bluuebunny/crossref_metadata_2025 huggingface dataset:
Similarities and differences in the neuronal death processes activated by 3OH‐kynurenine and quinolinic acid
Authors: Chiarugi Alberto , Meli Elena , Moroni Flavio
Published: March 2003

3OH‐Kynurenine and quinolinic acid are tryptophan metabolites able to cause, at relatively elevated concentrations, neuronal death in vitro and in vivo. In primary cultures of mixed cortical cells, the minimal concentration of these compounds leading to a significant degree of neurotoxicity decreased from 100 to 1 µm, when the exposure time was prolonged from 24 to 72 h. NMDA receptor antagonists and inhibitors of nitric oxide synthase or poly(ADP‐ribose) polymerase reduced quinolinic acid, but not 3OH‐kynurenine toxicity. In contrast, scavengers of free radicals, caspase inhibitors and cyclosporin preferentially reduced 3OH‐kynurenine neurotoxicity. These observations suggest that quinolinic acid causes necrosis, whereas 3OH‐kynurenine‐exposed neurons primarily die in apoptosis. In line with this possibility, we found that ATP levels decreased more rapidly in quinolinate‐ than in 3OH‐kynurenine‐exposed cultures and that poly(ADP‐ribose) polymer, the product of poly(ADP‐ribose) polymerase activity, was more abundant in the nuclei of quinolinic acid than in those of 3OH‐kynurenine‐exposed neurons. Because minor changes in the physiological concentrations of 3OH‐kynurenine and quinolinic acid may cause neuronal death, our data suggest that these metabolites play a key role in the pathogenesis of several neurological disorders.
Tau‐66: evidence for a novel tau conformation in Alzheimer's disease
Authors: Ghoshal Nupur , García‐Sierra Francisco , Fu Yifan , Beckett Laurel A. , Mufson Elliott J. , Kuret Jeff , Berry Robert W. , Binder Lester I.
Published: March 2003

We have characterized a novel monoclonal antibody, Tau‐66, raised against recombinant human tau. Immunohistochemistry using Tau‐66 reveals a somatic‐neuronal stain in the superior temporal gyrus (STG) that is more intense in Alzheimer's disease (AD) brain than in normal brain. In hippocampus, Tau‐66 yields a pattern similar to STG, except that neurofibrillary lesions are preferentially stained if present. In mild AD cases, Tau‐66 stains plaques lacking obvious dystrophic neurites (termed herein ‘diffuse reticulated plaques’) in STG and the hippocampus. Enzyme‐linked immunosorbent assay (ELISA) analysis reveals that Tau‐66 is specific for tau, as there is no cross‐reactivity with MAP2, tubulin, Aβ1−40, or Aβ1−42, although Tau‐66 fails to react with tau or any other polypeptide on western blots. The epitope of Tau‐66, as assessed by ELISA testing of tau deletion mutants, appears discontinuous, requiring residues 155–244 and 305–314. Tau‐66 reactivity exhibits buffer and temperature sensitivity in an ELISA format and is readily abolished by SDS treatment. Taken together these lines of evidence indicate that the Tau‐66 epitope is conformation‐dependent, perhaps involving a close interaction of the proline‐rich and the third microtubule‐binding regions. This is the first indication that tau can undergo this novel folding event and that this conformation of tau is involved in AD pathology.
Up‐regulation of hippocampal glutamate transport during chronic treatment with sodium valproate
Authors: Hassel Bjørnar , Iversen Evy Grini , Gjerstad Leif , Taubøll Erik
Published: March 2003

Excessive glutamatergic neurotransmission has been implicated in some neurodegenerative disorders. It would be of value to know whether glutamate transport, which terminates the glutamate signal, can be up‐regulated pharmacologically. Here we show that chronic treatment of rats with the anti‐epileptic drug sodium valproate (200 mg or 400 mg/kg bodyweight, twice per day for 90 days) leads to a dose‐dependent increase in hippocampal glutamate uptake capacity as measured by uptake of [3H]glutamate into proteoliposomes. The level of glutamate transporters EAAT1 and EAAT2 in hippocampus also increased dose‐dependently. No effect of sodium valproate on glutamate transport was seen in frontal or parietal cortices or in cerebellum. The hippocampal levels of glial fibrillary acidic protein and glutamine synthetase were unaffected by valproate treatment, whereas the levels of synapsin I and phosphate‐activated glutaminase were reduced by valproate treatment, suggesting that the increase in glutamate transporters was not caused by astrocytosis or increased synaptogenesis. A direct effect of sodium valproate on the glutamate transporters could be excluded. The results show that hippocampal glutamate transport is an accessible target for pharmacological intervention and that sodium valproate may have a role in the treatment of excitotoxic states in the hippocampus.
Identification of the region of non‐Aβ component (NAC) of Alzheimer's disease amyloid responsible for its aggregation and toxicity
Authors: Bodles Angela M. , Guthrie David J. S. , Greer Brett , Irvine G. Brent
Published: March 2003

The non‐beta‐amyloid (Aβ) component of Alzheimer's disease amyloid (NAC) and its precursor α‐synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and α‐synuclein both form β‐sheet structures upon ageing, aggregate to form fibrils, and are neurotoxic. We recently established that a peptide comprising residues 3–18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY‐5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8–18) and NAC(8–16) are toxic, whereas NAC(12–18), NAC(9–16) and NAC(8–15) are not. Circular dichroism indicates that none of the peptides displays β‐sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce β sheet, fragments NAC(8–18) and NAC(8–16) both form β‐sheet structure. Only NAC(8–18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8–16 of NAC, equivalent to residues 68–76 in α‐synuclein, comprise the region crucial for toxicity.
GABAa receptor α‐subunit proteins in human chronic alcoholics
Authors: Lewohl Joanne M. , Huygens Flavia , Crane Denis I. , Dodd Peter R.
Published: March 2003

