bluuebunny/crossref_metadata_2025 huggingface dataset:
The cortical serotonin2Areceptor and the pathology of schizophrenia: a likely accomplice
Authors: Dean Brian
Published: July 2010

A large body of evidence shows that there is a change in the density of cortical serotonin2Areceptors (5HT2AR) in post‐mortem CNS from subjects with schizophrenia. Furthermore, some antipsychotic drugs have also been shown to cause a decrease in the density of 5HT2AR in the rat CNS. Thus, it appeared possible that changes in this receptor in human post‐mortem CNS simply reflected an antipsychotic drug effect. However, a great deal of research on the 5HT2AR and schizophrenia now suggests that the changes in this receptor are complex and may be involved in both the pathology of the disorder and the effects of some antipsychotic drugs. Moreover, recent advances in basic research on the role of the 5HT2AR in the CNS add further support to the hypothesis that the receptor could be involved in the pathology of the illness. In particular, an argument will be developed that the changes in the 5HT2AR in schizophrenia are reflective of a real or perceived change in serotonergic tone and that this forms an important part of the pathology of the illness.
Valproic acid down‐regulates the conversion of arachidonic acid to eicosanoids via cyclooxygenase‐1 and ‐2 in rat brain
Authors: Bosetti Francesca , Weerasinghe Gayani R. , Rosenberger Thad A. , Rapoport Stanley I.
Published: July 2010

Sodium valproate, a mood stabilizer, when chronically administered to rats (200 mg/kg i.p. daily for 30 days) significantly reduced the brain protein levels of cyclooxygenase (COX)‐1 and COX‐2, without altering the mRNA levels of these enzymes. COX activity was decreased, as were the brain concentrations of 11‐dehydrothromboxane B2 and prostaglandin E2 (PGE2), metabolites of arachidonic acid (AA) produced via COX. In contrast, the brain protein level of 5‐lipoxygenase and the concentration of its AA metabolite leukotriene B4 were unchanged. In view of published evidence that lithium chloride administered chronically to rats, like chronic valproate, reduces AA turnover within brain phospholipids, and that lithium post‐transcriptionally down‐regulates COX‐2 but not COX‐1 protein level and enzyme activity, these observations suggest that mood stabilizers generally modulate the release and recycling of AA within brain phospholipids, and the conversion of AA via COX‐2 to PGE2 and related eicosanoids. If targeting this part of the ‘AA cascade’ accounts for their therapeutic action, non‐steroidal anti‐inflammatory drugs or selective COX‐2 inhibitors might prove effective in bipolar disorder.
Human spinal motoneurons express low relative abundance of GluR2 mRNA: an implication for excitotoxicity in ALS
Authors: Kawahara Yukio , Kwak Shin , Sun Hui , Ito Kyoko , Hashida Hideji , Aizawa Hitoshi , Jeong Seon‐Yong , Kanazawa Ichiro
Published: July 2010

AMPA receptor‐mediated neurotoxicity is currently the most plausible hypothesis for the etiology of amyotrophic lateral sclerosis (ALS). The mechanism initiating this type of neuronal death is believed to be exaggerated Ca2+‐influx through AMPA receptors, which is critically determined by the presence or absence of the glutamate receptor subunit 2 (GluR2) in the assembly. We have provided the first quantitative measurements of the expression profile of AMPA receptor subunits mRNAs in human single neurons by means of quantitative RT–PCR with a laser microdissector. Among the AMPA subunits, GluR2 shared the vast majority throughout the neuronal subsets and tissues examined. Furthermore, both the expression level and the proportion of GluR2 mRNA in motoneurons were the lowest among all neuronal subsets examined, whereas those in motoneurons of ALS did not differ from the control group, implying that selective reduction of the GluR2 subunit cannot be a mechanism of AMPA receptor‐mediated neurotoxicity in ALS. However, the low relative abundance of GluR2 might provide spinal motoneurons with conditions that are easily affected by changes of AMPA receptor properties including deficient GluR2 mRNA editing in ALS.
The human prion octarepeat fragment prevents and reverses the inhibitory action of copper in the P2X4receptor without modifying the zinc action
Authors: Lorca Ramón A. , Chacón Marcelo , Barría María Inés , Inestrosa Nibaldo C. , Huidobro‐Toro J. Pablo
Published: July 2010

Human prion protein fragments (PrP60−67or PrP59−91) prevented and reversed the inhibition elicited by 5 µmcopper on the P2X4receptor expressed inXenopus laevisoocytes. A 60‐s pre‐application of 5 µmcopper caused a 69.2 ± 2.6% inhibition of the 10 µmadenosine triphosphate (ATP)‐evoked currents, an effect that was prevented by mixing 5 µmcopper with 0.01–10 µmof the PrP fragments 1‐min prior to application. This interaction was selective, as PrP59−91did not alter the facilitatory action of zinc. The EC50of PrP60−67and PrP59−91for the reduction of the copper inhibition were 4.6 ± 1 and 1.3 ± 0.4 µm, respectively. A synthetic PrP59−91variant in which all four His were replaced by Ala was inactive. However, the replacement of Trp in each of the four putative copper‐binding domains by Ala slightly decreased its potency. Furthermore, the application of 10 µmPrP59−91reversed the copper‐evoked inhibition, restoring the ATP concentration curve to the same level as the non‐inhibited state. Fragment 139–157 of βA4 amyloid precursor protein also prevented the action of copper; its EC50was 1.6 ± 0.1 µm; the metal chelator penicillamine was equipotent with PrP60−67, but carnosine was significantly less potent. Our findings highlight the role of PrP in copper homeostasis and hint at its possible role as a modulator of synapses regulated by this trace metal.
A dual role for poly‐ADP‐ribosylation in spatial memory acquisition after traumatic brain injury in mice involving NAD+ depletion and ribosylation of 14‐3‐3γ
Authors: Satchell Margaret A. , Zhang Xiaopeng , Kochanek Patrick M. , Dixon C. Edward , Jenkins Larry W. , Melick John , Szabó Csaba , Clark Robert S. B.
Published: July 2010

