bluuebunny/crossref_metadata_2025 huggingface dataset:
Tau Promoter Confers Neuronal Specificity and Binds Sp1 and AP‐2
Authors: Heicklen‐Klein Alice , Ginzburg Irith
Published: March 2003

Abstract: Tau, a microtubule‐associated protein, is encoded by a
single gene, whose expression is primarily neuronal. In this work, we defined
an 80‐bp region of the tau promoter that confers tau protein with neuronal
expression. This fragment works in conjunction with an endogenous initiation
region to activate neuronal precursor‐specific transcription of the tau
promoter and works independently of this initiation region to confer nerve
growth factor inducibility. Furthermore, this 80‐bp fragment binds both Sp1
and AP‐2 proteins. DNase I foot‐print analysis revealed a third protein
binding region at the center of this 80‐bp fragment in neuronal cells.
Mutation within any of these three protein binding sites decreases
transcriptional activation of the tau gene. Comprehension of the interactions
that occur between cis‐ and trans‐regulatory elements of the tau promoter is important to understand the regulation of tau expression during normal development and changes that may occur in many cases of dementia, including Alzheimer's disease.
Protein Phosphatase 1 and 2A Inhibitors Prolong the Switch in the Control of Glutamate Release by Group I Metabotropic Glutamate Receptors
Authors: Sistiaga Alessandra , Sánchez‐Prieto José
Published: March 2003

Abstract: We have addressed the role of protein phosphatases (PPs)
in the modulation of the switch in glutamate release observed after repetitive
stimulation of group I metabotropic glutamate receptors (mGluRs). In
cerebrocortical nerve terminals the agonist
(S)‐3,5‐dihydroxyphenylglycine facilitated evoked glutamate release.
However, a second stimulation, 5 min later, reduced rather than facilitated
this release. This switch in the control of glutamate release was reversed
when a 30‐min interval was left between stimulations. Inhibition of the
endogenous PPs, PP1 and PP2A, with calyculin A and okadaic acid prevented the
recovery of the facilitatory response and maintained the receptor permanently
coupled to the inhibitory pathway. The inhibitors of PP2B, cyclosporin A and
cypermethrine, had no effect. The inhibition of glutamate release was
insensitive to pertussis toxin and was the result of the loss of the release
component coupled to N‐type Ca2+ channels. This inhibitory action was suppressed by addition of the protein kinase C activator 4β‐phorbol 12,13‐dibutyrate. We conclude that the balance between protein kinase and phosphatase activity at the nerve terminal plays a key role in accommodating the modulation of glutamate release by group I mGluRs.
Analysis of Three Ptx2 Splice Variants on Transcriptional Activity and Differential Expression Pattern in the Brain
Authors: Smidt Marten P. , Cox Joke J. , Schaick Hermien S. A. van , Coolen Marcel , Schepers Janneke , Kleij Arno M. van der , Burbach J. Peter H.
Published: March 2003

Abstract:Three different transcripts of the homeodomain gene termed pituitary homeobox (Ptx) 2 (Pitx2/Brx/Rieg/Solurshin/Arp) were cloned from different species encoding proteins belonging to the paired‐like family of homeodomain proteins. Ptx2a (324 amino acids), Ptx2b (271 amino acids), and Ptx2c (318 amino acids) share the C terminus, including the homeodomain, and have different N termini. Here we report the comparative analysis of all three different Ptx2 splice variants for their transcriptional activity and their expression pattern in the adult rat brain. Ptx2 is able totrans‐activate via different model promoters in different cell lines. A mild difference intrans‐activating potential is observed among the splice variants, but the underlying mechanism is at present unknown. It is surprising that all Ptx2 transcripts displayed an identical expression pattern in the brain. This markedly restricted pattern is limited to the following brain areas: the anterior and intermediate lobes of the pituitary gland, the subthalamic nucleus, the posterior hypothalamic nucleus, the mammillary bodies, the red nucleus, and the deep gray layer of the superior colliculus. The data presented suggest that all variants of Ptx2 are involved in the development and regulation of distinct neuronal cell groups and the pituitary gland.
Intracellular pH on Translocation of Protein Kinase C Isozymes in Rat Pinealocytes
Authors: Ho A. K. , Ling A. , Chik C. L.
Published: March 2003

Abstract: In rat pinealocytes, cytoplasmic alkalization causes
protein kinase C (PKC) translocation, but the isozyme involved is not known.
In this study, we investigated the effect of cytoplasmic alkalization on
membrane‐associated PKCα, δ, ε, and ζ, four isozymes
present in the rat pineal gland. Treatment with NH4Cl, which had no
effect on PKCζ, caused a sustained increase in membrane‐associated
PKCα, δ, and ε that lasted for at least 60 min. The effect of
NH4Cl on PKCα, δ, and ε was reduced by sodium
propionate, an agent that counteracts the effect of NH4Cl on
intracellular pH. Both sodium propionate and
5‐(N,N‐hexamethylene)amiloride (HMA), two treatments that abolished the effect of norepinephrine on cytoplasmic alkalization, also reduced norepinephrine‐mediated increases in membrane‐associated PKCα, δ, and ε. In contrast, these two treatments did not have an effect on the increase in membrane‐associated PKC isozymes caused by 4β‐phorbol 12‐myristate 13‐acetate (PMA), an active phorbol ester, even though HMA was effective in abolishing PMA‐mediated increases in intracellular pH. These results, apart from demonstrating that cytoplasmic alkalization by itself can cause translocation of PKCα, δ, and ε in rat pinealocytes, also indicate that the norepinephrine‐stimulated cytoplasmic alkalization plays an important role in transducing signals from the adrenergic receptor to selective PKC isozymes. However, PKC translocation stimulated directly by PMA does not appear to be sensitive to changes in intracellular pH.
Coordinated Expression of β‐Amyloid Precursor Protein and the Putative β‐Secretase BACE and α‐Secretase ADAM10 in Mouse and Human Brain
Authors: Marcinkiewicz Mieczyslaw , Seidah Nabil G.
Published: March 2003