Antibodies were raised against specific peptides from N‐terminal regions of the α1 and α3 isoforms of the GABAA receptor, and used to assess the relative expression of these proteins in the superior frontal and primary motor cortices of 10 control, nine uncomplicated alcoholic and six cirrhotic alcoholic cases were matched for age and post‐mortem delay. The regression of expression on post‐mortem delay was not statistically significant for either isoform in either region. In both cortical areas, the regression of α1 expression on age differed significantly between alcoholic cases, which showed a decrease, and normal controls, which did not. Age had no effect on α3 expression. The α1 and α3 isoforms were found to be expressed differentially across cortical regions and showed a tendency to be expressed differentially across case groups. In cirrhotic alcoholics, α1 expression was greater in superior frontal than in motor cortex, whereas this regional difference was not significant in controls or uncomplicated alcoholics. In uncomplicated alcoholics, α3 expression was significantly lower in superior frontal than in motor cortex. Expression of α1 was significantly different from that of α3 in the superior frontal cortex of alcoholics, but not in controls. In motor cortex, there were no significant differences in expression between the isoforms in any case group.
The Ras–ERK pathway is required for the induction of neuronal nitric oxide synthase in differentiating PC12 cells
Authors: Schonhoff Christopher M. , Bulseco Dylan A. , Brancho Deborah M. , Parada Luis F. , Ross Alonzo H.
Published: March 2003

We have studied the role of MAP kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In nerve growth factor (NGF)‐treated PC12 cells, we find nNOS induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRNA, nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ras adenovirus. We have also used drugs that block MAP kinase pathways and assessed their ability to inhibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we find that U0126, but not PD98059, blocks induction of nNOS protein and NOS activity in NGF‐treated PC12 cells. Also, the p38 kinase inhibitor, SB203580, does not block nNOS induction in our clone of PC12 cells. Since the JNK pathway is not activated in NGF‐treated PC12 cells, we conclude that the Ras–ERK pathway and not the p38 or JNK pathway is required for nNOS induction in NGF‐treated PC12 cells. We find that U0126 is much more effective than PD98059 in blocking the Ras–ERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the Ras–ERK pathway is required for nNOS induction.
Evidence of a role for cyclic ADP‐ribose in calcium signalling and neurotransmitter release in cultured astrocytes
Authors: Verderio Claudia , Bruzzone Santina , Zocchi Elena , Fedele Ernesto , Schenk Ursula , De Flora Antonio , Matteoli Michela
Published: March 2003

Astrocytes possess different, efficient ways to generate complex changes in intracellular calcium concentrations, which allow them to communicate with each other and to interact with adjacent neuronal cells. Here we show that cultured hippocampal astrocytes coexpress the ectoenzyme CD38, directly involved in the metabolism of the calcium mobilizer cyclic ADP‐ribose, and the NAD+ transporter connexin 43. We also demonstrate that hippocampal astrocytes can release NAD+ and respond to extracellular NAD+ or cyclic ADP‐ribose with intracellular calcium increases, suggesting the existence of an autocrine cyclic ADP‐ribose‐mediated signalling. Cyclic ADP‐ribose‐induced calcium changes are in turn responsible for an increased glutamate and GABA release, this effect being completely inhibited by the cyclic ADP‐ribose specific antagonist 8‐NH2‐cADPR. Furthermore, addition of NAD+ to astrocyte‐neuron co‐cultures results in a delayed intracellular calcium transient in neuronal cells, which is strongly but not completely inhibited by glutamate receptor blockers. These data indicate that an astrocyte‐to‐neuron calcium signalling can be triggered by the CD38/cADPR system, which, through the activation of intracellular calcium responses in astrocytes, is in turn responsible for the increased release of neuromodulators from glial cells.
High‐affinity sodium–vitamin C co‐transporters (SVCT) expression in embryonic mouse neurons
Authors: Castro Maite , Caprile Teresa , Astuya Allisson , Millán Carola , Reinicke Karin , Vera Juan Carlos , Vásquez Osmán , Aguayo Luis G , Nualart Francisco
Published: March 2003

The sodium–vitamin C co‐transporters SVCT1 and SVCT2 transport the reduced form of vitamin C, ascorbic acid. High expression of the SVCT2 has been demonstrated in adult neurons and choroid plexus cells by in situ hybridization. Additionally, embryonic mesencephalic dopaminergic neurons express the SVCT2 transporter. However, there have not been molecular and kinetic analyses addressing the expression of SVCTs in cortical embryonic neurons. In this work, we confirmed the expression of a SVCT2‐like transporter in different regions of the fetal mouse brain and in primary cultures of neurons by RT‐PCR. Kinetic analysis of the ascorbic acid uptake demonstrated the presence of two affinity constants, 103 µm and 8 µm. A Km of 103 µm corresponds to a similar affinity constant reported for SVCT2, while the Km of 8 µm might suggest the expression of a very high affinity transporter for ascorbic acid. Our uptake analyses also suggest that neurons take up dehydroascorbic acid, the oxidized form of vitamin C, through the glucose transporters. We consider that the early expression of SVCTs transporters in neurons is important in the uptake of vitamin C, an essential molecule for the fetal brain physiology. Vitamin C that is found at high concentration in fetal brain may function in preventing oxidative free radical damage, because antioxidant radical enzymes mature only late in the developing brain.
Thyroid hormone regulates oxidative phosphorylation in the cerebral cortex and striatum of neonatal rats
Authors: Martinez Bienvenida , Del Hoyo Pilar , Martin Miguel Angel , Arenas Joaquín , Perez‐Castillo Ana ,   Angel Santos
Published: March 2003

