bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Targeting
Authors: Burke Zoe , Wells Tim , Carter David , Klein David , Baler Ruben
Published: March 2003

Abstract : The arylalkylamine N‐acetyltransferase (AA‐NAT) gene is strongly expressed in the rat primarily in the pineal gland ; low levels of expression are also found in the retina. AA‐NAT catalyzes the key regulatory step controlling rhythmic melatonin output : the acetylation of serotonin. In the rat, the AA‐NAT gene is expressed at night. This is controlled partly by cyclic AMP (cAMP) acting through a composite cAMP‐responsive element‐CCAAT site located upstream of the transcription start point. In the present study, we have extended our previous in vitro findings and found that additional elements in the 5′ flanking region and first intron play an important role in the regulation of the AA‐NAT gene. This led us to test the influence of an AA‐NAT 5′ flanking segment on the expression of the bacterial chloramphenicol acetyltransferase gene in a rat transgenic model. The results of this study clearly demonstrate that the segment of the AA‐NAT gene that encompasses the minimal promoter and the first intron is able to confer the highly specific pineal/retinal and time‐of‐day patterns of AA‐NAT gene expression. This advance also provides a tool that selectively targets genetic expression to pinealocytes and retinal photoreceptors, providing new experimental opportunities to probe gene expression in these tissues.
Purification of Glutathione Reductase from Bovine Brain, Generation of an Antiserum, and Immunocytochemical Localization of the Enzyme in Neural Cells
Authors: Gutterer Jan Mirko , Dringen Ralf , Hirrlinger Johannes , Hamprecht Bernd
Published: March 2003

Abstract : Glutathione reductase (GR) is an essential enzyme for the glutathione‐mediated detoxification of peroxides because it catalyzes the reduction of glutathione disulfide. GR was purified from bovine brain 5,000‐fold with a specific activity of 145 U/mg of protein. The homogeneity of the enzyme was proven by sodium dodecyl sulfate‐polycrylamide gel electrophoresis and silver staining of the gel. The purified GR from bovine brain is a dimer of two subunits that have an apparent molecular mass of 55 kDa. The purified GR was used to generate a rabbit antiserum with the intention to localize GR in brain cells. The antiserum was useful for the detection of GR by double‐labeling immunocytochemical staining in astroglia‐rich and neuron‐rich primary cultures from rat brain. In homogenates of these cultures, no significant difference in the specific activities of GR was determined. However, not all cell types present in these cultures showed identical staining intensity for GR. In astrogliarich primary cultures, strong GR immunoreactivity was found in cells positive for the cellular markers galactocerebroside and C3b (antibody Ox42), indicating that oligodendroglial and microglial cells, respectively, contain GR. In contrast, only weak immunoreactivity for GR was found in cells positive for glial fibrillary acidic protein. In neuron‐rich primary cultures, GAP43‐positive cells stained with the antiserum against GR. These data demonstrate that, in cultures of neural cells, neurons, oligodendroglial cells, and microglial cells express high levels of GR.
Sodium Salicylate and 17β‐Estradiol Attenuate Nuclear Transcription Factor NF‐κB Translocation in Cultured Rat Astroglial Cultures Following Exposure to Amyloid Aβ1‐40 and Lipoplysaccharides
Authors: Dodel Richard , Du Yansheng , Bales Kelly R. , Gao Feng , Paul Steven
Published: March 2003