Abstract: To define the enzymes involved in the etiology of Alzheimer's disease, we compared in mouse and human brain the mRNA levels and cellular localization of the ubiquitous β‐amyloid precursor protein (β‐APP) with those of the putative α‐secretases ADAM10 and ADAM17 and the β‐secretases BACE and BACE2. In situ hybridization performed in mice during prenatal and postnatal development and in adulthood revealed the coexpression of β‐APP, BACE, and ADAM10. The patterns of BACE2 and ADAM17 only partially overlapped with that of β‐APP. β‐APP, BACE, and ADAM10 mRNAs have also been detected by northern blot in human brain cortex of normal subjects and in Alzheimer's disease subjects. In situ hybridization performed using combined biotin‐ and radiolabeled riboprobes provided evidence for the coexpression of β‐APP with BACE and ADAM10 in human cortical neurons. Our data provide cytochemical evidence supporting the role of ADAM10 and BACE as authentic α‐ and β‐secretases.
Association of Increased Bcl‐2 Expression with Rescue from Tumor Necrosis Factor‐α‐Induced Cell Death in the Oligodendrocyte Cell Line OLN‐93
Authors: Burgmaier Gruscha , Schönrock Lisa M. , Kuhlmann Tanja , Richter‐Landsberg Christiane , Brück Wolfgang
Published: March 2003

Abstract: The present study investigated the effects of flupirtine(Katadolon) on tumor necrosis factor (TNF)‐α‐mediated cell death andBcl‐2 expression in the permanent rat oligodendrocyte cell line OLN‐93 (OLNcells). TNF‐α (500 U/ml) induced apoptosis of OLN cells, which wasconfirmed by DNA fragmentation using an in situ end‐labeling technique andultrastructural analysis. Flupirtine significantly reduced the rate ofspontaneous cell death of OLN cells already at low concentrations;TNF‐α‐mediated apoptosis was suppressed only with higher concentrationsof flupirtine (100 μM). Expression of Bcl‐2 protein and mRNA inOLN cells was detected by immunocytochemistry, western blot, and RT‐PCR.Quantitative analysis of western blots revealed an ∼2.5‐fold up‐regulationof Bcl‐2 protein during TNF‐α treatment. Furthermore, addition of 10 or100 μM flupirtine before incubation with TNF‐α led to anapproximately threefold increase of Bcl‐2 expression. Exposure of OLN cells toflupirtine alone moderately augmented the expression of Bcl‐2 protein. Ourdata demonstrate that flupirtine up‐regulates the expression of Bcl‐2 proteinin OLN cells; this Bcl‐2 induction is associated with a reduced rate ofTNF‐α‐induced cell death.
Endothelins Regulate Arachidonic Acid Release and Mitogen‐Activated Protein Kinase Activity in Schwann Cells
Authors: Berti‐Mattera Liliana N. , Wilkins Pamela L. , Harwalkar Subash , Madhun Zuhayr , Almhanna Khaldoun , Mattera Rafael
Published: March 2003

Abstract: Immortalized rat Schwann cells (iSC) express endothelin(ET) receptors coupled to inhibition of adenylyl cyclase and stimulation ofphospholipase C (PLC). These effects precede phenotypic changes and increasedDNA synthesis. We have investigated the role of ETs in the regulation ofarachidonic acid (AA) release and mitogen‐activated protein kinases (MAPKs).Both ET‐1 and ET‐3 increased AA release in iSC. This effect was sensitive tothe phospholipase A2 (PLA2) inhibitorsE‐6‐(bromomethylene)tetrahydro‐3‐(1‐naphthalenyl)‐2H‐pyran‐2‐oneand arachidonyl‐trifluoromethyl ketone but was insensitive to inhibitors ofPLC or phospholipase D‐dependent diacylglycerol generation. ET‐1‐dependent AArelease was also unaffected by removal of extracellular Ca2+ andblocking the concomitant elevation in [Ca2+]i,consistent with participation of a Ca2+‐independentPLA2. Treatment of iSC with ETs also resulted in activation ofextracellular signal‐regulated kinase, c‐Jun‐NH2‐terminal kinase(JNK), and p38 MAPK. A cause‐effect relationship between agonist‐dependent AArelease and stimulation of MAPKs, but not the opposite, was suggested byactivation of JNK by exogenous AA and by the observation that inhibition ofMAPK kinase or p38 MAPK was inconsequential to ET‐1‐induced AA release.Similar effects of ETs on AA release and MAPK activity were observed incultures expanded from primary SC and in iSC. Regulation of these effectorsmay mediate the control of proliferation and differentiation of SC by ETsduring peripheral nerve development and regeneration.
Tauroursodeoxycholic Acid Partially Prevents Apoptosis Induced by 3‐Nitropropionic Acid
Authors: Rodrigues Cecilia M. P. , Stieers Cheryle Linehan‐ , Keene C. Dirk , Ma Xiaoming , Kren Betsy T. , Low Walter C. , Steer Clifford J.
Published: March 2003

Abstract: Ursodeoxycholic acid (UDCA) has been shown to be a strongmodulator of the apoptotic threshold in both hepatic and nonhepatic cells.3‐Nitropropionic acid (3‐NP), an irreversible inhibitor of succinatedehydrogenase, appears to cause apoptotic neuronal cell death in the striatum,reminiscent of the neurochemical and anatomical changes associated withHuntington's disease (HD). This study was undertaken (a) to characterizefurther the mechanism by which 3‐NP induces apoptosis in rat neuronal RN33Bcells and (b) to determine if and how the taurine‐conjugated UDCA,tauroursodeoxycholic acid (TUDCA), inhibits apoptosis induced by 3‐NP. Ourresults indicate that coincubation of cells with TUDCA and 3‐NP was associatedwith an ∼80% reduction in apoptosis (p < 0.001), whereasneither taurine nor cyclosporin A, a potent inhibitor of the mitochondrialpermeability transition (MPT), inhibited cell death. Moreover, TUDCA, as wellas UDCA and its glycine‐conjugated form, glycoursodeoxycholic acid, preventedmitochondrial release of cytochrome c (p < 0.001), whichprobably accounts for the observed inhibition of DEVD‐specific caspaseactivity and poly(ADP‐ribose) polymerase cleavage. 3‐NP decreasedmitochondrial transmembrane potential (p < 0.001) and increasedmitochondrial‐associated Bax protein levels (p < 0.001).Coincubation with TUDCA was associated with significant inhibition of thesemitochondrial membrane alterations (p < 0.01). The results suggestthat TUDCA inhibits 3‐NP‐induced apoptosis via direct inhibition ofmitochondrial depolarization and outer membrane disruption, together withmodulation of Bax translocation from cytosol to mitochondria. In addition,cell death by 3‐NP apparently occurs through pathways that are independent ofthe MPT.
Kynurenine 3‐Hydroxylase Inhibition in Rats
Authors: Urenjak Jutta , Obrenovitch Tihomir P.
Published: March 2003

