bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase C‐mediated down‐regulation of β2‐adrenergic receptor and gene expression in rat C6 glioma cells
Authors: Leavitt Maria , Setola Vincent , Fishman Peter H.
Published: March 2003

We investigated the regulation of β2‐adrenergic receptors (β2AR) by protein kinase C (PKC) in rat C6 glioma cells at the levels of receptor activity, protein expression and gene expression. Cells exposed to 4β‐phorbol‐12‐myristate‐13‐acetate (PMA), a potent activator of PKC, exhibited a time‐ and concentration‐dependent decrease in β2AR binding activity. Maximum down‐regulation was ∼50% by 24 h and western blot analysis revealed a parallel decrease in β2AR protein. In addition, PMA treatment resulted in an acute desensitization of β2AR‐stimulated cyclic AMP response prior to any reduction in receptor levels. PMA exposure also affected steady‐state β2AR mRNA levels in a time‐dependent, biphasic manner. During the first 4 h, levels decreased by ∼60% and then slowly recovered to ∼75% of control by 24 h. As the reduction in receptor mRNA was not due to a decrease in its stability, we examined β2AR gene transcription by nuclear run‐on assays. Transcriptional activity in nuclei from C6 cells treated with PMA for 2 h was reduced by 70% compared to controls. Thus PKC can regulate β2AR at least two levels: the first being an acute desensitization of receptor function, and the second being a more prolonged repression of receptor gene transcription that in turn results in decreased receptor expression.
Cannabinoids activate p38 mitogen‐activated protein kinases through CB1 receptors in hippocampus
Authors: Derkinderen Pascal , Ledent Catherine , Parmentier Marc , Girault Jean‐Antoine
Published: March 2003

Cannabinoid receptors (CB1‐R) are the target of a novel class of neuromodulators, the endocannabinoids. Yet, their signalling mechanisms in adult brain are poorly understood. We report that, in rat and mouse hippocampal slices, anandamide and 2‐arachidonoylglycerol, synthetic cannabinoids, and Δ9‐tetrahydrocannabinol activated p38 mitogen‐activated protein kinases (MAPK), but not c‐Jun N‐terminal kinase (JNK). In contrast, lysophosphatidic acid (LPA), a lipid messenger acting on different receptors, increased both p38‐MAPK and JNK phosphorylation. The effects of cannabinoids on p38‐MAPK were mediated through activation of CB1‐R because they were blocked in the presence of SR 141716 A and absent in CB1‐R knockout mice, two conditions that did not alter the effects of LPA. The activation of p38‐MAPK by cannabinoids was insensitive to inhibitors of Src. These results provide new insights into the cellular mechanisms by which cannabinoids exert their effects in hippocampus.
Proinflammatory cytokines promote glial heme oxygenase‐1 expression and mitochondrial iron deposition: implications for multiple sclerosis
Authors: Mehindate K. , Sahlas D. J. , Frankel D. , Mawal Y. , Liberman A. , Corcos J. , Dion S. , Schipper H. M.
Published: March 2003

Proinflammatory cytokines, pathological iron deposition, and oxidative stress have been implicated in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). HO‐1 mRNA levels and mitochondrial uptake of [55Fe]Cl3‐derived iron were measured in rat astroglial cultures exposed to interleukin‐1β (IL‐1β) or tumor necrosis factor‐α (TNF‐α) alone or in combination with the heme oxygenase‐1 (HO‐1) inhibitors, tin mesoporphyrin (SnMP) or dexamthasone (DEX), or interferon β1b (INF‐β). HO‐1 expression in astrocytes was evaluated by immunohistochemical staining of spinal cord tissue derived from MS and control subjects. IL‐1β or TNF‐α promoted sequestration of non‐transferrin‐derived 55Fe by astroglial mitochondria. HO‐1 inhibitors, mitochondrial permeability transition pore (MTP) blockers and antioxidants significantly attenuated cytokine‐related mitochondrial iron sequestration in these cells. IFN‐β decreased HO‐1 expression and mitochondrial iron sequestration in IL‐1β‐ and TNF‐α‐challenged astroglia. The percentage of astrocytes coexpressing HO‐1 in affected spinal cord from MS patients (57.3% ± 12.8%) was significantly greater (p < 0.05) than in normal spinal cord derived from controls subjects (15.4% ± 8.4%). HO‐1 is over‐expressed in MS spinal cord astroglia and may promote mitochondrial iron deposition in MS plaques. In MS, IFN‐β may attenuate glial HO‐1 gene induction and aberrant mitochondrial iron deposition accruing from exposure to proinflammatory cytokines.
Proteolysis of glutamate receptor‐interacting protein by calpain in rat brain: implications for synaptic plasticity
Authors: Lu Xiaoying , Wyszynski Michael , Sheng Morgan , Baudry Michel
Published: March 2003

Activation of the calcium‐dependent protease calpain has been proposed to be a key step in synaptic plasticity in the hippocampus. However, the exact pathway through which calpain mediates or modulates changes in synaptic function remains to be clarified. Here we report that glutamate receptor‐interacting protein (GRIP) is a substrate of calpain, as calpain‐mediated GRIP degradation was demonstrated using three different approaches: (i) purified calpain I digestion of synaptic membranes, (ii) calcium treatment of frozen–thawed brain sections, and (iii) NMDA‐stimulated organotypic hippocampal slice cultures. More importantly, calpain activation resulted in the disruption of GRIP binding to the GluR2 subunit of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptors. Because GRIP has been proposed to function as an AMPA receptor‐targeting and synaptic‐stabilizing protein, as well as a synaptic‐organizing molecule, calpain‐mediated degradation of GRIP and disruption of AMPA receptor anchoring are likely to play important roles in the structural and functional reorganization accompanying synaptic modifications in long‐term potentiation and long‐term depression.
1,2‐bis(2‐Aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid induces caspase‐mediated apoptosis and reactive oxygen species‐mediated necrosis in cultured cortical neurons
Authors: Han Kong Sook , Kang Hyo Jung , Kim Eun Young , Yoon Won Joo , Sohn Seonghyang , Kwon Hyuk Jae , Gwag Byoung Joo
Published: March 2003

