bluuebunny/crossref_metadata_2025 huggingface dataset:
Amphetamine‐induced c‐fosmRNA expression in the caudate‐putamen and subthalamic nucleus: interactions between dose, environment, and neuronal phenotype
Authors: Uslaner Jason M. , Norton Camille S. , Watson Stanley J. , Akil Huda , Robinson Terry E.
Published: July 2010

When administered in a novel environment relatively low doses of amphetamine induce c‐fosmRNA in the subthalamic nucleus (STN) and in preproenkephalin mRNA‐containing (ENK+) neurons in the caudate‐putamen (CPu). When administered at home, however, low doses of amphetamine do not produce these effects. Environmental novelty also facilitates the behavioral effects of acute and repeated amphetamine, but this is dose‐dependent. The purpose of the present experiment therefore was to determine if the effect of context on amphetamine‐induced c‐fosexpression is also dose‐dependent. It was found that: (i) No dose of amphetamine tested (1–10 mg/kg) induced c‐fosin many ENK+ cells when given at home. (ii) When given in a novel environment low to moderate doses of amphetamine (1–5 mg/kg) induced c‐fosin substantial numbers of ENK+ cells, but the highest dose examined (10 mg/kg) did not. (iii) Environmental novelty enhanced the ability of low to moderate doses of amphetamine to induce c‐fosin the STN, but the highest dose of amphetamine induced robust c‐fosmRNA expression in the STN regardless of context. The results do not support the idea that engaging ENK+ cells, at least as indicated by c‐fosmRNA expression, is critical to produce robust behavioral sensitization, but do suggest a possible role for the STN. Furthermore, the results highlight the importance of drug–environment interactions on the neurobiological effects of drugs, and have implications for thinking about the circuits by which context modulates the acute and long‐lasting consequences of amphetamine treatment.
The roles of calcium/calmodulin‐dependent and Ras/mitogen‐activated protein kinases in the development of psychostimulant‐induced behavioral sensitization
Authors: Licata Stephanie C. , Pierce R. Christopher
Published: July 2010

Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium‐stimulated second messengers, including the calcium/calmodulin‐dependent protein kinases and the Ras/mitogen‐activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium‐stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long‐term expression of psychostimulant‐induced behavioral sensitization.
Oligodendrocyte injury in multiple sclerosis: a role for p53
Authors: Wosik Karolina , Antel Jack , Kuhlmann Tanja , Brück Wolfgang , Massie Bernard , Nalbantoglu Josephine
Published: July 2010

Multiple sclerosis (MS) is a neurological disorder characterized by myelin destruction and a variable degree of oligodendrocyte death. We have previously shown that overexpression of the transcription factor p53 can induce oligodendrocyte apoptosis. We investigated the mechanism of p53‐induced apoptosis using primary cultures of central nervous system‐derived adult human oligodendrocytes. Adenovirus‐mediated p53 overexpression resulted in up‐regulation of the death receptors Fas, DR4 and DR5 with subsequent caspase‐mediated apoptosis of the oligodendrocytes. The oligodendrocytes were protected from p53‐induced cell death by blocking signaling through Fas and/or tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) receptors. Although lower levels of p53 did not induce apoptosis, the increase in death receptor expression was sufficient to render the oligodendrocytes susceptible to apoptosis in the presence of exogenous Fas ligand and TRAIL. These ligands are present in the inflammatory milieu of active MS lesions. In situ analysis of active MS lesions revealed increased p53 expression in oligodendrocytes in lesions that featured oligodendrocyte apoptosis and cell loss. Our data provide evidence for a novel role for p53 in the pathogenesis of MS.
Secretoneurin promotes pertussis toxin‐sensitive neurite outgrowth in cerebellar granule cells
Authors: Gasser M. C. , Berti I. , Hauser K. F. , Fischer‐Colbrie R. , Saria A.
Published: July 2010

The neuropeptide secretoneurin (SN) is an endoproteolytic product of the chromogranin secretogranin II. We investigated the effects of SN on the differentiation of immature cerebellar granule cells derived from the external granular layer (EGL). Secretoneurin caused concentration‐dependent increases in neurite outgrowth, reflecting differentiation. The maximum effect was reached at a concentration of 100 nm SN. Secretoneurin immunoneutralization using specific antiserum significantly decreased neurite outgrowth; however, neurite morphology was altered. An affinity chromatography‐purified antibody significantly inhibited the outgrowth response to SN (p < 0.001) without altering the morphology. Binding studies suggest the existence of specific G‐protein‐coupled receptors on the surface of monocytes that recognize SN. Assuming that SN promotes neurite outgrowth in EGL cells by acting through a similar G‐protein‐coupled mechanism, we treated SN‐stimulated EGL cultures with pertussis toxin. Exposure to pertussis toxin (0.1 µg/mL) showed a significant inhibition of the SN‐induced outgrowth. To establish a second messenger pathway we used the protein kinase C inhibitor staurosporine. We found that EGL cell viability was not enhanced following chronic SN treatment for 24 h. These data indicate that SN is a novel trophic substance that can affect cerebellar maturation, primarily by accelerating granule cell differentiation through a signalling mechanism that is coupled to pertussis toxin‐sensitive G‐proteins.
Fluorescence resonance energy transfer to probe human M1 muscarinic receptor structure and drug binding properties
Authors: Ilien Brigitte , Franchet Christelle , Bernard Philippe , Morisset Séverine , Odile Weill Claire , Bourguignon Jean‐Jacques , Hibert Marcel , Galzi Jean‐Luc
Published: July 2010