Abstract: Inhibition of kynurenine 3‐hydroxylase suppressesquinolinic acid synthesis and, therefore, shunts all kynurenine metabolismtoward kynurenic acid (KYNA) formation. This may be a pertinentantiexcitotoxic strategy because quinolinic acid is an agonist of NMDAreceptors, whereas kynurenic acid antagonises all ionotropic glutamatereceptors with preferential affinity for the NMDA receptor glycine site. Wehave examined whether the kynurenine 3‐hydroxylase inhibitor Ro 61‐8048increases extracellular (KYNA) sufficiently to control excessive NMDA receptorfunction. Microdialysis probes incorporating an electrode were implanted intothe striatum of anaesthetised rats, repeated NMDA stimuli were applied throughthe probe, and the resulting depolarisation was recorded. Changes inextracellular KYNA were assessed by HPLC analysis of consecutive dialysatesamples. Ro 61‐8048 (42 or 100 mg/kg) markedly increased the dialysate levelsof KYNA. The maximum increase (from 3.0 ± 1.0 to 31.0 ± 6.0nM; means ± SEM, n = 6) was observed 4 h after administrationof 100 mg/kg Ro 61‐8048, but the magnitude of the NMDA‐induced depolarisationswas not reduced. A separate study suggested that extracellular KYNA would needto be increased further by two orders of magnitude to become effective in thispreparation. These results challenge the notion that kynurenine 3‐hydroxylaseinhibition may be neuroprotective, primarily through accumulation of KYNA andsubsequent attenuation of NMDA receptor function.
Alterations in Lipid and Calcium Metabolism Associated with Seizure Activity in the Postischemic Brain
Authors: Katsura Ken‐ichiro , Turco Elena B. Rodriguez de , Kristián Tibor , Folbergrová Jaroslava , Bazan Nicolas G. , Siesjö Bo K.
Published: March 2003

Abstract: Transient ischemia is known to lead to a long‐lastingdepression of cerebral metabolic rate and blood flow and to an attenuatedmetabolic and circulatory response to physiological stimuli. However, thecorresponding responses to induced seizures are retained, demonstratingpreserved metabolic and circulatory capacity. The objective of the presentstudy was to explore how a preceding period of ischemia (15 min) alters therelease of free fatty acids (FFAs) and diacylglycerides (DAGs), the formationof cyclic nucleotides, and the influx/efflux of Ca2+, followingintense neuronal stimulation. For that purpose, seizure activity was inducedwith bicuculline for 30 s or 5 min at 6 h after the ischemia. ExtracellularCa2+ concentration (Ca2+e) was recorded, andthe tissue was frozen in situ for measurements of levels of FFAs, DAGs, andcyclic nucleotides. Six hours after ischemia, the FFA concentrations werenormalized, but there was a lowering of the content of 20:4 in the DAGfraction. Cyclic AMP levels returned to normal values, but cyclic GMP contentwas reduced. Seizures induced in postischemic animals showed similar changesin Ca2+e, as well as in levels of FFAs, DAGs, and cyclicnucleotides, as did seizures induced in nonischemic control animals, with theexception of an attenuated rise in 20:4 content in the DAG fraction. Weconclude that, at least in the neocortex, seizure‐induced phospholipidhydrolysis and cyclic cAMP/cyclic GMP formation are not altered by a precedingperiod of ischemia, nor is there a change in the influx/efflux ofCa2+ during seizure discharge or in associated spreadingdepression.
Acidic Amino Acid Clearance from CSF in the Neonatal Versus Adult Rat Using Ventriculo‐Cisternal Perfusion
Authors: Al‐Sarraf Hameed , Preston Jane E. , Segal Malcolm B.
Published: March 2003

Abstract: The acidic amino acids aspartate and glutamate are excitatory neurotransmitters in the CNS. The clearance of this group of amino acids from CSF of adult and neonatal (7‐day‐old) rats was investigated. Ventriculo‐cisternal perfusions with 14C‐amino acids and 3H‐dextran were carried out for up to 90 min. Uptake of the amino acids by the whole brain was measured, and the loss to blood was calculated. 3H‐Dextran was included in the perfusate for measurement of CSF secretion rate. After 90‐min perfusion, both aspartate and glutamate showed a similar uptake into the whole brain, and this did not change with age (p > 0.05). However, clearance from CSF was greater in the adult, as was entry into blood from CSF. Addition of 5 mM excess unlabelled amino acid resulted in reduction in the brain uptake of both 14C‐amino acids in the adult rat. In the neonate, addition of aspartate also reduced brain aspartate uptake, whereas addition of glutamate increased brain neonatal [14C]glutamate uptake. The rate of CSF secretion was significantly greater in the adult, 1.26 ± 0.18 μl · min‐1· g‐1, than in the neonate, 0.62 ± 0.08 μl · min‐1· g‐1, and the turnover of CSF was greater in adults (p < 0.01). In summary, both aspartate and glutamate showed greater clearances from CSF in the adult than the neonate. This clearance was found to be by carrier‐mediated mechanisms.
Intracellular Ca2+ Stores Regulate Muscarinic Receptor Stimulation of Phospholipase C in Cerebellar Granule Cells
Authors: Masgrau Roser , Servitja Joan‐Marc , Sarri Elisabet , Young Kenneth W. , Nahorski Stefan R. , Picatoste Fernando
Published: March 2003

