bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduction of glyceraldehyde‐3‐phosphate dehydrogenase activity in Alzheimer's disease and in Huntington's disease fibroblasts
Authors: Mazzola Jennifer L. , Sirover Michael A.
Published: March 2003

New functions have been identified for glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) including its role in neurodegenerative disease and in apoptosis. GAPDH binds specifically to proteins implicated in the pathogenesis of a variety of neurodegenerative disorders including the β‐amyloid precursor protein and the huntingtin protein. However, the pathophysiological significance of such interactions is unknown. In accordance with published data, our initial results indicated there was no measurable difference in GAPDH glycolytic activity in crude whole‐cell sonicates of Alzheimer's and Huntington's disease fibroblasts. However, subcellular‐specific GAPDH–protein interactions resulting in diminution of GAPDH glycolytic activity may be disrupted or masked in whole‐cell preparations. For that reason, we examined GAPDH glycolytic activity as well as GAPDH–protein distribution as a function of its subcellular localization in 12 separate cell strains. We now report evidence of an impairment of GAPDH glycolytic function in Alzheimer's and Huntington's disease subcellular fractions despite unchanged gene expression. In the postnuclear fraction, GAPDH was 27% less glycolytically active in Alzheimer's cells as compared with age‐matched controls. In the nuclear fraction, deficits of 27% and 33% in GAPDH function were observed in Alzheimer's and Huntington's disease, respectively. This evidence supports a functional role for GAPDH in neurodegenerative diseases. The possibility is considered that GAPDH : neuronal protein interaction may affect its functional diversity including energy production and as well as its role in apoptosis.
Protein phosphorylation is required for endocytosis in nerve terminals: potential role for the dephosphins dynamin I and synaptojanin, but not AP180 or amphiphysin
Authors: Cousin Michael A. , Tan Timothy C. , Robinson Phillip J.
Published: March 2003

Dynamin I and at least five other nerve terminal proteins, amphiphysins I and II, synaptojanin, epsin and eps15 (collectively called dephosphins), are coordinately dephosphorylated by calcineurin during endocytosis of synaptic vesicles. Here we have identified a new dephosphin, the essential endocytic protein AP180. Blocking dephosphorylation of the dephosphins is known to inhibit endocytosis, but the role of phosphorylation has not been determined. We show that the protein kinase C (PKC) antagonists Ro 31‐8220 and Go 7874 block the rephosphorylation of dynamin I and synaptojanin that occurs during recovery from an initial depolarizing stimulus (S1). The rephosphorylation of AP180 and amphiphysins 1 and 2, however, were unaffected by Ro 31‐8220. Although these dephosphins share a single phosphatase, different protein kinases phosphorylated them after nerve terminal stimulation. The inhibitors were used to selectively examine the role of dynamin I and/or synaptojanin phosphorylation in endocytosis. Ro 31‐8220 and Go 7874 did not block the initial S1 cycle of endocytosis, but strongly inhibited endocytosis following a second stimulus (S2). Therefore, phosphorylation of a subset of dephosphins, which includes dynamin I and synaptojanin, is required for the next round of stimulated synaptic vesicle retrieval.
Cyclic AMP and mitogen‐activated protein kinases are required for glutamate‐dependent cyclic AMP response element binding protein and Elk‐1 phosphorylation in the dorsal striatum in vivo
Authors: Choe Eun Sang , McGinty Jacqueline F.
Published: March 2003

Dopaminergic and glutamatergic signalling cascades are integrated in striatal medium spiny neurones by cyclic AMP response‐element binding protein and Elk‐1 phosphorylation. Phosphorylated cyclic AMP response‐element binding protein and phosphorylated Elk‐1 contribute to c‐fos expression by binding to the calcium and cyclic AMP response‐element and the serum response element, respectively, in the c‐fos promoter. The role of cyclic AMP and mitogen‐activated protein kinase signalling cascades in glutamate‐induced cyclic AMP response‐element binding protein and Elk‐1 phosphorylation and Fos expression was investigated using semiquantitative immunocytochemistry in vivo. Intracerebroventricular infusion of the sodium channel blocker, tetrodotoxin, decreased the glutamate‐induced increase in phosphorylated cyclic AMP response‐element binding protein, phosphorylated Elk‐1, and Fos immunoreactivity. Intracerebroventricular infusion of the mitogen‐activated and extracellular signal‐regulated kinase inhibitor, PD98059, the p38 mitogen‐activated protein kinase inhibitor, SB203580, or the cyclic AMP inhibitor, Rp‐8‐Br‐cAMPS, decreased glutamate‐induced phosphorylated cyclic AMP response‐element binding protein, phosphorylated Elk‐1, and Fos immunoreactivity. Simultaneous infusion of glutamate and Sp‐8‐Br‐cAMPS, a cyclic AMP analogue, augmented induction of Fos immunoreactivity but not phosphorylated cyclic AMP response‐element binding protein or phosphorylated Elk‐1 immunoreactivity. These data indicate that cyclic AMP and mitogen‐activated protein kinase signalling cascades are necessary for glutamate to induce cyclic AMP response‐element binding protein and Elk‐1 phosphorylation and Fos expression in the striatum. Furthermore, neuronal activity plays an important role in glutamate‐induced signalling cascades in vivo.
Possible linkage between glutamate transporter and mitogen‐activated protein kinase cascade in cultured rat cortical astrocytes
Authors: Abe Kazuho , Saito Hiroshi
Published: March 2003