Human M1 muscarinic receptor chimeras were designed (i) to allow detection of their interaction with the fluorescent antagonist pirenzepine labelled with Bodipy [558/568], through fluorescence resonance energy transfer, (ii) to investigate the structure of the N‐terminal extracellular moiety of the receptor and (iii) to set up a fluorescence‐based assay to identify new muscarinic ligands. Enhanced green (or yellow) fluorescent protein (EGFP or EYFP) was fused, through a linker, to a receptor N‐terminus of variable length so that the GFP barrel was separated from the receptor first transmembrane domain by six to 33 amino‐acids. Five fluorescent constructs exhibit high expression levels as well as pharmacological and functional properties superimposable on those of the native receptor. Bodipy‐pirenzepine binds to the chimeras with similar kinetics and affinities, indicating a similar mode of interaction of the ligand with all of them. From the variation in energy transfer efficiencies determined for four different receptor‐ligand complexes, relative donor (EGFP)‐acceptor (Bodipy) distances were estimated. They suggest a compact architecture for the muscarinic M1 receptor amino‐terminal domain which may fold in a manner similar to that of rhodopsin. Finally, this fluorescence‐based assay, prone to miniaturization, allows reliable detection of unlabelled competitors.
Regulation of neuritogenesis and synaptic transmission by msec7‐1, a guanine nucleotide exchange factor, in cultured Aplysia neurons
Authors: Huh Minjung , Han Jin‐Hee , Lim Chae‐Seok , Lee Seung‐Hee , Kim Seho , Kim Eunjoon , Kaang Bong‐Kiun
Published: July 2010

msec7‐1, a mammalian homologue of yeast sec7p, is known as a GDP/GTP exchange factor (GEF) for the ADP ribosylation factor (ARF) family of small GTPases. Here, we report that msec7‐1 overexpression in cultured Aplysia neurons leads to an extensive neuritogenesis in a GEF activity‐dependent manner through the modulation of the actin cytoskeleton. Similarly, the overexpression of ARNO, another mammalin GEF, produces extensive neuritogenesis in Aplysia neurons. In addition, msec7‐1 overexpression increases the number of varicosities with an altered size and shape in a GEF activity‐dependent manner. The overexpression of msec7‐1 in pre‐synaptic sensory neurons co‐cultured with post‐synaptic target motor neurons leads to an increase in the amplitude of the excitatory post‐synaptic potential through its GEF activity. Our results demonstrate that msec7‐1 regulates neuritogenesis and synaptic transmission.
Up‐regulation of cerebral carbonic anhydrase by anoxic stress in piglets
Authors: Nógrádi Antal , Domoki Ferenc , Dégi Rózsa , Borda Stefánia , Pákáski Magdolna , Szabó András , Bari Ferenc
Published: July 2010

The resuscitation of asphyxiated babies is associated with changes in cerebral protein synthesis that can influence the neurological outcome. Insufficient gas exchange results in rapid shifts in extracellular and intracellular pH. Carbonic anhydrase (CA) plays an important role in buffering acute changes in pH in the brain. We investigated whether asphyxia/re‐ventilation influences the expression of cerebral CA isoforms (CA‐II, CA‐III and CA‐IV) in anaesthetized newborn pigs. The cerebral cortex, hippocampus, cerebellum and retina were sampled, and prepared for either CA immunohistochemistry or CA immunoblotting from piglets subjected to asphyxia (10 min) followed by 2–4 h of re‐ventilation, and also from normoxic controls. The CA immunoreactivity (IR) of all the isoforms studied was weak in the controls, apart from staining of a few oligodendrocytes in the subcortical white matter, some astrocytes in the superficial layer of the cerebral cortex, the cerebellar Purkinje cells and the retinal Müller cells that possessed moderate CA‐II IR. However, asphyxia induced a marked increase in the CA IR of all isoforms in all the cerebral regions investigated and the retina after 4 h of survival. The pyramidal cells of the frontal cortex and hippocampus displayed the most conspicuous increase in CA IR. Immunoblotting confirmed increased levels of all the CA isoenzymes. We conclude that raised CA levels after asphyxia may contribute to the compensatory mechanisms that protect against the pathological changes in the neonatal CNS.
Immortalized olfactory ensheathing glia promote axonal regeneration of rat retinal ganglion neurons
Authors: Moreno‐Flores M. Teresa , Lim Filip , Martín‐Bermejo M. Jesús , Díaz‐Nido Javier , Ávila Jesús , Wandosell Francisco
Published: July 2010

Olfactory bulb ensheathing glia (OEG) have attracted special attention during the last few years because of their unique properties in promoting regeneration of adult mammalian central nervous system (CNS) components. However the molecular and cellular characteristics responsible for this capacity remain to be revealed. Such studies are presently hindered by the lack of a plentiful source of homogenous OEG. Thus the availability of immortalized OEG lines maintaining the regenerative characteristics of the primary cultures would represent an unlimited source of OEG for use not only in biochemical analyses of neuroregenerative mechanisms but also to characterize their regenerative properties in models in culture and in vivo. We have immortalized primary rat OEG using the SV40 large T antigen expressed from a constitutive cellular promotor, and report here the isolation and characterization of clonal lines. These OEG clonal lines were comparable to primary OEG and Schwann cells in the promotion of axonal regeneration of mature rat retinal ganglion neurons (RGN) but, significantly, this culture assay system more closely reflects the in vivo reparative properties of OEG on transected nerves than other assays of neuritogenesis in that it revealed OEG cells to promote the growth of a larger number of long axons than Schwann cells. Using this assay we were able to grade our OEG lines for their neuroregenerative capacity, opening the possibility of identifying molecules with correlative expression levels in these cells. Our preliminary characterization revealed that the expression level of a classical OEG marker, the p75‐NGF receptor, does not correlate with neuroregenerative capacity.
Efficient gene transfer from innervated muscle into rat peripheral and central nervous systems using a non‐viral haemagglutinating virus of Japan (HVJ)‐liposome method
Authors: Kato Naoki , Nakanishi Kuniaki , Nemoto Koichi , Morishita Ryuichi , Kaneda Yasufumi , Uenoyama Maki , Ikeda Tomosumi , Fujikawa Kyosuke
Published: July 2010

