 enkephalin with similar potency. However, enzyme kinetic analysis of agonist stimulation of GTPase activity demonstrated that the GTP turnover number produced in response to d‐ala2,d‐leu5 enkephalin was more than three times greater for Gi1α than for Go1α. As the effect of agonist in both cases was to increase Vmax without increasing the observed Km for GTP, this is consistent with receptor promoting greater guanine nucleotide exchange, and thus activation, of Gi1α compared with Go1α. An equivalent fusion protein between the human µ opioid receptor‐1 and Gi1α produced a similar d‐ala2,d‐leu5 enkephalin‐induced GTP turnover number as the δ opioid receptor−Gi1α fusion construct, consistent with agonist occupation of these two opioid receptor subtypes being equally efficiently coupled to activation of Gi1α.
Inhibition of vesicular glutamate storage and exocytotic release by Rose Bengal
Authors: Ogita Kiyokazu , Hirata Koji , Bole David G. , Yoshida Sumiko , Tamura Yutaka , Leckenby Anne Marie , Ueda Tetsufumi
Published: March 2003

It had been thought that quantal size in synaptic transmission is invariable. Evidence has been emerging, however, that quantal size can be varied under certain conditions. We present evidence that alteration in vesicular [3H]l‐glutamate (Glu) content within the synaptosome (a pinched‐off nerve ending preparation) leads to a change in the amount of exocytotically released [3H]Glu. We found that Rose Bengal, a polyhalogenated fluorescein derivative, is a quite potent membrane‐permeant inhibitor (Ki = 19 nm) of glutamate uptake into isolated synaptic vesicles. This vesicular Glu uptake inhibition was achieved largely without affecting H+‐pump ATPase. We show that various degrees of reduction elicited by Rose Bengal in [3H]Glu in synaptic vesicles inside the synaptosome result in a corresponding decrease in the amount of [3H]Glu released in a depolarization‐ (induced by 4‐aminopyridine) and Ca2+‐dependent manner. In contrast, fluorescein, the halogen‐free analog of Rose Bengal, which is devoid of inhibitory activity on vesicular [3H]Glu uptake, failed to change the amount of exocytotically released [3H]Glu. These observations suggest that glutamate synaptic transmission could be altered by pharmacological intervention of glutamate uptake into synaptic vesicles in the nerve terminal, a new mode of synaptic manipulation for glutamate transmission.
Gαolf is necessary for coupling D1 and A2a receptors to adenylyl cyclase in the striatum
Authors: Corvol J. C. , Studler J. M. , Schonn J. S. , Girault J. A. , Hervé D.
Published: March 2003

In the brain, dopamine and adenosine stimulate cyclic AMP (cAMP) production through D1 and A2a receptors, respectively. Using mutant mice deficient in the olfactory isoform of the stimulatory GTP‐binding protein α subunit, Gαolf, we demonstrate here the obligatory role of this protein in the adenylyl cyclase responses to dopamine and adenosine in the caudate putamen. Responses to dopamine were also dramatically decreased in the nucleus accumbens but remained unaffected in the prefrontal cortex. Moreover, in the caudate putamen of mice heterozygous for the mutation, the amounts of Gαolf were half of the normal levels, and the efficacy of dopamine‐ and CGS 21680 A2 agonist‐stimulated cAMP production was decreased. Together, these results identify Gαolf as a critical parameter in the responses to dopamine and adenosine in the basal ganglia.
Tyrosine phosphorylation of insulin receptor substrate‐1 (IRS‐1) by oxidant stress in cerebellar granule neurons: modulation by N‐methyl‐d‐aspartate through calcineurin activity
Authors: Hallak Hazem , Ramadan Bassel , Rubin Raphael
Published: March 2003

