bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Tryptophan Hydroxylase Activity in Xenopus laevis Photoreceptor Cells by Cyclic AMP
Authors: Valenciano A. I. , Alonso‐Gómez A. L. , Iuvone P. M.
Published: March 2003

Abstract: The aim of this study was to investigate the role of cyclic AMP in the regulation of tryptophan hydroxylase activity localized in retinal photoreceptor cells of Xenopus laevis, where the enzyme plays a key role in circadian melatonin biosynthesis. In photoreceptor‐enriched retinas that lack serotonergic neurons, tryptophan hydroxylase activity is markedly stimulated by treatments that increase intracellular levels of cyclic AMP or activate cyclic AMP‐dependent protein kinase, including forskolin, phosphodiesterase inhibitors, and cyclic AMP analogues. In contrast, cyclic AMP has no effect on tryptophan hydroxylase mRNA abundance. Experiments using cycloheximide and actinomycin D demonstrate that cyclic AMP exerts its regulatory effect via posttranslational mechanisms mediated by cyclic AMP‐dependent protein kinase. The effect of cyclic AMP is independent of the phase of the photoperiod, suggesting that the nucleotide is not a mediator of the circadian rhythm of tryptophan hydroxylase. Cyclic AMP accumulation is higher in darkness than in light, as is tryptophan hydroxylase activity. Furthermore, the stimulatory effect of forskolin and that of darkness are inhibited by H89, an inhibitor of cyclic AMP‐dependent protein kinase. In conclusion, cyclic AMP may mediate the acute effects of light and darkness on tryptophan hydroxylase activity of retinal photoreceptor cells.
Differential Regulation of Leukemia Inhibitory Factor‐Stimulated Neuronal Gene Expression by Protein Phosphatases SHP‐1 and SHP‐2 Through Mitogen‐Activated Protein Kinase‐Dependent and ‐Independent Pathways
Authors: Bartoe Joseph L. , Nathanson Neil M.
Published: March 2003

Abstract: The neurally active cytokine leukemia inhibitory factor (LIF) signals through a bipartite receptor complex composed of LIF receptor α (LIFR) and gp 130. gp 130 and LIFR contain consensus binding motifs for the protein tyrosine phosphatase SHP‐2 surrounding tyrosines 118 and 115 (Y118 and Y115) of their cytoplasmic domains, respectively. These sites are necessary for maximal activation of mitogen‐activated protein kinase (MAPK). Coexpression of catalytically inactive, but not wild‐type, SHP‐2 reduced LIFR‐ and gp 130‐mediated activation of MAPK up to 75%. Conversely, coexpression of the wild‐type, but not catalytically inactive, SHP‐1, a related phosphatase, reduced activity up to 80%, demonstrating that SHP‐2 and SHP‐1 have opposing effects on the MAPK pathway. Mutation of Y115 of the cytoplasmic domain of LIFR eliminates receptor‐mediated tyrosine phosphorylation of SHP‐2. In contrast, SHP‐1 association with gp 130 and LIFR is constitutive and independent of Y118 and Y115, respectively. SHP‐1 has a positive regulatory role on LIF‐stimulated vasoactive intestinal peptide (VIP) reporter gene expression in neuronal cells, whereas the effect of SHP‐2 is negative. Furthermore, LIF‐stimulated MAPK activation negatively regulates this VIP reporter gene induction. SHP‐2 also negatively regulates LIF‐dependent expression of choline acetyltransferase, but this regulation could be dissociated from its effects on MAPK activation. These data indicate that SHP‐1 and SHP‐2 are important regulators of LIF‐dependent neuronal gene expression via both MAPK‐dependent and ‐independent pathways.
Effect of N‐Glycosylation on Turnover and Subcellular Distribution of N‐Acetylgalactosaminyltransferase I and Sialyltransferase II in Neuroblastoma Cells
Authors: Bieberich Erhard , Tencomnao Tewin , Kapitonov Dmitri , Yu Robert K.
Published: March 2003

Abstract: Gangliosides are sialylated glycosphingolipids whose biosynthesis is catalyzed by a series of endoplasmic reticulum (ER)‐ and Golgi‐resident glycosyltransferases. Protein expression, processing, and subcellular localization of the key regulatory enzymes for ganglioside biosynthesis, sialyltransferase II (ST‐II) and N‐acetylgalactosaminyltransferase I (GalNAcT), were analyzed upon transient expression of the two enzymes in the neuroblastoma cell lines NG108‐15 and F‐11. The enzymes were endowed with a C‐terminal epitope tag peptide (FLAG) for immunostaining and immunoaffinity purification using a FLAG‐specific antibody. Mature ST‐II‐FLAG and GalNAcT‐FLAG were expressed as N‐glycoproteins with noncomplex oligosaccharides. ST‐II‐FLAG was distributed to the Golgi apparatus, whereas GalNAcT‐FLAG was found in the ER and Golgi. Inhibition of early N‐glycoprotein processing with castanospermine resulted in a distribution of ST‐II‐FLAG to the ER, whereas that of GalNAcT‐FLAG remained unaltered. In contrast to GalNAcT, the activity of ST‐II and the amount of immunostained enzyme were reduced concomitantly by 75% upon incubation with castanospermine. This was due to a fourfold increased turnover of ST‐II‐FLAG, which was not found with GalNAcT‐FLAG. The ER retention and increased turnover of ST‐II‐FLAG were most likely due to its inability to bind to calnexin upon inhibition of early N‐glycoprotein processing. Calnexin binding was not observed for GalNAcT‐FLAG, indicating a differential effect of N‐glycosylation on the turnover and subcellular localization of the two glycosyltransferases.
Neuronal Nicotinic Acetylcholine Receptors from Drosophila
Authors: Schulz Regine , Bertrand Sonia , Chamaon Kathrin , Smalla Karl‐Heinz , Gundelfinger Eckart D. , Bertrand Daniel
Published: March 2003