We evaluated the feasibility of gene delivery into the peripheral and central nervous systems via retrograde axonal transport following injection of a haemagglutinating virus of Japan (HVJ)‐liposome‐DNA complex vector into an innervated muscle. Transfection efficiency was assessed by measuring luciferase activity, and was compared statistically with that achieved using a liposome‐DNA control vector. High luciferase activity was observed in the injected muscle, the ipsilateral sciatic nerve, and the ipsilateral dorsal root ganglia on day 1 after gene transfer. The spinal cord also showed luciferase activity, although this was lower than in the other tissues. However, no activity was observed in the contralateral sciatic nerve or the contralateral dorsal root ganglia. In addition, we performed gene transfer twice, with a 1‐week interval, to evaluate the feasibility of repeated therapeutic gene delivery. Again, a high transfection efficiency was observed immediately, even after the second gene transfer, and transfection efficiency was significantly higher at each defined time‐point using the HVJ‐liposome complex vector than using a control vector. These results indicate that this method could be used for repeated therapeutic gene delivery into muscle, nerve, dorsal root ganglia, and possibly spinal cord, without the need for a surgical approach, making it well suited to clinical applications.
Presynaptic control of striatal dopamine neurotransmission in adult vesicular monoamine transporter 2 (VMAT2) mutant mice
Authors: Patel Jyoti , Mooslehner Katrin A. , Chan Pok Man , Emson Piers C. , Stamford Jonathan A.
Published: July 2010

The vesicular monoamine transporter 2 (VMAT2) plays a pivotal role in regulating the size of vesicular and cytosolic dopamine (DA) storage pools within the CNS, and can thus influence extracellular DA neurotransmission. Transgenic mice have been generated with a dramatically reduced (by ∼95%) expression of the VMAT2 gene which, unlike complete knockout lines, survive into adulthood. We compared the pre‐synaptic regulation of both impulse‐dependent (exocytotic) and carrier‐mediated (via reversal of the DA transporter, DAT) DA release in the dorsolateral caudate putamen (CPu) of striatal slices derived from adult homozygous VMAT2 mutant and wild‐type mice using fast cyclic voltammetry. Impulse‐dependent DA release, evoked by a single electrical pulse, was lower in homozygous (116 nm) than wild‐type mice (351 nm) indicating smaller vesicular DA stores, an observation supported by the evanescent effect of amfonelic acid (300 nm) in homozygous mice. Amphetamine (2 μm) increased extracellular DA via DAT reversal in both wild‐type (by 459 nm) and VMAT2 mutant (by 168 nm,p < 0.01 vs. wild‐type) mice. In both cases, the effect was blocked by the DAT inhibitor GBR12935 (1 μm). Simultaneously, amphetamine decreased impulse‐dependent DA release, albeit less in homozygous (by 55%) than in wild‐type (by 78%) mice. In wild‐types, this decrement was largely reversed by GBR12935 but not by the D2/D3 autoreceptor antagonist (–)sulpiride (1 μm). Conversely, in homozygous VMAT2 mutant mice, it was attenuated by (–)sulpiride but not GBR12935. The D2/D3 receptor agonist quinpirole inhibited impulse‐dependent DA release with a lower EC50value in homozygous mice (12 nm) compared with wild‐types (34 nm), indicating the compensatory presence of functionally supersensitive release‐regulating autoreceptors. However, analysis of DA reuptake kinetics obtained in the absence and presence of DAT blockade (by cocaine and amfonelic acid) revealed only minor differences in DAT functionality. These results demonstrate that impaired vesicular DA storage constrains extracellular DA levels in the dorsolateral CPu whether induced by either impulse‐dependent or carrier‐mediated mechanisms and that the relative importance of the DAT and terminal autoreceptors as control mechanisms in the actions of amphetamine are reversed in VMAT2 mutant mice.
Amyloid precursor protein‐processing products affect mononuclear phagocyte activation: pathways for sAPP‐ and Aβ‐mediated neurotoxicity
Authors: Ikezu Tsuneya , Luo Xiaoguang , Weber Gregory A. , Zhao Jianxing , McCabe Laura , Buescher James L. , Ghorpade Anuja , Zheng Jialin , Xiong Huangui
Published: July 2010

