bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase C activation by 12–0‐tetradecanoylphorbol 13‐acetate in CG‐4 line oligodendrocytes stimulates turnover of choline and ethanolamine phospholipids by phospholipase D and induces rapid process contraction
Authors: Kobayashi Masakazu , Kidd Dan , Hutson Elizabeth , Grafton James , McNulty Shaun , Rumsby Martin
Published: March 2003

Treatment of [3H]‐choline‐ or [14C]‐ethanolamine‐labelled undifferentiated bipolar and differentiated multipolar CG‐4 line oligodendrocytes with 12–0‐tetradecanoylphorbol 13‐acetate (TPA) to activate protein kinase C stimulated the release of choline or ethanolamine metabolites to the medium over controls. Ro31–8220, a PKC inhibitor, reduced TPA‐stimulated release of choline‐ and ethanolamine‐metabolites to basal levels. TPA treatment of both bipolar and multipolar cells caused rapid contraction of processes leaving rounded up cells: this effect was blocked by Ro31–8220. After 12–15 h exposure to TPA, bipolar undifferentiated CG‐4 line cells extended short processes again and the cells became multipolar. Nocodozole, an agent which disrupts microtubules and caused CG‐4 line cells to round up, caused increased choline or ethanolamine‐metabolite release to the medium over basal levels suggesting that some release during TPA‐treatment might occur due to process fragmentation. However, the transphosphatidylation reaction confirmed that phospholipase D was active in these cells. Exposure of bipolar undifferentiated CG‐4 line cells to TPA resulted in down‐regulatation of PKC‐α and PKC‐βΙΙ which could not be detected by Western blotting after a few hours; PKC‐ε was down‐regulated much more slowly but PKCs δ, ζ and ι were not influenced by 48 h exposure of cells to TPA. Formation of phosphatidylethanol in the transphosphatidylation reaction was markedly reduced in TPA down‐regulated cells indicating a role for PKCs α and βΙΙ in phospholipase D activation in CG‐4 line oligodendrocytes.
Biphasic modulation of protein kinase C and enhanced cell toxicity by amyloid beta peptide and anoxia in neuronal cultures
Authors: Kuperstein Faina , Reiss Nachum , Koudinova Natalia , Yavin Ephraim
Published: March 2003

A major feature of Alzheimer's disease is the deposition of the amyloid beta peptide (Aβ) in the brain by mechanisms which remain unclear. One hypothesis suggests that oxidative stress and Aβ aggregation are interrelated processes. Protein kinase C, a major neuronal regulatory protein is activated after oxidative stress and is also altered in the Alzheimer's disease brain. Therefore, we examined the effects of Aβ1−40 peptide on the protein kinase C cascade and cell death in primary neuronal cultures following anoxic conditions. Treatment with Aβ1−40 for 48 h caused a significant increase in the content and activity of Ca2+‐dependent and Ca2+‐independent protein kinase C isoforms. By 72 h various protein kinase C isoforms were down‐regulated. Following 90 min anoxia and 6 h normoxia, a decrease in protein kinase C isoforms was noticed, independent of Aβ1−40 treatment. A combination of Aβ1−40 and 30‐min anoxia enhanced cytotoxicity as noticed by a marked loss in the mitochondrial ability to convert 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide and by enhanced 4′,6‐diamidino‐2‐phenylindole nuclear staining. Phosphorylation of two downstream protein kinase C substrates of apparent molecular mass 80 and 43 kDa, tentatively identified as the mirystoyl alanine‐rich C‐kinase substrate (MARCKS), were gradually elevated up to 72 h upon incubation with Aβ1−40. Anoxia followed by 30 min normoxia enhanced MARCKS phosphorylation in the membrane but not in the cytosolic fraction. In the presence of Aβ1−40, phosphorylation of MARCKS was reduced. After 6 h normoxia, MARCKS phosphorylability was diminished possibly because of protein kinase C down‐regulation. The data suggest that a biphasic modulation of protein kinase C and MARCKS by Aβ1−40 combined with anoxic stress may play a role in Alzheimer's disease pathology.1
N‐Methyl‐d‐aspartate receptors mediating hippocampal noradrenaline and striatal dopamine release display differential sensitivity to quinolinic acid, the HIV‐1 envelope protein gp120, external pH and protein kinase C inhibition
Authors: Pittaluga Anna , Pattarini Roberto , Feligioni Marco , Raiteri Maurizio
Published: March 2003

NMDA receptors regulating hippocampal noradrenaline (NA) and striatal dopamine (DA) release have been compared using superfused synaptosomes prelabelled with the [3H]catecholamines. Both receptors mediated release augmentation when exposed to NMDA plus glycine. Quinolinic acid (100 µm or 1 mm) plus glycine (1 µm)‐elicited [3H]NA, but not [3H]DA release. The NMDA (100 µm)‐evoked release of [3H]NA and [3H]DA was similar and concentration‐dependently enhanced by glycine or d‐serine (0.1–1 µm); in contrast, the HIV‐1 envelope protein gp120 potently (30–100 pm) enhanced the NMDA‐evoked release of [3H]NA, but not that of [3H]DA. Gp120 also potentiated quinolinate‐evoked [3H]NA release. Ifenprodil (0.1–0.5 µm) or CP‐101,606 (0.1–10 µm) inhibited the NMDA plus glycine‐evoked release of both [3H]catecholamines. Zinc (0.1–1 µm) was ineffective. Lowering external pH from 7.4 to 6.6 strongly inhibited the release of [3H]NA elicited by NMDA plus glycine, whereas the release of [3H]DA was unaffected. The protein kinase C inhibitors GF 109203X (0.1 µm) or H7 (10 µm) selectively prevented the effect of NMDA plus glycine on the release of [3H]NA. GF 109203X also blocked the release of [3H]NA induced by NMDA or quinolinate plus gp120. It is concluded that the hippocampal NMDA receptor and the striatal NMDA receptor are pharmacologically distinct native subtypes, possibly containing NR2B subunits but different splice variants of the NR1 subunit.
Circadian regulation of phospholipid metabolism in retinal photoreceptors and ganglion cells
Authors: Guido M. E. , Pico E. Garbarino , Caputto B. L.
Published: March 2003