We have previously shown that thyroid hormone (T3) regulates mitochondrial gene expression, morphology and transmembrane potential in the developing brain. Here, we have analysed the effect of thyroid hormone on mitochondrial function in different brain regions. For this purpose we have determined, in control, hypothyroid and T3‐treated hypothyroid neonatal rats, the rate of oxidative phosphorylation in isolated mitochondria and the activity of the respiratory complexes in tissue homogenates. Our results showed a decrease in oxidative phosphorylation rate (only in the presence of NADH‐generating substrates) and mitochondrial complexes I and III activity in the cerebral cortex and striatum of hypothyroid neonates, but not in the other areas analysed (hippocampus, cerebellum, thalamus, mid brain and brain stem). In parallel with mitochondrial activity, the levels of mitochondrially encoded transcripts were decreased only in the cerebral cortex and striatum of hypothyroid rats. The administration of T3 corrected all these parameters. In summary, this study showed a down‐regulation of mitochondrial gene expression accompanied by a decrease in mitochondrial activity in the cerebral cortex and striatum of developing hypothyroid neonatal rats.
γ‐Hydroxybutyrate modulation of glutamate levels in the hippocampus: an in vivo and in vitro study
Authors: Ferraro Luca , Tanganelli Sergio , O'Connor William Thomas , Francesconi Walter , Loche Antonella , Gessa Gian Luigi , Antonelli Tiziana
Published: March 2003

The effect of γ‐hydroxybutyric acid on extracellular glutamate levels in the hippocampus was studied by microdialysis in freely moving rats and in isolated hippocampal synaptosomes. Intra‐hippocampal (CA1) perfusion with γ‐hydroxybutyric acid (10 nm–1 mm) concentration‐dependently influenced glutamate levels: γ‐hydroxybutyric acid (100 and 500 nm) increased glutamate levels; 100 and 300 µm concentrations were ineffective; whereas the highest 1 mm concentration reduced local glutamate levels. The stimulant effect of γ‐hydroxybutyric acid (100 nm) was suppressed by the locally co‐perfused γ‐hydroxybutyric acid receptor antagonist NCS‐382 (10 µm) but not by the GABAB receptor antagonist CGP‐35348 (500 µm). Furthermore, the γ‐hydroxybutyric acid (1 mm)‐induced reduction in CA1 glutamate levels was counteracted by NCS‐382 (10 µm), and it was also reversed into an increase by CGP‐35348. Given alone, neither NCS‐382 nor CGP‐35348 modified glutamate levels. In hippocampal synaptosomes, γ‐hydroxybutyric acid (50 and 100 nm) enhanced both the spontaneous and K+‐evoked glutamate efflux, respectively, both effects being counteracted by NCS‐382 (100 nm), but not by CGP‐35348 (100 µm). These findings indicate that γ‐hydroxybutyric acid exerts a concentration‐dependent regulation of hippocampal glutamate transmission via two opposing mechanisms, whereby a direct γ‐hydroxybutyric acid receptor mediated facilitation is observed at nanomolar γ‐hydroxybutyric acid concentrations, and an indirect GABAB receptor mediated inhibition predominates at millimolar concentrations.
The pituitary adenylate cyclase‐activating polypeptide modulates glutamatergic calcium signalling: investigations on rat suprachiasmatic nucleus neurons
Authors: Kopp Michael D. A. , Meissl Hilmar , Dehghani Faramarz , Korf Horst‐Werner
Published: March 2003

Circadian rhythms generated by the hypothalamic suprachiasmatic nucleus (SCN) are synchronized with the external light/dark cycle by photic information transmitted directly from the retina via the retinohypothalamic tract (RHT). The RHT contains the neurotransmitters glutamate and pituitary adenylate cyclase‐activating polypeptide (PACAP), which code chemically for ‘light’ or ‘darkness’ information, respectively. We investigated interactions of PACAP and glutamate by analysing effects on the second messenger calcium in individual SCN neurons using the Fura‐2 technique. PACAP did not affect NMDA‐mediated calcium increases, but influenced signalling cascades of non‐NMDA glutamate receptors, which in turn can regulate NMDA receptors. On the one hand, PACAP amplified/induced glutamate‐dependent calcium increases by interacting with α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate signalling. This was not related to direct PACAPergic effects on the second messengers cAMP and calcium. On the other hand, PACAP reduced/inhibited calcium increases elicited by glutamate acting on metabotropic receptors. cAMP analogues mimicked this inhibition. Most neurons displaying PACAPergic neuromodulation were immunoreactive for vasoactive intestinal polypeptide, which is a marker for retinorecipient SCN neurons. The observed PACAPergic effects provide a broad range of interactions that allow a fine‐tuning of the endogenous clock by the integration of ‘light’ and ‘darkness’ information on the level of single SCN neurons.
Transcriptional regulation of the chicken hydroxyindole‐O‐methyltransferase gene by the cone–rod homeobox‐containing protein
Authors: Bernard Marianne , Dinet Virginie , Voisin Pierre
Published: March 2003