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a homeostatic enzyme that paradoxically contributes to disturbances in spatial memory acquisition after traumatic brain injury (TBI) in transgenic mice, thought to be related to depletion of its substrate nicotinamide adenine dinucleotide (NAD+). In this study, systemic administration of the PARP‐1 inhibitor 5‐iodo‐6‐amino‐1,2‐benzopyrone (INH2BP) after TBI preserved brain NAD+ levels and dose‐dependently reduced poly‐ADP‐ribosylation 24 h after injury. While moderate‐dose INH2BP improved spatial memory acquisition after TBI; strikingly, both injured‐ and sham‐mice receiving high‐dose INH2BP were unable to learn in the Morris‐water maze. Poly‐ADP‐ribosylated peptides identified using a proteomics approach yielded several proteins potentially associated with memory, including structural proteins (tubulin α and β, γ‐actin, and α‐internexin neuronal intermediate filament protein) and 14‐3‐3γ. Nuclear poly‐ADP‐ribosylation of 14‐3‐3γ was completely inhibited by the dose of INH2BP that produced profound memory disturbances. Thus, partial inhibition of poly‐ADP‐ribosylation preserves NAD+ and improves functional outcome after TBI, whereas more complete inhibition impairs spatial memory acquisition independent of injury, and is associated with ribosylation of 14‐3‐3γ.
Isofurans, but not F2‐isoprostanes, are increased in the substantia nigra of patients with Parkinson's disease and with dementia with Lewy body disease
Authors: Fessel Joshua P. , Hulette Christine , Powell Suzanne , Roberts L. Jackson , Zhang Jing
Published: July 2010

F2‐isoprostanes (F2‐IsoPs) are well‐established sensitive and specific markers of oxidative stressin vivo. Isofurans (IsoFs) are also products of lipid peroxidation, but in contrast to F2‐IsoPs, their formation is favored when oxygen tension is increasedin vitroorin vivo. Mitochondrial dysfunction in Parkinson's disease (PD) may not only lead to oxidative damage to brain tissue but also potentially result in increased intracellular oxygen tension, thereby influencing relative concentrations of F2‐IsoPs and IsoFs. In this study, we attempted to compare the levels of F2‐IsoPs and IsoFs esterified in phospholipids in the substantia nigra (SN) from patients with PD to those of age‐matched controls as well as patients with other neurodegenerative diseases, including dementia with Lewy body disease (DLB), multiple system atrophy (MSA), and Alzheimer's disease (AD). The results demonstrated that IsoFs but not F2‐IsoPs in the SN of patients with PD and DLB were significantly higher than those of controls. Levels of IsoFs and F2‐IsoPs in the SN of patients with MSA and AD were indistinguishable from those of age‐matched controls. This preferential increase in IsoFs in the SN of patients with PD or DLB not only indicates a unique mode of oxidant injury in these two diseases but also suggests different underlying mechanisms of dopaminergic neurodegeneration in PD and DLB from those of MSA.
Characterization of two novel proteins, NgRH1 and NgRH2, structurally and biochemically homologous to the Nogo‐66 receptor
Authors: Pignot V. , Hein A. E. , Barske C. , Wiessner C. , Walmsley A. R. , Kaupmann K. , Mayeur H. , Sommer B. , Mir A. K. , Frentzel S.
Published: July 2010

Nogo‐66 receptor (NgR) has recently been identified as the neuronal receptor of the myelin‐associated proteins Nogo‐A, oligodendrocyte protein (OMgp) and myelin‐associated glycoprotein (MAG), and mediates inhibition of axonal regeneration both in vitro and in vivo. Through database searches, we have identified two novel proteins (NgRH1 and NgRH2) that turned out to be homologous in their primary structures, biochemical properties and expression patterns to NgR. Like NgR, the homologues contain eight leucine‐rich repeats (LRR) flanked by a leucine‐rich repeat C‐terminus (LRRCT) and a leucine‐rich repeat N‐terminus (LRRNT), and also have a C‐terminal GPI signal sequence. Northern blot analysis showed predominant expression of NgRH1 and NgRH2 mRNA in the brain. In situ hybridization and immunohistochemistry on rat brain slices revealed neuronal expression of the genes. NgRH1 and NgRH2 were detected on the cell surface of recombinant cell lines as N‐glycosylated GPI anchored proteins and, consistent with other GPI anchored proteins, were localized within the lipid rafts of cellular membranes. In addition, an N‐terminal proteolytic fragment of NgR comprising the majority of the ectodomain was found to be constitutively secreted from cells. Our data indicate that NgR, NgRH1 and NgRH2 constitute a novel receptor protein family, which may play related roles within the CNS.
Differential regulation of NF‐κB, SRE and CRE by dopamine D1 and D2 receptors in transfected NG108‐15 cells
Authors: Takeuchi Yusuke , Fukunaga Kohji
Published: July 2010