Sustained alteration in [Ca2+]i triggers neuronal death. We examined morphological and signaling events of Ca2+‐deficiency‐induced neuronal death. Cortical cell cultures exposed to 20 µm 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA‐AM), an intracellular calcium chelator, underwent neuronal apoptosis within 12 h that was evident by shriveled cell bodies, aggregated and condensed nuclear chromatin, and disrupted nuclear membrane. Thereafter, surviving neurons revealed typical necrosis, accompanied by swelling of cell body and mitochondria, over 24 h. Both apoptosis and necrosis were prevented by inclusion of 1 µg/mL cycloheximide, a protein synthesis inhibitor. Treatment with BAPTA‐AM induced translocation of Bax into mitochondria within 4 h and release of cytochrome c from mitochondria over 4–12 h. An active fragment of caspase‐3, a downstream mediator of cytochrome c, was observed within 8 h and cleaved PHF‐1‐positive tau. Administration of zVAD‐fmk, a broad inhibitor of caspases, or DEVD‐amc, a selective inhibitor of caspase‐3, selectively prevented the apoptosis component of BAPTA‐AM neurotoxicity. In contrast, BAPTA‐AM‐induced necrosis was propagated through sequential production of superoxide, mitochondrial and cytoplasmic reactive oxygen species. Combined treatment with caspase inhibitors and antioxidants blocked BAPTA‐AM neurotoxicity. The present study suggests that neurons deficient in [Ca2+]i undergo caspase‐3‐mediated apoptosis and reactive oxygen species (ROS)‐mediated necrosis.
Oxidative stress mediates neuronal DNA damage and apoptosis in response to cytosine arabinoside
Authors: Geller Herbert M. , Cheng Ke‐Yi , Goldsmith Noriko K. , Romero Alejandro A. , Zhang Ai‐Ling , Morris Erick J. , Grandison Lindsey
Published: March 2003

Cytosine arabinoside (AraC) is a nucleoside analog that produces significant neurotoxicity in cancer patients. The mechanism by which AraC causes neuronal death is a matter of some debate because the conventional understanding of AraC toxicity requires incorporation into newly synthesized DNA. Here we demonstrate that AraC‐induced apoptosis of cultured cerebral cortical neurons is mediated by oxidative stress. AraC‐induced cell death was reduced by treatment with several different free‐radical scavengers (N‐acetyl‐l‐cysteine, dipyridamole, uric acid, and vitamin E) and was increased following depletion of cellular glutathione stores. AraC induced the formation of reactive oxygen species in neurons as measured by an increase in the fluorescence of the dye 5‐(6)‐carboxy‐2′,7′‐dichlorodihydrofluorescein diacetate. AraC produced DNA single‐strand breaks as measured by single‐cell gel electrophoresis and the level of DNA strand breakage was reduced by treatment with the free radical scavengers. These data support a model in which AraC induces neuronal apoptosis by provoking the generation of reactive oxygen species, causing oxidative DNA damage and initiating the p53‐dependent apoptotic program. These observations suggest the use of antioxidant therapies to reduce neurotoxicity in AraC chemotherapeutic regimens.
Down‐regulation of occludin expression in astrocytes by tumour necrosis factor (TNF) is mediated via TNF type‐1 receptor and nuclear factor‐κB activation
Authors: Wachtel Marco , Bolliger Marc F. , Ishihara Hideyuki , Frei Karl , Bluethmann Horst , Gloor Sergio M.
Published: March 2003

Tight junctions form the diffusion barrier of brain microcapillary endothelial cells and support cell polarity. Also astrocytes express tight junction components such as occludin, claudin‐1, ZO‐1 and ZO‐2, but do not establish a permeability barrier. However, little is known about the function and regulation of these molecules in astrocytes. We studied the impact of tumour necrosis factor (TNF) on occludin and ZO‐1 expression in astrocytes. TNF decreased occludin, but not ZO‐1 expression. In brain microcapillary endothelial cells, as well as in epithelial cells, occludin expression was not influenced by TNF. Removal of TNF from astrocytes restored the basal level of occludin. Down‐regulation was inhibited by caffeic acid phenethyl ester, a specific inhibitor of nuclear factor‐κB (NF‐κB) activation. Exposure of astrocytes isolated from mice deficient in either TNF type‐1 receptor (TNFR1), TNF type‐2 receptor (TNFR2), or both, respectively, revealed that down‐regulation was mediated entirely by TNFR1. ZO‐1, which can interact with occludin, was found to co‐precipitate connexin43, but not occludin. These findings demonstrate that TNF selectively down‐regulates occludin in astrocytes, but not in cells forming established tight junctions, through TNFR1 and suggest that NF‐κB is involved as a negative regulator.
Chronic ethanol exposure delays the ‘developmental switch’ of the NMDA receptor 2A and 2B subunits in cultured cerebellar granule neurons
Authors: Snell Lawrence D. , Bhave Sanjiv V. , Tabakoff Boris , Hoffman Paula L.
Published: March 2003