Functional differentiation of photoreceptor cells involves the expression of two sets of genes: those encoding the proteins of the phototransduction cascade and those encoding the enzymes of melatonin synthesis. The transcription factor Crx (cone−rod homeobox) plays a major role in the differentiation and maintenance of the photoreceptor phenotype. Previous studies have shown that this effect of Crx is correlated with its ability to transactivate several genes of the phototransduction cascade. Here, we show that Crx can also act on the gene encoding the melatonin‐synthesizing enzyme, hydroxyindole‐O‐methyltransferase (HIOMT, EC 2.1.1.4). Three of the six putative Crx‐binding sites found in the chicken HIOMT promoter interact directly with recombinant Crx and bound a pineal/retina‐specific protein showing DNA‐binding characteristics similar to those of Crx. In transient transfection experiments, Crx transactivated transcription of a reporter gene from the chicken HIOMT promoter. Transactivation was observed even with a portion of promoter carrying only one Crx‐binding site and it was abolished by a mutation in this cis‐regulatory element. These data indicate that Crx may participate in photoreceptor cell differentiation, not only by acting on the genes of the phototransduction cascade, but also by controlling the expression of the genes involved in melatonin synthesis.
Brain‐derived neurotrophic factor (BDNF) mediates bone morphogenetic protein‐2 (BMP‐2) effects on cultured striatal neurones
Authors: Gratacòs E. , Checa N. , Pérez‐Navarro E. , Alberch J.
Published: March 2003

Bone morphogenetic proteins are members of the transforming growth factor‐β superfamily that have multiple functions in the developing nervous system. One of them, bone morphogenetic protein‐2 (BMP‐2), promotes the differentiation of cultured striatal neurones, enhancing dendrite growth and calbindin‐positive phenotype. Bone morphogenetic proteins have been implicated in cooperative interactions with other neurotrophic factors. Here we examined whether the effects of BMP‐2 on cultured striatal neurones are mediated or enhanced by other neurotrophic factors. BMP‐2 had a cooperative effect with low doses of brain‐derived neurotrophic factor or neurotrophin‐3 (but not with other neurotrophic factors such as glial cell line‐derived neurotrophic factor, neurturin or transforming growth factor‐β2) on the number of calbindin‐positive striatal neurones. Moreover, BMP‐2 induced phosphorylated Trk immunoreactivity in cultured striatal neurones, suggesting that neurotrophins are involved in BMP‐2 neurotrophic effects. The addition of TrkB‐IgG or antibodies against brain‐derived neurotrophic factor abolished the effects of BMP‐2 on the number and degree of differentiation of calbindin‐positive striatal neurones. Indeed, BMP‐2 treatment increased brain‐derived neurotrophic factor protein levels in treated cultures media and BDNF immunocytochemistry revealed that this neurotrophin was produced by neuronal cells. Taken together, these results indicate that brain‐derived neurotrophic factor mediates the effects of BMP‐2 on striatal neurones.
The γ‐aminobutyric acid receptor B, but not the metabotropic glutamate receptor type‐1, associates with lipid rafts in the rat cerebellum
Authors: Becher Anja , White Julia H. , McIlhinney R. A. Jeffrey
Published: March 2003

Recent evidence suggests that specialized microdomains, called lipid rafts, exist within plasma membranes. These domains are enriched in cholesterol and sphingolipids and are resistant to non‐ionic detergent‐extraction at 4°C. They contain specific populations of membrane proteins, and can change their size and composition in response to cellular signals, resulting in activation of signalling cascades. Here, we demonstrate that both the metabotropic γ‐aminobutyric acid receptor B (GABAB receptor) and the metabotropic glutamate receptor‐1 from rat cerebellum are insoluble in the non‐ionic detergent Triton X‐100. However, only the GABAB receptor associates with raft fractions isolated from rat brain by sucrose gradient centrifugation. Moreover, increasing the stringency of isolation by decreasing the protein : detergent ratio caused an enrichment of the GABAB receptor in raft fractions. In contrast, depletion of cholesterol from cerebellar membranes by either saponin or methyl‐β‐cyclodextrin treatment, which solubilize known raft markers, also increased the solubility of the GABAB receptor. These properties are all consistent with an association of the GABAB receptor with lipid raft microdomains.
Gene expression differences in bipolar disorder revealed by cDNA array analysis of post‐mortem frontal cortex
Authors: Bezchlibnyk Yarema B. , Wang Jun‐Feng , McQueen Glenda M. , Young L. Trevor
Published: March 2003

Previous studies have implicated a number of biochemical pathways in the etiology of bipolar disorder (BD). However, the precise abnormalities underlying this disorder remain to be established. To investigate novel factors that may be important in the pathophysiology of BD, we utilized cDNA expression arrays to examine differences in expression of up to 1200 genes known to be involved in potentially relevant biochemical processes. This investigation was undertaken in post‐mortem samples of frontal cortex tissue from patients with BD and matched controls, obtained (n = 10/group) from the Stanley Foundation Neuropathology Consortium. Results include significant (greater than 35% change in signal intensity) differences between BD and controls in a number of genes (n = 24). Selected targets were analyzed by RT‐PCR, which confirmed a decrease in transforming growth factor‐beta1 (TGF‐β1), and an increase in both caspase‐8 precursor (casp‐8) and transducer of erbB2 (Tob) expression in BD. We further observed a significant decrease of TGF‐β1 mRNA levels in BD by RT‐PCR in individual post‐mortem samples. Given the neuroprotective role attributed to this inhibitory cytokine, our results suggest that the down‐regulation of TGF‐β1 may lead to various neurotoxic insults potentially involved in the etiology of certain mood disorders.
RGS14 is a bifunctional regulator of Gαi/o activity that exists in multiple populations in brain
Authors: Hollinger Susanne , Taylor Jackie B. , Goldman Erinn H. , Hepler John R.
Published: March 2003