To investigate transcriptional regulation by dopamine receptors, we established NG108‐15 cells stably expressing D1R, D2LR and D2SR (NGD1R, NGD2LR and NGD2SR) and evaluated the effects of these receptors on NF‐κB, SRE and CRE activity using luciferase reporter constructs. Stimulation with quinpirole, a selective D2R agonist, increased NF‐κB and SRE activity but decreased CRE activity in both NGD2R cell lines. By contrast, stimulation with SKF 38393, a selective D1R agonist, decreased NF‐κB and SRE activity but increased CRE activity in NGD1R cells. Stimulation with forskolin and overexpression of constitutively active PKA suppressed NF‐κB activity, likely due to D1R stimulation. D2R stimulation activated ERK, and treatment with U1026, a selective MEK inhibitor, eliminated D2R‐induced NF‐κB activation. D2R stimulation also activated the neural cell adhesion molecule (NCAM) promoter, which includes a potential NF‐κB site. Furthermore, by transfecting constitutively active CaM KII and MEKK, and dominant negative p38 MAPK, we show that the NCAM promoter is positively regulated by CaM KII but negatively regulated by p38 MAPK. These results indicate that D2R‐induced NF‐κB activation through ERK may be involved in activation of the NCAM promoter, and additionally that other protein kinases such as CaM KII and p38 MAPK also regulate NCAM expression.
Characterization of P2X3, P2Y1and P2Y4receptors in cultured HEK293‐hP2X3cells and their inhibition by ethanol and trichloroethanol
Authors: Fischer Wolfgang , Wirkner Kerstin , Weber Marco , Eberts Christoph , Köles Laszlo , Reinhardt Robert , Franke Heike , Allgaier Clemens , Gillen Clemens , Illes Peter
Published: July 2010

Membrane currents and changes in the intracellular Ca2+concentration ([Ca2+]i) were measured in HEK293 cells transfected with the human P2X3receptor (HEK293‐hP2X3). RT‐PCR and immunocytochemistry indicated the additional presence of endogenous P2Y1and to some extent P2Y4receptors. P2 receptor agonists induced inward currents in HEK293‐hP2X3cells with the rank order of potency α,β‐meATP ≈ ATP > ADP‐β‐S > UTP. A comparable rise in [Ca2+]iwas observed after the slow superfusion of ATP, ADP‐β‐S and UTP; α,β‐meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP‐ATP, PPADS and MRS2179 indicate that the current response to α,β‐meATP is due to P2X3receptor activation, while the ATP‐induced rise in [Ca2+]iis evoked by P2Y1and P2Y4receptor activation. TCE depressed the α,β‐meATP current in a manner compatible with a non‐competitive antagonism. The ATP‐induced increase of [Ca2+]iwas much less sensitive to the inhibitory effect of TCE than the current response to α,β‐meATP. The present study indicates that in HEK293‐hP2X3cells, TCE, but not ethanol, potently inhibits ligand‐gated P2X3receptors and, in addition, moderately interferes with G protein‐coupled P2Y1and P2Y4receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.
Contribution of bradykinin and nitric oxide to AT2receptor‐mediated differentiation in PC12 W cells
Authors: Zhao Yi , Biermann Torsten , Luther Claudia , Unger Thomas , Culman Juraj , Gohlke Peter
Published: July 2010

We investigated the effect of angiotensin II on intracellular cyclic GMP content and neurite outgrowth as an indicator of cell differentiation in PC12 W cells. Neurite outgrowth was examined by phase‐contrast microscopy. Outgrown neurites were classified as small, medium and large, and were expressed as neurites per 100 cells. Angiotensin II (10−7 m) increased the outgrowth of medium and large neurites by mean ± SEM 20.2 ± 2.3 and 6.6 ± 1.4 compared with 1.66 ± 0.5 and 0.1 ± 0.06 neurites per 100 cells in control. Cellular cyclic GMP content increased by 50–250% with angiotensin II at concentrations of 10−6–10−4 m. Both blockade of AT2receptors and of nitric oxide synthase markedly reduced angiotensin II‐induced neurite outgrowth and cyclic GMP production. In contrast, B2receptor blockade had no effect or even increased these angiotensin II effects. Sodium nitroprusside and 8‐bromo‐cyclic GMP both mimicked the effects of angiotensin II on cell differentiation. The protein kinase G inhibitor KT‐5823 inhibited the neurite outgrowth induced by both angiotensin II and 8‐bromo‐cyclic GMP. Our results demonstrate that angiotensin II can stimulate cell differentiation in PC12 W cells by nitric oxide‐related and cyclic GMP‐dependent mechanisms. The effects of angiotensin II on cell differentiation and cyclic GMP production were mediated via the AT2receptor and further enhanced by bradykinin B2receptor blockade.
Alternative splicing in single cells dissected from complex tissues: separate expression of prepro‐tachykinin A mRNA splice variants in sensory neurones
Authors: Springer Jochen , McGregor Gerard P. , Fink Ludger , Fischer Axel
Published: July 2010

Tachykinins play an important role in peripheral inflammatory diseases and disorders of the CNS. Most members of the tachykinin family are generated by alternative post‐transcriptional splicing of the prepro‐tachykinin (PPT) A gene. Here, we examined the simultaneous expression of PPT‐A splice variants in individual neurones of the nodose ganglion. In extracts of ganglia, the expression of the four PPT‐A mRNA splice variants and their four encoded peptides was shown by RT–PCR and combined HPLC and radioimmunoassay respectively. In order to examine prepro‐tachykinin A expression in individual cells, single neurones were isolated from the ganglia using laser‐assisted microdissection and processed for RT–PCR. Some 31.9% of the neurones investigated expressed a specific PPT‐A transcript. Each individual neurone was found to express only a single splice variant. This is the first study to analyse the differential expression of PPT‐A splice variants at the single‐cell level. In view of the large number of alternatively spliced genes in the human genome and the resulting profound physiological effects, including several diseases, the technique described here is useful for isolating cells without possible confounding effects of dissociated neuronal cultures. For PPT‐A, the results indicate that alternative post‐transcriptional splicing determines the tachykinergic phenotype and may therefore have important functional implications.
Protein kinase A and protein kinase C signaling pathway interaction in phenylethanolamineN‐methyltransferase gene regulation
Authors: Tai T. C. , Wong Dona L.
Published: July 2010