Chronic ethanol treatment of cultured neurons from various brain areas has been found to increase NMDA receptor function and to alter the levels of some NMDA receptor subunit proteins. Because the cultured neurons are exposed to ethanol during a period when the NMDA receptor is undergoing developmental changes in subunit expression, we wished to determine whether ethanol treatment alters this developmental pattern. We found that 3 days of treatment of cerebellar granule neurons with ethanol, which was previously reported to increase NMDA receptor function, resulted in a delay in the ‘developmental switch’ of the NR2A and NR2B subunits, i.e. the developmental decrease in NR2B and increase in NR2A protein expression. As a result, the level of NR2B was higher, and that of NR2A was lower, in the ethanol‐treated cells than in control cells. Cross‐linking experiments showed that the changes in total receptor subunit proteins levels were reflected in cell‐surface expressed proteins, indicating changes in the amount of functional receptors. These results were confirmed by a higher potency of glycine at the NMDA receptor in the ethanol‐treated cells, as determined by NMDA/glycine‐induced increases in intracellular Ca2+. The results suggest that the mechanism by which ethanol alters NMDA receptor expression in cultured neurons, where receptors are undergoing development, differs from the mechanism of ethanol's effect on NMDA receptors in adult brain. Changes in the proportion of NR2A and NR2B subunits may contribute to effects of ethanol on neuronal development.
Cyclic AMP modulates the response of central nervous system glia to fibroblast growth factor‐2 by redirecting signalling pathways
Authors: Bayatti Nadhim , Engele Jürgen
Published: March 2003

Fibroblast growth factor‐2 (FGF‐2) acts as both a potent mitogen and differentiation factor for CNS glia. In the present study, we provide evidence that intracellular cAMP determines the proliferation‐differentiation decision of astroglia to FGF‐2 by either facilitating FGF‐2 signalling to extracellular signal‐related protein kinase (ERK) or cAMP response element binding protein (CREB). Pharmacologically increasing intracellular cAMP levels in cultured cortical astroglia by treatment with dbcAMP or forskolin attenuated FGF‐2‐induced ERK phosphorylation and glial cell proliferation. Similarly, FGF‐2‐induced glial proliferation was attenuated in the presence of the MEK inhibitor, PD98059, thus, confirming a direct correlation between FGF‐2‐induced ERK activation and glial cell proliferation. On the other hand, increases in intracellular cAMP levels in cortical astroglia prolonged FGF‐2‐induced CREB phosphorylation and subsequently potentiated the cAMP response element‐dependent transcription of the immediate early gene, c‐fos. Moreover, the effects of cAMP on the time‐course of FGF‐2‐dependent CREB phosphorylation were mimicked by PD98059, suggesting that the cAMP‐induced redirection of FGF‐2‐signalling is linked to the RAF‐MEK‐ERK signalling pathway.
Retinal phospholipase C from squid is a regulator of Gqα GTPase activity
Authors: Mayeenuddin Linnia H. , Bamsey Christine , Mitchell Jane
Published: March 2003

The phospholipase C (PLC) pathway is the major signaling mechanism of photoactivation in invertebrate photoreceptors. Here we report the cloning of a cDNA encoding a 140‐kDa retinal PLC that is uniquely expressed in squid photoreceptors. This cDNA encodes a protein with multiple distinct modular domains: PH, X and Y catalytic, and C2 domains, as well as G‐ and P‐box motifs and two GTP/ATP binding motifs. The PLC was stimulated by activated squid Gqα but not by squid Gqβγ or mammalian βγ subunits. The PLC was inhibited by monophosphate, diphosphate and triphosphate nucleotides but not cyclic nucleosides. We also tested the ability of PLC‐140 to regulate the GTPase activity of Gqα in the rhabdomeric membranes. Depletion of PLC‐140 from the rhabdomeric membranes decreased the GTP hydrolysis but not GTPγS binding to the membranes. Reconstitution of purified PLC‐140 with membranes accelerated Gqα GTPase activity by fivefold at a concentration of 2.5 µm. Our data suggest that PLC‐140 plays an important role in both the activation and inactivation pathways of invertebrate visual transduction.
Expression of glucose transporter GLUT3 by endotoxin in cultured rat astrocytes: the role of nitric oxide
Authors: Cidad Pilar , Garcia‐Nogales Paula , Almeida Angeles , Bolaños Juan P.
Published: March 2003

The induction of nitric oxide (NO) synthase in astrocytes by endotoxin and/or cytokine treatment is associated with increased glucose consumption and glycolysis, but the mechanism whereby this phenomenon occurs remains obscure. In this work, we have addressed this issue and found that incubation of cultured rat astrocytes with lipopolysaccharide (LPS; 1 µg/mL) for 24 h increased the level of constitutively expressed GLUT1 glucose transporter mRNA, and triggered GLUT3 mRNA expression, which was absent in normal astrocytes. The occurrence of GLUT3 protein after LPS treatment was corroborated by western blotting and immunocytochemistry. A 4‐h incubation of astrocytes in the absence of glucose, or under an oxygen‐poor (3%) atmosphere also resulted in GLUT3 mRNA overexpression. Experiments performed with 2‐deoxy‐d‐[U‐14C]glucose (at 0.1 mm of d‐glucose) confirmed that LPS (0.1–10 µg/mL) dose‐dependently increased the rate of glucose uptake (by a factor of 1.6 at 1 µg/mL of LPS), which was paralleled with the increase in NO synthesis. Furthermore, blockade of NO synthase with 2‐amino‐5,6‐dihydro‐6‐methyl‐(4H)‐1,3‐thiazine (AMT; 50 µm) partially (by 45%) prevented the LPS‐mediated increase in glucose uptake. Finally, incubation of astrocytes with the NO donor 1‐[2‐(2‐aminoethyl)‐N‐(2‐ammonioethyl)amino]diazen‐1‐ium‐1,2‐diolate (DETA; 100 µm) increased by a factor of 1.4 the rate of glucose uptake. We conclude that the increase in GLUT3‐driven glucose uptake in astrocytes would have a neuroprotective role under conditions in which NO formation is combined with hypoglycaemia, such as in brain ischemia.
Agonists of the P2YAC‐receptor activate MAP kinase by a ras‐independent pathway in rat C6 glioma
Authors: Grobben Bert , Claes Patrik , Van Kolen Kristof , Roymans Dirk , Fransen Paul , Sys Stanislas U. , Slegers Herman
Published: March 2003

