bluuebunny/crossref_metadata_2025 huggingface dataset:
Nicotine protects against arachidonic‐acid‐induced caspase activation, cytochrome c release and apoptosis of cultured spinal cord neurons
Authors: Garrido Rosario , Mattson Mark P. , Hennig Bernhard , Toborek Michal
Published: March 2003

Hydrolysis of membrane phospholipids of spinal cord neurons is one of the first events initiated in spinal cord trauma. In this process, free fatty acids, and in particular arachidonic acid, are released. Exposure of spinal cord neurons to free arachidonic acid can compromise cell survival and initiate apoptotic cell death. In order to determine potential mechanisms of apoptosis induced by arachidonic acid, activation of caspases ‐3, ‐8, and ‐9, as well as the release of cytochrome c into the cytoplasm were measured in cultured spinal cord neurons exposed to 10 µm of this fatty acid. In addition, because nicotine can exert a variety of neuroprotective effects, we hypothesized that it can prevent arachidonic acid induced apoptosis of spinal cord neurons. To study this hypothesis, spinal cord neurons were pretreated with nicotine (10 µm for 2 h) before arachidonic acid exposure and caspase activation as well as markers of apoptotic cell death were studied. Treatment of spinal cord neurons with arachidonic acid for up to 24 h significantly increased cytoplasmic levels of cytochrome c, induced caspase activation and induced DNA laddering, a hallmark of apoptotic cell death. Nicotine pretreatment markedly attenuated all these effects. In addition, antagonist studies suggest that the α7 nicotinic receptor is primarily responsible for these anti‐apoptotic effects of nicotine. These results indicate that nicotine can exert potent neuroprotective effects by inhibiting arachidonic acid induced apoptotic cascades of spinal cord neurons.
Superoxide dismutase activity in organotypic midbrain–striatum co‐cultures is associated with resistance of dopaminergic neurons to excitotoxicity
Authors: Katsuki Hiroshi , Tomita Michiko , Takenaka Chikako , Shirakawa Hisashi , Shimazu Seiichiro , Ibi Masakazu , Kume Toshiaki , Kaneko Shuji , Akaike Akinori
Published: March 2003

We have previously demonstrated that dopaminergic neurons in midbrain–striatum slice co‐cultures are more resistant to NMDA cytotoxicity than the same neuronal population in single midbrain slice cultures. Here, we show that dopaminergic neurons in midbrain–striatum co‐cultures also exhibit resistance to the cytotoxicity of nitric oxide donors, 2,2′‐(hydroxynitrosohydrazono)bis‐ethanamine (NOC‐18) and 3‐morpholinosydnonimine (SIN‐1). The cytotoxicity of NMDA (30 µm) in single cultures was significantly attenuated by the nitric oxide synthase (NOS) inhibitor Nω‐nitro‐l‐arginine (100 µm), whereas the toxicity in co‐cultures was not. The levels of tyrosine residue nitration of tyrosine hydroxylase, a hallmark of the occurence of peroxynitrite anion in dopaminergic neurons, were lower in co‐cultures than those in single cultures. Single cultures and co‐cultures did not show appreciable differences in the number or distribution of NOS‐containing neurons as assessed by NADPH diaphorase histochemistry. On the other hand, midbrain slices cultured with striatal slices showed higher levels of superoxide dismutase (SOD) activity as well as increased protein levels of Cu,Zn‐SOD, than midbrain slices cultured alone. These results suggested that the generation of NO is involved in NMDA cytotoxicity on dopaminergic neurons, and that increased activity of SOD in co‐cultures renders dopaminergic neurons resistant to NMDA cytotoxicity by preventing the formation of peroxynitrite.
Reactive oxygen species regulate signaling pathways induced by M1 muscarinic receptors in PC12M1 cells
Authors: Mangelus Miriam , Kroyter Avital , Galron Ronit , Sokolovsky Mordechai
Published: March 2003

Activation of the m1 muscarinic receptor subtype in rat pheochromocytoma (PC12) cells stably expressing cloned m1 muscarinic acetylcholine receptors was previously shown to induce morphological changes and growth arrest. However, the signaling pathways which lead to these effects were not identified. In an attempt to characterize the intracellular signaling that might be involved in the muscarinic‐induced effects, we investigated the role of reactive oxygen species in the regulation of these processes. Stimulation of the muscarinic receptor in these cells increased the intracellular concentrations of reactive oxygen species. Muscarinic activation induced intracellular signaling pathways that involve activation of Ras, extracellular signal‐regulated kinase (ERK), and p38. These pathways were partially blocked when reactive oxygen species (ROS) production was prevented by the antioxidant N‐acetylcysteine. Other muscarinic‐induced signals, such as activation of c‐Jun NH2‐terminal kinase (JNK) or an increase in the binding activity of the transcription factors nuclear factor‐κB and activator protein‐1, were inhibited by the antioxidant dicoumarol. N‐Acetylcysteine also blocked the growth arrest and changes in cell shape induced by stimulation of the muscarinic receptor in PC12M1 cells. These findings suggest that ROS act as second messengers in muscarinic‐induced cellular signaling. Moreover, generation of ROS appears to be an early and critical intermediary event, which occurs immediately after stimulation of the muscarinic receptor and affects in a variety of mechanisms the muscarinic‐mediated cellular signaling.
The intracellular segment of the sodium channel β1 subunit is required for its efficient association with the channel α subunit
Authors: Meadows Laurence , Malhotra Jyoti Dhar , Stetzer Alisa , Isom Lori L. , Ragsdale David S.
Published: March 2003