Insulin receptor‐substrate‐1 (IRS‐1) is a docking protein for several tyrosine kinase receptors. Upon tyrosine phosphorylation, IRS‐1 binds to signaling molecules that express Src homology 2 (SH‐2) binding domains, including phosphatidylinositol 3‐kinase (PI 3‐kinase), phosphotyrosine phosphatase SHP‐2 (Syp), Nck, Crk and Grb‐2. Hydrogen peroxide (H2O2) induces tyrosine phosphorylation of key signaling mediators presumably by inhibition of tyrosine phosphatases. In many cell types, the activation of extracellular signal‐related kinases (e.g. MAPK) and other protein kinases by H2O2 leads to transcriptional activation. In the current study, we examined the effect of H2O2 on IRS‐1 tyrosine phosphorylation in primary cultured rat cerebellar granule neurons. H2O2 stimulated the rapid tyrosine phosphorylation of IRS‐1 and p42/p44 MAP kinase, and induced its association with PI 3‐kinase. H2O2‐induced IRS‐1 phosphorylation was rapidly reversible (5 min) whereas MAPK phosphorylation persisted for up to 1 h. NMDA reversed H2O2‐mediated tyrosine phosphorylation of IRS‐1 and its association with PI 3‐kinase. The dephosphorylation of IRS‐1 by NMDA was calcium‐dependent and was inhibited by the calcineurin inhibitor cyclosporine. Calmodulin‐dependent tyrosine phosphatase activity of calcineurin was observed in vitro using both immunoprecipitated and recombinant tyrosine‐phosphorylated IRS‐1 as substrates. These data highlight the role of multiple phosphatases in the regulation of IRS‐1 tyrosine phosphorylation and identify a novel functional property of calcineurin.
Enrichment of N‐methyl‐d‐aspartate NR1 splice variants and synaptic proteins in rat postsynaptic densities
Authors: Al‐Hallaq Rana A. , Yasuda Robert P. , Wolfe Barry B.
Published: March 2003

Previous studies have suggested that the localization of the NMDA receptor NR1 subunit may be determined by the splice variant form of NR1 present. Functional studies have also supported selective targeting of NR2A and NR2B to synaptic and extrasynaptic populations, respectively. We set out to determine whether rat cortical and cerebellar NR1 splice variants and NR2 subunits are differentially localized to the postsynaptic density. Using western blot techniques, we measured the percentage of NR1 containing each cassette and the enrichment of the different cassettes and other proteins in the preparations. The results indicate that: (1) no single cassette of NR1 is differentially enriched in the postsynaptic densities and (2) the NR2A and NR2B subunits are similarly enriched at the synapse. The enrichment profiles of postsynaptic density‐associated proteins demonstrated similar enrichment levels for postsynaptic density (PSD)‐95, the NMDA receptor subunits, chapsyn‐110, and the CaMKII α subunit. However, synaptophysin, SAP‐102, and the GABAA receptor β subunit exhibited lower enrichment levels compared to PSD‐95. Additionally, cerebellar but not cortical PSDs exhibited significantly lower enrichment of alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) GluR1. Thus, although postsynaptic densities are highly enriched in synaptic proteins, there appears to be no selective incorporation of specific NR1 splice variants or NR2 subunits into this structure.
Depletion of glutathione up‐regulates mitochondrial complex I expression in glial cells
Authors: Vásquez Osmán L. , Almeida Ángeles , Bolaños Juan P.
Published: March 2003

Glutathione deficiency is commonly associated with mitochondrial complex I dysfunction and loss of viability in neurones, but not in glia. In order to address the possible mechanism responsible for this cellular difference, the regulation of mitochondrial complex I expression by glutathione depletion was investigated in glial cells. Incubation of rat‐cultured astrocytes and C6 glioma cells with the specific γ‐glutamylcysteine synthetase inhibitor l‐buthionine‐(S,R)‐sulfoximine (l‐BSO; 0.1–1 mm) decreased the total specific content of glutathione in a dose‐ and time‐dependent fashion. Northern blot analyses revealed that glutathione deficiency caused by l‐BSO (0.1 mm) was associated with a twofold enhancement in complex I regulatory subunit ND6 (mitochondrially encoded) mRNA expression after 24–72 h. This effect was accompanied by a twofold increase in complex‐I activity at 72 h in l‐BSO‐treated cells, as compared with control cells, but complex II–III, complex IV and citrate synthase activities were unaltered. It is suggested that the oxidative stress caused by glutathione depletion in glial cells would up‐regulate complex‐I activity by enhancing the expression of the mitochondrially encoded regulatory subunit. These results could offer further insight into the different degree of cellular susceptibility observed in glial vs. neuronal cells against oxidative stress.
Group I metabotropic glutamate receptors are expressed in the chicken retina and by cultured retinal amacrine cells
Authors: Kreimborg Kathryn M. , Lester Martha L. , Medler Kathryn F. , Gleason Evanna L.
Published: March 2003