We have previously shown that an ecto‐NPPase modulates the ATP‐ and ADP‐mediated P2YAC‐receptor activation in rat C6 glioma. In the present study, 2MeSADP and Ap3A induced no detectable PI turnover and were identified as specific agonists of the P2YAC‐receptor with EC50 values of 250 ± 37 pm and 1 ± 0.5 µm, respectively. P2YAC‐receptor stimulation increased MAP kinase (ERK1/2) activation that returned to the basal level 4 h after stimulation and was correlated with a gradual desensitization of the P2YAC‐purinoceptor. The purinoceptor antagonists DIDS and RB2 blocked MAP kinase activation. An IP3‐independent Ca2+‐influx was observed after P2YAC‐receptor activation. Inhibition of this influx by Ca2+‐chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC‐inhibitors and a specific MEK‐inhibitor inhibited the 2MeSADP‐ and Ap3A‐induced MAP kinase activation. In addition, transfection with dominant negative RhoAAsn19 rendered C6 cells insensitive to P2YAC‐receptor‐mediated MAP kinase activation whereas dominant negative ras was without effect. Immunoprecipitation experiments indicated a significant increase in the phosphorylation of raf‐1 after P2YAC‐receptor activation. We may conclude that P2YAC‐purinoceptor agonists activate MAP kinase through a Gi‐RhoA‐PKC‐raf‐MEK‐dependent, but ras‐ and Ca2+‐independent cascade.
Role of capacitative calcium entry on glutamate‐induced calcium influx in type‐I rat cortical astrocytes
Authors: Pizzo Paola , Burgo Andrea , Pozzan Tullio , Fasolato Cristina
Published: March 2003

Capacitative calcium entry (CCE) has been described in a variety of cell types. To date, little is known about its role in the CNS, and in particular in the cross‐talk between glia and neurons. We have first analyzed the properties of CCE of astrocytes in culture, in comparison with that of the rat basophilic leukemia cell line (RBL‐2H3), a model where calcium release‐activated Ca2+ (CRAC) channels have been unambiguously correlated with CCE. We here show that (i) in astrocytes CCE activated by store depletion and Ca2+ influx induced by glutamate share the same pharmacological profile of CCE in RBL‐2H3 cells and (ii) glutamate‐induced Ca2+ influx in astrocytes plays a primary role in glutamate‐dependent intracellular Ca2+ concentration ([Ca2+]i) oscillations, being these latter reduced in frequency and amplitude by micromolar concentrations of La3+. Finally, we compared the expression of various mammalian transient receptor potential genes (TRP) in astrocytes and RBL‐2H3 cells. Despite the similar pharmacological properties of CCE in these cells, the pattern of TRP expression is very different. The involvement of CCE and TRPs in glutamate dependent activation of astrocytes is discussed.
Anandamide, but not 2‐arachidonoylglycerol, accumulates during in vivo neurodegeneration
Authors: Hansen Henrik H. , Schmid Patricia C. , Bittigau Petra , Lastres‐Becker Isabel , Berrendero Fernando , Manzanares Jorge , Ikonomidou Chrysanthy , Schmid Harald H. O. , Fernández‐Ruiz J. Javier , Hansen Harald S.
Published: March 2003

Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N‐arachidonoylethanolamine (anandamide) and 2‐arachidonoylglycerol (2‐AG), as well as other constituents of their lipid families, N‐acylethanolamines (NAEs) and 2‐monoacylglycerols (2‐MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46‐fold increase of cortical NAE concentrations (anandamide, 13‐fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2‐AG and other 2‐MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB1 receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up‐regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2‐AG, and CB1 receptor density.
ΔΨm‐Dependent and ‐independent production of reactive oxygen species by rat brain mitochondria
Authors: Votyakova Tatyana V. , Reynolds Ian J.
Published: March 2003

Mitochondria are widely believed to be the source of reactive oxygen species (ROS) in a number of neurodegenerative disease states. However, conditions associated with neuronal injury are accompanied by other alterations in mitochondrial physiology, including profound changes in the mitochondrial membrane potential ΔΨm. In this study we have investigated the effects of ΔΨm on ROS production by rat brain mitochondria using the fluorescent peroxidase substrates scopoletin and Amplex red. The highest rates of mitochondrial ROS generation were observed while mitochondria were respiring on the complex II substrate succinate. Under this condition, the majority of the ROS signal was derived from reverse electron transport to complex I, because it was inhibited by rotenone. This mode of ROS generation is very sensitive to depolarization of ΔΨm, and even the depolarization associated with ATP generation was sufficient to inhibit ROS production. Mitochondria respiring on the complex I substrates, glutamate and malate, produce very little ROS until complex I is inhibited with rotenone, which is also consistent with complex I being the major site of ROS generation. This mode of oxidant production is insensitive to changes in ΔΨm. With both substrates, ubiquinone‐derived ROS can be detected, but they represent a more minor component of the overall oxidant signal. These studies demonstrate that rat brain mitochondria can be effective producers of ROS. However, the optimal conditions for ROS generation require either a hyperpolarized membrane potential or a substantial level of complex I inhibition.
Chronic inhibition of cortex microsomal Mg2+/Ca2+ ATPase‐mediated Ca2+ uptake in the rat pilocarpine model following epileptogenesis
Authors: Parsons J. Travis , Churn Severn B. , DeLorenzo Robert J.
Published: March 2003