Abstract : In recent years inflammatory mechanisms have become

increasingly appreciated as important steps in the Alzheimer's pathogenic

pathway. There is accumulating evidence that amyloid β‐peptide (Aβ),

the peptide product of the cleavage of amyloid precursor protein, may promote

or exacerbate local inflammation by stimulating glial cells to release immune

mediators. In addition, clinical studies using nonsteroidal antiinflammatory

drugs have found a reduced risk for Alzheimer's disease with their use. Here

we show that the neurotoxic Aβ, a major plaque component, and

lipopolysaccharides (LPS), an immune reaction‐triggering portion of bacterial

membranes, are both potent activators of the nuclear transcription factor

NF‐κB in primary rat astroglial cells. The activation was found to be

concentration‐ and time‐dependent and could be attenuated in the presence of

NF‐κB decoy nucleotides. The pretreatment by either 17β‐estradiol

(1‐10 μg) or sodium salicylate (3‐30 mM) reduced the Aβ

(LPS)‐induced activation of NF‐κB by 48 (50%) and 60% (50%) of activated

levels, respectively. In addition, 17β‐estradiol (10 μM) and

sodium salicylate (10 mM) were able to attenuate the increase in

interleukin‐1β levels following exposure to 25 μM Aβ. Our data suggest that the aberrant gene expression is at least in part due to Aβ‐induced activation of NF‐κB, a potent immediatearly transcriptional regulator of numerous proinflammatory genes ; this event takes place in astroglial cells. The results of our experiments provide a further understanding of the effects of estrogen and aspirin on astroglial cells exposed to Aβ and LPS.
Increased Acute Cocaine Sensitivity and Decreased Cocaine Sensitization in GABAA Receptor β3 Subunit knockout Mice
Authors: Resnick Adam , Homanics Gregg E. , Jung Bruce J. , Peris Joanna
Published: March 2003

Abstract : The role of the GABAA receptor

β3 subunit in determining acute cocaine sensitivity and

behavioral sensitization to repeated cocaine was measured in mice missing both

(‐/‐), one (+/‐), or neither (+/+) allele of the β3 gene.

Locomotor stimulation induced by one cocaine injection (20 mg/kg, i.p.) was

found to be greater in ‐/‐ mice compared with +/+ mice, whereas

cocaine‐induced behaviors were intermediate in +/‐ mice. Amphetamine did not

cause greater locomotor responses in ‐/‐ mice, suggesting that the increased

sensitivity of ‐/‐ mice to cocaine does not generalize to other psychomotor

stimulants. GABA‐stimulated chloride uptake was 51% lower in striatum of ‐/‐

mice compared with +/+ mice, but only 27% lower in cortex. After 14 daily

cocaine injections, the behavioral response to cocaine was increased in +/+

and +/‐ mice, but was not increased further in ‐/‐ mice. Additionally,

repeated cocaine exposure decreased striatal GABAA receptor

function in +/+ and +/‐ mice. In ‐/‐ mice, GABAA receptor function

was not decreased any further by repeated cocaine injections. Thus,

alterations in the β3 subunit may be responsible for determining the behavioral responses induced by acute and repeated cocaine treatment, as well as mediating the neurochemical adaptation that occurs during sensitization to repeated cocaine.
Lithium Activates the c‐Jun NH2‐Terminal Kinases In Vitro and in the CNS In Vivo
Authors: Yuan Peixiong , Chen Guang , Manji Husseini K.
Published: March 2003

Abstract : The therapeutic efficacy of lithium in the treatment of

mood disorders is delayed and only observed after chronic administration, a

temporal profile that suggests alterations at the genomic level. Lithium has

been demonstrated to modulate AP‐1 DNA binding activity as well as the

expression of genes regulated by AP‐1, but the mechanisms underlying these

effects have not been fully elucidated. In the present study, we found that

the lithium‐induced increases in AP‐1 DNA binding activity were accompanied by

increases in p‐cJun and cJun levels in SH‐SY5Y cells. Lithium also increased

cJun‐mediated reporter gene expression in a dose‐dependent manner, with

significant effects observed at therapeutically relevant concentrations.

Lithium's effects on cJun‐mediated reporter gene expression in SH‐SY5Y cells

were more pronounced in the absence of myo‐inositol and were blocked

by protein kinase C (PKC) inhibitors and by cotransfection with a PKCα

dominant‐negative mutant. Chronic in vivo lithium administration increased

AP‐1 DNA binding activity in frontal cortex and hippocampus and also increased

the levels of the phosphorylated, active forms of c‐Jun

NH2‐terminal kinases (JNKs) in both brain regions. These results demonstrate that lithium activates the JNK signaling pathway in rat brain during chronic in vivo administration and in human cells of neuronal origin in vitro ; in view of the role of JNKs in regulating various aspects of neuronal function and their well‐documented role in regulating gene expression, these effects may play a major role in lithium's long‐term therapeutic effects.
Chronic ▵9‐Tetrahydrocannabinol Treatment Produces a Time‐Dependent Loss of Cannabinoid Receptors and Cannabinoid Receptor‐Activated G Proteins in Rat Brain
Authors: Breivogel Christopher S. , Childers Steven R. , Deadwyler Sam A. , Hampson Robert E. , Vogt Leslie J. , Sim‐Selley Laura J.
Published: March 2003

Abstract : Chronic treatment of rats with

▵9‐tetrahydrocannabinol (▵9‐THC) results in

tolerance to its acute behavioral effects. In a previous study, 21‐day

▵9‐THC treatment in rats decreased cannabinoid activation of G

proteins in brain, as measured by in vitro autoradiography of

guanosine‐5′‐O‐(3‐[35S]thiotriphosphate)

([35S]GTPγS) binding. The present study investigated the time

course of changes in cannabinoid‐stimulated [35S]GTPγS

binding and cannabinoid receptor binding in both brain sections and membranes,

following daily ▵9‐THC treatments for 3, 7, 14, and 21 days.