The neural retina is a key component of the vertebrate circadian system that is responsible for synchronizing the central circadian pacemaker to external light–dark (LD) cycles. The retina is itself rhythmic, showing circadian cycles in melatonin levels and gene expression. We assessed the in vivo incorporation of 32P‐phosphate and 3H‐glycerol into phospholipids of photoreceptor cells (PRCs) and retina ganglion cells (GCs) from chicks in constant illumination conditions (dark: DD or light: LL) over a 24‐h period. Our findings showed that in DD there was a daily oscillation in 32P‐labeling of total phospholipids synthesized in GCs and axonally transported to the brain. This metabolic fluctuation peaked during the subjective night (zeitgeber time [ZT] 20), persisted for several hours well into the subjective day and declined at subjective dusk (ZT 10–12). PRCs also exhibited an in vivo rhythm of 32P‐phospholipid synthesis in DD. This rhythm peaked around ZT 22, continued a few hours into the day and declined by the end of subjective dusk. The major individual species labeled 1 h after 32P administration was phosphatidylinositol (PI) in both PRCs and GCs. Rhythmic phospholipid biosynthesis was also observed in DD after 3H‐glycerol administration, with levels in GCs elevated from midday to early night. PRCs exhibited a similar rhythmic profile with the lowest levels of labeling during midnight. Phosphatidylcholine (PC) accounted for the individual species with the highest ratio of 3H‐glycerol incorporation in both cell populations at all phases examined. By contrast, in LL the rhythm of 3H‐glycerol labeling of phospholipids damped out in both cell layers. Our findings support the idea that, in constant darkness, the metabolism of retinal phospholipids, including their de novo biosynthesis, is regulated by an endogenous circadian clock.
β,γ‐Methylene ATP‐induced cAMP formation in C6Bu‐1 cells: involvement of local metabolism and subsequent stimulation of adenosine A2B receptor
Authors: Ohkubo Satoko , Kumazawa Keiko , Sagawa Koji , Kimura Junko , Matsuoka Isao
Published: March 2003

The mechanism underlying β,γ‐methylene ATP (β,γ‐MeATP)‐induced cAMP elevation was investigated in rat glioma C6Bu‐1 cells. β,γ‐MeATP increased forskolin‐stimulated cAMP formation in a manner sensitive to both the P1 antagonist xanthine amine congener (XAC) and the P2 antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS). Adenosine deaminase (ADA; 1 U/mL), which abolished the adenosine‐induced response, did not eliminate the β,γ‐MeATP‐induced response. However, combination of ADA with α,β‐methylene ADP (α,β‐MeADP), an ecto‐5′‐nucleotidase inhibitor, blocked the β,γ‐MeATP‐induced response. AMP, the substrate for ecto‐5′‐nucleotidase, also induced cAMP formation in a manner sensitive to XAC and α,β‐MeADP inhibition. However, the AMP‐induced response was not blocked by PPADS. HPLC analyses revealed that adenosine was generated from β,γ‐MeATP and AMP. In addition, α,β‐MeADP inhibited the conversion of β,γ‐MeATP and AMP to adenosine, whereas PPADS blocked adenosine formation from β,γ‐MeATP but not from AMP. [3H]Adenosine generated from [3H]AMP was preserved on the cell surface environment even in the presence of ADA. The mRNAs for ecto‐phosphodiesterase/pyrophosphatase 1 (EC 3.1.4.1), ecto‐5′‐nucleotidase (EC 3.1.3.5) and adenosine A2B receptor were detected by RT‐PCR. These results suggest that C6Bu‐1 cells possess ecto‐enzymes converting β,γ‐MeATP to adenosine, and the locally accumulated adenosine in this mechanism efficiently stimulates A2B receptors in a manner resistant to exogenous ADA.
Oxidative‐stress‐dependent up‐regulation of Bcl‐2 expression in the central nervous system of aged Fisher‐344 rats
Authors: Kaufmann Joel A. , Bickford Paula C. , Taglialatela Giulio
Published: March 2003

Oxidative stress has been shown to play a role in aging and in neurodegenerative disorders. Some of the consequences of oxidative stress are DNA base modifications, lipid peroxidation, and protein modifications such as formation of carbonyls and nitrotyrosine. These events may play a role in apoptosis, another factor in aging and neurodegeneration, in response to uncompensated oxidative stress. Bcl‐2 is a mitochondrial protein that protects neurons from apoptotic stimuli including oxidative stress. Using immunohistochemistry and western blot analysis, here we show that Bcl‐2 is up‐regulated in the hippocampus and cerebellum of aged (24 months) Fisher 344 rats. Treatment with the free radical spin trap N‐tert‐butyl‐α‐phenylnitrone (PBN) effectively reverses this age‐dependent Bcl‐2 up‐regulation indicating that this response is redox sensitive. This conclusion was further supported by inducing the same regional Bcl‐2 up‐regulation in young (3 months) Fisher 344 rats exposed to 100% normobaric O2 for 48 h. Our results indicate that Bcl‐2 expression is increased in the aged brain, possibly as a consequence of oxidative stress challenges. These results also illustrate the effectiveness of antioxidants in reversing age‐related changes in the CNS and support further research to investigate their use in aging and in age‐related neurodegenerative disorders.
Vitreal dihydroxyphenylacetic acid (DOPAC) as an index of retinal dopamine release
Authors: Megaw Pam , Morgan Ian , Boelen Meeuwis
Published: March 2003