The protein kinase A (PKA) and protein kinase C (PKC) signaling pathways appear to interact in regulating phenylethanolamineN‐methyltransferase (PNMT) promoter‐driven gene transcription in PC12 cells. Forskolin treatment of cells transfected with the rat PNMT promoter‐luciferase reporter gene construct pGL3RP893 increased promoter activity approximately two‐fold whereas phorbol‐12‐myristate‐13 acetate (PMA) treatment had no effect. However, simultaneous forskolin and PMA treatment synergistically activated the PNMT promoter approximately four‐fold, suggesting that PKC stimulation requires prior induction of the PKA pathway. Consistent with this possibility the adenylate cyclase inhibitor MDL12,330A, and the PKA inhibitor H‐89 prevented PNMT promoter stimulation by the combination of forskolin and PMA. PKA and PKC regulation seems to be mediated in part by Egr‐1 and Sp1 through their consensus elements in the PNMT promoter. Forskolin and PMA treatment of PC12 cells increased Egr‐1 protein and phosphorylated Egr‐1/DNA‐binding complex formation to the same extent but only increased phosphorylated Sp1/DNA binding complex formation without altering Sp1 protein levels. Mutation of the − 165 bp Egr‐1 and − 48 bp Sp1 sites, respectively, attenuated and abolished combined forskolin and PMA‐mediated promoter activation. PNMT promoter analysis further showed that synergistic stimulation by PKA and PKC involves DNA sequences between − 442 and − 392 bp, and potentially a GCM binding element lying within this region.
Cannabinoid–vanilloid receptor interactions in pain signaling
Authors: Di Marzo V.
Published: July 2010

Agents that activate cannabinoid CB1 receptors for marijuana's active principal, THC, or vanilloid VR1 receptors for red chilli peppers' pungent ingredient, capsaicin, modulate pain perception. Stimulation of presynaptic CB1 leads to inhibition of glutamate release in the spinal cord, whereas VR1 stimulation causes release of substance P and CGRP from DRG neurons. VR1 undergoes rapid desensitization by its agonists, which makes VR1‐expressing neurons insensitive to subsequent stimulation and results in analgesia. Thus, both CB1 and VR1, which are coexpressed in several spinal and DRG neurons, are targets for analgesic drug development. CB1 and VR1 also share endogenous agonists, namely anandamide, NADA and some of their analogs, and may be regarded as metabotropic and ionotropic receptors for the same family of mediators, with opposing roles in pain perception. The development of ‘hybrid’ CB1/VR1 agonists as potent analgesics and the functional relationships between CB1 and VR1 in sensory neurons will be discussed.
Molecular profiling of midbrain dopamine regions in cocaine overdose victims
Authors: Tang Wen‐Xue , Fasulo Wendy H. , Mash Deborah C. , Hemby Scott E.
Published: July 2010

Chronic cocaine use in humans and animal models is known to lead to pronounced alterations in neuronal function in brain regions associated with drug reinforcement. To evaluate whether the alterations in gene expression in cocaine overdose victims are associated with specific dopamine populations in the midbrain, cDNA arrays and western blotting were used to compare gene and protein expression patterns between cocaine overdose victims and age‐matched controls in the ventral tegmental area (VTA) and lateral substantia nigra (l‐SN). Array analysis revealed significant up‐regulation of numerous transcripts in the VTA, but not in the l‐SN, of cocaine overdose victims including NMDAR1, GluR2, GluR5 and KA2 receptor mRNA (p < 0.05). No significant alterations between overdose victims and controls were observed for GluR1, R3 or R4 mRNA levels. Correspondingly, western blot analysis revealed VTA‐selective up‐regulation of CREB (p < 0.01), NMDAR1 (p < 0.01), GluR2 (p < 0.05), GluR5 (p < 0.01) and KA2 (p < 0.05) protein levels of cocaine overdose victims. The present results indicate that selective alterations of CREB and certain ionotropic glutamate receptor (iGluR) subtypes appear to be associated with chronic cocaine use in humans in a region‐specific manner. Moreover, as subunit composition determines the functional properties of iGluRs, the observed changes may indicate alterations in the excitability of dopamine transmission underlying long‐term biochemical and behavioral effects of cocaine in humans.
The C‐type natriuretic peptide precursor of snake brain contains highly specific inhibitors of the angiotensin‐converting enzyme
Authors: Hayashi Mirian A. F. , Murbach Alessandra F. , Ianzer Danielle , Portaro Fernanda C. V. , Prezoto Benedito C. , Fernandes Beatriz L. , Silveira Paulo F. , Silva Carlos A. , Pires Raquel S. , Britto Luiz R. G. , Dive Vincent , Camargo Antonio C. M.
Published: July 2010

The bradykinin‐potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin‐converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin‐converting enzyme in blood pressure regulation. In the present study, seven bradykinin‐potentiating peptides were identified within the C‐type natriuretic peptide precursor cloned from snake brain. The bradykinin‐potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin‐converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin‐potentiating peptides, one of which displays high specificity toward the N‐domain active site of the somatic angiotensin‐converting enzyme. In situ hybridization studies revealed the presence of the bradykinin‐potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin‐potentiating peptides, their presence within the neuroendocrine regulator C‐type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.
Role of protein kinase A in GABAAreceptor dysfunction in CA1 pyramidal cells following chronic benzodiazepine treatment
Authors: Lilly Scott M. , Zeng X. J. , Tietz E. I.
Published: July 2010