In the rat pilocarpine model, 1 h of status epilepticus caused significant inhibition of Mg2+/Ca2+ ATPase‐mediated Ca2+ uptake in cortex endoplasmic reticulum (microsomes) isolated immediately after the status episode. The rat pilocarpine model is also an established model of acquired epilepsy. Several weeks after the initial status epilepticus episode, the rats develop spontaneous recurrent seizures, or epilepsy. To determine whether inhibition of Ca2+ uptake persists after the establishment of epilepsy, Ca2+ uptake was studied in cortical microsomes isolated from rats displaying spontaneous recurrent seizures for 1 year. The initial rate and total Ca2+ uptake in microsomes from epileptic animals remained significantly inhibited 1 year after the expression of epilepsy compared to age‐matched controls. The inhibition of Ca2+ uptake was not due to individual seizures nor an artifact of increased Ca2+ release from epileptic microsomes. In addition, the decreased Ca2+ uptake was not due to either selective isolation of damaged epileptic microsomes from the homogenate or decreased Mg2+/Ca2+ ATPase protein in the epileptic microsomes. The data demonstrate that inhibition of microsomal Mg2+/Ca2+ ATPase‐mediated Ca2+ uptake in the pilocarpine model may underlie some of the long‐term plasticity changes associated with epileptogenesis.
Potentiation of PGE2‐mediated cAMP production during neuronal differentiation of human neuroblastoma SK‐N‐BE(2)C cells
Authors: Choi, Se‐Young , Choi, Bo‐Hwa , Suh, Byung‐Chang , Chae, Hee‐Don , Kim, Jong‐So , Shin, Min‐Jung , Kang Shin‐Sung , Negishi Manabu , Kim Kyong‐Tai
Published: March 2003

The prostaglandin‐evoked cAMP production was studied in human neuroblastoma SK‐N‐BE(2)C cells during neuronal differentiation induced by all‐trans retinoic acid. The incubation with 5 µm all‐trans retinoic acid for 4–6 days promoted neurite outgrowth of cells. After differentiation, prostaglandin E2 (PGE2)‐induced cAMP production was dramatically increased, whereas forskolin‐ and AlF

‐induced cAMP productions were not changed. The increase reached maximum after 4‐days of incubation with all‐trans retinoic acid. The differentiation caused an increase in the maximal response and a decrease in the half‐maximal effective concentration of the PGE2‐induced cAMP production. In addition, the binding of [3H]PGE2 to membrane receptors was enhanced in differentiated cells. However, the order of potency of the various prostaglandins (PGE1 = PGE2 > PGD2 = PGF2α = PGI2) in cAMP production did not change during the differentiation, suggesting that mainly E‐prostanoid (EP) receptors were involved. Butaprost, an EP2 receptor specific agonist, increased the cAMP level in a concentration dependent manner and had a similar potentiating effect on cAMP production as PGE2 upon differentiation. Northern blot analysis using the human cDNA probes shows that the EP2 mRNA level was about seven times higher in differentiated cells, while the dopamine β‐hydroxylase (DBH) mRNA completely disappeared. Our results, thus, suggest that elevated gene expression of the prostanoid EP2 receptor results in an increase in the PGE2‐evoked cAMP production in SK‐N‐BE(2)C cells during neuronal differentiation.
SIN‐1‐induced cytotoxicity in mixed cortical cell culture: peroxynitrite‐dependent and ‐independent induction of excitotoxic cell death
Authors: Trackey Joseph L. , Uliasz Tracy F. , Hewett Sandra J.
Published: March 2003

3‐Morpholinosyndnomine (SIN‐1) has been reported to be a peroxynitrite (OONO−) donor because it produces both nitric oxide (NO) and superoxide (

O2−·) upon decomposition in aqueous solution. However, SIN‐1 can decompose to primarily NO in the presence of electron acceptors, including those found in biological tissues, making it necessary to determine the release product(s) formed in any given biological system. In a mixed cortical cell culture system, SIN‐1 caused a concentration‐dependent increase in cortical cell injury with a parallel increase in the release of cellular proteins containing 3‐nitrotyrosine into the culture medium. The increase in 3‐nitrotyrosine immunoreactivity, a footprint of OONO− production, was specific for SIN‐1 as exposure to neurotoxic concentrations of an NO donor (Z)‐1‐[2‐aminoethyl)‐N‐(2‐ammonioethyl) aminodiazen‐1‐ium‐1,2‐diolate (DETA/NO), or NMDA did not result in the nitration of protein tyrosine residues. Both SIN‐1‐induced injury and 3‐nitrotyrosine staining were prevented by the addition of either 5,10,15,20‐Tetrakis (4‐sulfonatophenyl) prophyrinato iron (III) [FeTPPS], an OONO− decomposition catalyst, or uric acid, an OONO− scavenger. Removal of NO alone was sufficient to inhibit the formation of OONO− from SIN‐1 as well as its cytotoxicity. Removal of
O2−· and the subsequently formed H2O2 by superoxide dismutase (SOD) plus catalase likewise prevented the nitration of protein‐bound tyrosine but actually enhanced the cytotoxicity of SIN‐1, indicating that cortical cells can cope with the oxidative but not the nitrosative stress generated. Finally, neural injury induced by SIN‐1 in unadulterated cortical cells was prevented by antagonism of AMPA/kainate receptors, while blockade of the NMDA receptor was without effect. In contrast, activation of both NMDA and non‐NMDA receptors contributed to the SIN‐1‐mediated neurotoxicity when cultures were exposed in the presence of SOD plus catalase. Thus, whether SIN‐1 initiates neural cell death in an OONO−‐dependent or ‐independent manner is determined by the antioxidant status of the cells. Further, the mode of excitotoxicity by which injury progresses is determined by the NO‐related species generated.
Depolarization‐stimulated catecholamine biosynthesis: involvement of protein kinases and tyrosine hydroxylase phosphorylation sites in situ
Authors: Salvatore Michael F. , Waymire Jack C. , Haycock John W.
Published: March 2003