Dopamine is generally accepted as a major neurotransmitter associated with light‐adaptive processes in the retina. However, little is known about its precise release pattern in vivo, largely due to the lack of an unambiguous method for the determination of dopamine release. We have found that vitreal levels of dihydroxyphenylacetic acid (DOPAC) reflect the rate of dopamine release in chickens. Blocking re‐uptake with nomifensine significantly lowered vitreal DOPAC and retinal dopamine, confirming the retinal origin and reliance of vitreal DOPAC on intact re‐uptake mechanisms. Further, inhibition of monoamine oxidase with pargyline reduced vitreal as well as retinal DOPAC levels, confirming that the DOPAC detected is generated by monoamine oxidase. Finally, we found that DOPAC diffused freely into and out of isolated vitreous bodies and we found the vitreous to be metabolically inert with respect to DOPAC, supporting the idea that vitreal levels of DOPAC are consequential to the retinal metabolism of dopamine. Exposure to light, which is known to increase retinal dopamine release, readily increased vitreal DOPAC levels. The accumulation of DOPAC in the vitreous over 6 h light fitted a mathematical model of DOPAC accumulation based on zero‐order influx (proportional to dopamine release rates) and diffusion driven, first‐order efflux.
Reduction of functional N‐methyl‐d‐aspartate receptors in neurons by RNase P‐mediated cleavage of the NR1 mRNA
Authors: Yen Laising , Gonzalez‐Zulueta Mirella , Feldman Alicia , Yuan Yan , Fryer Hugh , Dawson Ted , Dawson Valina , Kalb Robert G.
Published: March 2003

One approach to studying the functional role of individual NMDA receptor subunits involves the reduction in the abundance of the protein subunit in neurons. We have pursued a strategy to achieve this goal that involves the use of a small guide RNA which can lead to the destruction of the mRNA for a specific receptor subunit. We designed a small RNA molecule, termed ‘external guide sequence’ (EGS), which binds to the NR1 mRNA and directs the endonuclease RNase P to cleave the target message. This EGS has exquisite specificity and directed the RNase P‐dependent cleavage at the targeted location within the NR1 mRNA. To improve the efficiency of this EGS, an in vitro evolution strategy was employed which led to a second generation EGS that was 10 times more potent than the parent molecule. We constructed an expression cassette by flanking the EGS with self‐cleaving ribozymes and this permitted generation of the specified EGS RNA sequence from any promoter. Using a recombinant Herpes simplex virus (HSV), we expressed the EGS in neurons and showed the potency of the EGS to reduce NR1 protein within neurons. In an excitotoxicity assay, we showed that expression of the EGS in cortical neurons is neuroprotective. Our results demonstrate the utility of EGSs to reduce the expression of any gene (and potentially any splice variant) in neurons.
Brain‐derived neurotrophic factor superinduction parallels anti‐epileptic−neuroprotective treatment in the pilocarpine epilepsy model
Authors: Biagini Giuseppe , Avoli Massimo , Marcinkiewicz Jadwiga , Marcinkiewicz Mieczyslaw
Published: March 2003

Antiepileptic drugs provide neuroprotection in several animal models of brain damage, including those induced by status epilepticus (SE). The mechanisms involved in this action are unknown, but neurotrophic factors such as brain‐derived neurotrophic factor (BDNF) may play a role. In this study we investigated the changes in BDNF levels in rats in which SE had been induced by pilocarpine injection (400 mg/kg i.p.) and continued for several hours (unprotected group). In other animals (protected groups), SE was suppressed after 30 min by intraperitoneal injection of either diazepam (10 mg/kg) + pentobarbital (30 mg/kg) or paraldehyde (0.3 mg/kg). In diazepam + pentobarbital‐treated rats the hippocampal damage caused by SE was significantly lower (p < 0.05) than in unprotected animals. In addition, 2 and 24 h after pilocarpine injection, the levels of BDNF mRNA were moderately increased in the unprotected group, but ‘superinduced’ in protected animals, especially in the neocortex and hippocampus. A time‐dependent increase in BDNF immunoreactivity was also found by western blot analysis in rats treated with diazepam + pentobarbital. In contrast, a decrease of BDNF immunoreactivity occurred in the unprotected group. In conclusion, these results show that neuroprotection induced by anti‐epileptic drugs in pilocarpine‐treated rats is accompanied by strong potentiation of BDNF synthesis in brain regions involved in SE.
Increased expression of NR2A subunit does not alter NMDA‐evoked responses in cultured fetal trisomy 16 mouse hippocampal neurons
Authors: Klein Rebecca C. , Siarey Richard J. , Caruso Alessandra , Rapoport Stanley I. , Castellino Francis J. , Galdzicki Zygmunt
Published: March 2003

The trisomy 16 (Ts16) mouse is an animal model for human trisomy 21 (Down's syndrome). The gene encoding the NR2A subunit of the NMDA receptor has been localized to mouse chromosome 16. In the present study, western blot analysis revealed a 2.5‐fold increase of NR2A expression in cultured Ts16 embryonic hippocampal neurons. However, this increase did not affect the properties of NMDA‐evoked currents in response to various modulators. The sensitivity of NMDA receptors to transient applications of NMDA, spermine, and Zn2+ was investigated in murine Ts16 and control diploid cultured embryonic hippocampal neurons. Peak and steady‐state currents evoked by NMDA were potentiated by spermine at concentrations < 1 mm, and inhibited by Zn2+ in a dose‐dependent and voltage‐independent manner. No marked difference was observed between Ts16 and control diploid neurons for any of these modulators with regard to IC50 and EC50 values or voltage dependency. Additionally, inhibition by the NR2B selective inhibitor, ifenprodil, was similar. These results demonstrate that NMDA‐evoked currents are not altered in cultured embryonic Ts16 neurons and suggest that Ts16 neurons contain similar functional properties of NMDA receptors as diploid control neurons despite an increased level of NR2A expression.
Phosphoinositide 3‐kinase regulates crosstalk between Trk A tyrosine kinase and p75NTR‐dependent sphingolipid signaling pathways
Authors: Bilderback Tim R. , Gazula Valeswara‐Rao , Dobrowsky Rick T.
Published: March 2003