One‐week treatment with the benzodiazepine (BZ) flurazepam (FZP), results in anticonvulsant tolerance, associated with reduced GABAAreceptor (GABAR) subunit protein and miniature inhibitory post‐synaptic current (mIPSC) amplitude in CA1 neurons of rat hippocampus. Because protein kinase A (PKA) has been shown to modulate GABAR function in CA1 pyramidal cells, the present study assessed whether GABAR dysfunction is associated with changes in PKA activity. Two days after 1‐week FZP treatment, there were significant decreases in basal (− 30%) and total (− 25%) PKA activity, and a 40% reduction in PKA RIIβ protein in the insoluble fraction of CA1 hippocampus. The soluble component of CA1 showed a significant increase in basal (100%) but not total PKA activity. Whole‐cell recordingin vitroshowed a 50% reduction in mIPSC amplitude in CA1 pyramidal cells, with altered sensitivity to PKA modulators. Neurons from FZP‐treated rats responded to 8‐bromo‐cAMP with a significant increase (31%) in mIPSC amplitude. Likewise, vasoactive intestinal polypeptide (VIP), an endogenous PKA activator, caused a significant 36% increase in mIPSC amplitude in FZP‐treated cells. Neither agent had a significant effect on mIPSC amplitude in control cells. This study supports a role for PKA in GABAR dysfunction after chronic FZP treatment.
Biochemical and molecular studies using human autopsy brain tissue
Authors: Hynd Matthew R. , Lewohl Joanne M. , Scott Heather L. , Dodd Peter R.
Published: July 2010

The use of human brain tissue obtained at autopsy for neurochemical, pharmacological and physiological analyses is reviewed. RNA and protein samples have been found suitable for expression profiling by techniques that include RT‐PCR, cDNA microarrays, western blotting, immunohistochemistry and proteomics. The rapid development of molecular biological techniques has increased the impetus for this work to be applied to studies of brain disease. It has been shown that most nucleic acids and proteins are reasonably stable post‐mortem. However, their abundance and integrity can exhibit marked intra‐ and intercase variability, making comparisons between case‐groups difficult. Variability can reveal important functional and biochemical information. The correct interpretation of neurochemical data must take into account such factors as age, gender, ethnicity, medicative history, immediate ante‐mortem status, agonal state and post‐mortem and post‐autopsy intervals. Here we consider issues associated with the sampling of DNA, RNA and proteins using human autopsy brain tissue in relation to various ante‐ and post‐mortem factors. We conclude that valid and practical measures of a variety of parameters may be made in human brain tissue, provided that specific factors are controlled.
4‐Hydroxynonenal contributes to NGF withdrawal‐induced neuronal apoptosis
Authors: Bruckner Shane R. , Perry George , Estus Steven
Published: July 2010

Reactive oxygen species are a necessary triggering event for apoptosis of sympathetic neurons after nerve growth factor (NGF) withdrawal. Reactive oxygen species can lead to the generation of 4‐hydroxynonenal (HNE), a highly reactive aldehyde that forms adducts with proteins. This covalent modification can activate or inhibit signal transduction pathways involved in the induction of apoptosis. This process may be clinically relevant because HNE‐adduct immunoreactivity increases in several disease states. Here we evaluate the role of HNE‐adducts in sympathetic neurons undergoing NGF‐deprivation‐induced apoptosis, a model of developmental programmed cell death. We show that HNE‐adduct immunoreactivity is dramatically increased after NGF‐withdrawal in an NADPH oxidase‐dependent manner. Moreover, HNE‐adducts appear to contribute to NGF‐deprivation‐induced apoptotic signal transduction because microinjected HNE‐adduct antiserum protects sympathetic neurons from NGF withdrawal. In conclusion, this report suggests the direct contribution of endogenously generated HNE in the stimulation of apoptotic signal transduction in neurons.
Attenuation of signal flow from P2Y6receptor by protein kinase C‐α in SK‐N‐BE(2)C human neuroblastoma cells
Authors: Lee Hyun , Choi Bo‐Hwa , Suh Byung‐Chang , Lee Sun‐Kyong , Kim Kyong‐Tai
Published: July 2010

Extracellular nucleotides exert a variety of biological actions through several kinds of P2 receptors in many tissues and cell types. We found that treatment with nucleotides increases intracellular Ca2+concentration ([Ca2+]i) in SK‐N‐BE(2)C human neuroblastoma cells with a following order of potency: UDP > UTP > ADP >> ATP. Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis showed that specific mRNAs coding for human P2Y1, P2Y4, and P2Y6receptors were expressed in the cells, but Northern blot analysis revealed that P2Y6receptors were the predominant type. Activation of protein kinase C‐α by treatment with 1 µmphorbol 12‐myristate 13‐acetate dramatically inhibited both the UDP‐induced [Ca2+]irise and inositol 1,4,5‐trisphosphate (IP3) generation, whereas incubation with pertussis toxin had little effect on the responses. The UDP‐induced [Ca2+]irise and IP3production were maintained up to 30 min after stimulation, while bradykinin‐induced responses rapidly decreased to the basal level within 5 min of stimulation. Pretreatment of cells with the maximal effective concentration of UDP reduced the subsequent carbachol‐ or bradykinin‐induced [Ca2+]irise without inhibition of IP3generation. Neuronal differentiation of the cells by treatment with retinoic acid for 7 days did not change the expression level of P2Y6receptors. Taken together, the data indicate that P2Y6receptors highly responsive to diphosphonucleotide UDP are endogenously expressed in the human neuroblastoma SK‐N‐BE(2)C cells and that they are involved in the modulation of other phospholipase C‐coupled receptor‐mediated Ca2+mobilization by depleting the IP3‐sensitive Ca2+stores.
Differential subcellular localization of two dopamine D2receptor isoforms in transfected NG108‐15 cells
Authors: Takeuchi Yusuke , Fukunaga Kohji
Published: July 2010