Abstract: Muscarinic receptor activation of phosphoinositide phospholipase C (PLC) has been examined in rat cerebellar granule cells under conditions that modify intracellular Ca2+ stores. Exposure of cells to medium devoid of Ca2+ for various times reduced carbachol stimulation of PLC with a substantial loss (88%) seen at 30 min. A progressive recovery of responses was observed following the reexposure of cells to Ca2+‐containing medium (1.3 mM). However, these changes did not appear to result exclusively from changes in the cytosolic Ca2+ concentration ([Ca2+]i), which decreased to a lower steady level (∼25 nM decrease in 1‐3 min after extracellular omission) and rapidly returned (within 1 min) to control values when extracellular Ca2+ was restored. Only after loading of the intracellular Ca2+ stores through a transient 1‐min depolarization of cerebellar granule cells with 40 mM KCl, followed by washing in nondepolarizing buffer, was carbachol able to mobilize intracellular Ca2+. However, the same treatment resulted in an 80% enhancement of carbachol activation of PLC. In other experiments, partial depletion of the Ca2+ stores by pretreatment of cells with thapsigargin and caffeine resulted in an inhibition (18 and 52%, respectively) of the PLC response. Furthermore, chelation of cytosolic Ca2+ with BAPTA/AM did not influence muscarinic activation of PLC in either the control or predepolarized cells. These conditions, however, inhibited both the increase in [Ca2+]i and the PLC activation elicited by 40 mM KCl and abolished carbachol‐induced intracellular Ca2+ release in predepolarized cells. Overall, these results suggest that muscarinic receptor activation of PLC in cerebellar granule cells can be modulated by changes in the loading state of the Ca2+ stores.
The palmitoylation state of the β2‐adrenergic receptor regulates the synergistic action of cyclic AMP‐dependent protein kinase and β‐adrenergic receptor kinase involved in its phosphorylation and desensitization
Authors: Moffett Serge , Rousseau Guy , Lagacé Monique , Bouvier Michel
Published: March 2003

Although palmitoylation of the β2‐adrenergic receptor (β2AR), as well as its phosphorylation by the cyclic AMP‐dependant protein kinase (PKA) and the β‐adrenergic receptor kinase (βARK), are known to play important roles in agonist‐promoted desensitization, their relative contribution and mutual regulatory influences are still poorly understood. In this study, we investigated the role that the carboxyl tail PKA site (Ser345,346) of the β2AR plays in its rapid agonist‐promoted phosphorylation and desensitization. Mutation of this site (Ala345,346β2AR) significantly reduced the rate and extent of the rapid desensitization promoted by sustained treatment with the agonist isoproterenol. The direct contribution of Ser345,346 in desensitization was then studied by mutating all other putative PKA and βARK phosphorylation sites (Ala261,262βARK−β2AR). We found this mutant receptor to be phosphorylated upon receptor activation but not following direct activation of PKA, suggesting a role in receptor‐specific (homologous) but not heterologous phosphorylation. However, despite its phosphorylated state, Ala261,262βARK−β2AR did not undergo rapid desensitization upon agonist treatment, indicating that phosphorylation of Ser345,346 alone is not sufficient to promote desensitization. Taken with the observation that mutation of either Ser345,346 or of the βARK phosphorylation sites prevented both the hyper‐phosphorylation and constitutive desensitization of a palmitoylation‐less mutant (Gly341β2AR), our data suggest a concerted/synergistic action of the two kinases that depends on the palmitoylation state of the receptor. Consistent with this notion, in vitro phosphorylation of Gly341β2AR by the catalytic subunit of PKA facilitated further phosphorylation of the receptor by purified βARK. Our study therefore allows us to propose a coordinated mechanism by which sequential depalmitoylation, and phosphorylation by PKA and βARK lead to the functional uncoupling and desensitization of the β2AR.
Mutant NG108‐15 cells (NG‐CR72) deficient in GM1 synthase respond aberrantly to axonogenic stimuli and are vulnerable to calcium‐induced apoptosis: they are rescued with LIGA‐20
Authors: Wu Gusheng , Lu Zi‐Hua , Xie Xin , Li Libin , Ledeen Robert W.
Published: March 2003

The neuroblastoma × glioma NG108‐15 hybrid cell line, a widely used model for the study of neuronal differentiation, contains a variety of gangliosides including GM1 and its sialosylated derivative, GD1a. To investigate the role of these a‐series gangliotetraose gangliosides in neuritogenesis, we have obtained a mutated subclone of NG108‐15 that is deficient in that family of gangliosides. NG108‐15 cells were grown in the presence of cholera toxin, which killed the large majority of cells, and from the cholera‐resistant survivors we isolated a clone, NG‐CR72, that lacks GM1 and GD1a in the plasma and nuclear membranes. GM2 concentration was significantly higher in the plasma membrane. Enzyme assay indicated deficiency of UDP‐Gal:GM2 galactosyltransferase (GM1 synthase), which was confirmed by incorporation studies with [3H]sphingosine. These cells resembled wild‐type NG108‐15 in extending dendritic processes in response to dendritogenic agents (retinoic acid, dibutyryl cAMP) but responded aberrantly to axonogenic stimuli (KCl, ionomycin) by extending unstable neurites that showed the cytoskeletal staining characteristic of dendrites. Moreover, mutant cells treated with the Ca2+‐elevating axonogenic agents underwent apoptosis over time, attributed to dysfunction of Ca2+regulatory mechanisms normally mediated by GM1. Such agents caused dramatic and sustained elevation of intracellular Ca2+in mutant cells, in contrast to modest and temporary elevation in wild‐type cells. Exogenous GM1, inserted into the plasma membrane, had no discernable protective effect on NG‐CR72 cells whereas LIGA‐20, a membrane‐permeant derivative of GM1 that entered both plasma and nuclear membranes, blocked apoptosis, permitted extension of stable neurites, and attenuated the abnormal elevation of intracellular Ca2+.
Metabotropic glutamate receptor type 1α and tubulin assemble into dynamic interacting complexes
Authors: Ciruela Francisco , McIlhinney R. A. Jeffrey
Published: March 2003