The mechanism of crosstalk between signaling pathways coupled to the Trk A and p75NTR neurotrophin receptors in PC12 cells was examined. In response to nerve growth factor (NGF), Trk A activation inhibited p75NTR‐dependent sphingomyelin (SM) hydrolysis. The phosphoinositide 3‐kinase (PI 3‐kinase) inhibitor, LY294002, reversed this inhibition suggesting that Trk A activation of PI 3‐kinase is necessary to inhibit sphingolipid signaling by p75NTR. In contrast, SM hydrolysis induced by neurotrophin‐3 (NT‐3), which did not activate PI‐3 kinase, was uneffected by LY294002. However, transient expression of a constituitively active PI 3‐kinase inhibited p75NTR‐dependent SM hydrolysis by both NGF and NT‐3. Intriguingly, NGF induced an association of activated PI 3‐kinase with acid sphingomyelinase (SMase). This interaction localized to caveolae‐related domains and correlated with a 50% decrease in immunoprecipitated acid SMase activity. NGF‐stimulated PI 3‐kinase activity was necessary for inhibition of acid SMase but was not required for ligand–induced association of the p85 subunit of PI 3‐kinase with the phospholipase. Finally, this interaction was specific for NGF since EGF did not induce an association of PI 3‐kinase with acid SMase. In summary, our data suggest that PI 3‐kinase regulates the inhibitory crosstalk between Trk A tyrosine kinase and p75NTR‐dependent sphingolipid signaling pathways and that this interaction localizes to caveolae‐related domains.
Neurotrophic actions of novel compounds designed from cyclopentenone prostaglandins
Authors: Satoh Takumi , Furuta Kyoji , Tomokiyo Keiichiro , Namura Shobu , Nakatsuka Daisaku , Sugie Yutaka , Ishikawa Yasuyuki , Hatanaka Hiroshi , Suzuki Masaaki , Watanabe Yasuyoshi
Published: March 2003

Previously we found that some cyclopentenone prostaglandin derivatives promoted neurite outgrowth from PC12 cells and dorsal root ganglia explants in the presence of nerve growth factor; and so we referred to them as neurite outgrowth‐promoting prostaglandins (NEPPs). In this study, NEPPs protected HT22 cells against oxidative glutamate toxicity. NEPP6, one of the most effective promoters of neurite outgrowth in PC12 cells, protected the cells most potently among NEPPs 1–10. Several derivatives, NEPPs 11–19, were newly synthesized based on the chemical structure of NEPP6. NEPP11 had a more potent neuroprotective effect than NEPP6. NEPP11 also prevented the death of cortical neurons induced by various stimuli and reduced ischemic brain damage in mice. Biotinylated compounds of NEPPs were synthesized to investigate their cellular accumulation. NEPP6‐biotin protected the cells and emitted potent signals from the cells. In contrast, biotinylated non‐neuroprotective derivatives emitted much weaker signals. These results suggest that NEPPs are novel types of neurotrophic compounds characterized by their dual biological activities of promoting neurite outgrowth and preventing neuronal death and that their accumulation in the cells is closely associated with their neuroprotective actions.
Serotonin depletion produces long lasting increase in striatal glutamatergic transmission
Authors: Di Cara B. , Dusticier N. , Forni C. , Lievens J. C. , Daszuta A.
Published: March 2003

The ability of serotonin (5‐HT) to influence striatal glutamatergic transmission was examined by determining changes over time in glutamate extracellular levels, transporter expression and synaptosomal uptake in rats with lesion of serotonergic neurones. By 8 days after intraraphe injections of 5,7‐dihydroxytryptamine, producing 80% decreases in striatal tissue 5‐HT levels, no changes were observed in the glutamatergic transmission. When 5‐HT depletion was almost complete (21 days post‐lesion), high affinity glutamate uptake in striatal synaptosomal preparations was significantly increased (156% of control), although no changes in striatal GLT1, GLAST and EAAC1mRNAs, and GLT1 protein were detected by in situ hybridization and immunohistochemistry. Meanwhile, the serotonin lesion produced large increases in basal extracellular levels of glutamate and glutamine (364% and 259%, respectively) determined in awake rats by in vivo microdialysis, whereas no change was observed in dopamine levels as compared with control rats. High potassium depolarization as well as ltrans‐pyrrolidine‐2,4‐dicarboxylate, also induced larger increases in extracellular levels of glutamate in lesioned rats than in controls. Finally, similar changes in glutamate transmission were observed by 3 months post‐lesion. These results suggest that 5‐HT has a long lasting and tonic inhibitory influence on the striatal glutamatergic input, without affecting the basal dopaminergic transmission.
Copper neurotoxicity is dependent on dopamine‐mediated copper uptake and one‐electron reduction of aminochrome in a rat substantia nigra neuronal cell line
Authors: Paris Irmgard , Dagnino‐Subiabre Alexies , Marcelain Katherine , Bennett Lori B. , Caviedes Pablo , Caviedes Raúl , Azar Claudio Olea , Segura‐Aguilar Juan
Published: March 2003