Abstract: Although neuronal nicotinic acetylcholine receptors from insects have been reconstituted in vitro more than a decade ago, our knowledge about the subunit composition of native receptors as well as their functional properties still remains limited. Immunohistochemical evidence has suggested that two α subunits, α‐like subunit (ALS) and Drosophilaα2 subunit (Dα2), are colocalized in the synaptic neuropil of the Drosophila CNS and therefore may be subunits of the same receptor complex. To gain further understanding of the composition of these nicotinic receptors, we have examined the possibility that a receptor may imbed more than one α subunit using immunoprecipitations and electrophysiological investigations. Immunoprecipitation experiments of fly head extracts revealed that ALS‐specific antibodies coprecipitate Dα2, and vice versa, and thereby suggest that these two α subunits must be contained within the same receptor complex, a result that is supported by investigations of reconstituted receptors in Xenopus oocytes. Discrimination between binary (ALS/β2 or Dα2/β2) and ternary (ALS/Dα2/β2) receptor complexes was made on the basis of their dose‐response curve to acetylcholine as well as their sensitivity to α‐bungarotoxin or dihydro‐β‐erythroidine. These data demonstrate that the presence of the two α subunits within a single receptor complex confers new receptor properties that cannot be predicted from knowledge of the binary receptor’s properties.
The Catalytic Subunit of Telomerase Protects Neurons Against Amyloid β‐Peptide‐Induced Apoptosis
Authors: Zhu Haiyan , Fu Weiming , Mattson Mark P.
Published: March 2003

Abstract : The catalytic subunit of telomerase (TERT) is a specialized reverse transcriptase that has been associated with cell immortalization and cancer. It was reported recently that TERT is expressed in neurons throughout the brain in embryonic and early postnatal development, but is absent from neurons in the adult brain. We now report that suppression of TERT levels and function in embryonic mouse hippocampal neurons in culture using antisense technology and the telomerase inhibitor 3' ‐azido‐2' 3' ‐dideoxythymidine significantly increases their vulnerability to cell death induced by amyloid β‐peptide, a neurotoxic protein believed to promote neuronal degeneration in Alzheimer's disease. Neurons in which TERT levels were reduced exhibited increased levels of oxidative stress and mitochondrial dysfunction following exposure to amyloid β‐peptide. Overexpression of TERT in pheochromocytoma cells resulted in decreased vulnerability to amyloid β‐peptide‐induced apoptosis. Our findings demonstrate a neuroprotective function of TERT in an experimental model relevant to Alzheimer's disease, and suggest the possibility that restoration of TERT expression in neurons in the adult brain may protect against age‐related neurodegeneration.
Brain‐Derived Neurotrophic Factor Accelerates Nitric Oxide Donor‐Induced Apoptosis of Cultured Cortical Neurons
Authors: Ishikawa Yasuyuki , Ikeuchi Toshihiko , Hatanaka Hiroshi
Published: March 2003

Brain‐derived neurotrophic factor (BDNF) is known to have important functions in neuronal survival, differentiation, and plasticity. In addition to its role as a survival‐promoting factor, BDNF reportedly can enhance neuronal cell death in some cases, for example, the death caused by excitotoxicity or glucose deprivation. The cellular mechanism of the death‐enhancing effect of BDNF remains unknown, in contrast to that of its survival‐promoting effect. In this work, we found that BDNF markedly accelerated the nitric oxide (NO) donor‐induced death of cultured embryonic cortical neurons. BDNF increased the number of cells with nuclear condensation and DNA fragmentation 24 h after treatment with the NO donor, but it did not change the number of those cells 36 h after the treatment. The BDNF‐accelerated death of cortical neurons was inhibited by the addition of actinomycin D or cycloheximide. These results suggest that BDNF can accelerate apoptotic cell death elicited by NO donor. TrkB‐IgG and K252a blocked the BDNF‐induced acceleration of the death, indicating that the death‐accelerating effect by BDNF is mediated by TrkB. In addition, the BDNF‐accelerated apoptosis was inhibited by the addition of SB202190 and SB203580, specific inhibitors of p38 mitogen‐activated protein kinase (MAPK), and U0126, a specific inhibitor of MAPK/ERK kinase 1, indicating that the activation of both p38 MAPK and ERK is involved in the signaling cascade of the BDNF‐accelerated, NO donor‐induced apoptosis.
Facilitatory Roles of Novel Compounds Designed from Cyclopentenone Prostaglandins on Neurite Outgrowth‐Promoting Activities of Nerve Growth Factor
Authors: Satoh Takumi , Furuta Kyoji , Tomokiyo Keiichiro , Nakatsuka Daisaku , Tanikawa Motoki , Nakanishi Makoto , Miura Maki , Tanaka Shuuitsu , Koike Tetsuro , Hatanaka Hiroshi , Ikuta Kazuyoshi , Suzuki Masaaki , Watanabe Yasuyoshi
Published: March 2003

Abstract: Cyclopentenone prostaglandins (PGs) are known to arrest

the cell cycle at the G1 phase in vitro and to suppress tumor

growth in vivo. However, their effects on neurons are unclear. Here, we report

that some cyclopentenone PGs function as neurite outgrowth‐promoting factors.

They promoted neurite outgrowth from PC12 cells and from dorsal root ganglion

explants but only in the presence of nerve growth factor (NGF). We refer to

these PGs as neurite outgrowth‐promoting PGs (NEPPs). Through study of the structure‐function relationship of NEPP1‐10 and related compounds, we found that the cross‐conjugated dienone moiety of NEPPs was essential for promoting neurite outgrowth, and NEPP10 was concluded to be the best candidate for drug development. We also investigated the intracellular mechanism of the promotion by NEPPs and obtained evidence that immunoglobulin heavy chain binding protein/glucose‐regulated protein 78 (BiP/GRP78) plays a role in the promotion, based on the following observations: Antisense nucleotides for BiP/GRP78 gene blocked the promotion of neurite outgrowth; BiP/GRP78 protein level increased in response to NEPPs; and overexpression of BiP/GRP78 protein by adenoviral gene transfer promoted the neurite outgrowth by NGF.
Temporal Profile and Cell Subtype Distribution of Activated Caspase‐3 Following Experimental Traumatic Brain Injury
Authors: Beer R. , Franz G. , Srinivasan A. , Hayes R. L. , Pike B. R. , Newcomb J. K. , Zhao X. , Schmutzhard E. , Poewe W. , Kampfl A.
Published: March 2003