The mitogen‐activated protein kinases (MAPKs) play a pivotal role in the mediation of cellular responses to a variety of signalling molecules. In the present study, we investigated possible linkage between glutamate signalling and the MAPK cascade in cultured rat cortical astrocytes. Exposure of the cells to L‐glutamate (100–1000 µm) resulted in an increase in phosphorylated p44/42 MAPK (ERK1/2) in a concentration‐ and time‐dependent manner. The glutamate‐induced ERK1/2 phosphorylation was blocked by U0126 and PD98059, specific inhibitors of the MAPK‐activating enzyme MEK. Furthermore, L‐glutamate‐induced ERK1/2 phosphorylation was not mimicked by glutamate receptor agonists and was not blocked by glutamate receptor antagonists. In contrast, the effect of L‐glutamate was mimicked by d‐ and L‐aspartate and transportable glutamate uptake inhibitors. These results suggest that the MEK/ERK cascade is activated by a mechanism related to glutamate transporters. We propose that the glutamate transporter functions as a receptor transmitting extracellular glutamate signal to intracellular messengers.
A dinucleotide deletion in amyloid precursor protein (APP) mRNA associated with sporadic Alzheimer's disease results in efficient secretion of truncated APP isoforms from neuroblastoma cell cultures
Authors: Hersberger Martin , Santiago‐Garcia Juan , Patarroyo‐White Susannah , Yan Jimmy , Xu Xiao
Published: March 2003

Recently, two dinucleotide deletions were detected in the mRNA of the amyloid precursor protein (APP) from cerebral cortex neurons of patients with sporadic Alzheimer's disease (AD) or Down's syndrome. These deletions resulted in truncation of APP, producing an APP isoform with a 38‐kDa N‐terminus and a novel carboxyl terminus (APP+1). We investigated the subcellular localization and the processing of APP+1 in the neuroblastoma cell line B103. cDNA constructs were generated encoding fusion proteins of APP+1 or full‐length APP with the enhanced green fluorescent protein (eGFP). In transient transfection experiments using B103 cells, the APP+1–eGFP fusion protein showed a reticular localization with intense staining in the Golgi complex. Unlike full‐length APP fused to eGFP, the APP+1–eGFP fusion protein did not localize to the perinuclear area or to the plasma membrane. Western blot analysis of cell extracts confirmed the translation of the expected fusion proteins. Analysis of the supernatant by western blot indicated that the APP+1–eGFP fusion protein was efficiently secreted from B103 cells, whereas the secreted form of full‐length APP fusion protein (APPs) was hardly detectable. Thus, both dinucleotide deletions in the APP mRNA result in truncated APP+1 that is not membrane associated and is readily secreted from neurons.
Pharmacological characterization of threo‐3‐methylglutamic acid with excitatory amino acid transporters in native and recombinant systems
Authors: Eliasof Scott , McIlvain H. Beal , Petroski Robert E. , Foster Alan C. , Dunlop John
Published: March 2003

The glutamate analog (±) threo‐3‐methylglutamate (T3MG) has recently been reported to inhibit the EAAT2 but not EAAT1 subtype of high‐affinity, Na+‐dependent excitatory amino acid transporter (EAAT). We have examined the effects of T3MG on glutamate‐elicited currents mediated by EAATs 1–4 expressed in Xenopus oocytes and on the transport of radiolabeled substrate in mammalian cell lines expressing EAATs 1–3. T3MG was found to be an inhibitor of EAAT2 and EAAT4 but a weak inhibitor of EAAT1 and EAAT3. T3MG competitively inhibited uptake of

d‐[3H]‐aspartate into both cortical and cerebellar synaptosomes with a similar potency, consistent with its inhibitory activity on the cloned EAAT2 and EAAT4 subtypes. In addition, T3MG produced substrate‐like currents in oocytes expressing EAAT4 but not EAAT2. However, T3MG was unable to elicit heteroexchange of preloaded

d‐[3H]‐aspartate in cerebellar synaptosomes, inconsistent with the behavior of a substrate inhibitor. Finally, T3MG acts as a poor ionotropic glutamate receptor agonist in cultured hippocampal neurons: concentrations greater than 100 µm T3MG were required to elicit significant NMDA receptor‐mediated currents. Thus, T3MG represents a pharmacological tool for the study of not only the predominant EAAT2 subtype but also the EAAT4 subtype highly expressed in cerebellum.
ATP stimulates calcium‐dependent glutamate release from cultured astrocytes
Authors: Jeremic Aleksandar , Jeftinija Ksenija , Stevanovic Jelena , Glavaski Aleksandra , Jeftinija Srdija
Published: March 2003

ATP caused a dose‐dependent, receptor‐mediated increase in the release of glutamate and aspartate from cultured astrocytes. Using calcium imaging in combination HPLC we found that the increase in intracellular calcium coincided with an increase in glutamate and aspartate release. Competitive antagonists of P2 receptors blocked the response to ATP. The increase in intracellular calcium and release of glutamate evoked by ATP were not abolished in low Ca2+‐EGTA saline, suggesting the involvement of intracellular calcium stores. Pre‐treatment of glial cultures with an intracellular Ca2+ chelator abolished the stimulatory effects of ATP. Thapsigargin (1 µm), an inhibitor of Ca2+‐ATPase from the Ca2+ pump of internal stores, significantly reduced the calcium transients and the release of aspartate and glutamate evoked by ATP. U73122 (10 µm), a phospholipase C inhibitor, attenuated the ATP‐stimulatory effect on calcium transients and blocked ATP‐evoked glutamate release in astrocytes. Replacement of extracellular sodium with choline failed to influence ATP‐induced glutamate release. Furthermore, inhibition of the glutamate transporters p‐chloromercuri‐phenylsulfonic acid and

ltrans‐pyrolidine‐2,4‐dicarboxylate failed to impair the ability of ATP to stimulate glutamate release from astrocytes. However, an anion transport inhibitor, furosemide, and a potent Cl− channel blocker, 5‐nitro‐2(3‐phenylpropylamino)‐benzoate, reduced ATP‐induced glutamate release. These results suggest that ATP stimulates excitatory amino acid release from astrocytes via a calcium‐dependent anion‐transport sensitive mechanism.
Induction of human β‐defensin‐2 expression in human astrocytes by lipopolysaccharide and cytokines
Authors: Hao Hsiao‐Nan , Zhao Jiun , Lotoczky Gregory , Grever William E. , Lyman William D.
Published: March 2003