The mechanism of copper (Cu) neurotoxicity was studied in the RCSN‐3 neuronal dopaminergic cell line, derived from substantia nigra of an adult rat. The formation of a Cu–dopamine complex was accompanied by oxidation of dopamine to aminochrome. We found that the Cu–dopamine complex mediates the uptake of 64CuSO4 into the Raúl Caviedes substantia nigra‐clone 3 (RCSN3) cells, and it is inhibited by the addition of excess dopamine (2 m

m) (63%, p < 0.001) and nomifensine (2 µm) (77%, p < 0.001). Copper sulfate (1 m

m) alone was not toxic to RCSN‐3 cells, but was when combined with dopamine or with dicoumarol (95% toxicity; p < 0.001) which inhibits DPNH and TPNH (DT)‐diaphorase. Electron spin resonance (ESR) spectrum of the 5,5‐dimethylpyrroline‐N‐oxide (DMPO) spin trap adducts showed the presence of a C‐centered radical when incubating cells with dopamine, CuSO4 and dicoumarol. A decrease in the expression of CuZn‐superoxide dismutase and glutathione peroxidase mRNA was observed when RCSN‐3 cells were treated with CuSO4, dopamine, or CuSO4 and dopamine. However, the mRNA expression of glutathione peroxidase remained at control levels when the cells were treated with CuSO4, dopamine and dicoumarol. The regulation of catalase was different since all the treatments with CuSO4 increased the expression of catalase mRNA. Our results suggest that copper neurotoxicity is dependent on: (i) the formation of Cu–dopamine complexes with concomitant dopamine oxidation to aminochrome; (ii) dopamine‐dependent Cu uptake; and (iii) one‐electron reduction of aminochrome.
Amino acid determinants for NMDA receptor inhibition by conantokin‐T
Authors: Warder Scott E. , Blandl Tamas , Klein Rebecca C. , Castellino Francis J. , Prorok Mary
Published: March 2003

Several derivatives of conantokin‐T (con‐T), a naturally occurring, γ‐carboxyglutamate (Gla)‐containing peptide with NMDA receptor (NMDAR) antagonist properties, were synthesized and evaluated for their ability to displace [3H]MK‐801 from adult rat forebrain membranes. Analyses of progressive C‐terminal truncation analogs of the parent 21‐mer revealed gradual losses in activity with decreased chain length. In this series, con‐T[1–8] was identified as the shortest variant capable of manifesting inhibitory activity (< 1% of the parent peptide). Ala substitution studies of individual residues identified Gly1, Gla3, Met8 and Leu12 as important for activity, while Glu2, Gla4 and Tyr5 were shown to be essential in this regard. The effect of side‐chain length and charge in the N‐terminal region was probed by single amino acid replacements. No correlation was observed between potencies and circular dichroism‐derived helical contents of the con‐T derivatives. Further elaboration of structure‐function relationships in con‐T was effected through the design and synthesis of helically constrained and destabilized analogs. The results of the current study were compared with those of a previous investigation on con‐G, a related conantokin. Substantial differences in activity requirements were noted between the peptides, particularly in the C‐terminal regions. Chimeras of con‐T and con‐G were generated and revealed virtually no interchangeability of residues between these two peptides. Finally, single amino acid substitutions that resulted in analogs with enhanced inhibitory properties were combined to yield superior conantokin‐based NMDAR inhibitors.
Interaction of α‐synuclein and synphilin‐1: effect of Parkinson's disease‐associated mutations
Authors: Kawamata Hibiki , McLean Pamela J. , Sharma Nutan , Hyman Bradley T.
Published: March 2003

α‐Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease. Two α‐synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease. Recently, synphilin‐1, a protein found to interact with α‐synuclein by yeast two hybrid techniques, was detected in Lewy bodies. In this study we report the interaction of α‐synuclein and synphilin‐1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique. We demonstrate that the C‐terminus of α‐synuclein is closely associated with the C‐terminus of synphilin‐1. A weak interaction occurs between the N‐terminus of α‐synuclein and synphilin‐1. The familial Parkinson's disease associated mutations of α‐synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C‐terminal regions and synphilin‐1. However, compared with wild‐type α‐synuclein, significantly less energy transfer occurs between the C‐terminus of Ala53Thr α‐synuclein and synphilin‐1, suggesting that the Ala53Thr mutation alters the conformation of α‐synuclein in relation to synphilin‐1.
Agonist efficacy and receptor efficiency in heterozygous CB1 knockout mice: relationship of reduced CB1 receptor density to G‐protein activation
Authors: Selley Dana E. , Rorrer W. Kirk , Breivogel Christopher S. , Zimmer Anne M. , Zimmer Andreas , Martin Billy R. , Sim‐Selley Laura J.
Published: March 2003

Heterozygous CB1 receptor knockout mice were used to examine the effect of reduced CB1 receptor density on G‐protein activation in membranes prepared from four brain regions: cerebellum, hippocampus, striatum/globus pallidus (striatum/GP) and cingulate cortex. Results showed that CB1 receptor levels were approximately 50% lower in heterozygous mice in all regions examined. However, maximal stimulation of [35S]guanosine‐5′‐(γ‐O‐thio) triphosphate ([35S]GTPγS) binding by the high efficacy agonist WIN 55,212–2 was reduced by only 20–25% in most brain regions, with the exception of striatum/GP where the decrease in stimulation was as predicted (approximately 50%). Furthermore, although the efficacies of the cannabinoid partial agonists, methanandamide and Δ9‐tetrahydrocannabinol, were similarly lower in heterozygous mice, their relative efficacies compared with WIN 55,212–2 were generally unchanged. Saturation analysis of net WIN 55,212–2‐stimulated [35S]GTPγS binding showed that decreased stimulation by WIN 55,212–2 in striatum/GP of heterozygous mice was caused by a decrease in the apparent affinity of net‐stimulated [35S]GTPγS binding. The apparent maximal number of binding sites (Bmax) values of net WIN 55,212–2‐stimulated [35S]GTPγS binding were unchanged in cerebellum and striatum/GP of heterozygous mice, but decreased in cingulate cortex, with a similar trend in hippocampus. Moreover, in every region except cingulate cortex, the maximal number of net‐stimulated [35S]GTPγS binding sites per receptor was significantly increased in heterozygous mice. These results indicate region‐dependent increases in the apparent efficiency of CB1 receptor‐mediated G‐protein activation in heterozygous CB1 knockout mice.
Long‐term potentiation in the dentate gyrus of the rat hippocampus is accompanied by brain‐derived neurotrophic factor‐induced activation of TrkB
Authors: Gooney Martina , Lynch M. A.
Published: March 2003