Sodium channels consist of a pore‐forming α subunit and auxiliary β1 and β2 subunits. The subunit β1 alters the kinetics and voltage‐dependence of sodium channels expressed in Xenopus oocytes or mammalian cells. Functional modulation in oocytes depends on specific regions in the N‐terminal extracellular domain of β1, but does not require the intracellular C‐terminal domain. Functional modulation is qualitatively different in mammalian cells, and thus could involve different molecular mechanisms. As a first step toward testing this hypothesis, we examined modulation of brain NaV1.2a sodium channel α subunits expressed in Chinese hamster lung cells by a mutant β1 construct with 34 amino acids deleted from the C‐terminus. This deletion mutation did not modulate sodium channel function in this cell system. Co‐immunoprecipitation data suggest that this loss of functional modulation was caused by inefficient association of the mutant β1 with α, despite high levels of expression of the mutant protein. In Xenopus oocytes, injection of approximately 10 000 times more mutant β1 RNA was required to achieve the level of functional modulation observed with injection of full‐length β1. Together, these findings suggest that the C‐terminal cytoplasmic domain of β1 is an important determinant of β1 binding to the sodium channel α subunit in both mammalian cells and Xenopus oocytes.
Ca2+ changes induced by different presynaptic nicotinic receptors in separate populations of individual striatal nerve terminals
Authors: Nayak Sanjay V. , Dougherty John J. , McIntosh J. Michael , Nichols Robert A.
Published: March 2003

Presynaptic nicotinic acetylcholine receptors likely play a modulatory role in the nerve terminal. Using laser‐scanning confocal microscopy, we have characterized physiological responses obtained on activation of presynaptic nicotinic receptors by measuring calcium changes in individual nerve terminals (synaptosomes) isolated from the rat corpus striatum. Nicotine (500 nm) induced Ca2+ changes in a subset (10–25%) of synaptosomes. The Ca2+ responses were dependent on extracellular Ca2+ and desensitized very slowly (several minutes) on prolonged exposure to agonist. The nicotine‐induced Ca2+ responses were dose‐dependent and were completely blocked by dihydro‐β‐erythroidine (5 µm), differentially affected by mecamylamine (10 µm) and α‐conotoxin MII (100 nm), and not affected by α‐bungarotoxin (500 nm). Immunocytochemical studies using well‐characterized monoclonal antibodies revealed the presence of the α4 and α3/α5 nicotinic subunits. The nicotine‐induced responses were unaffected by prior depolarization or by a mixture of Ca2+ channel toxins including ω‐conotoxin MVIIC (500 nm), ω‐conotoxin GVIA (500 nm) and agatoxin TK (200 nm). Our results indicate that nicotinic receptors present on striatal nerve terminals induce Ca2+ entry largely without involving voltage‐gated Ca2+ channels, most likely by direct permeation via the receptor channel itself. In addition, at least two subpopulations of presynaptic nicotinic receptors reside on separate terminals in the striatum, suggesting distinct modulatory roles.
Peroxidative stress selectively down‐regulates the neuronal stress response activated under conditions of endoplasmic reticulum dysfunction
Authors: Paschen Wulf , Mengesdorf Thorsten , Althausen Sonja , Hotop Svenja
Published: March 2003

Oxidative stress has been implicated in mechanisms leading to neuronal cell injury in various pathological states of the brain. Here, we investigated the effect of peroxide exposure on the expression of genes coding for cytoplasmic and endoplasmic reticulum (ER) stress proteins. Primary neuronal cell cultures were exposed to H2O2 for 6 h and mRNA levels of hsp70, grp78, grp94, gadd153 were evaluated by quantitative PCR. In addition, peroxide‐induced changes in protein synthesis and cell viability were investigated. Peroxide treatment of cells triggered an almost 12‐fold increase in hsp70 mRNA levels, but a significant decrease in grp78, grp94 and gadd153 mRNA levels. To establish whether peroxide exposure blocks the ER‐resident stress response, cells were also exposed to thapsigargin (Tg, a specific inhibitor of ER Ca2+‐ATPase) which has been shown to elicit the ER stress response. Tg exposure induced 7.2‐fold, 3.6‐fold and 8.8‐fold increase in grp78, grp94 and gadd153 mRNA levels, respectively. However, after peroxide pre‐exposure, the Tg‐induced effect on grp78, grp94 and gadd153 mRNA levels was completely blocked. The results indicate that oxidative damage causes a selective down‐regulation of the neuronal stress response activated under conditions of ER dysfunction. This down‐regulation was only observed in cultures exposed to peroxide levels which induced severe suppression of protein synthesis and cell injury, implying a causative link between peroxide‐induced down‐regulation of ER stress response system and development of neuronal cell injury. These observations could have implications for our understanding of the mechanisms underlying neuronal cell injury in pathological states of the brain associated with oxidative damage, including Alzheimer's disease where the neuronal stress response activated under conditions of ER dysfunction has been shown to be down‐regulated. Down‐regulation of ER stress response may increase the sensitivity of neurones to an otherwise nonlethal form of stress.
Hypoxia regulates glutamate metabolism and membrane transport in rat PC12 cells
Authors: Kobayashi Shuichi , Millhorn David E.
Published: March 2003

We investigated the effect of hypoxia on glutamate metabolism and uptake in rat pheochromocytoma (PC12) cells. Various key enzymes relevant to glutamate production, metabolism and transport were coordinately regulated by hypoxia. PC12 cells express two glutamate‐metabolizing enzymes, glutamine synthetase (GS) and glutamate decarboxylase (GAD), as well as the glutamate‐producing enzyme, phosphate‐activated glutaminase (PAG). Exposure to hypoxia (1% O2) for 6 h or longer increased expression of GS mRNA and protein and enhanced GS enzymatic activity. In contrast, hypoxia caused a significant decrease in expression of PAG mRNA and protein, and also decreased PAG activity. In addition, hypoxia led to an increase in GAD65 and GAD67 protein levels and GAD enzymatic activity. PC12 cells express three Na+‐dependent glutamate transporters; EAAC1, GLT‐1 and GLAST. Hypoxia increased EAAC1 and GLT‐1 protein levels, but had no effect on GLAST. Chronic hypoxia significantly enhanced the Na+‐dependent component of glutamate transport. Furthermore, chronic hypoxia decreased cellular content of glutamate, but increased that of glutamine. Taken together, the hypoxia‐induced changes in enzymes related to glutamate metabolism and transport are consistent with a decrease in the extracellular concentration of glutamate. This may have a role in protecting PC12 cells from the cytotoxic effects of glutamate during chronic hypoxia.
Enhancement of 3,4‐methylenedioxymethamphetamine neurotoxicity by the energy inhibitor malonate
Authors: Nixdorf W. L. , Burrows K. B. , Gudelsky G. A. , Yamamoto B. K.
Published: March 2003