The dopamine D2receptor (D2R) is target for antipsychotic drugs and associated with several neuropsychiatric disorders. D2R has a long third cytoplasmic loop and a short carboxyl‐terminal cytoplasmic tail. It exists as two alternatively spliced isoforms, termed D2LR and D2SR, which differ in the presence and absence, respectively, of a 29 amino acid insert in the third cytoplasmic loop. To evaluate the differential roles of the two D2R isoforms, we transfected both isoforms into NG108‐15 cells and observed their subcellular localization by a confocal laser scanning light microscope. D2SR was predominantly localized at the plasma membrane, whereas D2LR was mostly retained in the perinuclear region around the Golgi apparatus. Using a yeast two hybrid system with a mouse brain cDNA library and coimmunoprecipitation assay, we found that heart‐type fatty acid binding protein (H‐FABP) interacts with D2LR but not with D2SR. H‐FABP is a cytosolic protein involved in binding and transport of fatty acids. Overexpressed H‐FABP and endogenous H‐FABP were colocalized with the intracellular D2LR in NG108‐15 cells. Furthermore, in the rat striatum, H‐FABP was detected in the D2R‐expressing neurons. From these results, H‐FABP is associated with D2LR, and may thereby modulate the subcellular localization and function of D2LR.
Increased synaptosomal dopamine content and brain concentration of paraquat produced by selective dithiocarbamates
Authors: Barlow Brian K. , Thiruchelvam Mona J. , Bennice Lisa , Cory‐Slechta Deborah A. , Ballatori Nazzareno , Richfield Eric K.
Published: July 2010

Exposure to pesticides may be a risk factor for Parkinson's disease based on epidemiologic data in humans, animal models andin vitrostudies. Different dithiocarbamate pesticides potentiate the toxicity of both 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine and paraquat in mouse models of Parkinsonism by an unknown mechanism. This study examined the effects of commercially used dithiocarbamates on [3H]dopamine transport in striatal synaptosomal vesicles and on the concentration of [14C]paraquatin vivoin mice. Different ethylenebis‐dithiocarbamates and diethyl‐dithiocarbamate increased dopamine accumulation in synaptosomes, whereas dimethyl‐dithiocarbamate and methyl‐dithiocarbamate did not. Increased dopamine accumulation in synaptosomes was dose dependent and was related to the carbon backbone of these molecules. The dithiocarbamates that increased accumulation of dopamine did not alter the influx of dopamine, but rather delayed the efflux out of synaptosomes. These same dithiocarbamates also increased the tissue content of [14C]paraquatin vivoby a mechanism that appeared to be distinct from the dopamine transporter. There was a consistent relationship between the dithiocarbamates that increased synaptosomal accumulation of dopamine and tissue content of paraquat, with those previously demonstrated to enhance paraquat toxicityin vivo. These results suggest that selective dithiocarbamates may alter the kinetics of different endogenous and exogenous compounds to enhance their neurotoxicity.
Systemic morphine‐induced release of serotonin in the rostroventral medulla is not mimicked by morphine microinjection into the periaqueductal gray
Authors: Taylor Bradley K. , Basbaum Allan I.
Published: July 2010

We usedin vivomicrodialysis in awake rats to test the hypothesis that intravenous morphine increases serotonin (5‐HT) release within the rostral ventromedial medulla (RVM). We also injected morphine into various sites along the rostrocaudal extent of the periaqueductal gray (PAG), and examined the extent of its diffusion to the RVM. Intravenous morphine (3.0 mg/kg) produced thermal antinociception and increased RVM dialysate 5‐HT, 5‐hydroxyindole acetic acid (5‐HIAA), and homovanillic acid (HVA) in a naloxone‐reversible manner. As neither PAG microinjection of morphine (5 µg/0.5 µL) nor RVM administration of fentanyl ord‐Ala(2),NMePhe(4),Gly‐ol(5)]enkephalin (DAMGO) increased RVM 5‐HT, we were unable to determine the precise site of action of morphine. Surprisingly, peak morphine levels in the RVM were higher after microinjection into the caudal PAG as compared to either intravenous injection or microinjection into more rostral sites within the PAG. Naloxone‐precipitated withdrawal in morphine‐tolerant rats not only increased extracellular 5‐HT in the RVM, but also dopamine (DA) and HVA. We conclude that substantial amounts of morphine diffuse from the PAG to the RVM, and speculate that opioid receptor interactions at multiple brain sites mediate the analgesic effects of PAG morphine. Further studies will be required to elucidate the contribution of 5‐HT and DA release in the RVM to opioid analgesia and opioid withdrawal.
Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state
Authors: Starkov Anatoly A. , Fiskum Gary
Published: July 2010

Mitochondrial production of reactive oxygen species (ROS) at Complex I of the electron transport chain is implicated in the etiology of neural cell death in acute and chronic neurodegenerative disorders. However, little is known regarding the regulation of mitochondrial ROS production by NADH‐linked respiratory substrates under physiologically realistic conditions in the absence of respiratory chain inhibitors. This study used Amplex Red fluorescence measurements of H2O2 to test the hypothesis that ROS production by isolated brain mitochondria is regulated by membrane potential (ΔΨ) and NAD(P)H redox state. ΔΨ was monitored by following the medium concentration of the lipophilic cation tetraphenylphosphonium with a selective electrode. NAD(P)H autofluorescence was used to monitor NAD(P)H redox state. While the rate of H2O2 production was closely related to ΔΨ and the level of NAD(P)H reduction at high values of ΔΨ, 30% of the maximal rate of H2O2 formation was still observed in the presence of uncoupler (p‐trifluoromethoxycarbonylcyanide phenylhydrazone) concentrations that provided for maximum depolarization of ΔΨ and oxidation of NAD(P)H. Our findings indicate that ROS production by mitochondria oxidizing physiological NADH‐dependent substrates is regulated by ΔΨ and by the NAD(P)H redox state over ranges consistent with those that exist at different levels of cellular energy demand.
Neuromelanin associated redox‐active iron is increased in the substantia nigra of patients with Parkinson's disease
Authors: Faucheux Baptiste A. , Martin Marie‐Elise , Beaumont Carole , Hauw Jean‐Jacques , Agid Yves , Hirsch Etienne C.
Published: July 2010