A role for neurotrophic factors, in particular brain‐derived neurotrophic factor (BDNF), in modulating synaptic plasticity in the adult brain has been described in recent years by several laboratories. A great deal of emphasis has been placed on establishing its precise role in the expression of long‐term potentiation (LTP) in the hippocampus. Here we attempt to address this question by investigating, first, its release following induction of LTP in perforant path‐granule cell synapses and, second, the signalling events which follow activation of the BDNF receptor, TrkB, in the presynaptic terminal. We report that BDNF release is increased from slices of dentate gyrus following tetanic stimulation of the perforant path and that TrkB activation is increased in synaptosomes prepared from tetanized dentate gyrus. These changes are accompanied by increased activation of one member of the family of mitogen‐activated protein kinases, extracellular signal‐regulated kinase (ERK) and the data indicate that these events play a role in modulating release of glutamate from perforant path‐granule cell synapses, because the Trk inhibitor K252a and the ERK inhibitor, UO126, both inhibited the BDNF‐induced enhancement of release. We propose that the increase in phosphorylation of the transcription factor cAMP response element binding protein and in protein synthesis might underlie the more persistent components of LTP in dentate gyrus.
Inhibitors of Na+/H+ and Na+/Ca2+ exchange potentiate methamphetamine‐induced dopamine neurotoxicity: possible role of ionic dysregulation in methamphetamine neurotoxicity
Authors: Callahan Brian T. , Cord Branden J. , Yuan Jie , McCann Una D. , Ricaurte George A.
Published: March 2003

Although the neurotoxic potential of methamphetamine (METH) is well established, underlying mechanisms have yet to be identified. In the present study, we sought to determine whether ionic dysregulation was a feature of METH neurotoxicity. In particular, we reasoned that if METH impairs the function of Na+/H+ and/or Na+/Ca2+ antiporters by compromising the inward Na+ gradient [via prolonged DA transporter (DAT) activation and Na+/K+ ATPase inhibition], then amiloride (AMIL) and other inhibitors of Na+/H+ and/or Na+/Ca2+ exchange would potentiate METH neurotoxicity. To test this hypothesis, mice were treated with METH alone or in combination with AMIL or one of its analogs; 1 week later, the animals were killed for studies of dopamine (DA) neuronal integrity. AMIL markedly potentiated the toxic effect of METH on DA neurons. Potentiation was not caused by increased core temperature, enhanced DAT activity or higher METH brain levels. The DAT inhibitor, WIN‐35,428, protected completely against METH‐induced DA neurotoxicity in AMIL pretreated animals, suggesting that the potentiating effects of AMIL require a METH/DAT interaction. Findings with METH and AMIL were extended to six other AMIL analogs (MIA, EIPA, DIMA, BENZ, BEP, DiCBNZ), another species (rats), and neuronal type (5‐HT neurons). These results support the notion that ionic dysregulation may play a role in METH neurotoxicity.
Functional and physical coupling of voltage‐sensitive calcium channels with exocytotic proteins: ramifications for the secretion mechanism
Authors: Atlas Daphne
Published: March 2003

The secretion of neurotransmitters is a rapid Ca2+‐regulated process that brings about vesicle fusion with the plasma membrane. This rapid process (< 100 µs) involves multiple proteins located at the plasma and vesicular membranes. Because of their homology to proteins participating in constitutive secretion and protein trafficking, they have been characterized extensively. The sequential events that lead these proteins to vesicle docking and fusion are still unclear. We will review recent studies that demonstrate the operative role played by voltage‐sensitive Ca2+ channels and discuss the relevance for the process of evoked transmitter release. The regulation of Ca2+ influx by syntaxin, synaptosome‐associated protein of 25 kDa (SNAP‐25) and synaptotagmin, and the reciprocity of these proteins in controlling the kinetic properties of the channel will be discussed. Calcium channel and synaptic proteins expressed in Xenopus oocytes demonstrate a strong functional interaction, which could be pertinent to the mechanism of secretion. First, the voltage‐sensitive Ca2+ channels are negatively modulated by syntaxin: this inhibition is reversed by synaptotagmin. Second, the modulation of N‐type Ca2+ channel activation kinetics strongly suggests that the vesicle could be docked at the plasma membrane through direct interaction with synaptotagmin. Finally, these interactions provide evidence for the assembly of the voltage‐sensitive Ca2+ channel with syntaxin 1A, SNAP‐25 and synaptotagmin into an excitosome complex: a putative fusion complex with a potential role in the final stages of secretion. Studies suggest that cross‐talk between the synaptic proteins and the channel in a tightly organized complex may enable a rapid secretory response to an incoming signal such as membrane depolarization.
Astrocytes protect neurons from nitric oxide toxicity by a glutathione‐dependent mechanism
Authors: Chen Yongmei , Vartiainen Nina E. , Ying Weihai , Chan Pak H. , Koistinaho Jari , Swanson Raymond A.
Published: March 2003

Nitric oxide (NO) contributes to neuronal death in cerebral ischemia and other conditions. Astrocytes are anatomically well positioned to shield neurons from NO because astrocyte processes surround most neurons. In this study, the capacity of astrocytes to limit NO neurotoxicity was examined using a cortical co‐culture system. Astrocyte‐coated dialysis membranes were placed directly on top of neuronal cultures to provide a removable astrocyte layer between the neurons and the culture medium. The utility of this system was tested by comparing neuronal death produced by glutamate, which is rapidly cleared by astrocytes, and N‐methyl‐d‐aspartate (NMDA), which is not. The presence of an astrocyte layer increased the LD50 for glutamate by approximately four‐fold, but had no effect on NMDA toxicity. Astrocyte effects on neuronal death produced by the NO donors S‐nitroso‐N‐acetyl penicillamine and spermine NONOate were examined by placing these compounds into the medium of co‐cultures containing either a control astrocyte layer or an astrocyte layer depleted of glutathione by prior exposure to buthionine sulfoximine. Neurons in culture with the glutathione‐depleted astrocytes exhibited a two‐fold increase in cell death over a range of NO donor concentrations. These findings suggest that astrocytes protect neurons from NO toxicity by a glutathione‐dependent mechanism.
Induction of NADPH cytochrome P450 reductase by the Alzheimer β‐protein. Amyloid as a ‘foreign body’
Authors: Pappolla Miguel A. , Omar Rawhi A. , Chyan Yau‐Jan , Ghiso Jorge , Hsiao Karen , Bozner Peter , Perry George , Smith Mark A. , Cruz‐Sanchez Felix
Published: March 2003