The acute and long‐term effects of the local perfusion of 3,4‐methylenedioxymethamphetamine (MDMA) and the interaction with the mitochondrial inhibitor malonate (MAL) were examined in the rat striatum. MDMA, MAL or the combination of MAL with MDMA was reverse dialyzed into the striatum for 8 h via a microdialysis probe while extracellular dopamine (DA) and serotonin (5‐HT) were measured. One week later, tissue immediately surrounding the probe was assayed for DA and 5‐HT tissue content. Local perfusion of MDMA increased DA and 5‐HT release but did not produce long‐term depletion of DA or 5‐HT in tissue. Malonate also increased both DA and 5‐HT release but, in contrast to MDMA, produced only long‐term depletion of DA. The combined perfusion of MDMA/MAL synergistically increased the release of DA and 5‐HT and produced long‐term depletion of both DA and 5‐HT in tissue. These results support the conclusion that DA, compared with 5‐HT, neurons are more susceptible to mitochondrial inhibition. Moreover, MDMA, which does not normally produce DA depletion in the rat, exacerbated MAL‐induced DA depletions. The effect of MDMA in combination with MAL to produce 5‐HT depletion suggests a role for bio‐energetic stress in MDMA‐induced toxicity to 5‐HT neurons. Overall, these results highlight the importance of energy balance to the function of DA and 5‐HT neurons and to the toxic effects of MDMA.
[35S]GTPγS autoradiography reveals a wide distribution of Gi/o‐linked ADP receptors in the nervous system: close similarities with the platelet P2YADP receptor
Authors: Laitinen Jarmo T. , Uri Asko , Raidaru Gerda , Miettinen Riitta
Published: March 2003

No Gi‐linked P2Y receptors have been cloned to date but the presence of such receptors is thought to be restricted to platelets and certain clonal cell lines. Using the functional approach of [35S]guanosine 5′‐[γ‐thio]‐triphosphate autoradiography, we uncovered the widespread presence of such receptors in the CNS. Under conditions in which the prominent signal due to tonic adenosine receptor activity is masked, ADP and ATP stimulated G‐protein activity in multiple grey and white matter regions. Localization in the grey matter suggests inhibitory auto‐/heteroreceptor function. In the white matter, activated G proteins appeared as ‘hot spots’ (presumed oligodendrocyte progenitors) with scattered distribution along the main fibre tracts. Responses to ATP were diminished under conditions that inhibited degradation, suggesting that prior conversion to ADP explained agonist action. Uracil nucleotides were ineffective but 2‐methylthio‐ADP activated G proteins ≈ 500‐fold more potently than ADP, although both were similarly degraded. Throughout the brain, ADP‐dependent G‐protein activity was reversed by 2‐hexylthio‐AdoOC(O)Asp2, a non‐phosphate ATP analogue, whereas selective P2Y1 receptor antagonists proved ineffective. A similar receptor was also disclosed from the adrenal medulla. These data witness a hitherto unrecognized abundance of Gi/o‐linked ADP receptors in the nervous system. Biochemical and pharmacological behaviour suggests striking similarities to the elusive platelet P2YADP receptor.
CEP‐1347/KT‐7515, an inhibitor of SAPK/JNK pathway activation, promotes survival and blocks multiple events associated with Aβ‐induced cortical neuron apoptosis
Authors: Bozyczko‐Coyne Donna , O'Kane Teresa M. , Wu Zhi‐Liang , Dobrzanski Pawel , Murthy Seetha , Vaught Jeffry L. , Scott Richard W.
Published: March 2003

Although the mechanism of neuronal death in Alzheimer's disease (AD) has yet to be elucidated, a putative role for c‐junin this process has emerged. Thus, it was of interest to delineate signal transduction pathway(s) which regulate the transcriptional activity of c‐jun, and relate these to alternate gene inductions and biochemical processes associated with beta‐amyloid (Aβ) treatment. In this regard, the survival promoting activity of CEP‐1347, an inhibitor of the stress‐activated/c‐junN‐terminal (SAPK/JNK) kinase pathway, was evaluated against Aβ‐induced cortical neuron deathin vitro. Moreover, CEP‐1347 was used as a pharmacologic probe to associate multiple biochemical events with Aβ‐induced activation of the SAPK/JNK pathway. CEP‐1347 promoted survival and blocked Aβ‐induced activation of JNK kinase (MKK4, also known as MEK‐4, JNKK and SEK1) as well as other downstream events associated with JNK pathway activation. CEP‐1347 also blocked Aβ‐induction of cyclinD1andDP5genes and blocked Aβ‐induced increases in cytoplasmic cytochrome c, caspase 3‐like activity and calpain activation. The critical time window for cell death blockade by CEP‐1347 resided within the peak of Aβ‐induced MKK4 activation, thus defining this point as the most upstream event correlated to its survival‐promoting activity. Together, these data link the SAPK/JNK pathway and multiple biochemical events associated with Aβ‐induced neuronal death and further delineate the point of CEP‐1347 interception within this signal transduction cascade.
Testosterone‐mediated neuroprotection through the androgen receptor in human primary neurons
Authors: Hammond Jennifer , Le Quynh , Goodyer Cynthia , Gelfand Morrie , Trifiro Mark , LeBlanc Andrea
Published: March 2003