Depolarizing stimuli increase catecholamine (CA) biosynthesis, tyrosine hydroxylase (TH) activity, and TH phosphorylation at Ser19, Ser31, and Ser40 in a Ca2+‐dependent manner. However, the identities of the protein kinases that phosphorylate TH under depolarizing conditions are not known. Furthermore, although increases in Ser31 or Ser40 phosphorylation increase TH activity in vitro, the relative influence of phosphorylation at these sites on CA biosynthesis under depolarizing conditions is not known. We investigated the participation of extracellular signal‐regulated protein kinase (ERK) and cAMP‐dependent protein kinase (PKA) in elevated K+‐stimulated TH phosphorylation in PC12 cells using an ERK pathway inhibitor, PD98059, and PKA‐deficient PC12 cells (A126‐B1). In the same paradigm, we measured CA biosynthesis. TH phosphorylation stoichiometry (PS) was determined by quantitative blot‐immunolabeling using site‐ and phosphorylation state‐specific antibodies. Treatment with elevated K+ (+ 58 mm) for 5 min increased TH PS at each site in a Ca2+‐dependent manner. Pretreatment with PD98059 prevented elevated K+‐stimulated increases in ERK phosphorylation and Ser31 PS. In A126‐B1 cells, Ser40 PS was not significantly increased by forskolin, and elevated K+‐stimulated Ser40 PS was three‐ to five‐fold less than that in PC12 cells. In both cell lines, CA biosynthesis was increased 1.5‐fold after treatment with elevated K+ and was prevented by pretreatment with PD98059. These results suggest that ERK phosphorylates TH at Ser31 and that PKA phosphorylates TH at Ser40 under depolarizing conditions. They also suggest that the increases in CA biosynthesis under depolarizing conditions are associated with the ERK‐mediated increases in Ser31 PS.
Neuronal differentiation is triggered by oleic acid synthesized and released by astrocytes
Authors: Tabernero Arantxa , Lavado Eva M. , Granda Begoña , Velasco Ana , Medina José M.
Published: March 2003

Unlike in the adult brain, the newborn brain specifically takes up serum albumin during the postnatal period, coinciding with the stage of maximal brain development. Here we report that albumin stimulates oleic acid synthesis by astrocytes from the main metabolic substrates available during brain development. Oleic acid released by astrocytes is used by neurons for the synthesis of phospholipids and is specifically incorporated into growth cones. Oleic acid promotes axonal growth, neuronal clustering, and expression of the axonal growth‐associated protein‐43, GAP‐43; all these observations indicating neuronal differentiation. The effect of oleic acid on GAP‐43 synthesis is brought about by the activation of protein kinase C, since it was prevented by inhibitors of this kinase, such as H‐7, polymyxin or sphingosine. The expression of GAP‐43 was significantly increased in neurons co‐cultured with astrocytes by the presence of albumin indicating that neuronal differentiation takes place in the presence of oleic acid synthesized and released by astrocytes in situ. In conclusion, during brain development the presence of albumin could play an important role by triggering the synthesis and release of oleic acid by astrocytes, which induces neuronal differentiation.
Brain iron pathways and their relevance to Parkinson's disease
Authors: Berg D , Gerlach M. , Youdim M. B. H. , Double K. L. , Zecca L. , Riederer P. , Becker G.
Published: March 2003

A central role of iron in the pathogenesis of Parkinson's disease (PD), due to its increase in substantia nigra pars compacta dopaminergic neurons and reactive microglia and its capacity to enhance production of toxic reactive oxygen radicals, has been discussed for many years. Recent transcranial ultrasound findings and the observation of the ability of iron to induce aggregation and toxicity of α‐synuclein have reinforced the critical role of iron in the pathogenesis of nigrostriatal injury. Presently the mechanisms involved in the disturbances of iron metabolism in PD remain obscure. In this review we summarize evidence from recent studies suggesting disturbances of iron metabolism in PD at possibly different levels including iron uptake, storage, intracellular metabolism, release and post‐transcriptional control. Moreover we outline that the interaction of iron with other molecules, especially α‐synuclein, may contribute to the process of neurodegeneration. Because many neurodegenerative diseases show increased accumulation of iron at the site of neurodegeneration, it is believed that maintenance of cellular iron homeostasis is crucial for the viability of neurons.
Differential effects of unaggregated and aggregated amyloid β protein (1–40) on K+ channel currents in primary cultures of rat cerebellar granule and cortical neurones
Authors: Ramsden Martin , Plant Leigh D. , Webster Nicola J. , Vaughan Peter F. T. , Henderson Zaineb , Pearson Hugh A.
Published: March 2003