Autoradiographic results showed time‐dependent decreases in WIN

55212‐2‐stimulated [35S]GTPγS and [3H]WIN 55212‐2

binding in cerebellum, hippocampus, caudate‐putamen, and globus pallidus, with

regional differences in the rate and magnitude of down‐regulation and

desensitization. Membrane binding assays in these regions showed qualitatively

similar decreases in WIn 55212‐2‐stimulated [35S]GTPγS

binding and cannabinoid receptor binding (using [3H]SR141716A), and

demonstrated that decreases in ligand binding were due to decreases in maximal

binding values, and not ligand affinities. These results demonstrated that

chronic exposure to ▵9‐THC produced time‐dependent and region‐specific down‐regulation and desensitization of brain cannabinoid receptors, which may represent underlying biochemical mechanisms of tolerance to cannabinoids.
Activation of AP‐1 and Nuclear Factor‐κB Transcription Factors Is Involved in Hydrogen Peroxide‐Induced Apoptotic Cell Death of Olligodendrocytes
Authors: Vollgraf Ulrich , Wegner Michael , Richter‐Landsberg Christiane
Published: March 2003

Abstract : H2O2‐induced onset and execution of

programmed cell death in mature rat brain oligodendrocytes in culture is

accompanied by the induction and nuclear translocation of the transcription

factors AP‐1 and nuclear factor‐κB (NF‐κB), both of which have

been discussed as regulators of cell death and survival. Supershift analysis

of nuclear extracts indicated that the AP‐1 complex consists of c‐Jun, c‐Fos,

JunD, and possibly JunB proteins, and that the NF‐κB complex contains

p50, p65, and c‐Rel proteins. The first signs of DNA fragmentation were seen

already during the first hour after the treatment. DNA fragmentation could be

prevented by the antioxidants pyrrolidine dithiocarbamate and vitamin E, by

the nuclease inhibitor aurintricarboxylic acid, and by preincubation with the

iron chelator deferoxamine (DFO). Additionally, DFO protected oligodendrocytes

from H2O2‐induced cytotoxic effects as assessed by the

MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay, and

suppressed the formation of free radicals. DFO alone led to a slight increase

and in combination with H2O2 synergistically induced

DNA‐binding activities of AP‐1 and NF‐κB in oligodendrocytes. Our data

suggest that although low levels of H2O2 directly activate AP‐1 and NF‐κB and might contribute to signal transduction pathways promoting cell survival, the formation and action of hydroxyl radicals promote cell death mechanisms that can be attenuated by the iron chelator DFO.
Characterization of Wild‐Type and Mutants of Recombinant Human GTP Cyclohydrolase I
Authors: Suzuki Takahiro , Ohye Tamae , Inagaki Hidehito , Nagatsu Toshiharu , Ichinose Hiroshi
Published: March 2003

Abstract : To explore the molecular etiology of two disorders caused

by a defect in GTP cyclohydrolase I—hereditary progressive dystonia with

marked diurnal flucturation (HPD), also known as dopa‐responsive dystonia

(DRD), and autosomal recessive GTP cyclohydrolase I deficiency—we

purified and analyzed recombinant human wild‐type and mutant GTP

cyclohydrolase I proteins expressed in Escherichia coli. Mutant proteins showed very low enzyme activities, and some mutants were eluted at a delayed volume on gel filtration compared with the recombinant wild‐type. Next, we examined the GTP cyclohydrolase I protein amount by western blot analysis in phytohemagglutinin‐stimulated mononuclear blood cells from HPD/DRD patients. We found a great reduction in the amount of the enzyme protein not only in one patient who had a frameshift mutation, but also in an HPD/DRD patient who had a missense mutation. These results suggest that a dominant‐negative effect of chimeric protein composed of wild‐type and mutant subunits is unlikely as a cause of the reduced enzyme activity in HPD/DRD patients. We suggest that reduction of the amount of the enzyme protein, which is independent of the mutation type, could be a reason for the dominant inheritance in HPD/DRD.
The Na‐G Ion Channel Is Transcribed from a Single Promoter Controlled by Distinct Neuron‐ and Schwann Cell‐Specific DNA Elements
Authors: Poiraud Etienne , Gruszczynski Carole , Porteu Arlette , Cambier Hélène , Escurat Michel , Koulakoff Annette , Kahn Axel , Berwald‐Netter Yoheved , Gautron Sophie
Published: March 2003