Defensins are cationic peptides with broad‐spectrum antimicrobial activity. They are members of a supergene family consisting of α and β subtypes and each subtype is comprised of a number of different isoforms. For example, human α‐defensin (HAD) has six isoforms, which are expressed by polymorphonuclear leukocytes and Paneth cells. In contrast, human β‐defensin (HBD) has two isoforms that are expressed by epithelial cells of the skin, gut, respiratory and urogenital tracts. Recently, HBD‐1 was detected in human brain biopsy tissue. However, little is known about the expression of HBD‐1 or HBD‐2 in the CNS and whether neural cells can secrete these peptides. For the present study, human astrocyte, microglial, meningeal fibroblast and neuronal cultures were probed for the expression of HBD‐1 and HBD‐2 mRNA and protein. Each cell type was either maintained in tissue culture medium alone or in medium containing lipopolysaccharide (LPS) at concentrations ranging from 0.1 to 1 μg/mL, interleukin‐1 beta (IL‐1β) at 1–50 ng/mL, or tumor necrosis factor alpha (TNF‐α) at the same concentrations. The expression of HBD‐1 and HBD‐2 mRNAs was monitored by RT‐PCR. The cDNA products were sequenced to characterize the gene product. HBD‐2 protein was detected by immunoblot, immunoprecipitation and immunocytochemistry. Results of these studies showed that HBD‐1 mRNA was detected in all cell cultures except in those enriched for neurons. In contrast, HBD‐2 mRNA was detected only in astrocyte cultures that were treated with LPS, IL‐1β or TNF‐α. The detection of the respective proteins correlated positively with the mRNA results. As such, these data represent the first demonstration of HBD‐2 expression by astrocytes and suggest that this peptide may play a role in host defense against bacterial CNS pathogenesis.
Absolute quantification of AMPA receptor subunit mRNAs in single hippocampal neurons
Authors: Tsuzuki Keisuke , Lambolez Bertrand , Rossier Jean , Ozawa Seiji
Published: March 2003

α‐Amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor subunit (GluR1–4) mRNAs expressed by single neurons in rat hippocampal cultures were quantified by single‐cell RT‐PCR using an internal standard RNA after whole‐cell patch‐clamp recording. The internal standard RNA, derived from GluR2 with a single nucleotide substitution, was reverse‐transcribed and PCR‐amplified with the same efficiency as GluR1–4 mRNAs. The mean mRNA numbers harvested in vitro from pyramidal‐like neurons on day 9 were 1150 ± 324 molecules of GluR1, 1080 ± 273 molecules of GluR2, 100 ± 20 molecules of GluR3, and 50 ± 10 molecules of GluR4 (mean ± SEM, n = 12). In a non‐pyramidal neuronal population that expresses AMPA receptors characterized by high Ca2+ permeability, the numbers of GluR1, GluR3 and GluR4 mRNA molecules harvested per cell were 354 ± 64, 25 ± 17 and 168 ± 36, respectively (n = 8). The GluR2 mRNA was not detected in this cell type. The calculated ratio of AMPAR mRNA molecules per total mRNA molecules was 1/240 in pyramidal‐like neurons (1/500 for GluR2), being in the range obtained with total RNA from rat forebrain and cerebellum (1/170 and 1/380, respectively). Finally, our results indicated that the proportion of GluR1–4 mRNA located in neurites reached ∼60% in pyramidal‐like neurons. However, we found no evidence of preferential subcellular distribution of a given subunit.
Functional striatal hypodopaminergic activity in mice lacking adenosine A2A receptors
Authors: Dassesse Donald , Massie Ann , Ferrari Rosaria , Ledent Catherine , Parmentier Marc , Arckens Lutgarde , Zoli Michele , Schiffmann Serge N.
Published: March 2003

Adenosine and caffeine modulate locomotor activity and striatal gene expression, partially through the activation and blockade of striatal A2A receptors, respectively. The elucidation of the roles of these receptors benefits from the construction of A2A receptor‐deficient mice (A2A‐R−/−). These mice presented alterations in locomotor behaviour and striatal expression of genes studied so far, which are unexpected regarding the specific expression of A2A receptor by striatopallidal neurones. To clarify the functions of A2A receptors in the striatum and to identify the mechanisms leading to these unexpected modifications, we studied the basal expression of immediate early and constitutive genes as well as dopamine and glutamate neurotransmission in the striatum. Basal zif268 and arc mRNAs expression was reduced in mutant mice by 60–80%, not only in the striatum but also widespread in the cerebral cortex and hippocampus. Striatal expression of substance P and enkephalin mRNAs was reduced by about 50% and 30%, respectively, whereas the expression of GAD67 and GAD65 mRNAs was slightly increased and unaltered, respectively. In vivo microdialysis in the striatum revealed a 45% decrease in the extracellular dopamine concentration and three‐fold increase in extracellular glutamate concentration. This was associated with an up‐regulation of D1 and D2 dopamine receptors expression but not with changes in ionotropic glutamate receptors. The levels of tyrosine hydroxylase and of striatal and cortical glial glutamate transporters as well as adenosine A1 receptors expression were indistinguishable between A2A‐R−/− and wild‐type mice. Altogether these results pointed out that the lack of A2A receptors leads to a functional hypodopaminergic state and demonstrated that A2A receptors are necessary to maintain a basal level in immediate early and constitutive genes expression in the striatum and cerebral cortex, possibly via their control of dopamine pathways.
Bidirectional modulation of P2X receptor‐mediated response by divalent cations in rat dorsal motor nucleus of the vagus neurons
Authors: Ueno Tsuyoshi , Ueno Shinya , Kakazu Yasuhiro , Akaike Norio , Nabekura Junichi
Published: March 2003