Abstract: This study investigated the temporal expression and cell subtype distribution of activated caspase‐3 following cortical impact‐induced traumatic brain injury in rats. The animals were killed and examined for protein expression of the proteolytically active subunit of caspase‐3, p18, at intervals from 6 h to 14 days after injury. In addition, we also investigated the effect of caspase‐3 activation on proteolysis of the cytoskeletal protein α‐spectrin. Increased protein levels of p18 and the caspase‐3‐specific 120‐kDa breakdown product to α‐spectrin were seen in the cortex ipsilateral to the injury site from 6 to 72 h after the trauma. Immunohistological examinations revealed increased expression of p18 in neurons, astrocytes, and oligodendrocytes from 6 to 72 h following impact injury. In contrast, no evidence of caspase‐3 activation was seen in microglia at all time points investigated. Quantitative analysis of caspase‐3‐positive cells revealed that the number of caspase‐3‐positive neurons exceeded the number of caspase‐3‐positive glia cells from 6 to 72 h after injury. Moreover, concurrent assessment of nuclear histopathology using hematoxylin identified p18‐immunopositive cells exhibiting apoptotic‐like morphological profiles in the cortex ipsilateral to the injury site. In contrast, no evidence of increased p18 expression or α‐spectrin proteolysis was seen in the ipsilateral hippocampus, contralateral cortex, or hippocampus up to 14 days after the impact. Our results are the first to demonstrate the concurrent expression of activated caspase‐3 in different CNS cells after traumatic brain injury in the rat. Our findings also suggest a contributory role of activated caspase‐3 in neuronal and glial apoptotic degeneration after experimental TBI in vivo.
Effect of Exogenous and Endogenous Antioxidants on 3‐Nitropionic Acid‐Inducedin vivo Oxidative Stress and Striatal Lesions
Authors: Fontaine Michael A. La , Geddes James W. , Banks Andrea , Butterfield D. Allan
Published: March 2003

Abstract: 3‐Nitropropionic acid (3‐NP) is an irreversible inhibitor
of complex II in the mitochondria. 3‐NP toxicity has gained acceptance as an
animal model of Huntington's disease (HD). In the present study, we confirmed
that rats injected with 3‐NP (20 mg/kg, i.p., daily for 4 days) exhibit
increased oxidative stress in both striatum and cortical synaptosomes as well
as lesions in the striatum. Synaptosomal membrane proteins from rats injected
with 3‐NP exhibited a decrease in W/S ratio, the relevant electron
paramagnetic resonance (EPR) parameter used to determine levels of protein
oxidation, and western blot analysis for protein carbonyls revealed direct
evidence of increased synaptosomal protein oxidation. Treatment of rats with
the brain‐accessible free radical spin trap
5‐diethoxyphosphoryl‐5‐methyl‐1‐pyrroline N‐oxide (DEPMPO; 30 mg/kg,
i.p., daily 2 h before 3‐NP injection) or with N‐acetylcysteine (NAC; 100 mg/kg, i.p., daily 2 h before 3‐NP injection), a known glutathione precursor, before 3‐NP treatments protects against oxidative damage induced by 3‐NP as measured by EPR and western blot analysis for protein carbonyls. Furthermore, both DEMPMPO and NAC treatments before 3‐NP administration significantly reduce striatal lesion volumes. These data suggest oxidative damage is a prerequisite for striatal lesion formation and that antioxidant treatment may be a useful therapeutic strategy against 3‐NP neurotoxicity and perhaps against HD as well.
Some Metabotropic Glutamate Receptor Ligands Reduce Kynurenate Synthesis in Rats by Intracellular Inhibition of Kynurenine Aminotransferase II
Authors: Battaglia Guiseppe , Rassoulpour Arash , Wu Hui‐Qiu , Hodgkins Paul S. , Kiss Csaba , Nicoletti Ferdinando , Schwarcz Robert
Published: March 2003

Abstract: Some metabotropic glutamate receptor (mGluR) ligands, such
as quisqualate, L‐(+)‐2‐amino‐4‐phosphonobutyric acid (L‐AP4),
4‐carboxy‐3‐hydroxyphenylglycine (4C3HPG), and L‐serine‐O‐phosphate
(L‐SOP), reduced the formation of the endogenous excitatory amino acid
receptor antagonist kynurenate in brain and liver slices. The use of novel,
subtype‐selective mGluR agonists and antagonists excluded a role for any known
mGluR subtype in this effect. The reduction of kynurenate formation was no
longer observed when slices were incubated with the active mGluR ligands in
the absence of extracellular Na+.
trans‐Pyrrolidine‐2,4‐dicarboxylate (trans‐PDC), a
broad‐spectrum ligand of Na+‐dependent glutamate transporters, was
also able to reduce kynurenate formation. Quisqualate, 4C3HPG, L‐AP4, and
L‐SOP did not further reduce kynurenate formation in the presence of
trans‐PDC, suggesting that the two classes of drugs may share the
same mechanism of action. Hence, we hypothesized that the active mGluR ligands
are transported inside the cell and act intracellularly to reduce kynurenate
synthesis. We examined this possibility by assessing the direct effect of
mGluR ligands on the activity of kynurenine aminotransferases (KATs) I and II,
the enzymes that transaminate kynurenine to kynurenate. In brain tissue
homogenates, KAT II (but not KAT I) activity was inhibited by quisqualate,
4C3HPG, L‐AP4, L‐SOP, and trans‐PDC. Drugs that were unable to reduce kynurenate formation in tissue slices were inactive. We conclude that some mGluR ligands act intracellularly, inhibiting KAT II activity and therefore reducing kynurenate formation. This effect should be taken into consideration when novel mGluR ligands are developed for the treatment of neurological and psychiatric diseases.
Selective Adrenergic/Cyclic AMP‐Dependent Switch‐Off of Proteasomal Proteolysis Alone Switches on Neural Signal Transduction
Authors: Schomerus Christof , Korf Horst‐Werner , Laedtke Elke , Weller Joan L. , Klein David C.
Published: March 2003