Estrogen is an active neuroprotectant and is presently investigated as a potential therapy against Alzheimer's disease for women. To determine if male hormones could also be neuroprotective, we investigated the effect of testosterone, methyltestosterone, and epitestosterone at physiological concentrations on primary cultures of human neurons induced to undergo apoptosis by serum deprivation. Serum deprivation significantly induces neuronal apoptosis in a protracted fashion. As expected, physiological concentrations of 17‐β‐estradiol and transcriptionally inactive 17‐α‐estradiol protect neurons against apoptosis. Similar to 17‐β‐estradiol, physiological concentrations of testosterone are also neuroprotective. Androgen receptors are present at 8 ± 2 fmol/mg protein in the neuron cultures. The non‐aromatizable androgen, mibolerone, is also neuroprotective and aromatase inhibitor, 4‐androsten‐4‐OL‐3,17‐dione, does not prevent testosterone‐mediated neuroprotection. In contrast, anti‐androgen, flutamide, eliminates testosterone‐mediated neuroprotection. Testosterone analog, methyltestosterone, showed androgen receptor‐dependent neuroprotection that was delayed in time indicating that a metabolite may be the active agent. The endogenous anti‐androgen, epitestosterone, also showed a slight neuroprotective effect but not through the androgen receptor. These results indicate that androgens induce neuroprotection directly through the androgen receptor. These data suggest that androgens may also be of therapeutic value against Alzheimer's disease in aging males.
Dual coupling of opioid receptor‐like (ORL1) receptors to adenylyl cyclase in the different layers of the rat main olfactory bulb
Authors: Onali Pierluigi , Ingianni Angela , Olianas Maria C.
Published: March 2003

The coupling of opioid receptor‐like (ORL1) receptors to adenylyl cyclase has been investigated in specific layers of the rat main olfactory bulb. Membranes prepared from the olfactory nerve‐glomerular layer (ON‐G layer), external plexiform layer (EP layer) and granule cell layer (GR layer) displayed specific binding sites for [3H]‐nociceptin/orphanin FQ ([3H]Noc/OFQ). In each layer, the presence of high‐and low‐affinity binding sites, with KD values in the picomolar and nanomolar range, respectively, was detected. The binding of [3H]Noc/OFQ was displaced by unlabelled Noc/OFQ, but not by opioid antagonists. In each layer, Noc/OFQ significantly stimulated [35S]GTPγS binding with nanomolar potencies. In ON‐G layer, Noc/OFQ inhibited basal adenylyl cyclase activity and the enzyme stimulations by corticotropin releasing hormone (CRH), Ca2+/calmodulin (Ca2+/CaM) and forskolin (FSK). In EP layer, Noc/OFQ inhibited Ca2+/CaM‐and FSK‐stimulated enzyme activities. Conversely, in GR layer the peptide stimulated basal cyclase activity and potentiated the enzyme activation by CRH. The Noc/OFQ stimulation was counteracted by the GDP‐bound form of the α subunit of transducin and was mimicked by transducin βγ subunits. In the same tissue layer, Ca2+/CaM‐and FSK‐stimulated enzyme activities were inhibited. Naloxone failed to antagonize all the actions of Noc/OFQ. Western blot and RT‐PCR analysis revealed the expression of Ca2+‐insensitive and ‐sensitive adenylyl cyclases in the three layers. These results demonstrate that in rat main olfactory bulb ORL1 receptors can differentially affect distinct forms of adenylyl cyclase in a layer specific manner.
Homocysteine potentiates β‐amyloid neurotoxicity: role of oxidative stress
Authors: Ho Pei I. , Collins Scott C. , Dhitavat Sirikarnt , Ortiz Daniela , Ashline David , Rogers Eugene , Shea Thomas B.
Published: March 2003

The cause of neuronal degeneration in Alzheimer's disease (AD) has not been completely clarified, but has been variously attributed to increases in cytosolic calcium and increased generation of reactive oxygen species (ROS). The β‐amyloid fragment (Aβ) of the amyloid precursor protein induces calcium influx, ROS and apoptosis. Homocysteine (HC), a neurotoxic amino acid that accumulates in neurological disorders including AD, also induces calcium influx and oxidative stress, which has been shown to enhance neuronal excitotoxicity, leading to apoptosis. We examined the possibility that HC may augment Aβ neurotoxicity. HC potentiated the Aβ‐induced increase in cytosolic calcium and apoptosis in differentiated SH‐SY‐5Y human neuroblastoma cells. The antioxidant vitamin E and the glutathione precursor N‐acetyl‐l‐cysteine blocked apoptosis following cotreatment with HC and Aβ, indicating that apoptosis is associated with oxidative stress. These findings underscore that moderate accumulation of excitotoxins at concentrations that alone do not appear to initiate adverse events may enhance the effects of other factors known to cause neurodegeneration such as Aβ.
The vanilloid receptor (VR1)‐mediated effects of anandamide are potently enhanced by the cAMP‐dependent protein kinase
Authors: De Petrocellis Luciano , Harrison Selena , Bisogno Tiziana , Tognetto Michele , Brandi Ines , Smith Graham D. , Creminon Cristophe , Davis John B. , Geppetti Pierangelo , Di Marzo Vincenzo
Published: March 2003