The modulatory effects of Zn2+ and other divalent cations on the ATP‐induced responses of preganglionic neurons acutely dissociated from the rat dorsal motor nucleus of the vagus (DMV) were examined using a nystatin‐perforated patch technique under voltage‐clamp. DMV neurons were identified by back‐filling of DiI placed on the vagal bundle at the neck. Zn2+ exerts a concentration‐dependent effect on P2X receptor‐mediated current (IATP): a potentiation by low concentrations of Zn2+ (≤ 50 µm) and an inhibition by high concentrations (> 50 µm). Inhibition of the ATP response was associated with a prolongation of the rising phase of IATP. Cu2+ mimicked Zn2+ regarding the biphasic modulation of IATP. On the other hand, Ni2+ potentiated, but failed to inhibit, the ATP response even at a concentration of 3 mm. Quantitative RT‐PCR revealed the similarity of P2X2 mRNA expression between the DMV and superior cervical ganglion (SCG) but not in the dorsal root ganglion (DRG) and hypoglossal nucleus (XII). The results from the electrophysiological and molecular approaches suggest that functional P2X receptors expressed in DMV neurons are characterized mainly by the P2X2 and P2X2/6 subtype. DMV neurons possess similar P2X receptor characteristics to SCG neurons.
Ca2+‐induced inhibition of apoptosis in human SH‐SY5Y neuroblastoma cells: degradation of apoptotic protease activating factor‐1 (APAF‐1)
Authors: Reimertz Claus , Kögel Donat , Lankiewicz Silke , Poppe Monika , Prehn Jochen H. M.
Published: March 2003

During apoptotic and excitotoxic neuron death, challenged mitochondria release the pro‐apoptotic factor cytochrome c. In the cytosol, cytochrome c is capable of binding to the apoptotic protease‐activating factor‐1 (APAF‐1). This complex activates procaspase‐9 in the presence of dATP, resulting in caspase‐mediated execution of apoptotic neuron death. Many forms of Ca2+‐mediated neuron death, however, do not lead to prominent activation of the caspase cascade despite significant release of cytochrome c from mitochondria. We demonstrate that elevation of cytosolic Ca2+ induced prominent degradation of APAF‐1 in human SH‐SY5Y neuroblastoma cells and in a neuronal cell‐free apoptosis system. Loss of APAF‐1 correlated with a reduced ability of cytochrome c to activate caspase‐3‐like proteases. Ca2+ induced the activation of calpains, monitored by the cleavage of full‐length α‐spectrin into a calpain‐specific 150‐kDa breakdown product. However, pharmacological inhibition of calpain activity indicated that APAF‐1 degradation also occurred via calpain‐independent pathways. Our data suggest that Ca2+ inhibits caspase activation during Ca2+‐mediated neuron death by triggering the degradation of the cytochrome c‐binding protein APAF‐1.
Characterization of GTP cyclohydrolase I gene expression in the human neuroblastoma SKN‐BE(2)M17: enhanced transcription in response to cAMP is conferred by the proximal promoter
Authors: Hirayama Kei , Shimoji Mika , Swick Lance , Meyer Amy , Kapatos Gregory
Published: March 2003

GTP cyclohydrolase I (GTPCH) gene expression was investigated in the human monoamine‐containing neuroblastoma cell line SK‐N‐BE(2)M17. Northern blot analysis revealed a single GTPCH mRNA transcript that was confirmed by RNase protection assay to encode for Type 1 GTPCH; no alternatively spliced forms of GTPCH mRNA were detected with this assay. Incubation with 8Br‐cAMP, but not nerve growth factor or leukemia inhibitory factor, produced a rapid increase in GTPCH mRNA and protein levels; protein levels remained elevated during the entire treatment period while mRNA content declined rapidly between 10 and 24 h. Treatment with 8Br‐cAMP did not significantly modify the stability of GTPCH mRNA but did increase GTPCH transcription as determined by transient transfection assays of a luciferase reporter construct containing 1171 bp of human GTPCH 5′‐flanking sequence. Cis‐acting elements required for maximal basal and cAMP‐dependent transcription were localized by deletion analysis to the 146 bp proximal promoter. DNase I footprint analysis of the proximal promoter using SK‐N‐BE(2)M17 nuclear extracts identified two protein binding domains: one an upstream Sp1‐like site and the other a combined CRE‐Sp1‐CCAAT‐box element. EMSA and supershift assays demonstrated that the combined CRE‐Sp1‐CCAAT‐box element recruits ATF‐2 and NF‐Y but not Sp1–4 or Egr‐1–3. NF‐Y binding was confirmed using pure recombinant human NF‐Y protein. Transcription of the human GTPCH gene in human SK‐N‐BE(2)M17 cells is thus enhanced by cAMP acting through regulatory elements located in the proximal promoter and may involve the transcription factors NF‐Y and ATF‐2.
GeneChip® analysis after acute spinal cord injury in rat
Authors: Song Guoqing , Cechvala Cate , Resnick Daniel K. , Dempsey Robert J. , Rao Vemuganti L. Raghavendra
Published: March 2003