Increasing evidence strongly supports the role of glial immunity in the pathogenesis of Alzheimer's disease (AD). To investigate such events we have developed cell systems mimicking the interactions between β‐amyloid precursor protein (APP)‐expressing neurons and brain mononuclear phagocytes (MP; macrophages and microglia). MP were co‐cultured with neuronal cells expressing wild type APP or familial AD‐linked APP mutants. The latter was derived from recombinant adenoviral constructs. Neuronal APP processing products induced MP activation, reactive oxygen species, and neurotoxic activities. These occurred without the addition of pro‐inflammatory cytokines and were reversed by depletion of amyloid β‐peptide (Aβ) and secreted APP (sAPP). Neurotoxic activities were diminished by superoxide dismutase mimetics and NMDA receptor inhibitors. Microglial glutamate secretion was suppressed by the cystine‐glutamate antiporter inhibitor and its levels paralleled the depletion of sAPP and Aβ from conditioned media prepared from APP‐expressing neurons. The excitotoxins from activated MP were potent enough to evoke recombinant NMDA receptor‐mediated inward currents expressed in vitro in the Xenopus oocytes. These results demonstrate that neuronal APP‐processing products can induce oxidative neurotoxicity through microglial activation.
Evaluation of the role of the retinal G protein‐coupled receptor (RGR) in the vertebrate retina in vivo
Authors: Maeda Tadao , Van Hooser J. Preston , Driessen Carola A. G. G. , Filipek Sławomir , Janssen Jacques J. M. , Palczewski Krzysztof
Published: July 2010

The retinal G protein‐coupled receptor (RGR) is a protein that structurally resembles visual pigments and other G protein‐coupled receptors. RGR may play a role as a photoisomerase in the production of 11‐cis‐retinal, the chromophore of the visual pigments. As the proposed function of RGR, in a complex with 11‐cis‐retinol dehydrogenase (RDH5), is to regenerate 11‐cis‐retinal under light conditions and RDH5 is expected to function in the light‐independent part of the retinoid cycle, we speculated that the simultaneous loss of function of both proteins should more severely affect the rhodopsin regeneration capacity. Here, we evaluated the role of RGR using rgr–/– single and rdh5–/–rgr–/– double knockout mice under a number of light conditions. The most striking phenotype of rgr–/– mice after a single flash of light includes light‐dependent formation of 9‐cis‐ and 13‐cis‐retinoid isomers. These isomers are not formed in wild‐type mice because either all‐trans‐retinal is bound to RGR and protected from isomerization to 9‐cis‐ or 13‐cis‐retinal or because RGR is able to eliminate these isomers directly or indirectly. After intense bleaching, a transient accumulation of all‐trans‐retinyl esters and an attenuated recovery of 11‐cis‐retinal were observed. Finally, even under conditions of prolonged light illumination, as investigated in vitro in biochemical assays or in vivo by electroretinogram (ERG) measurements, no evidence of catalytic‐like photoisomerization‐driven production of 11‐cis‐retinal could be attained. These and previous results suggest that RGR and RDH5 are likely to function in the retinoid cycle, although their role is not essential and regeneration of visual pigment is only mildly affected by the absence of both proteins in rod‐dominated mice.
Co‐ordinate transcriptional regulation of dopamine synthesis genes by α‐synuclein in human neuroblastoma cell lines
Authors: Baptista Melisa J. , O'Farrell Casey , Daya Sneha , Ahmad Rili , Miller David W. , Hardy John , Farrer Matthew J. , Cookson Mark R.
Published: July 2010

Abnormal accumulation of α‐synuclein in Lewy bodies is a neuropathological hallmark of both sporadic and familial Parkinson's disease (PD). Although mutations in α‐synuclein have been identified in autosomal dominant PD, the mechanism by which dopaminergic cell death occurs remains unknown. We investigated transcriptional changes in neuroblastoma cell lines transfected with either normal or mutant (A30P or A53T) α‐synuclein using microarrays, with confirmation of selected genes by quantitative RT‐PCR. Gene products whose expression was found to be significantly altered included members of diverse functional groups such as stress response, transcription regulators, apoptosis‐inducing molecules, transcription factors and membrane‐bound proteins. We also found evidence of altered expression of dihydropteridine reductase, which indirectly regulates the synthesis of dopamine. Because of the importance of dopamine in PD, we investigated the expression of all the known genes in dopamine synthesis. We found co‐ordinated downregulation of mRNA for GTP cyclohydrolase, sepiapterin reductase (SR), tyrosine hydroxylase (TH) and aromatic acid decarboxylase by wild‐type but not mutant α‐synuclein. These were confirmed at the protein level for SR and TH. Reduced expression of the orphan nuclear receptor Nurr1 was also noted, suggesting that the co‐ordinate regulation of dopamine synthesis is regulated through this transcription factor.
Levodopa treatment reverses endocannabinoid system abnormalities in experimental parkinsonism
Authors: Maccarrone Mauro , Gubellini Paolo , Bari Monica , Picconi Barbara , Battista Natalia , Centonze Diego , Bernardi Giorgio , Finazzi‐Agrò Alessandro , Calabresi Paolo
Published: July 2010

Cannabinoid receptors and their endogenous ligands are potent inhibitors of neurotransmitter release in the brain. Here, we show that in a rat model of Parkinson's disease induced by unilateral nigral lesion with 6‐hydroxydopamine (6‐OHDA), the striatal levels of the endocannabinoid anandamide (AEA) were increased, while the activity of its membrane transporter and hydrolase (fatty‐acid amide hydrolase, FAAH) were decreased. These changes were not observed in the cerebellum of the same animals. Moreover, the frequency and amplitude of glutamate‐mediated spontaneous excitatory post‐synaptic currents were augmented in striatal spiny neurones recorded from parkinsonian rats. Remarkably, the anomalies in the endocannabinoid system, as well as those in glutamatergic activity, were completely reversed by chronic treatment of parkinsonian rats with levodopa, and the pharmacological inhibition of FAAH restored a normal glutamatergic activity in 6‐OHDA‐lesioned animals. Thus, the increased striatal levels of AEA may reflect a compensatory mechanism trying to counteract the abnormal corticostriatal glutamatergic drive in parkinsonian rats. However, this mechanism seems to be unsuccessful, since spontaneous excitatory activity is still higher in these animals. Taken together, these data show that anomalies in the endocannabinoid system induced by experimental parkinsonism are restricted to the striatum and can be reversed by chronic levodopa treatment, and suggest that inhibition of FAAH might represent a possible target to decrease the abnormal cortical glutamatergic drive in Parkinson's disease.
Oligodendrocyte myelin glycoprotein growth inhibition function requires its conserved leucine‐rich repeat domain, not its glycosylphosphatidyl‐inositol anchor
Authors: Vourc'h Patrick , Moreau Thierry , Arbion Flavie , Marouillat‐Védrine Sylviane , Müh Jean‐Pierre , Andres Christian
Published: July 2010