The endogenous cannabinoid receptor ligand, anandamide (AEA), is a full agonist of the vanilloid receptor type 1 (VR1) for capsaicin. Here, we demonstrate that the potency and efficacy of AEA at VR1 receptors can be significantly increased by the concomitant activation of protein kinase A (PKA). In human embryonic kidney (HEK) cells over‐expressing human VR1, AEA induces a rise in cytosolic Ca2+ concentration that is mediated by this receptor. The EC50 for this effect was decreased five‐fold in the presence of forskolin (FRSK, 1–5 µm) or the cAMP analogue, 8‐Br‐cAMP (10–100 µm). The effects of 8‐Br‐cAMP and FRSK were blocked by a selective PKA inhibitor. The FRSK (10 nm) also potently enhanced the sensory neurone‐ and VR1‐mediated constriction by AEA of isolated guinea‐pig bronchi, and this effect was abolished by a PKA inhibitor. In rat dorsal root ganglia slices, AEA‐induced release of substance P, an effect mediated by VR1 activation, was enhanced three‐fold by FRSK (10 nm). Thus, the ability of AEA to stimulate sensory VR1, with subsequent neuropeptide release, appears to be regulated by the state of activation of PKA. This observation supports the hypothesis that endogenous AEA might stimulate VR1 under certain pathophysiological conditions.
Selective and biphasic effect of the membrane lipid peroxidation product 4‐hydroxy‐2,3‐nonenal on N‐methyl‐d‐aspartate channels
Authors: Lu Chengbiao , Chan Sic L. , Haughey Norman , Lee Way‐Tso , Mattson Mark P.
Published: March 2003

Increased oxyradical production and membrane lipid peroxidation occur in neurons under physiological conditions and in neurodegenerative disorders. Lipid peroxidation can alter synaptic plasticity and may increase the vulnerability of neurons to excitotoxicity, but the underlying mechanisms are unknown. We report that 4‐hydroxy‐2,3‐nonenal (4HN), an aldehyde product of lipid peroxidation, exerts a biphasic effect on NMDA‐induced current in cultured rat hippocampal neurons with current being increased during the first 2 h and decreased after 6 h. Similarly, 4HN causes an early increase and a delayed decrease in NMDA‐induced elevation of intracellular Ca2+ levels. In contrast, 4HN affects neither the ion current nor the Ca2+ response to α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA). The initial enhancement of NMDA‐induced current is associated with increased phosphorylation of the NR1 receptor subunit, whereas the delayed suppression of current is associated with cellular ATP depletion and mitochondrial membrane depolarization. Cell death induced by 4HN is attenuated by an NMDA receptor antagonist, but not by an AMPA receptor antagonist. A secreted form of amyloid precursor protein, previously shown to protect neurons against oxidative and excitotoxic insults, prevented each of the effects of 4HN including the early and late changes in NMDA current, delayed ATP depletion, and cell death. These findings show that the membrane lipid peroxidation product 4HN can modulate NMDA channel activity, suggesting a role for this aldehyde in physiological and pathophysiological responses of neurons to oxidative stress.
Changes in the phosphorylation of initiation factor eIF‐2α, elongation factor eEF‐2 and p70 S6 kinase after transient focal cerebral ischaemia in mice
Authors: Althausen Sonja , Mengesdorf Thorsten , Mies Günter , Oláh Laszlo , Nairn Angus C. , Proud Christopher G. , Paschen Wulf
Published: March 2003

Mice were subjected to 60 min occlusion of the left middle cerebral artery (MCA) followed by 1–6 h of reperfusion. Tissue samples were taken from the MCA territory of both hemispheres to analyse ischaemia‐induced changes in the phosphorylation of the initiation factor eIF‐2α, the elongation factor eEF‐2 and p70 S6 kinase by western blot analysis. Tissue sections from additional animals were taken to evaluate ischaemia‐induced changes in global protein synthesis by autoradiography and changes in eIF‐2α phosphorylation by immunohistochemistry. Transient MCA occlusion induced a persistent suppression of protein synthesis. Phosphorylation of eIF‐2α was slightly increased during ischaemia, it was markedly up‐regulated after 1 h of reperfusion and it normalized after 6 h of recirculation despite ongoing suppression of protein synthesis. Similar changes in eIF‐2α phosphorylation were induced in primary neuronal cell cultures by blocking of endoplasmic reticulum (ER) calcium pump, suggesting that disturbances of ER calcium homeostasis may play a role in ischaemia‐induced changes in eIF‐2α phosphorylation. Dephosphorylation of eIF‐2α was not paralleled by a rise in levels of p67, a glycoprotein that protects eIF‐2α from phosphorylation, even in the presence of active eIF‐2α kinase. Phosporylation of eEF‐2 rose moderately during ischaemia, but returned to control levels after 1 h of reperfusion and declined markedly below control levels after 3 and 6 h of recirculation. In contrast to the only short‐lasting phosphorylation of eIF‐2a and eEF‐2, transient focal ischaemia induced a long‐lasting dephosphorylation of p70 S6 kinase. The results suggest that blocking of elongation does not play a major role in suppression of protein synthesis induced by transient focal cerebral ischaemia. Investigating the factors involved in ischaemia‐induced suppression of the initiation step of protein synthesis and identifying the underlying mechanisms may help to further elucidate those disturbances directly related to the pathological process triggered by transient cerebral ischaemia and leading to neuronal cell injury.
Dystroglycan contributes to the formation of multiple dystrophin‐like complexes in brain
Authors: Moukhles Hakima , Carbonetto Salvatore
Published: March 2003

In muscle, dystrophin anchors a complex of proteins at the cell surface which includes α‐dystroglycan, β‐dystroglycan, syntrophins and dystrobrevins. Mutations in the dystrophin gene lead to muscular dystrophy and mental retardation. In contrast to muscle, little is known about the localization and the molecular interactions of dystrophin and dystrophin associated proteins (DAPs) in brain. In the present study, we show that α‐dystroglycan and dystrophin are localized to large neurones in cerebral cortex, hippocampus, cerebellum and spinal cord. Furthermore, we show that dystroglycan is a member of three distinct dystrophin‐containing complexes. Two of these complexes contain syntrophin and both dystrophin and syntrophin are enriched in post‐synaptic densities. These data suggest that dystrophin and DAPs may have a role in the organization of CNS synapses. Interestingly, the enrichment for syntrophin in post‐synaptic densities is not affected in mice mutant for all dystrophin isoforms. Thus in the brain, unlike in muscle, the association of syntrophin with dystrophin is not crucial for the DAP complex which suggests that it may be associated with other proteins.
Specific proteolysis of the NR2 subunit at multiple sites by calpain
Authors: Guttmann Rodney P. , Baker Dana L. , Seifert Kristen M. , Cohen Akiva S. , Coulter Douglas A. , Lynch David R.
Published: March 2003