The effects of amyloid β protein on voltage‐gated K+ channel currents were studied using the whole‐cell patch‐clamp technique. The 1–40 amino acid form of amyloid β protein was applied to primary cultures of rat cerebellar granule and cortical neurones for 24 h. Both the unaggregated and aggregated forms of the peptide, which have differing biological activities, were used. In cerebellar granule neurones, 24‐h pre‐incubation with 1 µm unaggregated amyloid β protein resulted in a 60% increase in the ‘A’‐type component of K+ current. Increased delayed rectifier activity was Cd2+‐sensitive and was presumed to be secondary to an increase in voltage‐gated Ca2+ channel current activity. Unaggregated amyloid β protein had no effect on any component of the K+ channel current in cortical neurones. One micromolar of aggregated amyloid β protein had no effect on K+ channel current in either cell type but reduced cell survival within 24 h as measured using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling (TUNEL) assays. The unaggregated form of amyloid β protein had no neurotoxic effects when applied to either neurone type for up to 72 h. These data indicate that the unaggregated, non‐pathological form of amyloid β protein causes changes in the ion channel function of neurones, possibly reflecting a physiological role for the peptide.
Functional relevance of carnitine transporter OCTN2 to brain distribution ofl‐carnitine and acetyl‐l‐carnitine across the blood–brain barrier
Authors: Kido Yasuto , Tamai Ikumi , Ohnari Aki , Sai Yoshimichi , Kagami Toru , Nezu Jun‐ichi , Nikaido Hiroko , Hashimoto Noriyoshi , Asano Masahide , Tsuji Akira
Published: March 2003

Transport ofl‐[3H]carnitine and acetyl‐l‐[3H]carnitine at the blood–brain barrier (BBB) was examined by usingin vivoandin vitromodels.In vivobrain uptake of acetyl‐l‐[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl‐l‐carnitine and in the absence of sodium ions. Similar transport properties forl‐[3H]carnitine and/or acetyl‐l‐[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes ofl‐[3H]carnitine and acetyl‐l‐[3H]carnitine by RBEC1 were sodium ion‐dependent, saturable withKmvalues of 33.1 ± 11.4 µmand 31.3 ± 11.6 µm, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT‐PCR method. Furthermore, the uptake of acetyl‐l‐[3H]carnitine by the BCECs ofjuvenile visceral steatosis(jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions ofl‐[3H]carnitine and acetyl‐l‐[3H]carnitine injvsmice were slightly lower than those of wild‐type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport ofl‐carnitine and acetyl‐l‐carnitine from the circulating blood to the brain across the BBB.
Oxidative impairment in scrapie‐infected mice is associated with brain metals perturbations and altered antioxidant activities
Authors: Wong Boon‐Seng , Brown David R. , Pan Tao , Whiteman Matthew , Liu Tong , Bu Xiaodong , Li Ruliang , Gambetti Pierluigi , Olesik John , Rubenstein Richard , Sy Man‐Sun
Published: March 2003

Prion diseases are characterized by the conversion of the normal cellular prion protein (PrPC) into a pathogenic isoform (PrPSc). PrPC binds copper, has superoxide dismutase (SOD)‐like activity in vitro, and its expression aids in the cellular response to oxidative stress. However, the interplay between PrPs (PrPC, PrPSc and possibly other abnormal species), copper, anti‐oxidation activity and pathogenesis of prion diseases remain unclear. In this study, we reported dramatic depression of SOD‐like activity by the affinity‐purified PrPs from scrapie‐infected brains, and together with significant reduction of Cu/Zn‐SOD activity, correlates with significant perturbations in the divalent metals contents. We also detected elevated levels of nitric oxide and superoxide in the infected brains, which could be escalating the oxidative modification of cellular proteins, reducing gluathione peroxidase activity and increasing the levels of lipid peroxidation markers. Taken together, our results suggest that brain metal imbalances, especially copper, in scrapie infection is likely to affect the anti‐oxidation functions of PrP and SODs, which, together with other cellular dysfunctions, predispose the brains to oxidative impairment and eventual degeneration. To our knowledge, this is the first study documenting a physiological connection between brain metals imbalances, the anti‐oxidation function of PrP, and aberrations in the cellular responses to oxidative stress, in scrapie infection.
Cachexia in MAC16 adenocarcinoma: suppression of hunger despite normal regulation of leptin, insulin and hypothalamic neuropeptide Y
Authors: Bing Chen , Taylor Sue , Tisdale Michael J. , Williams Gareth
Published: March 2003

Weight loss normally stimulates hunger, through mechanisms that include falls in circulating leptin and insulin, leading to stimulation of hypothalamic neuropeptide Y (NPY). Here, we investigated the leptin, insulin and NPY to clarify why hunger is suppressed in mice with severe cachexia due to the MAC16 adenocarcinoma. MAC16‐bearing mice progressively lost weight (19% below controls) and fat (− 61%) over 16 days after tumour transplantation, while total food intake fell by 10%. Pair‐fed mice showed less wasting, with final weight being 9% and fat mass 25% below controls. Plasma leptin fell by 85% in MAC16 and 51% in pair‐fed mice, in proportion to loss of fat. Plasma insulin was also reduced by 49% in MAC16 and 53% in pair‐fed groups. Hypothalamic leptin receptor (OB‐Rb) mRNA was significantly increased in both MAC16 (+ 223%) and pair‐fed (+192%) mice. Hypothalamic NPY mRNA was also significantly raised in MAC16 (+152%) and pair‐fed (+ 99%) groups, showing negative correlations with plasma leptin and insulin, and a positive association with OB‐Rb mRNA. In MAC16‐induced cachexia, leptin production and hypothalamic OB‐Rb and NPY expression are regulated appropriately in response to fat depletion. Therefore, suppression of hunger is probably due to tumour products that inhibit NPY transport or release, or that interfere with neuronal targets downstream of NPY.
The dopamine transporter gene (SLC6A3) variable number of tandem repeats domain enhances transcription in dopamine neurons
Authors: Michelhaugh Sharon K. , Fiskerstrand Carolyn , Lovejoy Elizabeth , Bannon Michael J. , Quinn John P.
Published: March 2003