Metabotropic glutamate receptors (mGlu receptors) are coupled to G‐protein second messenger pathways and modulate glutamate neurotransmission in the brain, where they are targeted to specific synaptic locations. Very recently, we identified tubulin as an interacting partner of the mGlu1α receptor in rat brain. Using BHK‐570 cells permanently expressing the receptor we have shown that this interaction occurs predominantly with soluble tubulin, following its translocation to the plasma membrane. In addition, treatment of the cells with the agonist quisqualic acid induce tubulin depolimerization and its translocation to the plasma membrane. Immunofluorescence detection of both the receptor and tubulin in agonist‐treated cells reveals a disruption of the microtubule network and an increased clustering of the receptor. Collectively these data demonstrate that the mGlu1α receptor interacts with soluble tubulin and that this association can take place at the plasma membrane.
C2‐ceramide and reactive oxygen species inhibit pituitary adenylate cyclase activating polypeptide (PACAP)‐induced cyclic‐AMP‐dependent signalling pathway
Authors: Sée V. , Koch B. , Loeffler J. P.
Published: March 2003

The pituitary adenylate cyclase activating polypeptide (PACAP) type I receptor, a seven‐domain transmembrane receptor, is positively coupled to both adenylate cyclase and phospholipase C. PACAP exerts neurotrophic effects which are mainly mediated through the cAMP/protein kinase A pathway. Here we show that the cell‐permeable C2‐ceramide selectively blocks PACAP‐activated cAMP production, without affecting phosphoinositide breakdown. Thus by blocking the neuroprotective cAMP signalling pathway, C2‐ceramide will reinforce its direct death‐inducing signalling. We found that a reactive oxygen species scavenger reversed the C2‐ceramide effect and that H2O2 mimicked it. Together these data indicate that reactive oxygen species (ROS) mediates C2‐ceramide‐induced cAMP pathway uncoupling. This uncoupling did not involve ATP supply or Gαs protein function but rather adenylate cyclase function per se. Further, the tyrosine phophatases inhibitors, but not the serine/threonine phosphatases inhibitors, prevent inhibition of cAMP production by ROS. This suggests that H2O2 requires a functional tyrosine phopsphatase(s) to block PACAP‐dependent cAMP production.
Glycine is taken up through GLYT1 and GLYT2 transporters into mouse spinal cord axon terminals and causes vesicular and carrier‐mediated release of its proposed co‐transmitter GABA
Authors: Raiteri Luca , Raiteri Maurizio , Bonanno Giambattista
Published: March 2003

Glycine and GABA are likely co‐transmitters in the spinal cord. Their possible interactions in presynaptic terminals have, however, not been investigated. We studied the effects of glycine on GABA release using superfused mouse spinal cord synaptosomes. Glycine concentration dependently elicited [3H]GABA release which was insensitive to strychnine or 5,7‐dichlorokynurenic acid, but was Na+ dependent and sensitive to the glycine uptake blocker glycyldodecylamide. The glycine effect was external Ca2+ independent, but was reduced when intraterminal Ca2+ was chelated with 1,2‐bis‐(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetracetic acid or depleted with thapsigargin, or when vesicular storage was impaired with bafilomycin. Glycine‐induced [3H]GABA release was prevented, in part, by blocking GABA transport. The glycine effect was halved by sarcosine, a GLYT1 substrate/inhibitor, or by amoxapine, a GLYT2 blocker, and abolished by a mixture of the two. The sensitivity to sarcosine, used as a transporter inhibitor or substrate, persisted in synaptosomes prelabelled with [3H]GABA in the presence of β‐alanine, excluding major gliasome involvement. To conclude, in mice spinal cord, transporters for glycine (both GLYT1 and GLYT2) and for GABA coexist on the same axon terminals. Activation of the glycine transporters elicits GABA release, partly by internal Ca2+‐dependent exocytosis and partly by transporter reversal.
Neurotrophins differentially enhance acetylcholine release, acetylcholine content and choline acetyltransferase activity in basal forebrain neurons
Authors: Auld Daniel S. , Mennicken Françoise , Day Jamie C. , Quirion Rémi
Published: March 2003

Several lines of evidence indicate that nerve growth factor is important for the development and maintenance of the basal forebrain cholinergic phenotype. In the present study, using rat primary embryonic basal forebrain cultures, we demonstrate the differential regulation of functional cholinergic markers by nerve growth factor treatment (24–96 h). Following a 96‐h treatment, nerve growth factor (1–100 ng/mL) increased choline acetyltransferase activity (168–339% of control), acetylcholine content (141–185%), as well as constitutive (148–283%) and K+‐stimulated (162–399%) acetylcholine release, but increased release was not accompanied by increased high‐affinity choline uptake. Enhancement of ACh release was attenuated by vesamicol (1 µm), suggesting a vesicular source, and was abolished under choline‐free conditions, emphasizing the importance of extracellular choline as the primary source for acetylcholine synthesized for release. A greater proportion of acetylcholine released from nerve growth factor‐treated cultures than from nerve growth factor‐naïve cultures was blocked by voltage‐gated Ca2+ channel antagonists, suggesting that nerve growth factor modified this parameter of neurotransmitter release. Cotreatment of NGF (20 ng/mL) with K252a (200 nm) abolished increases in ChAT activity and prevented enhancement of K+‐stimulated ACh release beyond the level associated with K252a, suggesting the involvement of TrkA receptor signaling. Also, neurotrophin‐3, neurotrophin‐4 and brain‐derived neurotrophic factor (all at 5–200 ng/mL) increased acetylcholine release, although they were not as potent as nerve growth factor and higher concentrations were required. High brain‐derived neurotrophic factor concentrations (100 and 200 ng/mL) did, however, increase release to a level similar to nerve growth factor. In summary, long‐term exposure (days) of basal forebrain cholinergic neurons to nerve growth factor, and in a less‐potent fashion the other neurotrophins, enhanced the release of acetylcholine, which was dependent upon a vesicular pool and the availability of extracellular choline.
The role of protein kinase C in the regulation of serotonin‐2A receptor expression
Authors: Anji Antje , Hanley Nicole R. Sullivan , Kumari Meena , Hensler Julie G.
Published: March 2003