The NMDA subtype of glutamate receptor plays an important role in the molecular mechanisms of learning, memory and excitotoxicity. NMDA receptors are highly permeable to calcium, which can lead to the activation of the calcium‐dependent protease, calpain. In the present study, the ability of calpain to modulate NMDA receptor function through direct proteolytic digestion of the individual NMDA receptor subunits was examined. HEK293t cells were cotransfected with the NR1a/2A, NR1a/2B or NR1a/2C receptor combinations. Cellular homogenates of these receptor combinations were prepared and digested by purified calpain I in vitro. All three NR2 subunits could be proteolyzed by calpain I while no actin or NR1a cleavage was observed. Based on immunoblot analysis, calpain cleavage of NR2A, NR2B and NR2C subunits was limited to their C‐terminal region. In vitro calpain digestion of fusion protein constructs containing the C‐terminal region of NR2A yielded two cleavage sites at amino acids 1279 and 1330. Although it has been suggested that calpain cleavage of the NMDA receptor may act as a negative feedback mechanism, the current findings demonstrated that calpain cleavage did not alter [125I]MK801 binding and that receptors truncated to the identified cleavage sites had peak intracellular calcium levels, 45Ca uptake rates and basal electrophysiological properties similar to wild type.
CREB DNA binding activity is inhibited by glycogen synthase kinase‐3β and facilitated by lithium
Authors: Grimes Carol A. , Jope Richard S.
Published: March 2003

The regulatory influences of glycogen synthase kinase‐3β (GSK3β) and lithium on the activity of cyclic AMP response element binding protein (CREB) were examined in human neuroblastoma SH‐SY5Y cells. Activation of Akt (protein kinase B) with serum‐increased phospho‐serine‐9‐GSK3β (the inactive form of the enzyme), inhibited GSK3β activity, and increased CREB DNA binding activity. Inhibition of GSK3β by another paradigm, treatment with the selective inhibitor lithium, also increased CREB DNA binding activity. The inhibitory regulation of CREB DNA binding activity by GSK3β also was evident in differentiated SH‐SY5Y cells, indicating that this regulatory interaction is maintained in non‐proliferating cells. These results demonstrate that inhibition of GSK3β by serine‐9 phosphorylation or directly by lithium increases CREB activation. Conversely, overexpression of active GSK3β to 3.5‐fold the normal levels completely blocked increases in CREB DNA binding activity induced by epidermal growth factor, insulin‐like growth factor‐1, forskolin, and cyclic AMP. The inhibitory effects due to overexpressed GSK3β were reversed by treatment with lithium and with another GSK3β inhibitor, sodium valproate. Overall, these results demonstrate that GSK3β inhibits, and lithium enhances, CREB activation.
Activation of the mitogen‐activated protein kinase cascade through nitric oxide synthesis as a mechanism of neuritogenic effect of genipin in PC12h cells
Authors: Yamazaki Matsumi , Chiba Kenzo , Mohri Tetsuro , Hatanaka Hiroshi
Published: March 2003

Prominent neurite outgrowth induced by genipin, a plant‐derived iridoid, was substantially inhibited by addition of NG‐nitro‐l‐arginine methyl ester (l‐NAME), a nitric oxide (NO) synthase (NOS) inhibitor, and carboxy‐PTIO, an NO scavenger, in PC12h cells. Increases of the NADPH‐diaphorase activity and neuronal and inducible NOS proteins in cells preceded the neurite outgrowth after addition of genipin to medium. NO donors could induce the neurite outgrowth dose‐dependently in the cells. On the other hand, an inhibitor of soluble guanylate cyclase (SGC), which is known to be a stimulatory target of NO, abolished greatly the genipin‐induced neurite outgrowth. Addition of extracellular signal‐regulated kinase (ERK) kinase inhibitors could almost completely abolish the neurite induction. l‐NAME remarkably depressed genipin‐stimulated phosphorylation of ERK‐1 and ‐2. A neuritogenic effect of nerve growth factor (NGF) in PC12h cells was also remarkably inhibited by the NOS inhibitor, NO scavenger and SGC inhibitor. These findings suggest that induced NO production followed by cyclic GMP‐mediated stimulation of the mitogen‐activated protein kinase (MAPK) cascade is implicated in the neuritogenesis by genipin and NGF in PC12h cells.
Tetracycline derivatives and ceftriaxone, a cephalosporin antibiotic, protect neurons against apoptosis induced by ionizing radiation
Authors: Tikka Tiina , Usenius Taina , Tenhunen Mikko , Keinänen Riitta , Koistinaho Jari
Published: March 2003

DNA damage induced by low doses of ionizing radiation causes apoptosis, which is partially mediated via the generation of free radicals. Both free radicals and apoptosis are involved in the majority of brain diseases, including stroke, Alzheimer's disease and amyotrophic lateral sclerosis. Because previous studies have shown that tetracycline derivatives doxycycline and minocycline have anti‐inflammatory effects and are protective against brain ischemia, we studied whether minocycline and doxycycline or ceftriaxone, a cephalosporin antibiotic with the potential to inhibit excitotoxicity, protect neurons against ionizing radiation in primary cortical cultures. A single dose of 1 Gy significantly increased lactate dehydrogenase release, induced DNA fragmentation in neurons and triggered microglial proliferation. Treatment with minocycline (20 nm), doxycycline (20 nm) and ceftriaxone (1 µm) significantly reduced irradiation‐induced lactate dehydrogenase release and DNA fragmentation. The most efficient protection was achieved by minocycline treatment, which also inhibited the irradiation‐induced increase in microglial cell number. Our results suggest that some tetracycline derivatives, such as doxycycline and minocycline, and ceftriaxone, a cephalosporin derivative, protect neurons against apoptotic death.
Electrical stimulation of cerebellar fastigial nucleus protects rat brain, in vitro, from staurosporine‐induced apoptosis
Authors: Zhou Ping , Qian Liping , Glickstein Sara B. , Golanov Eugene V. , Pickel Virginia M. , Reis Donald J.
Published: March 2003