Spinal cord injury (SCI) leads to induction and/or suppression of several genes, the interplay of which governs the neuronal death and subsequent loss of motor function. Using GeneChip®, the present study analyzed changes in the mRNA abundance at 3 and 24 h after SCI in adult rats. SCI was induced at T9 level by the New York University impactor by dropping a 10‐g weight from a height of 25 mm. Several transcription factors, immediate early genes, heat‐shock proteins, pro‐inflammatory genes were up‐regulated by 3 h, and persisted at 24 h, after SCI. On the other hand, some neurotransmitter receptors and transporters, ion channels, kinases and structural proteins were down‐regulated by 3 h, and persisted at 24 h, after SCI. Several genes that play a role in growth/differentiation, survival and neuroprotection were up‐regulated at 24 h after SCI. Using real‐time quantitative PCR, the changes observed by GeneChip® were confirmed for seven up‐regulated (interleukin‐6, heat‐shock protein‐70, heme oxygenase‐1, suppressor of cytokine signaling 2, suppressor of cytokine signaling 3, interferon regulatory factor‐1, neuropeptide Y), two down‐regulated (vesicular GABA transporter and cholecystokinin precursor) and two unchanged (Cu/Zn‐superoxide dismutase and phosphatidyl inositol‐3‐kinase) genes. The present study shows that inflammation, neurotransmitter dysfunction, increased transcription, ionic imbalance and cytoskeletal damage starts as early as 3 h after SCI. In addition to these effects, 24 h after SCI the repair and regeneration process begins in an attempt to stabilize the injured spinal cord.
Transcriptional down‐regulation of MARCKS gene expression in immortalized hippocampal cells by lithium
Authors: Wang Le , Liu Xingge , Lenox Robert H.
Published: March 2003

The gene (Macs) for the mouse myristoylated alanine‐rich C kinasesubstrate (MARCKS) encodes a prominent substrate for protein kinase C that has been implicated in processes requiring signal dependent changes in actin‐membrane plasticity and cytoskeletal restructuring. We have previously demonstrated that MARCKS protein is significantly down‐regulated in rat hippocampus and in an immortalized hippocampal cell line (HN33.dw) following long‐term exposure to lithium at clinically relevant concentrations (1 mm). Our current studies have examined transcriptional and post‐transcriptional events that may underlie the lithium‐induced down‐regulation of MARCKS protein in the cultured hippocampal cell model system. MARCKS mRNA and protein expression were found to be concomitantly down‐regulated following exposure of the HN33.dw cells to chronic lithium. Whereas the stability of MARCKS mRNA remained unchanged in the presence of lithium, nuclear run‐off assay indicated that the transcription of nascent MARCKS mRNA was significantly reduced (≈50%) in the cells that had been treated with lithium for 7 days. Transient transfection of HN33.dw cells with a mouse clonedMacspromoter (993‐bp) showed that theMacspromoter activity was attenuated to the same extent after chronic (7–10 days), but not subacute (24 h), lithium exposure. The inhibition of theMacspromoter was found to be dependent upon the presence of a 280‐bp promoter region between −993‐bp and −713‐bp relative to the translation start site, suggesting that this region is a potential lithium‐responsive region ofMacspromoter (LRR). Mutant promoter lacking the LRR not only did not respond to chronic lithium exposure but also had significantly reduced promoter activity, suggesting that chronic lithium exposure represses the transcriptional activity of activator(s) bound to the promoter. Taken together, our data indicate that transcriptional inhibition of theMacsgene underlies the lithium‐induced down‐regulation of MARCKS expression in the immortalized hippocampal cells.
In vivo activation of N‐methyl‐d‐aspartate receptors in the rat hippocampus increases prostaglandin E2 extracellular levels and triggers lipid peroxidation through cyclooxygenase‐mediated mechanisms
Authors: Pepicelli O. , Fedele E. , Bonanno G. , Raiteri M. , Ajmone‐Cat M. A. , Greco A. , Levi G. , Minghetti L.
Published: March 2003

Cyclooxygenases (COX) are a family of enzymes involved in the biosynthesis of prostaglandin (PG) and thromboxanes. The inducible enzyme cyclooxygenase‐2 (COX‐2) is the major isoform found in normal brain, where it is constitutively expressed in neurons and is further up‐regulated during several pathological events, including seizures and ischaemia. Emerging evidence suggests that COX‐2 is implicated in excitotoxic neurodegenerative phenomena. It remains unclear whether PGs or other products associated to COX activity take part in these processes. Indeed, it has been suggested that reactive oxygen species, produced by COX, could mediate neuronal damage. In order to obtain direct evidence of free radical production during COX activity, we undertook an in vivo microdialysis study to monitor the levels of PGE2 and 8‐epi‐PGF2α following infusion of N‐methyl‐d‐aspartate (NMDA). A 20‐min application of 1 mm NMDA caused an immediate, MK‐801‐sensitive increase of both PGE2 and 8‐epi‐PGF2α basal levels. These effects were largely prevented by the specific cytosolic phospholipase A2 (cPLA2) inhibitor arachidonyl trifluoromethyl ketone (ATK), by non‐ selective COX inhibitors indomethacin and flurbiprofen or by the COX‐2 selective inhibitor NS‐398, suggesting that the NMDA‐evoked prostaglandin synthesis and free radical‐mediated lipid peroxidation are largely dependent on COX‐2 activity. As several lines of evidence suggest that prostaglandins may be potentially neuroprotective, our findings support the hypothesis that free radicals, rather than prostaglandins, mediate the toxicity associated to COX‐2 activity.
The mutation of two amino acid residues in the N‐terminus of tyrosine hydroxylase (TH) dramatically enhances the catalytic activity in neuroendocrine AtT‐20 cells
Authors: Nakashima Akira , Kaneko Yoko S. , Mori Keiji , Fujiwara Kentaro , Tsugu Toshimitsu , Suzuki Takahiro , Nagatsu Toshiharu , Ota Akira
Published: March 2003