Abstract : Na‐G is a putative sodium (or cationic) channel expressed in neurons and glia of the PNS, in restricted neuronal subpopulations of the brain, and in several tissues outside the nervous system, like lung and adrenal medulla. To analyze the mechanisms underlying tissue‐specific expression of this channel, we isolated the 5′ region of the correspsonding gene and show that Na‐G mRNA transcription proceeds from a single promoter with multiple initiation sites. By transgenic mice studies, we demonstrate that 600 bp containing the Na‐G proximal promoter region and the first exon are sufficient to drive the expression of a β‐galactosidase reporter gene in neurons of both CNS and PNS, whereas expression in Schwann cells depends on more remote DNA elements lying in the region between ‐6,500 and ‐1,050 bp upstream of the main transcription initiation sites. Crucial elements for lung‐specific expression seem to be located in the region between ‐1,050 and ‐375 bp upstream of the promoter. Using in vivo footprint experiments, we demonstrate that several sites of the Na‐G proximal promoter region are bound specifically by nuclear proteins in dorsal root ganglion neurons, as compared with nonexpressing hepatoma cells.
Mediation by Membrane Protein Kinase C of Zinc‐Induced Oxidative Neuronal Injury in Mouse Cortical Cultures
Authors: Noh Kyung‐Min , Kim Yang Hee , Koh Jae‐Young
Published: March 2003

Abstract: Transsynaptic movement of endogenous zinc may play a key role in selective neuronal death after brain ischemia and prolonged seizures. As to the mechanism, we have reported recently that zinc‐induced neuronal death occurs mainly by oxidative stress in cortical cultures. Here we present evidence supporting the idea that activation of membrane protein kinase C (PKC) in neurons is likely to play a key role in zinc‐induced oxidative neuronal injury. Exposure of cortical cultures to 300 μM zinc for 15 min induced increases in the activity, without changing the amount, of membrane PKC to two‐ to threefold of control values, followed by neuronal death over the next day. Addition of a zinc chelator, Ca‐EDTA, or PKC inhibitors with zinc completely abolished the zinc‐induced increase in the membrane PKC activity. Indicating the participation of PKC in zinc‐induced oxidative stress and neuronal death, the selective PKC inhibitor GF109203X attenuated both. Furthermore, as in zinc‐induced neuronal death, activation of PKC with phorbol esters induced free radical generation and neuronal death, which were blocked by GF109203X or an antioxidant, Trolox. The present results support the idea that zinc influx activates PKC in the membrane, which contributes to free radical generation and neuronal death. As an increasing body of evidence suggests that zinc neurotoxicity is an important mechanism of pathological neuronal death, timely prevention of PKC activation after acute brain insult may prove useful in ameliorating this type of neuronal death.
Neuronal Nicotinic Receptor Deficits in Alzheimer Patients with the Swedish Amyloid Precursor Protein 670/671 Mutation
Authors: Marutle Amelia , Warpman Ulrika , Bogdanovic Nenad , Lannfelt Lars , Nordberg Agneta
Published: March 2003