The oligodendrocyte myelin glycoprotein (OMgp) inhibits neurite outgrowth and axonal regeneration after brain injury, but its normal function remains unknown. Several observations suggest its implication in cell growth regulation. Here we report an analysis of the domain requirement in OMgp proliferation inhibitory function. We first studied the OMgp protein sequence in 14 mammal species and observed a high conservation of its leucine‐rich repeat (LRR) domain. The deletion of this LRR domain is responsible for a total loss of function in an in vitro expression system. The possible three‐dimensional structure of the LRR domain of OMgp was modelled using the structure of Yersinia pestis YopM cytotoxin as a template. The predicted arrangement of the LRR segments is compatible with a function of OMgp as a binding protein. The OMgp is a glycosylphosphatidyl‐inositol‐linked protein anchored in the plasma membrane of oligodendrocytes and neurones. Using deletion mutagenesis, we demonstrated the dispensability of the glycosylphosphatidyl‐inositol anchor for OMgp proliferation inhibition function. Our results suggest that OMgp is part of a receptor complex, either as a coreceptor or as a membrane‐bound or soluble ligand, involved in the transmission of a growth suppressive signal.
The synergistic effects of NGF and IGF‐1 on neurite growth in adult sensory neurons: convergence on the PI 3‐kinase signaling pathway
Authors: Jones David M. , Tucker Budd A. , Rahimtula Masuma , Mearow Karen M.
Published: July 2010

Nerve growth factor (NGF) and insulin‐like growth factor‐1 (IGF‐1) play an important role in promoting axonal growth from dorsal root ganglion (DRG) neurons. Adult DRG neurons exhibit neurotrophin‐independent survival, providing an excellent system with which to study trophic factor effects on neurite growth in the absence of significant survival effects. Using young adult rat DRG neurons we have demonstrated a synergistic effect of NGF plus IGF (N + I), compared with either factor alone, in promoting neurite growth. Not only does the presence of NGF and IGF‐1 enhance neurite initiation, it also significantly augments the extent of neurite branching and elongation. We have also examined potential mechanism(s) underlying this synergistic effect. Immunoblotting experiments of classical growth factor intermediary signalling pathways (PI 3‐K‐Akt‐GSK‐3 and Ras‐Raf‐MAPK) were performed using phospho‐specific antibodies to assess activation state. We found that activation of Akt and MAPK correlated with neurite elongation and branching. However, using pharmacological inhibitors, we observed that a PI 3‐K pathway involving both Akt and GSK‐3 appeared to be more important for neurite extension and branching than MAPK‐dependent signalling. In fact, inhibition of activation of MAPK with U0126 resulted in increased neuritic branching, possibly as a result of the concomitant increase observed in phospho‐Akt. Furthermore, inhibition of GSK3 (which is negatively regulated by phosphorylation on S9/S21) also resulted in increased growth. Our data point to signalling convergence upon the PI 3‐K‐Akt‐GSK‐3 pathway that underlies the NGF plus IGF synergism. In addition, to our knowledge, this is the first report in primary neurons that inhibition of GSK3 results in an enhanced neurite growth.
Activity of the rat GluR4 promoter in transfected cortical neurons and glia
Authors: Borges Karin , Myers Scott J. , Zhang Sunan , Dingledine Raymond
Published: July 2010

AMPA (alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate) receptors are assembled from four subunits, GluR1‐4. Although GluR4 is widely expressed in brain its abundance is less than GluR1‐3. We have isolated ∼5 kb of the rat GluR4 promoter region and analyzed its capacity to drive expression of a luciferase reporter gene in transfected rat cortical neurons and glia, and C6 glioma cells. Multiple transcriptional start sites were identified in a GC‐rich region lacking TATA‐boxes between − 1090 and − 1011 bp from ATG. In transfected mixed cortical cultures, luciferase expression driven by GluR4 promoter segments were found predominantly in TuJ1‐positive neurons, indicating neuronal preference of GluR4. The GluR4 promoter fragments were 6–12‐fold more active in neurons than glia, compared with a 30‐fold neuronal selectivity of GluR2. Deletion of the GluR4 transcriptional initiation region decreased luciferase activity in neurons, but increased activity in C6 cells, suggesting that regulatory elements governing neuronal expression reside in this region. An intron within the 5′‐untranslated region and Sp1, IK2 and E‐box sites are conserved in the rat, mouse and human GluR4 promoters. The relative activity of GluR4 and GluR2 promoters in transfected cells correlates with their expression in brain, and in both promoters regulatory elements for neuronal expression reside near the initiation sites.
Cellular distribution and bioactivity of the key steroidogenic enzyme, cytochrome P450side chain cleavage, in sensory neural pathways
Authors: Patte‐Mensah Christine , Kappes Vincent , Freund‐Mercier Marie‐José , Tsutsui Kazuyoshi , Mensah‐Nyagan Ayikoe G.
Published: July 2010