We have investigated in C6 glioma cells the involvement of protein kinase C (PKC) in the regulation of serotonin‐2A receptor (5‐HT2A receptor) expression by agonist treatment. Comparison of the time‐courses of agonist‐induced downregulation of receptor number and mRNA indicate that a decrease in the number of 5‐HT2A receptor binding sites in response to serotonin (5‐HT) treatment is preceded by a decrease in 5‐HT2A receptor mRNA. This decrease in 5‐HT2A receptor mRNA as a result of agonist exposure was not due to a change in the stability or half‐life of the transcript. Pretreatment of cells with the PKC inhibitor bisindolylmaleimide blocked the decrease in 5‐HT2A receptor mRNA levels, and attenuated the down‐regulation of 5‐HT2A receptor binding sites induced by treatment with 5‐HT. Experiments performed with the PKC inhibitors calphostin C and Gö 6976 confirmed that PKC was involved in the regulation of 5‐HT2A receptor mRNA by agonist and implicate the conventional subgroup of PKC isoforms. Western blot analysis, using isoform‐specific anti‐PKC antibodies showed that under our culture conditions C6 glioma cells express the conventional isoforms PKC α, PKC γ, as well as the novel isoforms PKC δ, PKC ε, and the atypical isoforms PKC λ and PKC ι. Upon treatment with 5‐HT for 10 min levels of the conventional isoforms PKC α and PKC γ increased in the nuclear fraction. Taken together, our results implicate PKC α and/or PKC γ in the regulation of 5‐HT2A mRNA receptor and binding sites in response to agonist treatment.
Compensatory mechanisms enhance hippocampal acetylcholine release in transgenic mice expressing human acetylcholinesterase
Authors: Erb Christina , Troost Joachim , Kopf Silvia , Schmitt Ulrich , Löffelholz Konrad , Soreq Hermona , Klein Jochen
Published: March 2003

Central cholinergic neurotransmission was studied in learning‐impaired transgenic mice expressing human acetylcholinesterase (hAChE‐Tg). Total catalytic activity of AChE was approximately twofold higher in synaptosomes from hippocampus, striatum and cortex of hAChE‐Tg mice as compared with controls (FVB/N mice). Extracellular acetylcholine (ACh) levels in the hippocampus, monitored by microdialysis in the absence or presence of 10−8−10−3m neostigmine in the perfusion fluid, were indistinguishable in freely moving control and hAChE‐Tg mice. Muscarinic receptor functions were unchanged as indicated by similar effects of scopolamine on ACh release and of carbachol on inositol phosphate formation. However, when the mice were anaesthetized with halothane (0.8 vol. %), hippocampal ACh reached significantly lower levels in AChE‐Tg mice as compared with controls. Also, the high‐affinity choline uptake (HACU) in hippocampal synaptosomes from awake hAChE‐Tg mice was accelerated but was reduced by halothane anaesthesia. Moreover, hAChE‐Tg mice displayed increased motor activity in novel but not in familiar environment and presented reduced anxiety in the elevated plus‐maze test. Systemic application of a low dose of physostigmine (100 µg/kg i.p.) normalized all of the enhanced parameters in hAChE‐Tg mice: spontaneous motor activity, hippocampal ACh efflux and hippocampal HACU, attributing these parameters to the hypocholinergic state due to excessive AChE activity. We conclude that, in hAChE‐Tg mice, hippocampal ACh release is up‐regulated in response to external stimuli thereby facilitating cholinergic neurotransmission. Such compensatory phenomena most likely play important roles in counteracting functional deficits in mammals with central cholinergic dysfunctions.
Antibodies against c‐Jun N‐terminal peptide cross‐react with neo‐epitopes emerging after caspase‐mediated proteolysis during apoptosis
Authors: Casas Celia , Ribera Joan , Esquerda Josep E.
Published: March 2003

In previous studies it has been shown that neural cells undergoing programmed cell death display strongly positive cytoplasmic immunoreactivity to polyclonal antibodies directed against a c‐Jun N‐terminal peptide. It was later found that c‐Jun‐like immunoreactivity in apoptosis was due to cross‐reactivity with proteins other than c‐Jun. We have analysed the biochemical counterpart of this property in neuroblastoma cell lines treated to induce apoptosis. Using the c‐Jun/sc‐45 antibody, several bands with apparent molecular masses distinct from c‐Jun were detected in extracts in parallel with both the degree of apoptosis and the appearance of the cytoplasmic signal after immunostaining. c‐Jun/sc‐45 immunostaining was prevented by caspase inhibitors and did not require de novo protein synthesis. One of the antigens recognized by the c‐Jun/sc‐45 antibody was identified as seryl‐tRNA synthetase. We provide evidence that seryl‐tRNA synthetase is a substrate of caspase‐3 in vitro and that the digested form turns highly immunoreactive towards the antibody. A carboxy‐terminus epitope of the protein that constitutes a consensus site for caspase‐3 is involved in c‐Jun/sc‐45 recognition. This epitope shares some amino acids with the peptide used as the immunogen and this could explain the cross‐reactivity observed. In conclusion, we demonstrate here that cytoplasmic c‐Jun/sc‐45‐like immunoreactivity specific to apoptosis is due to post‐translational changes which occur in seryl‐tRNA synthetase and probably also in other proteins as a consequence of caspase mediated proteolysis.
Interaction of phosphorylated tyrosine hydroxylase with 14‐3‐3 proteins: evidence for a phosphoserine 40‐dependent association
Authors: Kleppe Rune , Toska Karen , Haavik Jan
Published: March 2003