Glutamate is well established as an excitatory neurotransmitter in the vertebrate retina. Its role as a modulator of retinal function, however, is poorly understood. We used immunocytochemistry and calcium imaging techniques to investigate whether metabotropic glutamate receptors are expressed in the chicken retina and by identified GABAergic amacrine cells in culture. Antibody labeling for both metabotropic glutamate receptors 1 and 5 in the retina was consistent with their expression by amacrine cells as well as by other retinal cell types. In double‐labeling experiments, most metabotropic glutamate receptor 1‐positive cell bodies in the inner nuclear layer also label with anti‐GABA antibodies. GABAergic amacrine cells in culture were also labeled by metabotropic glutamate receptor 1 and 5 antibodies. Metabotropic glutamate receptor agonists elicited Ca2+ elevations in cultured amacrine cells, indicating that these receptors were functionally expressed. Cytosolic Ca2+ elevations were enhanced by metabotropic glutamate receptor 1‐selective antagonists, suggesting that metabotropic glutamate receptor 1 activity might normally inhibit the Ca2+ signaling activity of metabotropic glutamate receptor 5. These results demonstrate expression of group I metabotropic glutamate receptors in the avian retina and suggest that glutamate released from bipolar cells onto amacrine cells might act to modulate the function of these cells.
Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord detected using gridded cDNA arrays
Authors: Malaspina Andrea , Kaushik Narendra , De Belleroche Jackie
Published: March 2003

In order to obtain insight into the aetiology and pathogenesis of amyotrophic lateral sclerosis (ALS), high‐density gene discovery arrays (GDA human version 1.2) containing 18 400 non‐redundant EST cDNAs pooled from different tissue libraries have been used to monitor gene expression in lumbar spinal cord from ALS cases compared with controls. Quantitative filter analysis revealed differential expression of cDNAs normalized to internal standards. These candidates have been further investigated and their expression in spinal cord characterized in a panel of ALS and control subjects. Significant differential expression was obtained for 14 genes, 13 being elevated (up to six‐fold) and one decreased (by 80%) in ALS. Amongst those elevated in ALS were thioredoxin and glial fibrilary acid protein, which have already been shown to be up‐regulated in ALS, thus supporting the reliability of this approach. The other differentially regulated transcripts confirmed in the expression studies represent potential candidates in ALS pathogenesis being involved in antioxidant systems, neuroinflammation, the regulation of motor neurone function, lipid metabolism, protease inhibition and protection against apoptosis. The use of the GDA system has greatly facilitated the screening and retrieval of sequence information and has generated useful information on the cascade of molecular events occurring in ALS and potentially may highlight new candidates playing a role in the aetiology and progression of this disease.
Amyloid‐β peptide fragments p3 and p4 induce pro‐inflammatory cytokine and chemokine production in vitro and in vivo
Authors: Szczepanik Ann Marie , Rampe David , Ringheim Garth E.
Published: March 2003