Members of the regulators of G protein signaling (RGS) family modulate Gα‐directed signals as a result of the GTPase‐activating protein (GAP) activity of their conserved RGS domain. In addition to its RGS domain, RGS14 contains a Rap binding domain (RBD) and a GoLoco motif. To define the cellular and biochemical properties of RGS14 we utilized two different affinity purified antisera that specifically recognize recombinant and native RGS14. In brain, we observed two RGS14‐like immunoreactive bands of distinct size (60 kDa and 55 kDa). Both forms are present in brain cytosol and in two, biochemically distinct, membrane subpopulations: one detergent‐extractable and the other detergent‐insensitive. Recombinant RGS14 binds specifically to activated Gαi/o, but not Gαq/11, Gα12/13, or Gαs in brain membranes. In reconstitution studies, we found that RGS14 is a non‐selective GAP for Gαi1 and Gαo and that full‐length RGS14 is an approximately 10‐fold more potent stimulator of Gα GTPase activity than the RGS domain alone. In contrast, neither full‐length RGS14 nor the isolated RBD domain is a GAP for Rap1. RGS14 is also a highly selective guanine nucleotide dissociation inhibitor (GDI) for Gαi but not Gαo, and this activity is restricted to the C‐terminus containing the GoLoco domain. These findings highlight previously unknown biochemical properties of RGS14 in brain, and provide one of the first examples of an RGS protein that is a bifunctional regulator of Gα actions.
The I1‐imidazoline receptor in PC12 pheochromocytoma cells activates protein kinases C, extracellular signal‐regulated kinase (ERK) and c‐jun N‐terminal kinase (JNK)
Authors: Edwards Lincoln , Fishman Daniel , Horowitz Peleg , Bourbon Nicole , Kester Mark , Ernsberger Paul
Published: March 2003

We sought to further elucidate signal transduction pathways for the I1‐imidazoline receptor in PC12 cells by testing involvement of protein kinase C (PKC) isoforms (βII, ε, ζ), and the mitogen‐activated protein kinases (MAPK) ERK and JNK. Stimulation of I1‐imidazoline receptor with moxonidine increased enzymatic activity of the classical βII isoform in membranes by about 75% and redistributed the atypical isoform into membranes (40% increase in membrane‐bound activity), but the novel isoform of PKC was unaffected. Moxonidine and clonidine also increased by greater than two‐fold the proportion of ERK‐1 and ERK‐2 in the phosphorylated active form. In addition, JNK enzymatic activity was increased by exposure to moxonidine. Activation of ERK and JNK followed similar time courses with peaks at 90 min. The action of moxonidine on ERK activation was blocked by the I1‐receptor antagonist efaroxan and by D609, an inhibitor of phosphatidylcholine‐selective phospholipase C (PC‐PLC), previously implicated as the initial event in I1‐receptor signaling. Inhibition or depletion of PKC blocked activation of ERK by moxonidine. Two‐day treatment of PC12 cells with the I1/α2‐agonist clonidine increased cell number by up to 50% in a dose related manner. These data suggest that ERK and JNK, along with PKC, are signaling components of the I1‐receptor pathway, and that this receptor may play a role in cell growth.
Modulation of microglial superoxide production by α‐tocopherol in vitro: attenuation of p67phox translocation by a protein phosphatase‐dependent pathway
Authors: Egger Tamara , Hammer Astrid , Wintersperger Andrea , Goti Daniel , Malle Ernst , Sattler Wolfgang
Published: March 2003

As in other phagocytic cells, the NADPH‐oxidase system in microglia is thought to be primarily responsible for the production of superoxide anion radicals (O2−·), a potentially cytotoxic reactive oxygen species. The assembly of a functional NADPH‐oxidase complex at the plasma membrane depends on the phosphorylation and subsequent translocation of several cytosolic subunits. Immunocytochemical and subcellular fractionation experiments performed during the present study revealed that the NADPH‐oxidase subunit p67phox translocates from the cytosol to the plasma membrane upon stimulation. Pre‐incubation of microglia in α‐tocopherol (αTocH) containing medium decreased O2−· production in a time‐ and concentration‐dependent manner, findings attributed to attenuated p67phox translocation to the plasma membrane. Moreover, αTocH‐supplementation of the culture medium resulted in decreased microglial protein kinase C (PKC) activities, an effect that could be partially or completely reversed by the addition of protein phosphatase inhibitors (okadaic acid and calyculin A). The addition of the PKC‐inhibitor staurosporine inhibited the microglial respiratory burst in a manner comparable to αTocH. The addition of okadaic acid or calyculin A completely restored O2−· production in αTocH‐supplemented cells. The present findings suggest that αTocH inactivates PKC via a PP1 or PP2A‐mediated pathway and, as a consequence, blocks the phosphorylation‐dependent translocation of p67phox to the plasma membrane. As a result, O2−· production by the microglial NADPH‐oxidase system is substantially inhibited.
Identification and characterization of a novel Nogo‐interacting mitochondrial protein (NIMP)
Authors: Hu Wen‐Hui , Hausmann Oliver N. , Yan Ming‐Shan , Walters Winston M. , Wong Paul K. Y. , Bethea John R.
Published: March 2003