Electrical stimulation of the cerebellar fastigial nucleus (FN) elicits a prolonged (∼10 days) and substantial (50–80%) protection against ischemic and excitotoxic injuries. The mechanism(s) of protection are unknown. We investigated whether FN stimulation directly protects brain cells against apoptotic cell death in an in vitro rat brain slice culture model. Rats were electrically stimulated in FN or, as control, the cerebellar dentate nucleus (DN). Coronal slices through the forebrain were explanted, exposed to staurosporine, harvested, and analyzed for caspase‐3 activity by a fluorescence assay. FN, but not DN, stimulation significantly reduced staurosporine‐induced caspase‐3 activity by 39 ± 7% at 3 h, 31 ± 3% at 6 h and 26 ± 4% at 10 h of incubation. Immunocytochemistry revealed FN‐specific reductions in activated caspase‐3 mainly in glial‐like cells throughout the forebrain. FN stimulation also results in a 56.5% reduction in cytochrome c release upon staurosporine incubation. We conclude that neuroprotection elicited from FN stimulation can directly modify the sensitivity of brain cells to apoptotic stimuli and thereby suppress staurosporine induced apoptosis in adult rat brain slices. This model indicates that neuroprotection can be studied in vitro and provides new insight into the potential role of glial cells in ischemic protection of neurons induced by FN stimulation.
Brain‐derived neurotrophic factor enhances depolarization‐evoked glutamate release in cultured cortical neurons
Authors: Matsumoto Tomoya , Numakawa Tadahiro , Adachi Naoki , Yokomaku Daisaku , Yamagishi Satoru , Takei Nobuyuki , Hatanaka Hiroshi
Published: March 2003

Brain‐derived neurotrophic factor (BDNF) has been reported to play an important role in neuronal plasticity. In this study, we examined the effect of BDNF on an activity‐dependent synaptic function in an acute phase. First, we found that short‐term treatment (10 min) with BDNF enhanced depolarization‐evoked glutamate release in cultured cortical neurons. The enhancement diminished gradually according to the length of BDNF treatment. The BDNF‐enhanced release did not require the synthesis of protein and mRNA. Both tetanus toxin and bafilomycin abolished the depolarization‐evoked glutamate release with or without BDNF, indicating that BDNF acted via an exocytotic pathway. Next, we investigated the effect of BDNF on intracellular Ca2+. BDNF potentiated the increase in intracellular Ca2+ induced by depolarization. The Ca2+ was derived from intracellular stores, because thapsigargin completely inhibited the potentiation. Furthermore, both thapsigargin and xestospongin C inhibited the effect of BDNF. These results suggested that the release of Ca2+ from intracellular stores mediated by the IP3 receptor was involved in the BDNF‐enhanced glutamate release. Last, it was revealed that the enhancement of glutamate release by BDNF was dependent on the TrkB‐PLC‐γ pathway. These results clearly demonstrate that short‐term treatment with BDNF enhances an exocytotic pathway by potentiating the accumulation of intracellular Ca2+ through intracellular stores.
S100A12 protein is a strong inducer of neurite outgrowth from primary hippocampal neurons
Authors: Mikkelsen Sanne E. , Novitskaya Vera , Kriajevska Marina , Berezin Vladimir , Bock Elisabeth , Norrild Bodil , Lukanidin Eugene
Published: March 2003

Several members of the S100 family of Ca2+ binding proteins are at present known to be secreted and to have extracellular activities. We have investigated the neurite inducing potential of extracellularly added S100A12. Human recombinant S100A12 was found to dramatically induce neuritogenesis of hippocampal cells isolated from 17 to 19 days old rat embryos. The response to S100A12 was dependent on the dose in a bell‐shaped manner. A 10‐fold increase in neurite outgrowth was observed upon treatment with S100A12 in concentrations between 0.1 and 2.0 µm already after 24 h. Exposure to S100A12 for only 15 min was enough to induce neuritogenesis when measured after 24 h, but to obtain a maximal response, S100A12 had to be present in the culture for at least 4 h. The response to S100A12 was abolished by inhibitors of phospholipase C (PLC), protein kinase C (PKC), Ca2+ flux, Ca2+/calmodulin dependent kinase II (CaMKII) or mitogen‐activated protein kinase kinase (MEK). Therefore, we suggest that extracellular S100A12 triggers intracellular signal transduction in neurons, involving the classical mitogen‐activated protein (MAP) kinase pathway and a phospholipase C‐generated second messenger pathway leading to an increase in intracellular Ca2+ and activation of PKC, ultimately resulting in neuronal differentiation.
Proliferative and morphological effects of endothelins in Schwann cells: roles of p38 mitogen‐activated protein kinase and Ca2+‐independent phospholipase A2
Authors: Berti‐Mattera Liliana N. , Harwalkar Subash , Hughes Benjamin , Wilkins Pamela L. , Almhanna Khaldoun
Published: March 2003