Abstract: The molecular processes underlying neural transmission are
central issues in neurobiology. Here we describe a novel mechanism through
which noradrenaline (NA) activates its target cells, using the mammalian
pineal organ as a model. In this neuroendocrine transducer, NA stimulates
arylalkylamine N‐acetyltransferase (AANAT; EC 2.3.1.87), the key enzyme regulating the nocturnal melatonin production. In rodents, AANAT protein accumulates as a result of enhanced transcription, but in primates and ungulates, the AANAT mRNA level fluctuates only marginally, indicating that other mechanisms regulate AANAT protein and activity. These were investigated in cultured bovine pinealocytes. AANAT mRNA was readily detectable in unstimulated pinealocytes, and levels did not change following NA treatment. In contrast, NA increased AANAT protein levels in parallel with AANAT activity, apparently through a cyclic AMP‐mediated mechanism. Immunocytochemistry revealed that the changes in AANAT protein levels occurred in virtually all pinealocytes. Inhibition of AANAT degradation by proteasomal proteolysis alone was found to switch‐on enzyme activity by increasing AANAT protein levels five‐ to 10‐fold. Accordingly, under unstimulated conditions AANAT protein is continually synthesized and immediately destroyed by proteasomal proteolysis. NA appears to act via cyclic AMP to protect AANAT from proteolytic destruction, resulting in accumulation of the protein. These findings show that tightly regulated control of proteasomal proteolysis of a specific protein alone can play a pivotal role in neural regulation.
Selective Solubilization of High‐Molecular‐Mass Neurofilament Subunit During Nerve Regeneration
Authors: Tsuda Morihiro , Tashiro Tomoko , Komiya Yoshiaki
Published: March 2003

Abstract: A reduction in neurofilament (NF) protein synthesis and changes in their phosphorylation state are observed during nerve regeneration. To investigate how such metabolic changes are involved in the reorganization of the axonal cytoskeleton, we studied the injury‐induced changes in the solubility and axonal transport of NF proteins as well as their phosphorylation states in the rat sciatic nerve. In the control nerve, 15‐25% of high‐molecular‐mass NF subunit (NF‐H) was recovered in the 1% Triton‐soluble fraction when fractionated in the presence of phosphatase inhibitors. After a complete loss of NF proteins distal to the injury site (70‐75 mm from the spinal cord) 1 week after injury, NF‐H detected in the regenerating sprouts at 2 weeks or later exhibited higher solubility (>50%) and lower C‐terminal phosphorylation level than NF‐H in the control nerve. Solubility increase was also apparent with L‐[35S]methionine‐labeled NF‐H that was in transit in the proximal axon at the time of injury. The low‐molecular‐mass subunit remained in the insoluble fraction in both the normal and the regenerating nerves, indicating that selective solubilization of NF‐H rather than total filament disassembly occurs during regeneration.
Tubulin stimulates adenylyl cyclase activity in C6 glioma cells by bypassing the β‐adrenergic receptor: a potential mechanism of G protein activation
Authors: Yan Kun , Popova Juliana S , Moss Amy , Shah Bindu , Rasenick Mark M.
Published: March 2003

While the cytoskeleton is known to play several roles in the biology of the cell, one role, which has been revealed only recently, is that of a participant in the signal transduction process. Tubulin binds specifically to the α subunits of Gs (stimulatory GTP‐binding regulatory protein of adenylyl cyclase), Gi1 (inhibitory protein of adenylyl cyclase), and Gq and transactivates those molecules through direct transfer of GTP. The relevance of this transactivation process to G proteins which are normally activated by a neurotransmitter‐occupied receptor is the subject of this study. C6 glioma cells, made permeable with saponin, retained tight coupling between Gs and the β‐adrenergic receptor. Although 5‐guanylylimidodiphosphate (GppNHp) was incapable of activating Gs (and subsequently, adenylyl cyclase) in the absence of agonist, tubulin with GppNHp bound (tubulin‐GppNHp) activated adenylyl cyclase with an EC50 of 30 nm. Desensitization of β‐adrenergic receptors by isoproterenol exposure had no effect on the ability of tubulin‐GppNHp to activate Gs and adenylyl cyclase. When the photoaffinity GTP analog, azidoanilido GTP (AAGTP; P3(4‐azidoanilido)‐P1‐5'‐GTP), was added to C6 membranes or permeable C6 cells, it was only weakly incorporated by Gαs in the absence of isoproterenol. When the same concentration of dimeric tubulin with AAGTP bound was introduced, AAGTP was transferred from tubulin to Gαs, activating the latter species. Similar ‘preferential’ activation of Gαs by tubulin‐AAGTP versus the free nucleotide was seen using purified components. Thus, membrane‐associated tubulin may serve to activate Gαs, independent of signals not normally coupled to that protein. Tubulin may act as an agent to link a variety of membrane‐associated signalling systems.
Nicotinic agonists stimulate acetylcholine release from mouse interpeduncular nucleus: a function mediated by a different nAChR than dopamine release from striatum
Authors: Grady Sharon R. , Meinerz Natalie M. , Cao Jian , Reynolds Andrew M. , Picciotto Marina R. , Changeux Jean‐Pierre , McIntosh J. Michael , Marks Michael J. , Collins Allan C.
Published: March 2003