Nogo is a potent inhibitor of regeneration following spinal cord injury. To develop a better understanding of the mechanisms responsible for regenerative failure we used a yeast two‐ hybrid approach to try and identify proteins that interact with Nogo. We identified a novel mitochondrial protein designated Nogo‐interacting mitochondrial protein (NIMP) in a screen of an adult human brain cDNA library. This interaction was confirmed by co‐immunoprecipitation in both brain tissue (endogenous) and transfected HEK293T cells (overexpressed). In support of these studies we demonstrate that Nogo interacts with the UQCRC1 and UQCRC2 components of complex III, within the mitochondrial respiratory chain. The mitochondrial localization of NIMP was evidenced by confocal image analysis and western blot analysis of isolated mitochondria. NIMP is highly conserved and ubiquitously expressed in mitochondria‐enriched tissues. Within the CNS, NIMP‐like immunoreactivity is present in neurons and astrocytes. These data suggest that NIMP is a novel mitochondrial protein that interacts with Nogo. The interaction of Nogo with mitochondrial proteins may provide insight into the mechanisms for Nogo‐induced inhibition of neurite growth.
The cytotoxicity of dopamine may be an artefact of cell culture
Authors: Clement Marie‐Véronique , Long Lee Hua , Ramalingam Jeyakumar , Halliwell Barry
Published: March 2003

Administration of l‐DOPA is commonly used to treat Parkinson's disease, yet controversy continues as to whether the dopamine arising from it aggravates neuronal loss. Several authors have reported cytotoxic effects of l‐DOPA and dopamine on cultured cells, but others have not. In this report using the rat pheochromocytoma cell line PC12 and the M14 human melanoma cell line we show that dopamine‐mediated cell death is not specific for neuronal cells. Moreover, our data show that both l‐DOPA and dopamine interact with commonly used cell culture media, undergoing oxidation to generate hydrogen peroxide and dopamine semiquinones/quinones. Catalase and reduced glutathione could protect against cytotoxicity. These results suggest that caution needs to be employed when using cell culture studies to predict effects of l‐DOPA and/or dopamine in vivo because of the extracellular generation of reactive species in the culture media.
Sepsis inhibits reduction of dehydroascorbic acid and accumulation of ascorbate in astroglial cultures: intracellular ascorbate depletion increases nitric oxide synthase induction and glutamate uptake inhibition
Authors: Korcok Jasminka , Wu Feng , Tyml Karel , Hammond Robert R. , Wilson John X.
Published: March 2003

Sepsis is associated with oxidative stress and impaired glutamatergic transmission in brain. We investigated whether sepsis impairs accumulation of the antioxidant, ascorbate, and uptake of glutamate by astrocytes. Bacterial endotoxin (Escherichia colilipopolysaccharide, LPS) and the inflammatory cytokine, interferon‐γ (IFNγ), were applied to primary astrocyte cultures to model sepsis. In the absence of ascorbate, the combination of LPS and IFNγ (LPS + IFNγ) up‐regulated inducible nitric oxide synthase (iNOS) and decreased the initial rate of glutamate uptake by 50% within 24 h. Cell viability and facilitated glucose transport activity were not affected at 24 h. Pre‐treatment with ascorbate‐2‐O‐phosphate increased intracellular ascorbate concentration and attenuated the induction of iNOS and inhibition of glutamate uptake caused by LPS + IFNγ. Subsequent experiments examined the mechanisms by which cells accumulate ascorbate. LPS + IFNγ decreased slightly the initial rate of uptake of ascorbate and inhibited markedly the rate with which intracellular dehydroascorbic acid (DHAA) was reduced to ascorbate. We conclude that septic insult impairs astrocytic clearance of DHAA from the extracellular fluid and decreases intracellular ascorbate concentration. Furthermore, sepsis induces iNOS and inhibits glutamate uptake by astrocytes through mechanisms that can be modulated by intracellular ascorbate. These results indicate treatments that increase intracellular ascorbate concentration may be beneficial for patients at risk for neurologic complication in sepsis.
Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5
Authors: Holmberg Sara K. S. , Mikko Sofia , Boswell Timothy , Zoorob Rima , Larhammar Dan
Published: March 2003

The neuropeptide Y (NPY) receptor subtypes Y1 and Y5 are involved in the regulation of feeding and several other physiological functions in mammals. To increase our understanding of the origin and mechanisms of the complex NPY system, we report here the cloning and pharmacological characterization of receptors Y1 and Y5 in the first non‐mammal, chicken (Gallus gallus). The receptors display 80–83% and 64–72% amino acid sequence identity, respectively, with their mammalian orthologues. The three endogenous ligands NPY, peptide YY (PYY) and pancreatic polypeptide (PP) have similar affinities as in mammals, i.e. NPY and PYY have subnanomolar affinity for both receptors whereas chicken PP bound with nanomolar affinity to Y5 but not to Y1. A notable difference to mammalian receptor subtypes is that the Y1 antagonist SR120819A does not bind chicken Y1, whereas BIBP3226 does. The Y5 antagonist CGP71863A binds to the chicken Y5 receptor. Anatomically, both Y1 and Y5 have high mRNA expression levels in the infundibular nucleus which is the homologous structure of the hypothalamic arcuate nucleus in mammals. These results suggest that some of the selective Y1 and Y5 antagonists developed in mammals can be used to study appetite regulation in chicken.
Intracellular Ca2+ signals evoked by stimulation of nicotinic acetylcholine receptors in SH‐SY5Y cells: contribution of voltage‐operated Ca2+ channels and Ca2+ stores
Authors: Dajas‐Bailador Federico A. , Mogg Adrian J. , Wonnacott Susan
Published: March 2003