The regulation of Schwann cell (SC) proliferation and morphology is critical to nerve homeostasis. We have previously reported that endothelins (ETs) regulate the activity of different effectors in SC including adenylyl cyclase, phospholipases C and A2 and mitogen‐activated protein kinases (MAPKs). These effects imply a possible participation of ETs in the regulation of SC phenotype. We have now investigated the effects of endothelins on the proliferation and morphology of SC, and compared them with the responses to platelet‐derived growth factor (PDGF), a known mitogen in these cells. Both endothelin‐1 (ET‐1) and PDGF increased the incorporation of [3H]thymidine and the proportion of SC in S and G2/M, with a concomitant decrease in the G0/G1 stage cells. Treatment with ET‐1 produced rapid changes in the morphology of the SC, characterized by the appearance of cell spreading with shorter processes. The response to ET‐1 was considered to represent a proliferative phenotype, in contrast to the effects of forskolin, which decreased [3H]thymidine incorporation in immortalized SC (iSC) and lead to a differentiated morphology with longer extensions. While both ET‐1 and PDGF displayed a proliferative effect on SC, treatment with PDGF did not affect the morphology of these cells to a significant extent. A role for p38 MAPK and Ca2+‐independent phospholipase A2 in the changes in morphology and proliferation of iSC driven by ET‐1 was suggested by the effects of selective inhibitors of these pathways [SB202190 and HELSS, respectively]. The unique pattern of signaling pathways recruited by ET‐1 and its combined effects on regulation of phenotype and proliferation of SC suggest an important role for this peptide during nerve degeneration/regeneration.
CREB/CBP and SRE‐interacting transcriptional regulators are fast on–off switches: duration of calcium transients specifies the magnitude of transcriptional responses
Authors: Chawla Sangeeta , Bading Hilmar
Published: March 2003

Transient increases in the intracellular calcium concentration, which are associated with electrical activation of neurones, control synapse‐to‐nucleus communication. Calcium signals differ in time and space but it is unclear exactly how this translates into stimulus‐specific gene expression. Analysis of transcription induced by calcium transients with defined durations revealed that the evoked genomic responses, unlike those following neurotrophin exposure, are not all‐or‐none but graded events. The CRE‐binding protein CREB, its coactivator CREB‐binding protein (CBP), and SRE‐interacting transcriptional regulators are fast on–off switches: their activities are induced by short‐lasting calcium signals, remain active for the duration of the signal and are rapidly shut‐off after calcium concentrations have returned to basal levels. CREB is switched on by a fast, nuclear calmodulin (CaM) kinase‐dependent mechanism that mediates CREB phosphorylation on serine 133 within 30 s of calcium entry. The second calcium‐activated route to CREB involves the MAP kinase/extracellular signal‐regulated kinase (ERK1/2) cascade. This pathway can be triggered by brief, 30–60 s calcium transients. ERK1/2 activity peaks several minutes after calcium entry and can outlast the calcium transient. The shut‐off of CREB and ERK1/2 involves rapid dephosphorylation of their activator sites. These properties of transcription factors and their regulating kinases and phosphatases provide a mechanism through which the duration of calcium signals specifies the magnitude of the transcriptional response. The decoding of temporal features of calcium transients is likely to contribute to impulse‐specific gene expression.
Repression of myelin proteolipid protein gene expression is mediated through both general and cell type‐specific negative regulatory elements in nonexpressing cells
Authors: Li Shenyang , Dobretsova Anna , Kokorina Natalia A. , Wight Patricia A.
Published: March 2003

The myelin proteolipid protein gene (Plp) is expressed primarily in oligodendrocytes. Yet how the gene remains repressed in nonexpressing cells has not been defined, and potentially could cause adverse effects in an organism if the mechanism for repression was impaired. Previous studies suggest that the first intron contains element(s), which suppress expression in nonexpressing cells, although the identity of these elements within the 8 kb intron was not characterized. Here we report the localization of multiple negative regulatory elements that repress Plp gene expression in nonexpressing cells (+/+ Li). Two of these elements (regions) correspond to those used by Plp expressing cells (N20.1), whilst another acts in a cell type‐specific manner (i.e. operational in +/+ Li liver cells, but not N20.1 cells). By gel‐shift and DNase I footprinting analyses, the factor(s) that bind to the cell type‐specific negative regulatory region appear to be far more abundant in +/+ Li cells than in N20.1 cells. Thus, Plp gene repression is mediated through the combinatorial action of both ‘general’ and cell type‐specific negative regulatory elements. Additionally, repression in +/+ Li cells cannot be overcome via an antisilencer/enhancer element, which previously has been shown to function in N20.1 cells.
Glial cell line‐derived neurotrophic factor up‐regulates the expression of tyrosine hydroxylase gene in human neuroblastoma cell lines
Authors: Xiao Hengyi , Hirata, Yoko , Isobe Ken‐ichi , Kiuchi Kazutoshi
Published: March 2003

The role of glial cell line‐derived neurotrophic factor (GDNF) in the survival of dopaminergic neurons has been well documented, but its effect on dopamine biosynthesis remains to be elucidated. In this study, the effect of GDNF on the gene expression of tyrosine hydroxylase (TH), the rate‐limiting enzyme of dopamine biosynthesis, was investigated. We found that GDNF elevated the expression of the TH gene at both mRNA and protein levels in TGW cells, a human neuroblastoma cell line. GDNF significantly enhances the transcription rate of the TH gene as actinomycin D prevented the induction of TH mRNA and GDNF increased the activity of the TH promoter. In addition, GDNF exerts a relatively weak but significant effect on the stability of TH mRNA, because GDNF delayed the degradation of TH mRNA and strengthened a special TH mRNA/protein interaction known to be relevant with TH mRNA stability. By comparing several human neurogenic cell lines, we found that GDNF‐induced TH expression was only observed in the cells possessing Ret protein and coincided with the expression levels. Taken together, these results indicate that GDNF up‐regulates the expression of the TH gene by promoting the transcription of the TH gene and the stability of TH mRNA with the Ret receptor dependency in some neuroblastoma cell lines. Thus, GDNF exerts its neurotrophic role not only in promoting cells survival, but also in affecting dopamine biosynthesis.