A large body of data suggests that the Alzheimer's amyloid peptide (Aβ) causes degeneration and death of neurons by mechanisms that involve reactive oxygen species. The pathways involved in Aβ‐mediated oxidative injury are only partially understood. We theorized that abnormal microaggregates and/or pathological conformations of Aβ peptides may behave as xenobiotics and trigger the induction of NADPH cytochrome P450 reductase (CP450r), an enzyme which, if induced by non‐physiological substrates (such as xenobiotics like drugs or other ‘foreign molecules’), is known to cause oxidative stress. In order to test this hypothesis, i.e. that Aβ can increase the expression of CP450r, SK‐N‐SH human neuroblastoma cells were exposed to Aβ25‐35 and Aβ1‐42 and then examined for induction of this enzyme in immunoblots, using specific antibodies. Following exposure to Aβ peptides, neuroblastoma cells showed a clear‐cut induction of CP450r. To determine whether this mechanism is operational in vivo, we investigated the expression of CP450r in a transgenic mouse model of Alzheimer's disease (AD) and in brains from patients afflicted with AD, using an immunocytochemical approach. Tissue sections from brains of transgenic mice exhibited strong immunoreactivity for CP450r, surrounding amyloid deposits. The pattern of expression of CP450r was similar to that exhibited by neuritic and oxidative stress markers. Sections from non‐transgenic mice showed no detectable immunoreactivity. Immunostaining of sections from four brains with neuropathologically confirmed AD showed a pattern of abnormality different from transgenic mice that was characterized by abnormal immunoreactivity for CP450r within the cytoplasm of cortical neurons. No labeling was seen in sections from aged‐matched control brains. The data showed that CP450r is induced by Alzheimer amyloid peptide and that such a response must be considered as one possible mechanism whereby Aβ causes oxidative stress.
Inhibitory effect of somatostatin on neutral amino acid transport in isolated brain microvessels
Authors: Cardelli Patrizia , Fiori Anna , Corleto Vito D. , Savi Maria Rosaria , Granata Filippo , Ceci Fabrizio , Ferraguti Giampiero , Potenza Rosa Luisa , Fave Gianfranco Delle , Jensen Robert T. , Strom Roberto
Published: March 2003

In the presence of somatostatin‐14 or some of its receptorial agonists, the uptake of large neutral amino acids by isolated brain microvessels was found to be inhibited up to 50%, no other transport system being affected. Although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only uptake from the abluminal side appears to be inhibited by somatostatin. The involvement of a type‐2 somatostatin receptor was suggested by assays with a series of receptor‐specific somatostatin agonists, and was confirmed by the release of inhibition caused by a specific type‐2 receptor antagonist. A type‐2‐specific mRNA was indeed shown to be present in both bovine brain microvessels ex vivo and primary cultures of endothelial cells from rat brain microvessels.
A significant increase in both basal and maximal calcineurin activity in the rat pilocarpine model of status epilepticus
Authors: Kurz Jonathan E. , Sheets Dan , Parsons J. Travis , Rana Aniruddha , Delorenzo Robert J. , Churn Severn B.
Published: March 2003

This study focused on the effects of status epilepticus on the activity of calcineurin, a neuronally enriched, calcium‐dependent phosphatase. Calcineurin is an important modulator of many neuronal processes, including learning and memory, induction of apoptosis, receptor function and neuronal excitability. Therefore, a status epilepticus‐induced alteration of the activity of this important phosphatase would have significant physiological implications. Status epilepticus was induced by pilocarpine injection and allowed to continue for 60 min. Brain region homogenates were then assayed for calcineurin activity by dephosphorylation of p‐nitrophenol phosphate. A significant status epilepticus‐dependent increase in both basal and Mn2+‐dependent calcineurin activity was observed in homogenates isolated from the cortex and hippocampus, but not the cerebellum. This increase was resistant to 150 nm okadaic acid, but sensitive to 50 µm okadaic acid. The increase in basal activity was also resistant to 100 µm sodium orthovanadate. Both maximal dephosphorylation rate and substrate affinity were increased following status epilepticus. However, the increase in calcineurin activity was not found to be due to an increase in calcineurin enzyme levels. Finally, increase in calcineurin activity was found to be NMDA‐receptor activation dependent. The data demonstrate that status epilepticus resulted in a significant increase in both basal and maximal calcineurin activity.
Intrathecally delivered glial cell line‐derived neurotrophic factor produces electrically evoked release of somatostatin in the dorsal horn of the spinal cord
Authors: Issa Peter Charbel , Lever Isobel J. , Michael Gregory J. , Bradbury Elizabeth J. , Malcangio Marzia
Published: March 2003