Neuronal nicotinic acetylcholine receptors (nAChR) can regulate several neuronal processes through Ca2+‐dependent mechanisms. The versatility of nAChR‐mediated responses presumably reflects the spatial and temporal characteristics of local changes in intracellular Ca2+ arising from a variety of sources. The aim of this study was to analyse the components of nicotine‐evoked Ca2+ signals in SH‐SY5Y cells, by monitoring fluorescence changes in cells loaded with fluo‐3 AM. Nicotine (30 µm) generated a rapid elevation in cytoplasmic Ca2+ that was partially and additively inhibited (40%) by α7 and α3β2* nAChR subtype selective antagonists; α3β4* nAChR probably account for the remaining response (60%). A substantial blockade (80%) by CdCl2 (100 µm) indicates that voltage‐operated Ca2+ channels (VOCC) mediate most of the nicotine‐evoked response, although the α7 selective antagonist α‐bungarotoxin (40 nm) further decreased the CdCl2‐ resistant component. The elevation of intracellular Ca2+ levels provoked by nicotine was sustained for at least 10 min and required the persistent activation of nAChR throughout the response. Intracellular Ca2+ stores were implicated in both the initial and sustained nicotine‐evoked Ca2+ responses, by the blockade observed after ryanodine (30 µm) and the inositoltriphosphate (IP3)‐receptor antagonist, xestospongin‐c (10 µm). Thus, nAChR subtypes are differentially coupled to specific sources of Ca2+: activation of nAChR induces a sustained elevation of intracellular Ca2+ levels which is highly dependent on the activation of VOCC, and also involves Ca2+ release from ryanodine and IP3‐dependent intracellular stores. Moreover, the α7, but not α3β2* nAChR, are responsible for a fraction of the VOCC‐independent nicotine‐evoked Ca2+ increase that appears to be functionally coupled to ryanodine sensitive Ca2+ stores.
Modulation of synaptic signalling complexes by Homer proteins
Authors: Thomas Ulrich
Published: March 2003

The number of neurotransmitter receptors in the postsynaptic membrane and their functional coupling to intracellular signalling cascades are important determinants of synaptic strength – and hence potential targets for plasticity related modulation. In this context, Homer/Vesl proteins have gained particular interest for three main reasons: (i) they constitute part of the molecular scaffold at postsynaptic densities of excitatory synapses in the mammalian brain; (ii) they physically link type‐I metabotropic glutamate receptors to the postsynaptic density and to inositol 1,4,5‐triphosphate receptors in the subsynaptic endoplasmic reticulum; and (iii) Homer‐1a, which has been categorized as an immediate early gene isoform, exerts dominant‐negative activity, suggesting that it is involved in activity dependent rearrangements at synaptic junctions. Although these fundamental aspects have been reviewed previously by Xiao et al., this review will address primarily more recent studies on the regulation of Homer 1a expression and on the role of Homer/Vesl proteins in spine morphogenesis and receptor targeting and signalling.
Histamine H2‐like receptors in chick cerebral cortex: effects on cyclic AMP synthesis and characterization by [3H]tiotidine binding
Authors: Zawilska Jolanta B. , Woldan‐Tambor Agata , Nowak Jerzy Z.
Published: March 2003

In this study, histamine (HA) receptors in chick cerebral cortex were characterized using two approaches: (1) analysis of the effects of HA‐ergic drugs on the cAMP‐generating system, and (2) radioreceptor binding of [3H]tiotidine, a selective H2antagonist. HA was a weak activator of adenylyl cyclase in a crude membrane preparation of chick cerebrum. On the other hand, HA (0.1–1000 μm) potently and concentration dependently stimulated cAMP production in [3H]adenine pre‐labelled slices of chick cerebral cortex, displaying an EC50value (concentration that produces 50% of maximum response) of 2.65 μm. The effect of HA was mimicked by agonists of HA receptors with the following rank order of potency: HA ≥  4‐methylHA (H2) ≥ Nα,Nα‐dimethylHA (H3 ≫ H2 = H1) ≫  2‐methylHA (H1) ≫ 2‐thiazolylethylamine (H1) ≥ Rα‐methylHA (H3) ≫ amthamine, dimaprit (H2), immepip (H3, H4). The HA‐evoked increase in cAMP production in chick cerebral cortex was antagonized by selective H2receptor blockers (aminopotentidine ≥ tiotidine > ranitidine ≫ zolantidine), and not significantly affected by mepyramine and thioperamide, selective H1and H3/H4receptor blockers, respectively. A detailed analysis of the antagonistic action of aminopotentidine (vs. HA) revealed a non‐competitive mode of action. The binding of [3H]tiotidine to chick cortical membranes was rapid, stable and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of receptor binding site with high affinity [equilibrium dissociation constant (Kd) = 4.42 nm] and high capacity [maximum number of binding sites (Bmax) = 362 fmol/mg protein]. The relative rank order of HA‐ergic drugs to inhibit [3H]tiotidine binding to chick cerebrum was: antagonists – tiotidine ≫ aminopotentidine = ranitidine ≥ zolantadine ≫ thioperamide ≈ triprolidine; agonists – HA ≥ 4‐methylHA ≫ 2‐methylHA ≥Rα‐methylHA ≈ dimaprit. In conclusion, chick cerebral cortex contains H2‐like HA receptors that are linked to the cAMP‐generating system and are labelled with [3H]tiotidine. The pharmacological profile of these receptors is different from that described for their mammalian counterpart. It is suggested that the studied receptors represent either an avian‐specific H2‐like HA receptors or a novel subtype of HA receptors.
Chronic nicotine treatment reduces β‐amyloidosis in the brain of a mouse model of Alzheimer's disease (APPsw)
Authors: Nordberg Agneta , Hellström‐Lindahl Ewa , Lee Mandy , Johnson Mary , Mousavi Malahat , Hall Ros , Perry Elaine , Bednar Ivan , Court Jennifer
Published: March 2003