Alzheimer's disease (AD) pathology is characterized by senile plaques containing amyloid‐β (Aβ) peptide, a protein with neurotoxic and glial immune activating potential. In addition to the highly amyloidogenic peptides Aβ(1–40/42), plaques contain amino‐terminal truncated Aβ peptides including the alpha secretase‐generated p3 fragments Aβ(17–40/42). In the present study, Aβ(17–40/42), Aβ(1–40/42), Aβ(1–16), and Aβ(25–35) aged in different solvents exhibited varying capacity to activate the murine microglia cell line MG‐7 depending on solvent, peptide ‘aging’, and peptide sequence that did not strictly correlate with β‐sheet formation. Aβ(17–40/42) or Aβ(1–42) stimulated production of the pro‐inflammatory cytokines interleukin (IL)‐1α, IL‐1β, IL‐6 and tumor necrosis factor‐α (TNF‐α), and the chemokine MCP‐1 from differentiated human monocytes (THP‐1) while little or no stimulation was observed with the other Aβ fragments. MG7 cells also produced these five pro‐inflammatory proteins in response to Aβ(1–42) whereas Aβ(17–40/42) elicited mainly TNF‐α and MCP‐1. Murine and human astrocyte cell lines (D30 and U373, respectively) were generally less responsive to Aβ fragments producing mainly IL‐6 and MCP‐1 in response to Aβ(1–42) or Aβ(17–40/42) fragments. In mice, an intracerebroventricular infusion of Aβ(1–42) significantly increased IL‐1α, IL‐1β, IL‐6 and MCP‐1 while Aβ(17–40/42) increased MCP‐1 and Aβ(17–40) increased IL‐1β. These results demonstrate that p3 and p4 Aβ fragments are pro‐inflammatory glial modulators and thus may play a role in development of the immunopathology observed in AD.
Neuronal nicotinic acetylcholine receptor α3 subunit protein in rat brain and sympathetic ganglion measured using a subunit‐specific antibody: regional and ontogenic expression
Authors: Yeh Jenny J. , Yasuda Robert P. , Dávila‐García Martha I. , Xiao Yingxian , Ebert Steven , Gupta Tara , Kellar Kenneth J. , Wolfe Barry B.
Published: March 2003

A synthetic peptide corresponding to the C‐terminus of the α3 subunit of the rat neuronal nicotinic acetylcholine receptor (nAChR) was used to generate a rabbit polyclonal α3 antibody. The specificity of this antibody was characterized by immunoblotting, immunohistochemical and immunoprecipitation techniques. Using this antibody, the relative densities of the α3 subunit were quantitatively determined in different brain regions and in superior cervical ganglion (SCG). Among these regions, SCG, interpeduncular nucleus (IPN) and pineal gland showed the highest levels of α3 protein expression. Habenula and superior colliculi had intermediate levels of expression. Low levels were found in cerebral cortex, hippocampus and cerebellum. The ontogenic profile of the α3 subunit in the SCG was also determined. The α3 protein level is low at postnatal day (P 1), but increases rapidly during the first seven postnatal days. This level then plateaus and remains stable through postnatal day 35. These findings suggest that neuronal nAChRs containing the α3 subunit participate in important roles in specific regions of the rat brain and the SCG.
Estradiol protects against ATP depletion, mitochondrial membrane potential decline and the generation of reactive oxygen species induced by 3‐nitroproprionic acid in SK‐N‐SH human neuroblastoma cells
Authors: Wang J. , Green P. S. , Simpkins J. W.
Published: March 2003

Mitochondria are recognized as modulators of neuronal viability during ischemia, hypoxia and toxic chemical exposure, wherein mitochondria dysfunction leading to ATP depletion may be a common pathway of cell death. Estrogens have been reported to be neuroprotective and proposed to play a role in the modulation of cerebral energy/glucose metabolism. To address the involvement of 17β‐estradiol preservation of mitochondrial function, we examined various markers of mitochondrial activity in human SK‐N‐SH neuroblastoma cells exposed to 3‐nitroproprionic acid (3‐NPA), a succinate dehydrogenase inhibitor which uncouples oxidative phosphorylation. 3‐NPA (10 mm) significantly increased ATP levels at 2 h then caused a 40% and a 50% decrease in ATP levels from baseline when treated for 12 h and 24 h, respectively. 3‐NPA also induced significant increases in levels of cellular hydrogen peroxide and peroxynitrite at 2 h and a 60% decrease in mitochondrial membrane potential (MMP) at 12 h exposure. 17β‐Estradiol (17β‐E2) pretreatment restored the ATP level back to 80% at 12 h of that in control cells treated with 3‐NPA but without E2, blunted the effect of 3‐NPA on MMP and reactive oxygen species levels. The present study indicates that 17β‐E2 can preserve mitochondrial function in the face of inhibition of oxidative phosphorylation.