Acetylcholine release stimulated by nicotinic agonists was measured as radioactivity released from perfused synaptosomes prepared from mouse interpeduncular nucleus (IPN) that had been loaded with [3H]choline. Agonist‐stimulated release was dependent upon external calcium and over 90% of released radioactivity was acetylcholine. The release process was characterized by dose response curves for 13 agonists and inhibition curves for six antagonists. α‐Conotoxin MII did not inhibit this release, while α‐conotoxin AuIB inhibited 50% of agonist‐stimulated release. Comparison of this process with [3H]dopamine release from mouse striatal synaptosomes indicated that different forms of nicotinic acetylcholine receptors (nAChRs) may mediate these processes. This was confirmed by assays using mice homozygous for the β2 subunit null mutation. The deletion of the β2 subunit had no effect on agonist‐stimulated acetylcholine release, but abolished agonist‐stimulated release of dopamine from striatal synaptosomes. Mice heterozygous for the β2 subunit null mutation showed decreased dopamine release evoked by l‐nicotine with no apparent change in EC50 value, as well as similar decreases in both transient and persistent phases of release with no changes in desensitization rates.
Effect of subchronic lithium treatment on citalopram‐induced increases in extracellular concentrations of serotonin in the medial prefrontal cortex
Authors: Muraki Ihoko , Inoue Takeshi , Hashimoto Shinji , Izumi Takeshi , Ito Koichi , Koyama Tsukasa
Published: March 2003

We investigated the effect of citalopram [a selective serotonin (5‐HT) reuptake inhibitor; SSRI] and MKC‐242 (a selective 5‐HT1A agonist), following treatment with subchronic lithium (p.o., 1 week) on extracellular 5‐HT concentrations in the medial prefrontal cortex (mPFC). Acute treatment with citalopram (3 and 30 mg/kg) led to significant increases in extracellular 5‐HT concentrations. The subchronic lithium group showed significantly higher basal levels of extracellular 5‐HT than normal diet controls. Acute citalopram (3 and 30 mg/kg) treatment together with subchronic lithium treatment showed significant increases in the extracellular 5‐HT concentrations, compared with citalopram treatment alone. Acute MKC‐242 (1 mg/kg) treatment showed significant decreases in extracellular 5‐HT concentrations, in both the normal diet and lithium diet groups to the same extent. The addition of lithium did not change the effect of the 5‐HT1A agonist on extracellular 5‐HT concentrations. This study suggests that lithium augmentation of the antidepressant effect of SSRI is mediated by the additional increases in extracellular 5‐HT concentrations following the co‐administrations of lithium and SSRI.
A cGMP‐dependent protein kinase is implicated in wild‐type motility in C. elegans
Authors: Stansberry John , Baude Eric J. , Taylor Merritt K. , Chen Pei‐Jiun , Jin Suk‐Won , Ellis Ronald E. , Uhler Michael D.
Published: March 2003

In mammals, cyclic GMP and cGMP‐dependent protein kinases (cGKs) have been implicated in the regulation of many neuronal functions including long‐term potentiation and long‐term depression of synaptic efficacy. To develop Caenorhabditis elegans as a model system for studying the neuronal function of the cGKs, we cloned and characterized the cgk‐1 gene. A combination of approaches showed that cgk‐1 produces three transcripts, which differ in their first exon but are similar in length. Northern analysis of C. elegans RNA, performed with a probe designed to hybridize to all three transcripts, confirmed that a major 3.0 kb cgk‐1 transcript is present at all stages of development. To determine if the CGK‐1C protein was a cGMP‐dependent protein kinase, CGK‐1C was expressed in Sf9 cells and purified. CGK‐1C shows a Ka of 190 ± 14 nm for cGMP and 18.4 ± 2 µm for cAMP. Furthermore, CGK‐1C undergoes autophosphorylation in a cGMP‐dependent manner and is inhibited by the commonly used cGK inhibitor, KT5823. To determine which cells expressed CGK‐1C, a 2.4‐kb DNA fragment from the promoter of CGK‐1C was used to drive GFP expression. The CGK‐1C reporter construct is strongly expressed in the ventral nerve cord and in several other neurons as well as the marginal cells of the pharynx and intestine. Finally, RNA‐mediated interference of CGK‐1 resulted in movement defects in nematode larvae. These results provide the first demonstration that cGMP‐dependent protein kinase is present in neurons of C.elegans and show that this kinase is required for normal motility.
Axonal transport of VR1 capsaicin receptor mRNA in primary afferents and its participation in inflammation‐induced increase in capsaicin sensitivity
Authors: Tohda Chihiro , Sasaki Miwa , Konemura Takashi , Sasamura Takashi , Itoh Masayuki , Kuraishi Yasushi
Published: March 2003

Capsaicin receptors are expressed in primary sensory neurons and excited by heat and protons. We examined the inflammation‐induced changes of the level of VR1 capsaicin receptor mRNA in sensory neurons and the sensitivity of primary afferents to capsaicin. Carrageenan treatment induced axonal transport of VR1 mRNA, but not that of preprotachykinin mRNA, from the dorsal root ganglia to central and peripheral axon terminals. The sensitivity of central terminals to capsaicin, which was estimated by measuring the capsaicin‐evoked release of glutamate from the dorsal horn, was increased by peripheral inflammation, and such an increase was suppressed by inhibiting the RNA translation in the dorsal horn with cycloheximide and an intrathecal injection of VR1 antisense oligonucleotides. Thus, peripheral inflammation induces the axonal transport of VR1 mRNA, which may be involved in the hypersensitivity of primary afferents to capsaicin and the production of inflammatory hyperalgesia.
Isolation of 2000‐kDa complexes of N‐methyl‐d‐aspartate receptor and postsynaptic density 95 from mouse brain
Authors: Husi Holger , Grant Seth G. N.
Published: March 2003

Neurotransmitter receptors in vivo are linked to intracellular adaptor proteins and signalling molecules driving downstream pathways. Methods for physical isolation are essential to answer fundamental questions about the size, structure and composition of in vivo complexes and complement the widely used yeast 2‐hybrid method. The N‐methyl‐d‐aspartate receptor (NMDAR) binds postsynaptic density 95 (PSD‐95) protein; both are required for synaptic plasticity and learning and participate in other important pathophysiological functions. Here we describe the development and optimization of novel methods for large‐scale isolation of NMDAR–PSD‐95 complexes from mouse brain including immunoaffinity, immunoprecipitation, ligand‐affinity and immobilized PSD‐95 binding peptides. Short PDZ binding peptides modelled on NMDAR subunits were shown to isolate NMDAR complexes. Gel filtration indicated the native NMDAR–PSD‐95 complexes were 2000 kDa, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) revealed a complexity suggesting a huge network of both structural components and signalling enzymes. These methods can be used to define the structure of the complexes at different synapses and in mice carrying gene mutations as well as new tools for drug discovery.
Selective small‐molecule inhibitors of glycogen synthase kinase‐3 activity protect primary neurones from death
Authors: Cross Darren A. E. , Culbert Ainsley A. , Chalmers Katy A. , Facci Laura , Skaper Stephen D. , Reith Alastair D.
Published: March 2003