Tyrosine hydroxylase (TH) has been reported to require binding of 14‐3‐3 proteins for optimal activation by phosphorylation. We examined the effects of phosphorylation at Ser19, Ser31 and Ser40 of bovine TH and human TH isoforms on their binding to the 14‐3‐3 proteins BMH1/BMH2, as well as 14‐3‐3 ζ and a mixture of sheep brain 14‐3‐3 proteins. Phosphorylation of Ser31 did not result in 14‐3‐3 binding, however, phosphorylation of TH on Ser40 increased its affinity towards the yeast 14‐3‐3 isoforms BMH1/BMH2 and sheep brain 14‐3‐3, but not for 14‐3‐3 ζ. On phosphorylation of both Ser19 and Ser40, binding to the 14‐3‐3 ζ isoform also occurred, and the binding affinity to BMH1 and sheep brain 14‐3‐3 increased. Both phosphoserine‐specific antibodies directed against the 10 amino acids surrounding Ser19 or Ser40 of TH, and the phosphorylated peptides themselves, inhibited the association between phosphorylated TH and 14‐3‐3 proteins. This was also found when heparin was added, or after proteolytic removal of the N‐terminal 37 amino acids of Ser40‐phosphorylated TH. Binding of BMH1 to phosphorylated TH decreased the rate of dephosphorylation by protein phosphatase 2A, but no significant change in enzymatic activity was observed in the presence of BMH1. These findings further support a role for 14‐3‐3 proteins in the regulation of catecholamine biosynthesis and demonstrate isoform specificity for both TH and 14‐3‐3 proteins.
Expression of cyclin‐dependent kinase 5 and its activator p35 in models of induced apoptotic death in neurons of the substantia nigra in vivo
Authors: Neystat Michael , Rzhetskaya Margarita , Oo Tinmarla F. , Kholodilov Nikolai , Yarygina Olga , Wilson Alexandria , El‐Khodor Bassem F. , Burke Robert E.
Published: March 2003

Cyclin‐dependent kinase 5 is predominantly expressed in postmitotic neurons and plays a role in neurite elongation during development. It has also been postulated to play a role in apoptosis in a variety of cells, including neurons, but little is known about the generality and functional significance of cdk5 expression in neuronal apoptosis in living brain. We have therefore examined its expression and that of its known activators, p35, p39 and p67, in models of induced apoptosis in neurons of the substantia nigra. We find that cdk5 is expressed in apoptotic profiles following intrastriatal injection of 6‐hydroxydopamine and axotomy. It is expressed exclusively in profiles which are in late morphologic stages of apoptosis. In these late stages, derivation of the profiles from neurons, and localization of expression to the nucleus, can be demonstrated by co‐labeling with a neuron‐specific nuclear marker, NeuN. In another model of induced apoptotic death in nigra, produced by developmental striatal lesion, kinase activity increases in parallel with cell death. While mRNAs for all three cdk5 activators are expressed in nigra during development, only p35 protein is expressed in apoptotic profiles. We conclude that cdk5/p35 expression is a general feature of apoptotic neuron death in substantia nigra neurons in vivo.
Soluble forms of NCAM and F3 neuronal cell adhesion molecules promote Schwann cell migration: identification of protein tyrosine phosphatases ζ/β as the putative F3 receptors on Schwann cells
Authors: Thomaidou Dimitra , Coquillat Delphine , Meintanis Stathis , Noda Masaharu , Rougon Genevieve , Matsas Rebecca
Published: March 2003

Neural cell adhesion molecule (NCAM) and F3 are both axonal adhesion molecules which display homophilic (NCAM) or heterophilic (NCAM, F3) binding activities and participate in bidirectional exchange of information between neurones and glial cells. Engineered Fc chimeric molecules are fusion proteins that contain the extracellular part of NCAM or F3 and the Fc region of human IgG1. Here, we investigated the effect of NCAM‐Fc and F3‐Fc chimeras on Schwann cell (SC) migration. Binding sites were identified at the surface of cultured SCs by chimera coated fluorospheres. The functional effect of NCAM‐Fc and F3‐Fc binding was studied in two different SC migration models. In the first, migration is monitored at specific time intervals inside a 1‐mm gap produced in a monolayer culture of SCs. In the second, SCs from a dorsal root ganglion explant migrate on a sciatic nerve cryosection. In both systems addition of the chimeras significantly increased the extent of SC migration and this effect could be prevented by the corresponding anti‐NCAM or anti‐F3 blocking antibodies. Furthermore, antiproteoglycan‐type protein tyrosine phosphatase ζ/β (RPTPζ/β) antibodies identified the presence of RPTPζ/β on SCs and prevented the enhancing effect of soluble F3 on SC motility by 95%. The F3‐Fc coated Sepharose beads precipitated RPTPζ/β from SC lysates. Altogether these data point to RPTPζ/β is the putative F3 receptor on SCs. These results identify F3 and NCAM receptors on SC as potential mediators of signalling occurring between axons and glial cells during peripheral nerve development and regeneration.
The enhancement of glucose uptake caused by the collapse of gap junction communication is due to an increase in astrocyte proliferation
Authors: Tabernero Arantxa , Jiménez Cristina , Velasco Ana , Giaume Christian , Medina José M.
Published: March 2003

We have previously shown that several gap junction uncouplers increase the uptake of glucose in astrocytes. The aim of the present work was to study whether the increase in glucose uptake was a consequence of the inhibition of gap junction communication and the purpose of this effect. Our results show that α‐glycyrrhetinic acid and endothelin‐1 increase the uptake of glucose in highly, but not in poorly, coupled astrocytes. This effect depended on connexin 43 levels and was abolished when the inhibition of gap junction communication was prevented by tolbutamide or ouabain. The inhibition of gap junctions increased the rate of glucose incorporation into DNA and RNA, which was inhibited by treatment with dehydroepiandrosterone, an inhibitor of glucose‐6‐phosphate dehydrogenase, the regulatory enzyme of the pentose phosphate pathway. The inhibition of gap junctions significantly increased astrocyte proliferation, which was counteracted by tolbutamide. These effects were not observed in poorly coupled astrocytes expressing low levels of connexin 43. The increase in astrocyte proliferation caused by gap junction inhibition was prevented when either glucose uptake or the pentose phosphate pathway were inhibited. We conclude that the inhibition of gap junction communication induces astrocyte proliferation, resulting in an enhancement of glucose uptake and its utilization through the pentose phosphate pathway to provide ribose‐5‐phosphate for the synthesis of nucleic acids.
Ca2+‐dependent K+ current and exocytosis in responses to caffeine and muscarine in voltage‐clamped guinea‐pig adrenal chromaffin cells
Authors: Ohta Toshio , Wakade Arun R. , Nakazato Yoshikazu , Ito Shigeo
Published: March 2003