Abstract: The influence of β‐amyloid on cholinergic neurotransmission was studied by measuring alterations in nicotinic acetylcholine receptors (nAChRs) in autopsy brain tissue from subjects carrying the Swedish amyloid precursor protein (APP) 670/671 mutation. Significant reductions in numbers of nAChRs were observed in various cortical regions of the Swedish 670/671 APP mutation family subjects (‐73 to ‐87%) as well as in sporadic Alzheimer's disease (AD) cases (‐37 to ‐57%) using the nicotinic agonists [3H]epibatidine and [3H]nicotine, which bind with high affinity to both α3 and α4 and to α4 nAChR subtypes, respectively. Saturation binding studies with [3epibatidine revealed two binding sites in the parietal cortex of AD subjects and controls. A significant decrease inBmax(‐82%) for the high‐affinity site was observed in APP 670/671 subjects with no change inKDcompared with controls (0.018 nMAPP 670/671 ; 0.036 nMcontrol). The highest load of neuronal plaques (NPs) was observed in the parietal cortex of APP 670/671 brains, whereas the number of [3H]nicotine binding sites was less impaired compared with other cortical brain regions. Except for a positive significant correlation between the number of [3H]nicotine binding sites and number of NPs in the parietal cortex, no strict correlation was observed between nAChR deficits and the presence of NPs and neurofibrillary tangles, suggesting that these different processes may be closely related but not strictly dependent on each other.
In Vivo Effects of Adenosine A1 Receptor Agonist and Antagonist on Neuronal and Astrocytic Intermediary Metabolism Studied with Ex Vivo 13C NMR Spectroscopy
Authors: Håberg Asta , Qu Hong , Haraldseth Olav , Unsgård Geirmund , Sonnewald Ursula
Published: March 2003

Abstract: Adenosine is a neuromodulator, and it has been suggested that cerebral acetate metabolism induces adenosine formation. In the present study the effects that acetate has on cerebral intermediary metabolism, compared with those of glucose, were studied using the adenosine A1 receptor agonist 2‐chloro‐N6‐cyclopentyladenosine (CCPA) and antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX). Fasted rats received an intravenous injection of CCPA, DPCPX, or vehicle. Fifteen minutes later either [1,2‐13C]acetate or [1‐13C]glucose was given intraperitoneally; after another 30 min the rats were decapitated. Cortical extracts were analyzed with 13C NMR spectroscopy and HPLC analysis. DPCPX affected neuronal and astrocytic metabolism. De novo synthesis of GABA from neuronal and astrocytic precursors was significantly reduced. De novo syntheses of glutamate and aspartate were at control levels, but their degradation was significantly elevated. In glutamine the anaplerotic activity and the amount of label in the position representing the second turn in the tricarboxylic acid cycle were significantly increased, suggesting elevated metabolic activity in astrocytes. CCPA did not influence GABA, aspartate, or glutamine synthesis. In glutamate the contribution from the astrocytic anaplerotic pathway was significantly decreased. In the present study the findings in the [1,2‐13C]acetate and [1‐13C]glucose control, CCPA, and DPCPX groups were complementary, and no adenosine A1 agonist effects arising from cerebral acetate metabolism were detected.
Regulation of Mouse δ‐Opioid Receptor Gene Transcription
Authors: Wöltje Michael , Kraus Jürgen , Höllt Volker
Published: March 2003

Abstract: The aim of this study was to examine the effects of the
phorbol ester O‐tetradecanoylphorbol 13‐acetate (TPA) and forskolin
on δ‐opioid receptor gene transcription. Treatment of NG 108‐15 cells
with TPA (100 nM) for 48 h increased δ‐opioid receptor mRNA levels, whereas different concentrations of forskolin induced a transient down‐regulation of mRNA 5 h after treatment, followed by increased mRNA levels after 48 h. Reporter gene assays in transiently transfected NG 108‐15 cells in combination with electrophoretic mobility shift assays indicate that the increase of δ‐opioid receptor mRNA after stimulation with TPA is mediated by transcription factor AP‐1, which binds 355 bp upstream of the start codon within the gene promoter. The forskolin‐induced mRNA increase is mediated neither by a cyclic AMP‐response element nor indirectly by AP‐1 up‐regulation. Reporter gene assays, mutational analysis, and electrophoretic mobility shift assays revealed that δ‐opioid receptor gene regulation by forskolin is mediated by transcription factor AP‐2, which binds to an element 157 bp upstream of the start codon.
A Mechanism for the Neuroprotective Effect of Apolipoprotein E
Authors: Pedersen Ward A. , Chan Sic L. , Mattson Mark P.
Published: March 2003