Alzheimer's disease neuropathology is characterised by β‐amyloid plaques and neurofibrillary tangles. Inhibition of β‐amyloid accumulation may be essential for effective therapy in Alzheimer's disease. In this study we have treated transgenic mice carrying the Swedish mutation of human amyloid precursor protein [Tg(Hu.APP695.K670N‐M671L)2576], which develop brain β‐amyloid deposits, with nicotine in drinking fluid (200 µg/mL) from 9–14.5 months of age (5.5 months). A significant reduction in amyloid β peptide 1–42 positive plaques by more than 80% (p < 0.03) was observed in the brains of nicotine treated compared to sucrose treated transgenic mice. In addition, there was a selective reduction in extractable amyloid β peptides in nicotine treated mice; cortical insoluble 1–40 and 1–42 peptide levels were lower by 48 and 60%, respectively (p < 0.005), whilst there was no significant change in soluble 1–40 or 1–42 levels. The expression of glial fibrillary acidic protein was not affected by nicotine treatment. These results indicate that nicotine may effectively reduce amyloid β peptide aggregation in brain and that nicotinic drug treatment may be a novel protective therapy in Alzheimer's disease.
Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress
Authors:   Mauro Piacentini,, Grazia Farrace Maria , Piredda Lucia , Matarrese Paola , Ciccosanti Fabiola , Falasca Laura , Rodolfo Carlo , Giammarioli Anna Maria , Verderio Elisabetta , Griffin Martin , Malorni Walter
Published: March 2003

‘Tissue’ transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK‐N‐BE (2)) in which the tTG‐expression was driven by a constitutive promoter. Furthermore, a tet‐off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared ‘sensitized’ to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial‐mediated apoptosis by stauro‐ sporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a ‘sensitizer’ towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.
Progressive decrease of amyloid precursor protein carboxy terminal fragments (APP‐CTFs), associated with tau pathology stages, in Alzheimer's disease
Authors: Sergeant Nicolas , David Jean‐Philippe , Champain Danie , Ghestem Antoine , Wattez Annick , Delacourte André
Published: March 2003

Amyloid precursor protein (APP) dysfunction is a key aetiologic agent in Alzheimer's disease (AD). The processing of this transmembrane protein generates carboxy terminal fragments (CTFs) upstream of β‐amyloid peptide (Aβ) production. The physiologic significance of APP‐CTFs is still poorly understood, as well as the relationship that could link APP dysfunction and tau pathology in familial and non‐familial AD (non‐FAD). In the present study, we have investigated the quantitative and qualitative changes of APP‐CTFs in different brain areas of non‐demented and demented patients from a prospective and multidisciplinary study. A significant decrease of the five APP‐CTFs was observed, which correlated well with the progression of tau pathology, in most cases with infraclinical AD and AD, either familial or non‐FAD. Furthermore, solubility properties and the ratio between the five bands were also modified, both in the Triton‐soluble and/or ‐insoluble fractions. Together, we show here for the first time a modification directly observed on APP‐CTFs upstream of Aβ products and its relationship with tau pathology, which could reflect the basic aetiological mechanisms of AD.
Myelin‐associated glycoprotein modulates expression and phosphorylation of neuronal cytoskeletal elements and their associated kinases
Authors: Dashiell Suzanne M. , Tanner Sandra L. , Pant Harish C. , Quarles Richard H.
Published: March 2003

Decreased phosphorylation of neurofilaments in mice lacking myelin‐associated glycoprotein (MAG) was shown to be associated with decreased activities of extracellular‐signal regulated kinases (ERK1/2) and cyclin‐dependent kinase‐5 (cdk5). These in vivo changes could be caused directly by the absence of a MAG‐mediated signaling pathway or secondary to a general disruption of the Schwann cell–axon junction that prevents signaling by other molecules. Therefore, in vitro experimental paradigms of MAG interaction with neurons were used to determine if MAG directly influences expression and phosphorylation of cytoskeletal proteins and their associated kinases. COS‐7 cells stably transfected with MAG or with empty vector were co‐cultured with primary dorsal root ganglion (DRG) neurons. Total amounts of the middle molecular weight neurofilament subunit (NF‐M), microtubule‐associated protein 1B (MAP1B), MAP2, and tau were up‐regulated significantly in DRG neurons in the presence of MAG. There was also increased expression of phosphorylated high molecular weight neurofilament subunit (NF‐H), NF‐M, and MAP1B. Additionally, in similar in vitro paradigms, total and phosphorylated NF‐M were increased significantly in PC12 neurons co‐cultured with MAG‐expressing COS cells or treated with a soluble MAG Fc‐chimera. The increased expression of phosphorylated cytoskeletal proteins in the presence of MAG in vitro was associated with increased activities of ERK 1/2 and cdk5. We propose that interaction of MAG with an axonal receptor(s) induces a signal transduction cascade that regulates expression of cytoskeletal proteins and their phosphorylation by these proline‐directed protein kinases.