The phosphatidylinositol 3‐kinase (PI 3‐kinase)/protein kinase B (PKB; also known as Akt) signalling pathway is recognized as playing a central role in the survival of diverse cell types. Glycogen synthase kinase‐3 (GSK‐3) is a ubiquitously expressed serine/threonine protein kinase that is one of several known substrates of PKB. PKB phosphorylates GSK‐3 in response to insulin and growth factors, which inhibits GSK‐3 activity and leads to the modulation of multiple GSK‐3 regulated cellular processes. We show that the novel potent and selective small‐molecule inhibitors of GSK‐3; SB‐415286 and SB‐216763, protect both central and peripheral nervous system neurones in culture from death induced by reduced PI 3‐kinase pathway activity. The inhibition of neuronal death mediated by these compounds correlated with inhibition of GSK‐3 activity and modulation of GSK‐3 substrates tau and β‐catenin. Thus, in addition to the previously assigned roles of GSK‐3, our data provide clear pharmacological and biochemical evidence that selective inhibition of the endogenous pool of GSK‐3 activity in primary neurones is sufficient to prevent death, implicating GSK‐3 as a physiologically relevant principal regulatory target of the PI 3‐kinase/PKB neuronal survival pathway.
A transient inhibition of mitochondrial ATP synthesis by nitric oxide synthase activation triggered apoptosis in primary cortical neurons
Authors: Almeida Angeles , Bolaños Juan P.
Published: March 2003

In order to investigate the relationship between nitric oxide‐mediated regulation of mitochondrial function and excitotoxicity, the role of mitochondrial ATP synthesis and intracellular redox status on the mode of neuronal cell death was studied. Brief (5 min) glutamate (100 µm) receptor stimulation in primary cortical neurons collapsed the mitochondrial membrane potential (Δψm) and transiently (30 min) inhibited mitochondrial ATP synthesis, causing early (1 h) necrosis or delayed (24 h) apoptosis. The transient inhibition of ATP synthesis was paralleled to a loss of NADH, which was fully recovered shortly after the insult. In contrast, NADPH and the GSH/GSSG ratio were maintained, but progressively decreased thereafter. Twenty‐four hours after glutamate treatment, ATP was depleted, a phenomenon associated with a persistent inhibition of mitochondrial succinate–cytochrome c reductase activity and delayed necrosis. Blockade of either nitric oxide synthase (NOS) activity or the mitochondrial permeability transition (MPT) pore prevented Δψm collapse, the transient inhibition of mitochondrial ATP synthesis, early necrosis and delayed apoptosis. However, blockade of NOS activity, but not the MPT pore, prevented the inhibition of succinate–cytochrome c reductase activity and delayed ATP depletion and necrosis. From these results, we suggest that glutamate receptor‐mediated NOS activation would trigger MPT pore opening and transient inhibition of ATP synthesis leading to apoptosis in a neuronal subpopulation, whereas other groups of neurons would undergo oxidative stress and persistent inhibition of ATP synthesis leading to necrosis.
Glatiramer acetate inhibition of tumor necrosis factor‐α‐induced RANTES expression and release from U‐251 MG human astrocytic cells
Authors: Li Qingdi Q. , Burt David R. , Bever Christopher T.
Published: March 2003

Glatiramer acetate is an approved drug for the treatment of multiple sclerosis (MS). RANTES is a β‐family chemokine that manifests chemoattractant activity for T lymphocytes and monocytes/macrophages implicated in the pathogenesis of MS lesions. However, the effect of glatiramer acetate on the regulation of RANTES secretion in glial cells is unknown. In the present study, we demonstrate for the first time that treatment of human U‐251 MG astrocytic cells with glatiramer acetate blocks tumor necrosis factor‐α (TNF‐α)‐induced RANTES mRNA and protein in a dose‐ and time‐dependent manner. This effect is attributed to inhibition of transcription and a 40% decrease in transcript stability. Furthermore, our electrophoretic mobility shift assays of nuclear extracts from TNF‐α‐treated cells reveal an increase in DNA‐binding activity specific for the nuclear factor‐kappa B (NF‐κB) binding site, in the 5′‐flanking promoter region of the human RANTES gene, and that this increase in NF‐κB binding activity is prevented by pretreatment with glatiramer acetate or the NF‐κB inhibitors. These findings suggest that glatiramer acetate may exert its therapeutic effect in MS partially through inhibiting NF‐κB activation and chemokine production.
The glial glutamate transporter, GLT‐1, is oxidatively modified by 4‐hydroxy‐2‐nonenal in the Alzheimer's disease brain: the role of Aβ1–42
Authors: Lauderback Christopher M. , Hackett Janna M. , Huang Feng F. , Keller Jeffrey N. , Szweda Luke I. , Markesbery William R. , Butterfield D. Allan
Published: March 2003