The sequence Arg37‐Arg38 of tyrosine hydroxylase (TH) is known to play a significant role in the feedback inhibition by the end product DA. To clarify how deeply the sequence Arg37‐Arg38 and the phosphorylated Ser40 of human TH type 1 (hTH1) are involved in the regulation of this feedback inhibition in mammalian cells, we generated the following mutants: (i) RR‐GG, Arg37‐Arg38 replaced by Gly37‐Gly38; (ii) RR‐EE, Arg37‐Arg38 replaced by Glu37‐Glu38; (iii) S40D, Ser40 replaced by Asp40; and (iv) S40A, Ser40 replaced by Ala40. In a cell‐free system, the level of the DA inhibition of the RR‐EE mutant enzyme was to the same or smaller degree than that of the phosphorylation‐mimicking S40D. Next, AtT‐20 neuroendocrine cells were transfected with wild‐type and mutated TH genes because these cells were earlier shown to be capable of fully converting L‐3,4‐dihydroxyphenylalanine into DA, whereby the catalytic activity of TH would be expected to be inhibited by the end product DA accumulating in the cells. The level of DA accumulation in AtT‐20 cells expressing the TH gene was in the order: RR‐EE > S40D > S40A = RR‐GG > wild‐type, which was in accordance with the observations for the cell‐free system. These results suggest that the sequence Arg37‐Arg38 of hTH1 is a more potent determinant of the efficient production of DA in mammalian cells than is the phosphorylated Ser40‐hTH1.
Native group‐III metabotropic glutamate receptors are coupled to the mitogen‐activated protein kinase/phosphatidylinositol‐3‐kinase pathways
Authors: Iacovelli L. , Bruno V. , Salvatore L. , Melchiorri D. , Gradini R. , Caricasole A. , Barletta E. , De Blasi A. , Nicoletti F.
Published: March 2003

We used cultured cerebellar granule cells to examine whether native group‐III metabotropic glutamate (mGlu) receptors are coupled to the mitogen‐activated protein kinase (MAPK) and phosphatidylinositol‐3‐kinase (PI‐3‐K) pathways. Cultured granule cells responded to the group‐III mGlu receptor agonist, L‐2‐amino‐4‐phosphonobutanoate (l‐AP4), with an increased phosphorylation and activity of MAPKs (ERK‐1 and ‐2) and an increased phosphorylation of the PI‐3‐K target, protein kinase B (PKB/AKT). These effects were attenuated by the group‐III antagonists, α‐methyl‐serine‐O‐phosphate (MSOP) and (R,S)‐α‐cyclopropyl‐4‐phosphonophenylglycine (CPPG), or by pretreatment of the cultures with pertussis toxin. l‐AP4 also induced the nuclear translocation of β‐catenin, a downstream effector of the PI‐3‐K pathway. To assess the functional relevance of these mechanisms we examined the ability of l‐AP4 to protect granule cells against apoptosis by trophic deprivation, induced by lowering extracellular K+ from 25 to 10 mm. Neuroprotection by l‐AP4 was attenuated by MSOP and abrogated by the compounds PD98059 and UO126, which inhibit the MAPK pathway, or by the compound LY294002, which inhibits the PI‐3‐K pathway. Taken together, these results show for the first time that native group‐III mGlu receptors are coupled to MAPK and PI‐3‐K, and that activation of both pathways is necessary for neuroprotection mediated by this particular class of receptors.
Quantitative changes in mRNA expression of glutamate receptors in the rat peripheral and central vestibular systems following hypergravity
Authors: Uno Yoshihiro , Horii Arata , Uno Atsuhiko , Fuse Yuka , Fukushima Munehisa , Doi Katsumi , Kubo Takeshi
Published: March 2003

In order to investigate the mechanisms responsible for adaptation to altered gravity, we assessed the changes in mRNA expression of glutamate receptors in vestibular ganglion cells, medial vestibular nucleus, spinal vestibular nucleus/lateral vestibular nucleus, cerebellar flocculus, and uvula/nodulus from rats exposed to hypergravity for 2 h to 1 week using real‐time quantitative RT‐PCR methods. The mRNA expression of GluR2 and NR1 receptors in the uvula/nodulus and NR1 receptors in the medial vestibular nucleus increased in animals exposed to 2 h of hypergravity, and it decreased gradually to the control level. The mRNA expression of GluR2 receptors in vestibular ganglion cells decreased in animals exposed to 1 week of hypergravity. Neither the metabotropic glutamate receptor 1 nor δ2 glutamate receptor in flocculus and uvula/nodulus was affected by a hypergravity load for 2 h to 1 week. It is suggested that the animals adapted to the hypergravity by enhancing the cerebellar inhibition of the vestibular nucleus neurons through activation of the NR1 and GluR2 receptors on the Purkinje cells in uvula/nodulus especially at the early phase following hypergravity. In the later phase following hypergravity, the animals adapted to the hypergravity by reducing the neurotransmission between the vestibular hair cells and the primary vestibular neurons via down‐regulation of the postsynaptic GluR2 receptors in the vestibular periphery.
Interferon‐β activates multiple signaling cascades in primary human microglia
Authors: Kim Mee‐Ohk , Si Qiusheng , Zhou Jian Nian , Pestell Richard G. , Brosnan Celia F. , Locker Joseph , Lee Sunhee C.
Published: March 2003