Neurosteroids are steroids produced within the nervous system. Based on behavioural responses evoked in animals by synthetic steroid injections, several studies suggested neurosteroid involvement in important neurophysiological processes. These observations should be correlated only to neuroactive effects of the injected steroids. Neurosteroids mostly control the CNS activity through allosteric modulation of neurotransmitter receptors within concentration ranges used by neurotransmitters themselves. Therefore, neurosteroid production within pathways controlling a neurophysiological process is necessary to consider neurosteroid involvement in that process. Because of the increasing speculation about pain modulation by neurosteroids based on pharmacological observations, we decided to clarify the situation by investigating neurosteroidogenesis occurrence in sensory pathways, particularly in nociceptive structures. We studied the presence and activity of cytochrome P450side chain cleavage (P450scc) in rat pain pathways. P450scc‐immunoreactive cells were localized in dorsal root ganglia (DRG), spinal cord (SC) dorsal horn, nociceptive supraspinal nuclei (SSN) and somatosensory cortex. Incubation of DRG, SSN or SC tissue homogenates with [3H]cholesterol yielded the formation of radioactive metabolites including [3H]pregnenolone of which the synthesis was reduced in presence of aminogluthetimide, a P450scc inhibitor. These first neuroanatomical and neurochemical results demonstrate the occurrence of neurosteroidogenesis in nociceptive pathways and strongly suggest that neurosteroids may control pain mechanisms.
Glucocorticoid‐regulated human serotonin transporter (5‐HTT) expression is modulated by the 5‐HTT gene‐promotor‐linked polymorphic region
Authors: Glatz K. , Mössner R. , Heils A. , Lesch K. P.
Published: July 2010

Mood, emotion and cognition are modulated by serotonergic neurotransmission, while the physiological function of serotonergic synapses depends on serotonin reuptake, which is mediated by the serotonin transporter (5‐HTT). Allelic variation of 5‐HTT expression in humans is caused by a functional gene‐promoter polymorphism with two predominant variant alleles, which are associated with variations in anxiety measures as previously reported. Here we report that administration of dexamethasone, a potent glucocorticosteroid hormone, results in an increase in 5‐HTT expression in immortalized human B‐lymphoblastoid cells, which express the human 5‐HTT. Functional reporter gene assays as well as 5‐HT uptake and inhibitor binding measures revealed a genotype‐dependent dose–response to glucocorticosteroid administration, which was antagonized by RU 38486, a non‐specific glucocorticosteroid hormone antagonist. The allele‐specific differences after administration of dexamethasone depended on the repetitive GC‐rich sequence located approximately 1.4 kb upstream of the 5‐HTT gene transcription site because of absence of a significant steroid effect after transfecting a deletional mutant reporter gene construct, which lacks this repetitive promoter sequence. Our findings may contribute to explain the vulnerability to stress‐related disorders in susceptible individuals, in whom further clinical studies should follow up on these in vitro findings.
Overactivation of glycogen synthase kinase‐3 by inhibition of phosphoinositol‐3 kinase and protein kinase C leads to hyperphosphorylation of tau and impairment of spatial memory
Authors: Liu Shi Jie , Zhang Ai Hong , Li Hong Lian , Wang Qun , Deng Heng Mei , Netzer William J. , Xu Huaxi , Wang Jian Zhi
Published: July 2010

Neurofibrillary tangles (NFTs) consisting of the hyperphosphorylated microtubule‐associated protein tau are a defining pathological characteristic of Alzheimer's disease (AD). Hyperphosphorylation of tau is hypothesized to impair the microtubule stabilizing function of tau, leading to the formation of paired helical filaments and neuronal death. Glycogen synthase kinase‐3 (GSK‐3) has been shown to be one of several kinases that mediate tau hyperphosphorylation in vitro. However, molecular mechanisms underlying overactivation of GSK‐3 and its potential linkage to AD‐like pathologies in vivo remain unclear. Here, we demonstrate that injection of wortmannin (a specific inhibitor of phosphoinositol‐3 kinase) or GF‐109203X (a specific inhibitor of protein kinase C) into the left ventricle of rat brains leads to overactivation of GSK‐3, hyperphosphorylation of tau at Ser 396/404/199/202 and, most significantly, impaired spatial memory. The effects of wortmannin and GF‐109203X are additive. Significantly, specific inhibition of GSK‐3 activity by LiCl prevents hyperphosphorylation of tau, and spatial memory impairment resulting from PI3K and PKC inhibition. These results indicate that in vivo inhibition of phosphoinositol‐3 kinase and protein kinase C results in overactivation of GSK‐3 and tau hyperphosphorylation and support a direct role of GSK‐3 in the formation of AD‐like cognitive deficits.
Molecular cloning, characterization and brain mapping of the melanocortin 5 receptor in the goldfish
Authors: Cerdá‐Reverter José Miguel , Ling Maria Kristina , Schiöth Helgi Birgir , Peter Richard Ector
Published: July 2010