Abstract: Inheritance of the apolipoprotein E (apoE) ε4 allele increases the risk for Alzheimer's disease and may also influence the pathogenesis of other neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). The influence of apoE genotype on disease susceptibility must ultimately be explained by the fact that apoE proteins differ in only two amino acids: apoE2 has two cysteine residues, apoE3 has one cysteine residue, and apoE4 has none. We previously reported increased protein modification by the lipid peroxidation product 4‐hydroxynonenal (HNE), which covalently binds to proteins on cysteine residues, in human ALS lumbar spinal cord. We now report increased levels of HNE‐modified apoE in lumbar spinal cord samples from mice expressing an ALS‐linked mutation in Cu/Zn‐superoxide dismutase relative to controls. Studies of interactions of pure apoE proteins with HNE showed that the isoforms differ in the amount of HNE they can bind, with the order E2 > E3 > E4. This correlated with the differential ability of apoE isoforms to protect against apoptosis induced by HNE in cultures of mouse spinal cord motor neurons and by the amyloid β‐peptide in cultures of rat hippocampal neurons. These data suggest that apoE plays a major role in detoxifying HNE, and the differential neuroprotective effect of its isoforms may help explain the relationship between apoE genotype and the susceptibility to neurodegenerative diseases.
Dopamine Receptor‐Mediated Regulation of Striatal Cholinergic Activity
Authors: Ding Yu‐Shin , Logan Jean , Bermel Rob , Garza Victor , Rice Onarae , Fowler Joanna S. , Volkow Nora D.
Published: March 2003

Abstract: Large numbers of in vitro studies and microdialysis
studies suggest that dopaminergic regulation of striatal acetylcholine (ACh)
output is via inhibitory dopamine D2 receptors and stimulatory
dopamine D1 receptors. Questions remain as to the relative
predominance of dopamine D2 versus D1 receptor
modulation of striatal ACh output under physiological conditions.
Using positron emission tomography, we first demonstrate that
norchloro[18F]fluoroepibatidine ([18F]NFEP), a selective
nicotinic ACh receptor (nAChR) ligand, was sensitive to changes of striatal
ACh concentration. We then examined the effect of quinpirole (D2
agonist), raclopride (D2 antagonist), SKF38393 (D1
agonist), and SCH23390 (D1 antagonist) on striatal binding of
[18F]NFEP in the baboon. Pretreatment with quinpirole increased the
striatum (ST) to cerebellum (CB) ratio by 26 ± 6%, whereas pretreatment
with raclopride decreased the ST/CB ratio by 22 ± 2%. The ratio of the
distribution volume of [18F]NFEP in striatum to that in cerebellum,
which corresponds to (Bmax/KD) + 1
(index for nAChR availability), also showed a significant increase (29 and
20%; n = 2) and decrease (20 ± 3%; n = 3) after pretreatment with
quinpirole and raclopride, respectively. However, both the D1
agonist and antagonist had no significant effect. This suggests that under
physiological conditions the predominant influence of endogenous
dopamine on striatal ACh output is dopamine D2, not D1, receptor‐mediated.
Activation of Poly(ADP‐Ribose) Polymerase in the Rat Hippocampus May Contribute to Cellular Recovery Following Sublethal Transient Global Ischemia
Authors: Nagayama Tetsuya , Simon Roger P. , Chen Dexi , Henshall David C. , Pei Wei , Stetler R. Anne , Chen Jun
Published: March 2003

Abstract: We have investigated the role of poly(ADP‐ribose)
polymerase (PARP) activation in rat brain in a model of sublethal transient
global ischemia. Adult male rats were subjected to 15 min of ischemia with
brain temperature reduced to 34°C, followed by 1, 2, 4, 8, 16, 24, and 72
h of reperfusion. PARP mRNA expression was examined in the hippocampus using
quantitative RT‐PCR, northern blot analysis, and in situ hybridization.
Protein expression was assessed using western blot analysis. PARP enzymatic
activity was investigated by measuring nuclear [3H]NAD
incorporation. The presence of poly(ADP‐ribose) polymers was assessed
immunocytochemically. Although PARP mRNA and protein expressions were not
altered after ischemia, enzymatic activity was increased 4.37‐fold at 1 h
(p < 0.05 vs. sham) and 1.73‐fold (p < 0.05 vs. sham)
at 24 h of reperfusion. Immunostaining demonstrated the presence of
poly‐(ADP‐ribose) polymers in CA1 neurons. Cellular NAD+ levels
were not significantly altered at any time point. Furthermore, systemic
administration of 3‐aminobenzamide (30 mg/kg), a PARP inhibitor, prevented the
increase in PARP activity at 1 and 24 h of reperfusion, significantly
decreased the number of surviving neurons in the hippocampal CA1 region 72 h
after ischemia (p < 0.01 vs. sham), and increased DNA
single‐strand breaks assessed as DNA polymerase I‐mediated biotin‐dATP
nick‐translation (PANT)‐positive cells (p < 0.01 vs. sham).
Furthermore, using an in vitro DNA repair assay, 3‐aminobenzamide (30 mg/kg)
was shown to block DNA base excision repair activity. These data suggest that
the activation of PARP, without subsequent NAD+ depletion, following mild transient ischemia may be neuroprotective in the brain.
Purification and Characterization of a Trypsin‐Like Serine Proteinase from Rat Brain Slices that Degrades Laminin and Type IV Collagen and Stimulates Protease‐Activated Receptor‐2
Authors: Sawada Ken , Nishibori Masahiro , Nakaya Naoki , Wang Zhao , Saeki Kiyomi
Published: March 2003