Glial cell line‐derived neurotrophic factor (GDNF) is a trophic factor with an established role in sensory neuron development. More recently it has also been shown to support adult sensory neuron survival and exert a neuroprotective effect on damaged sensory neurons. Some adult small‐sized dorsal root ganglion (DRG) cells that are GDNF‐sensitive sensory neurons express the inhibitory peptide somatostatin (SOM). Thus, we tested the hypothesis that prolonged GDNF administration would regulate SOM expression in sensory neuron cell bodies in the dorsal root ganglia (DRG) and activity‐induced release of SOM from axon terminals in the dorsal horn. Continuous intrathecal delivery of GDNF for 11–13 days significantly increased the number of small DRG cells that expressed SOM. Furthermore, GDNF treatment evoked SOM release in the isolated dorsal horn following electrical stimulation of the dorsal roots that was otherwise undetectable in control rats. Conversely capsaicin‐induced release of SOM (EC50 50 nm) was not modified by GDNF treatment. These results show that GDNF can regulate central synaptic function in SOM‐containing sensory neurons.
Transgenic SOD1 G93A mice develop reduced GLT‐1 in spinal cord without alterations in cerebrospinal fluid glutamate levels
Authors: Bendotti Caterina , Tortarolo Massimo , Suchak Sachin K. , Calvaresi Novella , Carvelli Lucia , Bastone Antonio , Rizzi Massimo , Rattray Marcus , Mennini Tiziana
Published: March 2003

Glutamate‐induced excitotoxicity is suggested to play a central role in the development of amyotrophic lateral sclerosis (ALS), although it is still unclear whether it represents a primary cause in the cascade leading to motor neurone death. We used western blotting, immunocytochemistry and in situ hybridization to examine the expression of GLT‐1 in transgenic mice carrying a mutated (G93A) human copper–zinc superoxide dismutase (TgSOD1 G93A), which closely mimic the features of ALS. We observed a progressive decrease in the immunoreactivity of the glial glutamate transporter (GLT‐1) in the ventral, but not in the dorsal, horn of lumbar spinal cord. This effect was specifically found in 14‐ and 18‐week‐old mice that had motor function impairment, motor neurone loss and reactive astrocytosis. No changes in GLT‐1 were observed at 8 weeks of age, before the appearance of clinical symptoms. Decreases in GLT‐1 were accompanied by increased glial fibrillary acidic protein (GFAP) levels and no change in the levels of GLAST, another glial glutamate transporter. The glutamate concentration in the cerebrospinal fluid (CSF) of TgSOD1 G93A mice was not modified at any of the time points examined, compared with age‐matched controls. These findings indicate that the loss of GLT‐1 protein in ALS mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis and it is not associated with increases of glutamate levels in CSF. The lack of changes in GLT‐1 at the presymptomatic stage suggests that glial glutamate transporter reduction is not a primary event leading to motor neurone loss.
Activation of extracellular signal‐regulated kinases potentiates hemin toxicity in astrocyte cultures
Authors: Regan Raymond F. , Wang Yizheng , Ma Xin , Chong Andrew , Guo Yaping
Published: March 2003

Hemin is present in intracranial hematomas in high micromolar concentrations and is a potent, lipophilic oxidant. Growing evidence suggests that heme‐mediated injury may contribute to the pathogenesis of CNS hemorrhage. Extracellular signal‐regulated kinases (ERKs) are activated by oxidants in some cell types, and may alter cellular vulnerability to oxidative stress. In this study, the effect of hemin on ERK activation was investigated in cultured murine cortical astrocytes, and the consequence of this activation on cell viability was quantified. Hemin was rapidly taken up by astrocytes, and generated reactive oxygen species (ROS) within 30 min. Increased immunoreactivity of dually phosphorylated ERK1/2 was observed in hemin‐treated cultures at 30–120 min, without change in total ERK. Surprisingly, ERK activation was not attenuated by concomitant treatment with antioxidants (U74500A or 1,10‐phenanthroline) at concentrations that blocked ROS generation. Cell death commenced after 2 h of hemin exposure and was reduced by antioxidants and by the caspase inhibitor Z‐VAD‐FMK. Cytotoxicity was also attenuated by MEK inhibition with PD98059 or U0126 at concentrations that were sufficient to prevent ERK activation. Whereas the effect of Z‐VAD‐FMK on cell survival was transient, the effect of MEK inhibitors was long‐lasting. MEK inhibitors had no effect on cellular hemin uptake or subsequent ROS generation. The present results suggest that hemin activates ERK in astrocytes via a mechanism that is independent of ROS generation. This activation sensitizes astrocytes to hemin‐mediated oxidative injury.
Effects of chronic hypoxia on Ca2+stores and capacitative Ca2+entry in human neuroblastoma (SH‐SY5Y) cells
Authors: Smith I. F. , Boyle J. P. , Vaughan P. F. T. , Pearson H. A. , Peers C.
Published: March 2003

Microfluorimetric measurements of intracellular calcium ion concentration [Ca2+]iwere employed to examine the effects of chronic hypoxia (2.5% O2, 24 h) on Ca2+stores and capacitative Ca2+entry in human neuroblastoma (SH‐SY5Y) cells. Activation of muscarinic receptors evoked rises in [Ca2+]iwhich were enhanced in chronically hypoxic cells. Transient rises of [Ca2+]ievoked in Ca2+‐free solutions were greater and decayed more slowly following exposure to chronic hypoxia. In control cells, these transient rises of [Ca2+]iwere also enhanced and slowed by removal of external Na+, whereas the same manoeuvre did not affect responses in chronically hypoxic cells. Capacitative Ca2+entry, observed when re‐applying Ca2+following depletion of intracellular stores, was suppressed in chronically hypoxic cells. Western blots revealed that presenilin‐1 levels were unaffected by chronic hypoxia. Exposure of cells to amyloid β peptide (1–40) also increased transient [Ca2+]irises, but did not mimic any other effects of chronic hypoxia. Our results indicate that chronic hypoxia causes increased filling of intracellular Ca2+stores, suppressed expression or activity of Na+/Ca2+exchange and reduced capacitative Ca2+entry. These effects are not attributable to increased amyloid β peptide or presenilin‐1 levels, but are likely to be important in adaptive cellular remodelling in response to prolonged hypoxic or ischemic episodes.