Degeneration of dopaminergic neurones during Parkinson's disease is most extensive in the subpopulation of melanized‐neurones located in the substantia nigra pars compacta. Neuromelanin is a dark pigment produced in the dopaminergic neurones of the human substantia nigra and has the ability to bind a variety of metal ions, especially iron. Post‐mortem analyses of the human brain have established that oxidative stress and iron content are enhanced in association with neuronal death. As redox‐active iron (free Fe2+ form) and other transition metals have the ability to generate highly reactive hydroxyl radicals by a catalytic process, we investigated the redox activity of neuromelanin (NM)‐aggregates in a group of parkinsonian patients, who presented a statistically significant reduction (− 70%) in the number of melanized‐neurones and an increased non‐heme (Fe3+) iron content as compared with a group of matched‐control subjects. The level of redox activity detected in neuromelanin‐aggregates was significantly increased (+ 69%) in parkinsonian patients and was highest in patients with the most severe neuronal loss. This change was not observed in tissue in the immediate vicinity of melanized‐neurones. A possible consequence of an overloading of neuromelanin with redox‐active elements is an increased contribution to oxidative stress and intraneuronal damage in patients with Parkinson's disease.
A new G protein‐coupled receptor from a primitive metazoan shows homology with vertebrate aminergic receptors and displays constitutive activity in mammalian cells
Authors: Bouchard Christelle , Ribeiro Paula , Dubé François , Anctil Michel
Published: July 2010

Biogenic amine receptors mediate wide‐ranging hormonal and modulatory functions in vertebrates, but are largely unknown in primitive invertebrates. In a representative of the most basal multicellular animals possessing a nervous system, the cnidarianRenilla koellikeri, aminergic‐like receptors were previously characterized pharmacologically and found to engender control of the animal's bioluminescent and peristaltic reactions. Using degenerate oligonucleotides in a RT‐PCR strategy, we obtained a full‐length cDNA encoding a polypeptide with typical G protein‐coupled receptor (GPCR) characteristics and which displayed a significant degree of sequence similarity (up to 45%) to biogenic amine receptors, particularly dopamine and adrenergic receptors. The new receptor, named Ren1, did not resemble any one specific type of amine GPCR and thus could not be identified on the basis of sequence. Ren1 was expressed transiently and stably in cultured mammalian cells, as demonstrated by immunocytochemistry and western blotting. Functional analysis of transfected HEK293, LTK‐ and COS‐7 cells, based on both cAMP and Ca2+signalling assays, revealed that Ren1 was not activated by any of the known biogenic amines tested and several related metabolites. The results indicated, however, that cells stably expressing Ren1 contained, on average, an 11‐fold higher level of cAMP than the controls, in the absence of agonist stimulation. The high basal cAMP levels were shown to be specific for Ren1 and to vary proportionally with the level of Ren1 expressed in the transfected cells. Taken together, the data suggested that Ren1 was expressed as a constitutively active receptor. Its identification provides a basis for examination of the early evolutionary emergence of GPCRs and their functional properties.
Cytochrome P450‐dependent metabolism ofl‐deprenyl in monkey (Cercopithecus aethiops) and C57BL/6 mouse brain microsomal preparations
Authors: Stefania Dragoni , Lydia Bellik , Maria Frosini , Giacomo Matteucci , Giampietro Sgaragli , Massimo Valoti
Published: July 2010

The aim of the present investigation was to characterize the cytochrome P450 (CYP)‐dependent metabolism ofl‐deprenyl by brain microsomal preparations obtained from two different animal models that have been extensively used in Parkinson's disease studies, namely monkey (Cercopithecus aethiops) and C57BL/6 mouse. In monkey brain microsomal fractions, the apparentKmvalues for methamphetamine formation froml‐deprenyl were 67.8 ± 1.0 and 72.0 ± 1.6 μm, in the cortex and striatum, respectively. Similarly, for nordeprenyl formation froml‐deprenyl,Kmvalues in cortex and striatum were 21.3 ± 3.2 and 27.3 ± 4.0 μm, respectively. Both metabolic pathways appear to be more efficient in the cortex than in the striatum as theVmaxfor microsomal preparation was lower in the striatum for the formation of both metabolites. The formation rate ofl‐methamphetamine was up to one order of magnitude greater than that of nordeprenyl. Inhibition analysis of both pathways in monkey brain suggested thatl‐methamphetamine formation is catalysed by CYP2A and CYP3A, whereas only CYP3A appears to be involved in nordeprenyl formation. With microsomal preparations from whole brain of C57BL/6 mice, the onlyl‐deprenyl metabolite that could be detected was methamphetamine and theKmandVmaxvalues were similar to those determined in monkey cortex (53.6 ± 2.9 μmand 33.9 ± 0.4 pmol/min/mg protein, respectively). 4‐Methylpyrazole selectively inhibited methamphetamine formation, suggesting the involvement of CYP2E1. In conclusion, the present study indicates thatl‐deprenyl is effectively metabolised by CYP‐dependent oxidases in the brain, giving rise mainly to the formation of methamphetamine, which has been suggested to play a role in the pharmacological effects of the parent drug. The results also demonstrate that there are differences between species in CYP‐dependent metabolism ofl‐deprenyl.
Green tea polyphenols enhance sodium nitroprusside‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells
Authors: Zhang Yueting , Zhao Baolu
Published: July 2010