Abstract: A trypsin‐like serine proteinase was purified from the
incubation medium of rat brain slices by gelatin zymography. The purification
consisted of ammonium sulfate precipitation, benzamidine‐Sepharose 6B affinity
chromatography, and carboxymethyl‐cellulose and gel filtration
chromatographies. The gelatinolytic activity, identified at 22 kDa (P22) by
sodium dodecyl sulfate‐polyacrylamide gel electrophoresis under nonreducing
conditions, was eluted as one active peak throughout the purification, and the
final preparation gave a single protein peak on reverse‐phase HPLC.
Diisopropyl fluorophosphate, benzamidine, p‐toluenesulfonyl‐L‐lysine
chloromethyl ketone, and aprotinin completely inhibited the activity of P22,
whereas phenanthroline, p‐toluenesulfonyl‐L‐phenylalanine
chloromethyl ketone, and elastinal did not. P22 efficiently digested the
extracellular matrix proteins laminin and type IV collagen. P22 produced an
increase in intracellular Ca2+ concentration in A172 glioblastoma, which was desensitized through prior stimulation with protease‐activated receptor‐2 agonist peptide SLIGKV, indicating that P22 can stimulate protease‐activated receptor‐2. Rat brain penetration injury induced gelatinolytic activity in the lesioned area whose molecular size was consistent with that of P22. These results indicated that on incubation of rat brain slices, a trypsin‐like serine proteinase was secreted into the medium that was capable of digesting extracellular matrix and stimulating protease‐activated receptor‐2. It is suggested that the gelatinolytic activity induced by brain injury might be that of P22.
Modification of Cysteines Reveals Linkage to Acetylcholine and Vesamicol Binding Sites in the Vesicular Acetylcholine Transporter of Torpedocalifornica
Authors: Keller James E. , Bravo Dawn T. , Parsons Stanley M.
Published: March 2003

Abstract: Properties of cysteinyl residues in the vesicular
acetylcholine transporter (VAChT) of synaptic vesicles isolated from
Torpedo californica were probed. Cysteinespecific reagents of
different size and polarity were used and the effects on
[3H]vesamicol binding determined. The vesamicol dissociation
constant increased 1,000‐fold after reaction with
p‐chloromercuriphenylsulfonate or phenylmercury acetate, but only
severalfold after reaction with relatively small methylmercury chloride or
methylmethanethiosulfonate (MMTS). Methylmercury chloride, but not MMTS,
protected binding from phenylmercury acetate. Thus, two classes of cysteines
react to affect vesamicol binding. Class 1 reacts with only organomercurials,
and class 2 reacts with both organomercurials and MMTS. Quantitative analysis
of the competition between p‐chloromercuriphenylsulfonate and VAChT
ligands was possible after defining second‐order reaction conditions. The
results indicate that each cysteinyl class probably contains a single residue.
Acetylcholine protects cysteine 1, but apparently does not protect cysteine 2.
Vesamicol, which binds to a different site than acetylcholine does, apparently
protects both cysteines, suggesting that it induces a conformational change.
The relatively large reagent glutathione removes a substituent from cysteine
1, but not cysteine 2, suggesting that cysteine 2 is deeper in the transporter
than cysteine 1 is. The complete sequence of T. californica VAChT is given, and possible identities of cysteines 1 and 2 are discussed.
Mutant Presenilin 1 Increases the Levels of Alzheimer Amyloid β‐Peptide Aβ42 in Late Compartments of the Constitutive Secretory Pathway
Authors: Petanceska Suzana S. , Seeger Mary , Checler Frederic , Gandy Sam
Published: March 2003