We characterized changes in membrane currents and the cytosolic Ca2+ concentration, [Ca2+]i, in response to caffeine, and compared them with those in response to muscarine using the perforated patch‐clamp technique and fura‐2 microfluorimetry in guinea‐pig adrenal chromaffin cells. Catecholamine release from single voltage‐clamped cells was monitored with amperometry using carbon microelectrodes. Caffeine produced a transient outward current (Iout) at holding potentials over − 60 mV, increasing in amplitude with increasing the potentials. It also evoked a rapid increase of [Ca2+]i at all potentials examined. The current‐voltage relation revealed that the activation of K+ channels was responsible for the Iout evoked by caffeine. Both current and [Ca2+]i responses were reversibly abolished by cyclopiazonic acid, an inhibitor of Ca2+‐pump ATPase. At − 30 mV, the caffeine‐induced Iout, but not [Ca2+]i, was partly inhibited by either charybdotoxin or apamin. In the majority of cells tested, caffeine induced a larger Iout but a smaller [Ca2+]i increase than muscarine. Caffeine and muscarine increased catecholamine release from voltage‐clamped single cells concomitant with the transient increase of [Ca2+]i, and there was a positive correlation between them. These results indicate that caffeine activates Ca2+‐dependent K+ channels and catecholamine secretion due to the release of Ca2+ from internal stores in voltage‐clamped adrenal chromaffin cells of the guinea‐pig. There seems to be a spatial difference between [Ca2+]i increased by Ca2+ release from caffeine‐sensitive stores and that released from muscarine (inositol 1,4,5‐trisphosphate)‐sensitive ones.
Stress proteins in oligodendrocytes: differential effects of heat shock and oxidative stress
Authors: Goldbaum Olaf , Richter‐Landsberg Christiane
Published: March 2003

Heat shock proteins (HSP) or stress proteins serve as biomarkers to identify the contribution of stress situations underlying the pathogenesis of degenerative diseases of the CNS. We have analyzed by immunoblot technique the constitutive and inducible occurance of stress proteins in cultured rat brain oligodendrocytes subjected to heat shock or oxidative stress exerted by hydrogen peroxide, or a combination of both. The data demonstrate that oligodendrocytes constitutively express HSP32, HSP60 and the cognate form of the HSP70 family of proteins, HSC70. After heat shock, HSP25, αB‐crystallin and HSP70 were up‐regulated, while after oxidative stress the specific induction of HSP32 and αB‐crystallin was observed. HSP32 represents heme oxygenase 1 (HO‐1), a small stress protein with enzymatic activity involved in the oxidative degradation of heme which participates in iron metabolism. The presence of the iron chelators phenanthroline or deferoxamine (DFO), which previously has been shown to protect oligodendrocytes from oxidative stress‐induced onset of apoptosis, caused a marked stimulation of HSP32 without affecting HSP70. This indicates that DFO possibly exerts its protective role by directly influencing the antioxidant capacity of HO‐1. In summary, HSP in oligodendrocytes are differentially stimulated by heat stress and oxidative stress. Heme oxygenase‐1 has been linked to inflammatory processes and oxidative stress, its specific up‐regulation after oxidative stress in oligodendrocytes suggests that it is an ideal candidate to investigate the involvement of oxidative stress in demyelinating diseases.
Oxidative stress induces proorphanin FQ and proenkephalin gene expression in astrocytes through p38‐ and ERK‐MAP kinases and NF‐κB
Authors: Rosenberger John , Petrovics Gyorgy , Buzas Beata
Published: March 2003

Oxidative stress has been implicated in the pathogenesis of stroke, traumatic brain injuries, and neurodegenerative diseases affecting both neuronal and glial cells in the CNS. In this study we have demonstrated that reactive oxygen species (ROS) dramatically induce the expression of two neuropeptide genes, the opioid proenkephalin (pENK) and the opioid‐related proorphanin FQ (pOFQ; also known as pronociceptin) in primary astrocytes. Hydrogen peroxide (H2O2) treatment dose‐dependently increased pENK and pOFQ mRNA levels with a maximal effect (∼15‐fold increase) being detected at 50 µm concentration. Exposing the astrocyte cultures to hypoxia and subsequent re‐oxygenation also led to a profound elevation of pOFQ and pENK mRNA levels. Western blot analysis and immunocytochemistry revealed that H2O2 treatment elicited the phosphorylation and nuclear translocation of ERK 1/2 and p38 MAP kinases. Blockade of the p38 or the ERK MAP kinase pathways (by SB202190 and PD98059, respectively) prevented the H2O2‐induced increase in pENK and pOFQ mRNA levels indicating a central role for these cascades in the regulation of pOFQ and pENK genes in response to oxidative stress. Regulation of pOFQ and pENK gene expression by ERK and p38 activation may be mediated through the transcription factor cAMP‐response element binding protein (CREB). We observed CREB phosphorylation in response to H2O2, which was also prevented by SB202190 and PD98059. The nuclear factor‐κB (NF‐κB) pathway appears to be involved exclusively in the induction of pOFQ transcription by H2O2, as NF‐κB inhibitors antagonized the effect of oxidative stress on pOFQ, but not on pENK expression. The profound induction of these genes by oxidative stress and these other factors may suggest a role for orphanin FQ and enkephalin in injury and stress responses of the CNS and neuropathophysiological conditions involving reactive oxygen species.