The melanocortin 5 receptor (MC5R) is activated by melanocyte‐stimulating hormones (MSHs) and has a widespread tissue distribution, while its detailed central expression pattern and brain functions are fairly unknown. We report cloning, pharmacological characterization, tissue distribution and detailed brain mapping of melanocortin 5 receptor in goldfish (gMC5R). The goldfish orthologue protein is 69% identical to human MC5R and is conserved in important functional domains. The gMC5R showed similar potency to α‐, β‐ and γ‐MSH peptides in radioligand binding as the mammalian orthologues, while MTII and HS024 were both agonists at this receptor. The gMC5R‐mRNA was found in the peripheral tissues including kidney, spleen, skin and retina, with low expression levels in the intestine, fat, muscle, gill, pituitary and ovary. In situ hybridization studies demonstrated that gMC5R transcripts are widely distributed in the goldfish brain. The gMC5R expression was found in ventral telencephalon, pre‐optic area, dorsal and ventral thalamus, infundibular hypothalamus, posterior tuberculum, tectum and tegmentum mesencephali, reticular formation, vagal and facial lobes and spinal cord. The cloning and characterization of this receptor provides an important tool to elucidate its participation in neuroendocrine and behavioural control.
The expression of brain cyclooxygenase‐2 is down‐regulated in the cytosolic phospholipase A2 knockout mouse
Authors: Bosetti Francesca , Weerasinghe Gayani R.
Published: July 2010

We examined brain phospholipase A2 (PLA2) activity and the expression of enzymes metabolizing arachidonic acid (AA) in cytosolic PLA2 knockout () mice to see if other brain PLA2 can compensate for the absence of cPLA2 alpha and if cPLA2 couples with specific downstream enzymes in the eicosanoid biosynthetic pathway. We found that the rate of formation of prostaglandin E2 (PGE2), an index of net cyclooxygenase (COX) activity, was decreased by 62% in the compared with the control mouse brain. The decrease was accompanied by a 50–60% decrease in mRNA and protein levels of COX‐2, but no change in these levels in COX‐1 or in PGE synthase. Brain 5‐lipoxygenase (5‐LO) and cytochrome P450 epoxygenase (cyp2C11) protein levels were also unaltered. Total and Ca2+‐dependent PLA2 activities did not differ significantly between and control mice, and protein levels of type VI iPLA2 and type V sPLA2, normalized to actin, were unchanged. These results show that type V sPLA2 and type VI iPLA2 do not compensate for the loss of brain cPLA2 alpha, and that this loss has significant downstream effects on COX‐2 expression and PGE2 formation, sparing other AA oxidative enzymes. This suggests that cPLA2 is critical for COX‐2‐derived eicosanoid production in mouse brain.
Long‐term alterations in glutamate receptor and transporter expression following early‐life seizures are associated with increased seizure susceptibility
Authors: Zhang Guojun , Raol Yogendra Sinh H. , Hsu Fu‐Chun , Brooks‐Kayal Amy R.
Published: July 2010

Prolonged seizures in early childhood are associated with an increased risk of development of epilepsy in later life. The mechanism(s) behind this susceptibility to later development of epilepsy is unclear. Increased synaptic activity during development has been shown to permanently alter excitatory neurotransmission and could be one of the mechanisms involved in this increased susceptibility to the development of epilepsy. In the present study we determine the effect of status‐epilepticus induced by lithium/pilocarpine at postnatal day 10 (P10 SE) on the expression of glutamate receptor and transporter mRNAs in hippocampal dentate granule cells and protein levels in dentate gyrus of these animals in adulthood. The results revealed a decrease in glutamate receptor 2 (GluR2) mRNA expression and protein levels as well as an increase in protein levels for the excitatory amino acid carrier 1 (EAAC1) in P10 SE rats compared to controls. Expression of glutamate receptor 1 (GluR1) mRNA was decreased in both P10 SE rats and identically handled, lithium‐injected littermate controls compared to naive animals, and GluR1 protein levels were significantly lower in lithium‐controls than in naive rats, suggesting an effect of either the handling or the lithium on GluR1 expression. These changes in EAA receptors and transporters were accompanied by an increased susceptibility to kainic acid induced seizures in P10 SE rats compared to controls. The current data suggest that early‐life status‐epilepticus can result in permanent alterations in glutamate receptor and transporter gene expression, which may contribute to a lower seizure threshold.
The voltage‐gated calcium channel UNC‐2 is involved in stress‐mediated regulation of tryptophan hydroxylase
Authors: Estevez Miguel , Estevez Annette O. , Cowie Robin H. , Gardner Kathy L.
Published: July 2010

Migraine is an episodic pain disorder whose pathophysiology is related to deficiency of serotonin signaling and abnormal function of the P/Q‐type calcium channel, CACNA1A. Because the relationship of the CACNA1A channel to serotonin signaling is unknown and potentially of therapeutic interest we have used genetic analysis of the Caenorhabditis elegans ortholog of this calcium channel, UNC‐2, to help identify candidate downstream effectors of the human channel. By genetic dissection of the lethargic mutant phenotype of unc‐2, we have established an epistasis pathway showing that UNC‐2 function antagonizes a transforming growth factor (TGF)‐β pathway influencing movement rate. This same UNC‐2/TGF‐β pathway is required for accumulation of normal serotonin levels and stress‐induced modulation of tryptophan hydroxylase (tph) expression in the serotonergic chemosensory ADF neurons, but not the NSM neurons. We also show that transgenic expression of the migraine‐associated Ca2+ channel, CACNA1A, in unc‐2 animals can functionally substitute for UNC‐2 in stress‐activated regulation of tph expression. The demonstration that these evolutionarily related channels share a conserved ability to modulate tph expression through their effects on TGF‐β signaling provides the first specific example of how CACNA1A function may influence levels of the critical migraine neurotransmitter serotonin.
Identification of gene structure and subcellular localization of human centaurin α2, and p42IP4, a family of two highly homologueous, Ins 1,3,4,5‐P4‐/PtdIns 3,4,5‐P3‐binding, adapter proteins
Authors: Hanck Theodor , Stricker Rolf , Sedehizade Fariba , Reiser Georg
Published: July 2010