The dopamine (DAT) and serotonin (SERT) transporter genes both contain variable number of tandem repeats (VNTR) in non‐coding gene regions which have been correlated with a predisposition to a variety of CNS disorders. There is considerable homology between individual DAT and SERT repeat DNA sequences, which is reflected in their ability to compete with each other for specific protein binding as demonstrated by electrophoretic mobility shift assay. The SERT VNTR has recently been shown to act as a transcriptional enhancer. Because of the similarities between SERT and DAT VNTRs, the DAT VNTR may also enhance transcription. This study demonstrates by lipid transfection into an immortalized dopaminergic cell line and biolistic transfection into dopamine neurons in neonatal rat midbrain slices that the human nine‐repeat DAT VNTR can enhance transcription. This enhancing activity suggests that the DAT VNTR may play a role in regulation of DAT gene expression.
Increased oxidative damage to DNA in a transgenic mouse model of Huntington's disease
Authors: Bogdanov Misha B. , Andreassen Ole A. , Dedeoglu Alpaslan , Ferrante Robert J. , Beal M. Flint
Published: March 2003

Mitochondrial dysfunction and oxidative damage may play a role in the pathogenesis of Huntington's disease (HD). We examined concentrations of 8‐hydroxy‐2‐deoxyguanosine (OH8dG), a well‐established marker of oxidative damage to DNA, in a transgenic mouse model of HD (R6/2). Increased concentrations of OH8dG were found in the urine, plasma and striatal microdialysates of the HD mice. Increased concentrations were also observed in isolated brain DNA at 12 and 14 weeks of age. Immunocytochemistry showed increased OH8dG staining in late stages of the illness. These results suggest that oxidative damage may play a role in the pathogenesis of neuronal degeneration in the R6/2 transgenic mouse model of HD.
The role of dopamine in motor symptoms in the R6/2 transgenic mouse model of Huntington's disease
Authors: Hickey Miriam A. , Reynolds Gavin P. , Morton A. Jennifer
Published: March 2003

In both Huntington's disease (HD) patients and genetic mouse models of HD, there is a pre‐symptomatic loss of dopamine (DA) receptors, suggesting that dysfunctional dopaminergic neurotransmission may be involved in early HD presentation. However, the role of DA in HD symptoms is not fully understood. In this study, we examined the possibility that dysfunctional dopaminergic neurotransmission contributes to the progressive decline in motor function of a transgenic mouse model of HD (R6/2 line). We found that R6/2 mice display an age‐dependent abnormal behavioural response to (+)‐methamphetamine (METH) and a dose‐dependent increase in sensitivity to METH toxicity compared with wildtype (WT) mice. R6/2 mice also showed an attenuated response to cocaine, indicating that DA release may be compromised. Striatal DA levels were reduced in R6/2 mice by 9 weeks of age. Replacement of DA by chronic treatment with laevodopa (l‐DOPA, administered as Sinemet) caused short‐term improvements in activity and rearing behaviour, and abolished abnormal spontaneous hindlimb grooming. However, long‐term treatment with l‐DOPA had deleterious effects on survival and rotarod performance of R6/2 mice. These results suggest that dysfunctional DA neurotransmission contributes to phenotype development in R6/2 mice and thus also may be important in symptom progression in HD.
GM1 ganglioside induces phosphorylation and activation of Trk and Erk in brain
Authors: Duchemin Anne‐Marie , Ren Qun , Mo Lili , Neff Norton H. , Hadjiconstantinou Maria
Published: March 2003

We investigated the ability of GM1 to induce phosphorylation of the tyrosine kinase receptor for neurotrophins, Trk, in rat brain, and activation of possible down‐stream signaling cascades. GM1 increased phosphorylated Trk (pTrk) in slices of striatum, hippocampus and frontal cortex in a concentration‐ and time‐dependent manner, and enhanced the activity of Trk kinase resulting in receptor autophosphorylation. The ability of GM1 to induce pTrk was shared by other gangliosides, and was blocked by the selective Trk kinase inhibitors K252a and AG879. GM1 induced phosphorylation of TrkA > TrkC > TrkB in a region‐specific distribution. Adding GM1 to brain slices activated extracellular‐regulated protein kinases (Erks) in all three brain regions studied. In striatum, GM1 elicited activation of Erk2 > Erk1 in a time‐and concentration‐dependent manner. The GM1 effect on Erk2 was mimicked by other gangliosides, and was blocked by the Trk kinase inhibitors K252a and AG879. Pertussis toxin, as well as Src protein tyrosine kinase and protein kinase C inhibitors, did not prevent the GM1‐induced activation of Erk2, apparently excluding the participation of Gi and Gq/11 protein‐coupled receptors. Intracerebroventricular administration of GM1 induced a transient phosphorylation of TrkA and Erk1/2 in the striatum and hippocampus complementing the in situ studies. These observations support a role for GM1 in modulating Trk and Erk phosphorylation and activity in brain.