Glutamate transporters are involved in the maintenance of synaptic glutamate concentrations. Because of its potential neurotoxicity, clearance of glutamate from the synaptic cleft may be critical for neuronal survival. Inhibition of glutamate uptake from the synapse has been implicated in several neurodegenerative disorders. In particular, glutamate uptake is inhibited in Alzheimer's disease (AD); however, the mechanism of decreased transporter activity is unknown. Oxidative damage in brain is implicated in models of neurodegeneration, as well as in AD. Glutamate transporters are inhibited by oxidative damage from reactive oxygen species and lipid peroxidation products such as 4‐hydroxy‐2‐nonenal (HNE). Therefore, we have investigated a possible connection between the oxidative damage and the decreased glutamate uptake known to occur in AD brain. Western blots of immunoprecipitated HNE‐immunoreactive proteins from the inferior parietal lobule of AD and control brains suggest that HNE is conjugated to GLT‐1 to a greater extent in the AD brain. A similar analysis of beta amyloid (Aβ)‐treated synaptosomes shows for the first time that Aβ1–42 also increases HNE conjugation to the glutamate transporter. Together, our data provide a possible link between the oxidative damage and neurodegeneration in AD, and supports the role of excitotoxicity in the pathogenesis of this disorder. Furthermore, our data suggests that Aβ may be a possible causative agent in this cascade.
Intracellular neuronal calcium sensor (NCS) protein VILIP‐1 modulates cGMP signalling pathways in transfected neural cells and cerebellar granule neurones
Authors: Braunewell Karl‐Heinz , Brackmann Marian , Schaupp Michael , Spilker Christina , Anand Rene , Gundelfinger Eckart D.
Published: March 2003

The family of intracellular neuronal calcium‐sensors (NCS) belongs to the superfamily of EF‐hand proteins. Family members have been shown by in vitro assays to regulate signal cascades in retinal photoreceptor cells. To study the functions of NCS proteins not expressed in photoreceptor cells we examined Visinin‐like protein‐1 (VILIP‐1) effects on signalling pathways in living neural cells. Visinin‐like protein‐1 expression increased cGMP levels in transfected C6 and PC12 cells. Interestingly, in transfected PC12 cells stimulation was dependent on the subcellular localization of VILIP‐1. In cells transfected with membrane‐associated wild‐type VILIP‐1 particulate guanylyl cyclase (GC) was stimulated more strongly than soluble GC. In contrast, deletion of the N‐terminal myristoylation site resulted in cytosolic localization of VILIP‐1 and enhanced stimulation of soluble GC. To study the molecular mechanisms underlying GC stimulation VILIP‐1 was examined to see if it can physically interact with GCs. A direct physical interaction of VILIP‐1 with the recombinant catalytic domain of particulate GCs‐A, B and with native GCs enriched from rat brain was observed in GST pull‐down as well as in surface plasmon resonance interaction studies. Furthermore, following trituration of recombinant VILIP‐1 protein into cerebellar granule cells the protein influenced only signalling by GC‐B. Together with the observed colocalization of GC‐B, but not GC‐A, with VILIP‐1 in cerebellar granule cells, these results suggest that VILIP‐1 may be a physiological regulator of GC‐B.
Nitroxyl anion regulation of the NMDA receptor
Authors: Colton C. A. , Gbadegesin M. , Wink D. A. , Miranda K. M. , Espey M. G. , Vicini S.
Published: March 2003

Nitric oxide (NO) is an important regulator of NMDA channel function in the CNS. Recent findings suggest that nitroxyl anion (NO−) may also be generated by nitric oxide synthase, which catalyzes production of NO. Using recombinant NMDA receptors (NMDA‐r) transfected into human embryonic kidney cells, our data demonstrate that the nitroxyl anion donor, Angeli's salt (AS; Na2N2O3) dramatically blocked glycine‐independent desensitization in NMDA‐r containing NR1‐NR2A subunits. AS did not affect glycine‐dependent desensitization, calcium dependent inactivation or glutamate affinity for the NMDA‐r. This effect could be mimicked by treatment with DPTA, a metal chelator and was not evident under hypoxic conditions. In contrast, receptors containing the NR1‐NR2B subunits demonstrated an approximate 25% reduction in whole cell currents in the presence of AS with no apparent change in desensitization. Our data suggest that the regulation of NMDA‐r function by nitroxyl anion is distinctly different from NO and may result in different cellular outcomes compared with NO.
Interaction of a glucocorticoid‐responsive element with regulatory sequences in the promoter region of the mouse tyrosine hydroxylase gene
Authors: Hagerty Timothy , Fernandez Elizabeth , Lynch Kelly , Wang Shou‐Shu , Morgan William W. , Strong Randy
Published: March 2003

The purpose of the work reported here was to determine whether the tyrosine hydroxylase glucocorticoid‐responsive element (TH‐GRE) interacts with the cyclic AMP pathway and the CRE in regulating mouse TH promoter activity, and whether an additional, previously identified downstream GRE‐like element also participates in the function of the TH‐GRE and CRE. To determine the role of the cAMP pathway on TH‐GRE function, we compared the effects of forskolin and dexamethasone on TH mRNA, TH gene transcription and TH promoter activity in a mutant PC12 cell line (A126–1B2) deficient in cAMP‐dependent protein kinase A (PKA) with their effects in the wild‐type parental strain. Forskolin treatment increased TH mRNA content, transcriptional activity and the activity of a chimeric gene with 3.6 kb of the TH promoter in wild‐type cells, but not in PKA‐deficient cells. In contrast, dexamethasone treatment stimulated equivalent increases in TH mRNA, TH gene transcription and TH promoter activity in each cell type. Mutation of the CRE in chimeric constructs containing 3.6 kb of the 5′ flanking sequence of the mouse TH gene or coexpression of a dominant‐negative mutant of CREB prevented the stimulation of TH promoter activity by forskolin. However, neither the mutation of the CRE nor inhibition of CREB influenced basal or dexamethasone‐stimulated promoter activity. Site‐directed mutagenesis of the TH‐GRE eliminated the response of the promoter to dexamethasone. However, the mutagenesis of a more proximal 15‐bp region with a GRE‐like sequence had no demonstrable effect on the ability of dexamethasone to stimulate TH promoter activity. Neither mutagenesis of the TH‐GRE or the downstream GRE‐like sequence had an effect on the ability of forskolin to activate this chimeric gene. Taken together, these results provide evidence that a single GRE is sufficient for maximal induction of transcriptional activity by glucocorticoids and that the CRE is not required for either partial or full activity of this upstream GRE sequence.
Angiotensin II promotes the phosphorylation of cyclic AMP‐responsive element binding protein (CREB) at Ser133 through an ERK1/2‐dependent mechanism
Authors: Cammarota Martín , Bevilaqua Lia R. M. , Dunkley Peter R. , Rostas John A. P.
Published: March 2003