Proteins which recognize the two messengers phosphatidylinositol 3,4,5‐trisphosphate (PtdInsP3), a membrane lipid, and inositol 1,3,4,5‐tetrakisphosphate (InsP4), a water‐soluble ligand, play important roles by integrating external stimuli, which lead to differentiation, cell death or survival. p42IP4, a PtdInsP3/InsP4‐binding protein, is predominantly expressed in brain. The recently described centaurin α2 of similar molecular mass which is 58% identical and 75% homologous to the human p42IP4 orthologue, is expressed rather ubiquitously in many tissues. Here, elucidating the gene structure for both proteins, we found the human gene for centaurin α2 located on chromosome 17, position 17q11.2, near to the NF1 locus, and human p42IP4 on chromosome 7, position 7p22.3. The two isoforms, which both have 11 exons and conserved exon/intron transitions, seem to result from gene duplication. Furthermore, we studied binding of the two second messengers, PtdInsP3 and InsP4, and subcellular localization of the two proteins. Using recombinant baculovirus we expressed centaurin α2 and p42IP4 in Sf9 cells and purified the proteins to homogeneity. Recombinant centaurin α2 bound both InsP4 and PtdInsP3 equally well in vitro. Furthermore, fusion proteins of centaurin α2 and p42IP4, respectively, with the green fluorescent protein (GFP) were expressed in HEK 293 cells to visualize subcellular distribution. In contrast to p42IP4, which was distributed throughout the cell, centaurin α2 was concentrated at the plasma membrane already in unstimulated cells. The protein centaurin α2 was released from the membrane upon addition of wortmannin, which inhibits PI3‐kinase. p42IP4, however, translocated to plasma membrane upon growth factor stimulation. Thus, in spite of the high homology between centaurin α2 and p42IP4 and comparable affinities for InsP4 and PtdInsP3, both proteins showed clear differences in subcellular distribution. We suggest a model, which is based on the difference in phosphoinositide binding stoichiometry of the two proteins, to account for the difference in subcellular localization.
Mitochondrial glutaminase enhances extracellular glutamate production in HIV‐1‐infected macrophages: Linkage to HIV‐1 associated dementia
Authors: Zhao Jianxing , Lopez Alicia L. , Erichsen David , Herek Shelley , Cotter Robin L. , Curthoys Norman P. , Zheng Jialin
Published: July 2010

Dysfunction in mononuclear phagocyte (MP, macrophages and microglia) immunity is thought to play a significant role in the pathogenesis of HIV‐1 associated dementia (HAD). In particular, elevated extracellular concentrations of the excitatory neurotransmitter glutamate, produced by MP as a consequence of viral infection and immune activation, can induce neuronal injury. To determine the mechanism by which MP‐mediated neuronal injury occurs, the concentration and rates of production of extracellular glutamate were measured in human monocyte‐derived macrophage (MDM) supernatants by reverse phase high‐performance liquid chromatography (RP‐HPLC). Measurements were taken of supernatants from MDM infected with multiple HIV‐1 strains including ADA and DJV (macrophage tropic, M‐tropic), and 89.6 (dual tropic). High levels of glutamate were produced by MDM infected with M‐tropic viruses. AZT, an inhibitor of HIV‐1 replication, inhibited glutamate generation, demonstrating a linkage between HIV‐1 infection and enhanced glutamate production. In our culture system, glutamate production was dependent upon the presence of glutamine and was inhibited by 6‐diazo‐5‐oxo‐l‐norleucine, a glutaminase inhibitor. Supernatants collected from HIV‐1‐infected MP generated more glutamate following glutamine addition than supernatants isolated from uninfected MP. These findings implicate the involvement of a glutamate‐generating enzyme, such as phosphate‐activated mitochondrial glutaminase (PMG) in MP‐mediated glutamate production.
Delayed but sustained induction of mitogen‐activated protein kinase activity is associated with β‐adrenergic receptor‐mediated morphological differentiation of astrocytes
Authors: Gharami Kusumika , Das Sumantra
Published: July 2010

Astroglial β‐adrenergic receptors (β‐ARs) are functionally linked to regulate cellular morphology. In primary cultures, the β‐AR agonist isoproterenol (ISP) can transform flat polygonal astrocytes into process‐bearing, mature stellate cells by 48 h, an effect that can be blocked by the β‐AR antagonist, propranolol. ISP induced immediate activation of protein kinase A (PKA) which persisted up to 2 h, with no visible change in cell morphology. However, activation of PKA was sufficient to drive the process of transformation to completion, suggesting the involvement of downstream regulators of PKA. In addition to PKA inhibitors, the mitogen‐activated protein kinase (MAPK) kinase inhibitor PD098059 also blocked ISP‐induced morphological transformation. ISP treatment resulted in a biphasic response of cellular phosphorylated MAPK (phosphorylated extracellular signal‐regulated kinase; p‐ERK) level: an initial decline in p‐ERK level followed by a sustained induction at 12–24 h, both of which were blocked by PKA inhibitor. The induction in pERK level coincided with initiation of morphological differentiation of the astrocytes and nuclear translocation of p‐ERK. A long‐lasting activation of p‐ERK activity by ISP, at a later stage, appears to be critical for the transformation of astrocytes.