Microglia, the resident brain macrophages, are the principal cells involved in the regulation of inflammatory and antimicrobial responses in the CNS. Interferon‐β (IFNβ) is an antiviral cytokine induced by viral infection or following non‐specific inflammatory challenges of the CNS. Because of the well‐known anti‐inflammatory properties of IFNβ, it is also used to treat multiple sclerosis, an inflammatory CNS disease. Despite the importance of IFNβ signaling in CNS cells, little has been studied, particularly in microglia. In this report, we investigated the molecular mechanisms underlying IFNβ‐induced β‐chemokine expression in primary human fetal microglia. Multiple signaling cascades are activated in microglia by IFNβ, including nuclear factor‐κB (NF‐κB), activator protein‐1 (AP‐1) and Jak/Stat. IFNβ induced IκBα degradation and NF‐κB (p65:p50) DNA binding. Inhibition of NF‐κB by either adenoviral transduction of a super repressor IκBα, or an antioxidant inhibitor of NF‐κB reduced expression of the β‐chemokines, regulated upon activation, normal T‐cell expressed and secreted (RANTES) and macrophage inflammatory protein (MIP)‐1β. IFNβ also induced phosphorylation of extracellular signal‐regulated kinase (ERK) mitogen‐activated protein kinase, and the MAP kinase kinase 1 (MEK1) inhibitor PD98059 dose‐dependently inhibited β‐chemokine mRNA and protein expression. PD98059 did not inhibit NF‐κB binding, demonstrating that ERK was not responsible for NF‐κB activation. Two downstream targets of ERK were identified in microglia: AP‐1 and Stat1. IFNβ induced AP‐1 nuclear binding activity in microglia and this was suppressed by PD98059. Additionally, IFNβ induced Stat1 phosphorylation at both tyrosine 701 (Y701) and serine 727 (S727) residues. S727 phosphorylation of Stat1, which is known to be required for maximal transcriptional activation, was inhibited by PD98059. Our results demonstrating multiple signaling cascades initiated by IFNβ in primary human microglia are novel and have implications for inflammatory and infectious diseases of the CNS.
The binding of prion proteins to serum components is affected by detergent extraction conditions
Authors: Shaked Yuval , Engelstein Roni , Gabizon Ruth
Published: March 2003

As many GPI anchored proteins, PrPC and its abnormal conformer PrPSc, are inserted into membrane microdomains known as rafts. Upon raft disruption, PrPC becomes soluble, while PrPSc aggregates into insoluble structures. It was recently published that, as opposed to PrPC, PrPSc, as well as its protease resistant core PrP27‐30, can bind specifically to plasminogen and other serum components. These findings were suggested to have important physiological implications in transmissible spongiform encephalopathies (TSE) diagnosis and pathogenesis. In this work, we show that the binding of PrPSc or PrP 27–30 to serum proteins occurs only at specific detergent combinations, in which disease associated PrPs are present in aggregated structures. At detergent conditions in which rafts are intact, it is actually PrPC. that binds to blood proteins, albeit not directly, but through neighboring rafts components. Our results therefore indicate that the binding of PrPSc to blood components has no physiological relevance.
GABA is toxic for mouse striatal neurones through a transporter‐mediated process
Authors: Maus Marion , Glowinski Jacques , Premont Joel
Published: March 2003

In the present study, GABA was shown to induce a necrotic neuronal death in cultured striatal neurones from mouse embryos. This effect did not depend on the activation of GABAA, GABAB or GABAC receptors as it was neither antagonized by bicuculline, saclofen or picrotoxin, respectively, nor reproduced by the GABA receptor agonists, muscimol and baclofen. Excluding the participation of glutamate, GABA neurotoxicity persisted in the presence of either the antagonists of ionotropic and metabotropic glutamate receptors or glutamate pyruvate transaminase, which induces an immediate catabolism of glutamate. A GABA transport‐associated process is involved in GABA neurotoxicity as nipecotic acid and NO 711, two inhibitors of the high‐affinity neuronal GABA transporters (GAT‐1, in particular), completely prevented the neurotoxic effect of GABA. The activation of a subset of G proteins is also implicated in the GABA transport‐mediated neuronal death as GABA neurotoxicity was completely suppressed when striatal neurones were pre‐treated with pertussis toxin. Further demonstrating the specificity of this neurotoxic process, GABA‐induced neurotoxicity was not observed in cortical neurones which, in contrast to striatal neurones, are largely represented by glutamatergic neurones. In conclusion, our study suggests that glutamate is not the sole neurotransmitter that can be responsible for brain damage and that GABA neurotoxicity involves both GABA transport and G protein transduction pathways.
Identification and functional characterization of nadrin variants, a novel family of GTPase activating protein for rho GTPases
Authors: Furuta Birei , Harada Ayako , Kobayashi Yoko , Takeuchi Ken‐ichi , Kobayashi Tetsuyuki , Umeda Masato
Published: March 2003

Nadrin is a GTPase‐activating protein (GAP) for the rho family of GTPases that controls Ca2+‐dependent exocytosis in nerve endings. In this study, three novel splice variants of nadrin were identified and the variants were designated as nadrin‐102, ‐104, ‐116 and ‐126 according to their relative molecular masses. All nadrin variants share the GAP domain, coiled‐coil domain, serine/threonine/proline‐rich domain, SH3‐binding motif, and a successive repeat of 29 glutamines. Tissue distribution analyses using polyclonal antibodies that can discriminate each variant showed that the expression of nadrin‐102, ‐104 and ‐116 was dominant in neuronal tissues and correlates well with the differentiation of neurons while nadrin‐126 was strongly expressed in embryonic brain. Expression of nadrin‐116 in PC12 cells strongly inhibited NGF‐dependent neurite outgrowth and this effect was dependent on its GAP activity. In contrast, no significant effect on either cell morphology or neurite outgrowth was observed with other variants. All variants showed punctate appearance throughout the cytoplasm, while the 66‐kDa carboxyl‐terminal fragment of nadrin‐102 and/or nadrin‐116 was localized to the nucleus and its nuclear translocation was accelerated by NGF‐induced differentiation of the cells. These results suggested that nadrin variants are different in their ability to regulate rho‐mediated signaling and that, in addition to being aGTPase‐activating protein, nadrin‐102 and ‐116 have otherdistinct functions in the nucleus of the cell, implying apossible role in the cross‐talk between the cytoskeleton andthe nucleus.
Okadaic acid disrupts synaptic vesicle trafficking in a ribbon‐type synapse
Authors: Guatimosim Cristina , Hull Court , Von Gersdorff Henrique , Prado Marco A. M.
Published: March 2003