In cells from the adrenal medulla, angiotensin II (AII) regulates both the activity and mRNA levels of catecholamine biosynthetic enzymes whose expression is thought to be under the control of cAMP‐responsive element (CRE) binding protein (CREB). In this study, we evaluated the effect of AII stimulation on CREB phosphorylation at Ser133 (pCREB) in bovine adrenal chromaffin cells (BACC). We found that AII produces a rapid and AII type‐1 receptor (AT1)‐dependent increase in pCREB levels, which is blocked by the MEK1/2 inhibitor U0126 but not by H‐89, SB203580 or KN‐93, suggesting that it is mediated by the extracellular‐regulated protein kinases 1 and 2 (ERK1/2) and not by cAMP‐dependent protein kinase (PKA), p38 mitogen‐activated protein kinase (p38MAPK) or Ca2+/calmodulin‐dependent protein kinases (CaMKs) dependent pathways. Gel‐shift experiments showed that the increase in pCREB levels is accompanied by an ERK1/2‐dependent upregulation of CRE‐binding activity. We also found that AII promotes a rapid and reversible increase in the activity of the non‐receptor tyrosine kinase Src and that the inhibition of this enzyme completely blocks the AII‐induced phosphorylation of ERK1/2, the CREB kinase p90RSK and CREB. Our data support the hypothesis that in BACC, AII upregulates CREB functionality through a mechanism that requires Src‐mediated activation of ERK 1/2 and p90RSK.
Impact of endogenous nitric oxide on microglial cell energy metabolism and labile iron pool
Authors: Chénais Benoît , Morjani Hamid , Drapier Jean‐Claude
Published: March 2003

Microglial activation is common in several neurodegenerative disorders. In the present study, we used the murine BV‐2 microglial cell line stimulated with γ‐interferon and lipopolysaccharide to gain new insights into the effects of endogenously produced NO on mitochondrial respiratory capacity, iron regulatory protein activity, and redox‐active iron level. Using polarographic measurement of respiration of both intact and digitonin‐permeabilized cells, and spectrophotometric determination of individual respiratory chain complex activity, we showed that in addition to the reversible inhibition of cytochrome‐c oxidase, long‐term endogenous NO production reduced complex‐I and complex‐II activities in an irreversible manner. As a consequence, the cellular ATP level was decreased in NO‐producing cells, whereas ATPase activity was unaffected. We show that NO up‐regulates RNA‐binding of iron regulatory protein 1 in microglial cells, and strongly reduces the labile iron pool. Together these results point to a contribution of NO derived from inflammatory microglia to the misregulation of energy‐producing reactions and iron metabolism, often associated with the pathogenesis of neurodegenerative disorders.
Overexpression of the signaling adapter FRS2 reconstitutes the cell cycle deficit of a nerve growth factor non‐responsive TrkA receptor mutant
Authors: Zeng Guoqian , O. Meakin Susan
Published: March 2003

We have characterized the cell cycle deficit of a novel TrkA receptor mutant (TrkAS3) that fails to support nerve growth factor (NGF)‐dependent cell cycle arrest and neurite outgrowth. TrkAS3 receptors fail to support an NGF‐dependent increase in the expression of cyclin D1 and the cell cycle inhibitor, p21Waf1/Cip1, two important regulators of G1/S transition, and do not down‐regulate expression of the G2/M phase marker, cdc2/cdk1, or the S phase marker, proliferating cell nuclear antigen. Moreover, NGF‐activated TrkAS3 receptors do not down‐regulate cyclin‐dependent kinase 4 phosphorylation of the retinoblastoma protein, essential for G1 arrest, in comparison to NGF‐activated wild‐type TrkA. Collectively these data indicate that TrkAS3 receptors fail to support NGF‐dependent G1 arrest. Interestingly, ectopic expression of regulators of G1/S arrest, such as cyclin D1 or inhibitors of cell cycle (p21Waf1/Cip1, p16INK4A), or the fibroblast growth factor (FGF) receptor substrate‐2 (FRS2) in cells expressing TrkAS3 reconstitutes NGF‐dependent neurite outgrowth. Collectively, these data suggest a model in which NGF‐stimulated TrkA‐dependent activation of FRS2 supports neurite outgrowth through a mechanism that likely involves the induction of p21Waf1/Cip1 expression and the arrest of cells at G1/S.
Potentiation of a sodium–calcium exchanger in the nuclear envelope by nuclear GM1 ganglioside
Authors: Xie Xin , Wu Gusheng , Lu Zi‐Hua , Ledeen Robert W.
Published: March 2003

Calcium is recognized as an important intracellular messenger with a pivotal role in the regulation of many cytosolic and nuclear processes. Gangliosides of various types, especially GM1, are known to have a role in some aspects of Ca2+regulation, operating through a variety of mechanisms that are gradually coming to light. The present study provides evidence for a sodium–calcium exchanger in the nuclear envelope of NG108‐15 neuroblastoma cells that is potently and specifically activated by GM1. Immunoblot analysis revealed an unusually tight association of GM1 with the exchanger in the nuclear envelope but not with that in the plasma membrane. Exchanger and associated GM1 were located in the inner membrane of the nuclear envelope, suggesting this system could function to transfer Ca2+between nucleoplasm and the envelope lumen. The GM1‐enhanced exchange was blocked by cholera toxin B subunit while C2‐ceramide, a recently discovered inhibitor of the exchanger, blocked all transfer. Exchanger activity was significantly elevated in nuclei isolated from cells that were induced to differentiate by KCl + dibutyryl‐cAMP, a treatment previously shown to promote up‐regulation of nuclear GM1 in conjunction with axonogenesis. Similar enhancement was achieved by addition of exogenous GM1 to nuclei from undifferentiated cells. These results suggest a prominent role for nuclear GM1 in regulation of nuclear Ca2+homeostasis.