Oxidative stress is a main mediator in nitric oxide (NO) ‐induced neurotoxicity and has been implicated in the pathogenesis of many neurodegenerative disorders. Green tea polyphenols are usually expected as potent chemo‐preventive agents due to their ability of scavenging free radicals and chelating metal ions. However, not all the actions of green tea polyphenols are necessarily beneficial. In the present study, we demonstrated that higher‐concentration green tea ployphenols significantly enhanced the neurotoxicity by treatment of sodium nitroprusside (SNP), a nitric oxide donor. SNP induced apoptosis in human neuroblastoma SH‐SY5Y cells in a concentration and time‐dependent manner, as estimated by cell viability assessment, FACScan analysis and DNA fragmentation assay, whereas treatment with green tea polyphenols alone had no effect on cell viability. Pre‐treatment with lower‐dose green tea polyphenols (50 and 100 μm) had only a slightly deleterious effect in the presence of SNP, while higher‐dose green tea polyphenols (200 and 500 μm) synergistically damaged the cells severely. Further research showed that co‐incubation of green tea polyphenols and SNP caused loss of mitochondrial membrane potential, depletion of intracellular GSH and accumulation of reactive oxygen species, and exacerbated NO‐induced neuronal apoptosis via a Bcl‐2 sensitive pathway.
A novel function of Goα: mediation of extracellular signal‐regulated kinase activation by opioid receptors in neural cells
Authors: Zhang Wen‐Bo , Zhang Zhe , Ni Yan‐Xiang , Wu Ya‐Lan , Pei Gang
Published: July 2010

Go is the most abundant G protein expressed in brain but its function is less known. Here we show a novel function of Goα as a mediator of opioid receptor‐induced extracellular signal‐regulated kinase activation in neural cells. The current study found that, in neuroblastoma × glioma NG108‐15 hybrid cells, activation of extracellular signal‐regulated kinase through delta opioid receptors was mediated by pertussis toxin‐sensitive G protein and independent of Gβγ subunits, PI3 kinase and receptor internalization. Overexpression of a dominant negative form of Goα1, but not Giα2, completely blocked delta opioid receptor‐induced extracellular signal‐regulated kinase activity. Decreasing Goα expression by RNA interference greatly reduced delta opioid receptor‐induced extracellular signal‐regulated kinase activity and extracellular signal‐regulated kinase‐dependent gene expression, while knocking down Giα2did not. By taking advantage of differences between human and mouse Goα gene sequences, we simultaneously knocked down endogenous Goα expression and expressed exogenous human Goα subunits. We found that both human Goα1and Goα2could mediate delta opioid receptor‐induced extracellular signal‐regulated kinase activation. This study suggests that one of the functions of Goα in the brain is to mediate extracellular signal‐regulated kinase activation by G protein‐coupled receptors.
Both apoptosis and necrosis occur early after intracerebral grafting of ventral mesencephalic tissue: a role for protease activation
Authors: Emgård M. , Hallin U. , Karlsson J. , Bahr B. A. , Brundin P. , Blomgren K.
Published: July 2010

Neural transplantation is an experimental treatment for Parkinson's disease. Widespread clinical application of the grafting technique is hampered by a relatively poor survival (around 10%) of implanted embryonic dopamine neurones. Earlier animal studies have indicated that a large proportion of the grafted cells die during graft tissue preparation and within the first few days after intracerebral implantation. The present study was designed to reveal the prevalence of cell death in rat intrastriatal grafts at 90 min, 1, 3, 6 and 42 days after implantation. We examined apoptotic cell death using semi‐thin and paraffin sections stained with methylene blue and an antibody against activated caspase 3, respectively. We identified abundant apoptotic cell death up to 3 days after transplantation. In addition, we studied calpain activation using an antibody specific for calpain‐cleaved fodrin. We report a peak in calpain activity 90 min after grafting. Surprisingly, we did not observe any significant difference in the number of dopaminergic neurones over time. The present results imply that grafted cells may be victims of either an early necrotic or a later apoptotic cell death and that there is substantial cell death as early as 90 min after implantation.
Hydrogen peroxide induces loss of dopamine transporter activity: a calcium‐dependent oxidative mechanism
Authors: Huang Chuen‐Lin , Huang Nai‐Kuei , Shyue Song‐Kun , Chern Yijuang
Published: July 2010

H2O2dose dependently inhibited dopamine uptake in PC12 cells and in striatal synaptosomes. Treatment with H2O2resulted in a reversible reduction inVmax, with no effect on itsKmvalue. This suppressive effect of H2O2could be relieved by reducing agents (dithiothreitol and cysteine). Furthermore, an oxidizer (dithiodipyridine) also markedly suppressed the dopamine transporter (DAT). Oxidative stress therefore might contribute to the action of H2O2. H2O2appeared to modify DAT at both extracellular and intracellular sites because cumene‐H2O2(a radical generator mostly restricted to plasma membranes) at high concentrations also slightly suppressed DAT activity and the intracellular overexpression of catalase ameliorated the inhibitory effect of H2O2. Internalization was unlikely to be involved because concanavalin A, which blocked endocytosis, did not prevent the H2O2‐evoked inhibition of DAT activity. Interestingly, H2O2treatment evoked a Ca2+influx in PC12 cells. Moreover, removal of external calcium by EGTA or reduction in the intracellular calcium level using BAPTA‐AM reversed the inhibitory effect of H2O2. Conversely, depletion of intracellular calcium stores using thapsigargin did not affect the reduction in DAT activity by H2O2. Collectively, our results indicate that the DAT, one of the most important proteins controlling the dopaminergic system, is also a redox sensor. In addition, H2O2might suppress the DAT by a Ca2+‐dependent oxidative pathway.