Protein phosphorylation plays an essential role in regulating synaptic transmission and plasticity. However, regulation of vesicle trafficking towards and away from the plasma membrane is poorly understood. Furthermore, the extent to which phosphorylation modulates ribbon‐type synapses is unknown. Using the phosphatase inhibitor okadaic acid (OA), we investigated the influence of persistent phosphorylation on vesicle cycling in goldfish bipolar cells. We followed uptake of FM1‐43 during vesicle recycling in control and OA‐treated cells. FM1‐43 fluorescence spread to the center of control synaptic terminals after depolarization elicited Ca2+ influx. However, OA (1–50 nm) impaired this spatial spread of FM1‐43 in a dose‐dependent manner. Capacitance measurements revealed that OA (50 nm) did not modify either the amount or kinetics of exocytosis and endocytosis evoked by depolarizing pulses. The extremely low concentrations of OA (1–5 nm) sufficient to observe the inhibition of vesicle mobility implicate phosphatase 2A (PP2A) as a major regulator of vesicle trafficking after endocytosis. These results contrast with those at the neuromuscular junction where OA enhances lateral movement of vesicles between distinct vesicle clusters. Thus, our results suggest that phosphatases regulate vesicle translocation at ribbon synapses in a different manner than conventional active zones.
1‐Methyl‐4‐phenylpridinium (MPP+)‐induced functional run‐down of GABAA receptor‐mediated currents in acutely dissociated dopaminergic neurons
Authors: Wu Jie , Chan Piu , Schroeder Katherine M. , Ellsworth Kevin , Partridge L. Donald L.
Published: March 2003

We have evaluated GABAA receptor function during treatment of 1‐methyl‐4‐phenylpridinium (MPP+) using patch‐clamp perforated whole‐cell recording techniques in acutely dissociated dopaminergic (DAergic) neurons from rat substantia nigra compacta (SNc). γ‐Aminobutyric acid (GABA), glutamate or glycine induced inward currents (IGABA, IGlu, IGly) at a holding potential (VH) of −45 mV. The IGABA was reversibly blocked by the GABAA receptor antagonist, bicuculline, suggesting that IGABA is mediated through the activation of GABAA receptors. During extracellular perfusion of MPP+ (1–10 μm), IGABA, but neither IGlu nor IGly, declined (termed run‐down) with repetitive agonist applications, indicating that the MPP+‐induced IGABA run‐down occurred earlier than IGly or IGlu under our experimental conditions. The MPP+‐induced IGABA run‐down can be prevented by a DA transporter inhibitor, mazindol, and can be mimicked by a metabolic inhibitor, rotenone. Using conventional whole‐cell recording with different concentrations of ATP in the pipette solution, IGABA run‐down can be induced by decreasing intracellular ATP concentrations, or prevented by supplying intracellular ATP, indicating that IGABA run‐down is dependent on intracellular ATP concentrations. A GABAA receptor positive modulator, pentobarbital (PB), potentiated the declined IGABA and eliminated IGABA run‐down. Corresponding to these patch‐clamp data, tyrosine hydroxylase (TH) immunohistochemical staining showed that TH‐positive cell loss was protected by PB during MPP+ perfusion. It is concluded that extracellular perfusion of MPP+ induces a functional run‐down of GABAA receptors, which may cause an imbalance of excitation and inhibition of DAergic neurons.
Neuroserpin regulates neurite outgrowth in nerve growth factor‐treated PC12 cells
Authors: Parmar Parmjeet K. , Coates Leigh C. , Pearson John F. , Hill Rena M. , Birch Nigel P.
Published: March 2003

Neuroserpin is a serine protease inhibitor widely expressed in the developing and adult nervous systems and implicated in the regulation of proteases involved in processes such as synaptic plasticity, neuronal migration and axogenesis. We have analysed the effect of neuroserpin on growth factor‐induced neurite outgrowth in PC12 cells. We show that small changes in neuroserpin expression result in changes to the number of cells extending neurites and total neurite length following NGF treatment. Increased expression of neuroserpin resulted in a decrease in the number of cells extending neurites and a reduction in total free neurite length whereas reduced levels of neuroserpin led to a small increase in the number of neurite extending cells and a significant increase in total free neurite length compared to the parent cell line. Neuroserpin also altered the response of PC12 cells to bFGF and EGF treatment. Neuroserpin was localised to dense cored secretory vesicles in PC12 cells but was unable to complex with its likely enzyme target, tissue plasminogen activator at the acidic pH found in these vesicles. These data suggest that modulation of neuroserpin levels at the extending neurite growth cone may play an important role in regulating axonal growth.
Altered chemokine expression in the spinal cord and brain contributes to differential interleukin‐1β‐induced neutrophil recruitment
Authors: Campbell Sandra J. , Wilcockson David C. , Butchart Angus G. , Perry V. Hugh , Anthony Daniel C.
Published: March 2003

The pattern of neutrophil recruitment that accompanies inflammation in the CNS depends on the site of injury and the stage of development. The adult brain parenchyma is refractory to neutrophil recruitment and associated damage as compared to the spinal cord or juvenile brain. Using quantitative Taqman RT–PCR and enzyme‐liked immunosorbent assay (ELISA), we compared mRNA and protein expression of the rat neutrophil chemoattractant chemokines (CINC) in spinal cord and brain of adult and juvenile rats to identify possible association with the observed differences in neutrophil recruitment. Interleukin‐1β (IL‐1β) injection resulted in up‐regulated chemokine expression in both brain and spinal cord. CINC‐3 mRNA was elevated above CINC‐1 and CINC‐2α, with expression levels for each higher in spinal cord than in brain. By ELISA, IL‐1β induced greater CINC‐1 and CINC‐2α expression compared to CINC‐3, with higher protein levels in spinal cord than in brain. In the juvenile brain, significantly higher levels of CINC‐2α protein were observed in response to IL‐1β injection than in the adult brain following an equivalent challenge. Correspondingly, neutrophil recruitment was observed in the juvenile brain and adult spinal cord, but not in the adult brain. No expression of CINC‐2β mRNA was detected. Thus differential chemokine induction may contribute to variations in neutrophil recruitment in during development and between the different CNS compartments.