Abstract: Mutations in the presenilin 1 (PS1) gene are associated with autosomal dominant, early‐onset, familial Alzheimer's disease and result in increased release of the hyperaggregatable 42‐amino acid form of the amyloid β‐peptide (Aβ42). To determine which subcellular compartments are potential source(s) of released Aβ42, we compared the levels and spatial segregation of intracellular Aβ40 and Aβ42 peptides between N2a neuroblastoma cells doubly transfected with the “Swedish” familial Alzheimer's disease‐linked amyloid precursor protein variant and either wild‐type PS1 (PS1wt) or familial Alzheimer's disease‐linked Δ9 mutant PS1 (PS1Δ9). As expected, PS1Δ9‐expressing cells had dramatically higher levels of intracellular Aβ42 than did cells expressing PS1wt. However, the highest levels of Aβ42 colocalized not with endoplasmic reticulum or Golgi markers but with rab8, a marker for trans‐Golgi network (TGN)‐to‐plasma membrane (PM) transport vesicles. We show that PS1 mutants are capable of causing accumulation of Aβ42 in late compartments of the secretory pathway, generating there a readily releasable source of Aβ42. Our findings indicate that PS1 “bioactivity” localizes to the vicinity of the TGN and/or PM and reconcile the apparent discrepancy between the preponderant concentration of PS1 protein in proximal compartments of the secretory pathway and the recent findings that PS1 “bioactivity” can control γ‐secretase‐like processing of another trans‐membrane substrate, Notch, at or near the PM.
Development and Characterisation of a Glutamate‐Sensitive Motor Neurone Cell Line
Authors: Eggett Christopher J. , Crosier Stephen , Manning Philip , Cookson Mark R. , Menzies Fiona M. , McNeil Calum J. , Shaw Pamela J.
Published: March 2003

Abstract: Modification of the growth conditions of NSC‐34 mouse neuroblastoma × motor neurone cells by serum depletion promotes the expression of functional glutamate receptors as the cells mature into a form that bears the phenotypic characterisation of motor neurones. Immunocytochemical studies demonstrated the presence of the glutamate receptor proteins NMDAR1, NMDAR2A/B, GluR1, GluR2, GluR2/3, GluR4, GluR6/7, and KA2. Toxicity assays using cell counting techniques demonstrated a mild but significant cell death (∼30%, p < 0.01) following a 24‐h exposure to 1 mM glutamate that could be prevented by the presence of the glutamate receptor antagonists (+)‐5‐methyl‐10, 11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5, 10‐imine maleate (10 μM) and 2,3‐dioxo‐6‐nitro‐1,2,3,4‐tetrahydrobenzo[f]quinoxaline‐7‐sulphonamide (1 μM). As an indication of glutamate receptor functional activity a novel approach was used to detect the production of free radicals following stimulation with glutamate receptor agonists. The release of superoxide free radicals was detected using a microelectrochemical sensor following addition of glutamate receptor agonists to the cell bathing solution. Alterations in intracellular calcium concentrations were examined using fura‐2 imaging. Exposure of the differentiated NSC‐34 cells to glutamate leads to an increase in intracellular calcium concentrations that is prevented by the presence of glutamate receptor antagonists. The motor neurone origin of these cells makes them particularly useful for investigating the potential role of glutamatergic toxicity in motor neurone degeneration.
Neurotrophin‐3 Controls Proliferation of Granular Precursors as Well as Survival of Mature Granule Neurons in the Developing Rat Cerbellum
Authors: Katoh‐Semba Ritsuko , Takeuchi Ikuo K. , Semba Reiji , Kato Kanefusa
Published: March 2003

Abstract: Levels of neurotrophin‐3 markedly decrease in the rat cerebellum after the first 10 days of life, suggesting an importance during early development. To further examine the effect of neurotrophin‐3 on the developing cerebellum, we injected a monoclonal antibody against neurotrophin‐3 into the lateral ventricle of 7.5‐day‐old rats. The resultant depletion of neurotrophin‐3 caused a significant decrease in cerebellar wet weights noted at 7 and 23 days thereafter. Other changes noted 48 h after injection of monoclonal antibodies against neurotrophin‐3 included reduced incorporation of bromodeoxyuridine into granule neurons in the external germinal layer, an elevated density of atrophic neurons that had just migrated under the Purkinje cell layer, and an increased number of apoptotic neurons in the internal granule cell layer. These changes were limited to the central lobe. The concentration of neurotrophin‐3 protein in the posterior region, including the central lobe, was about four‐ and threefold higher than that in the anterior region of the cerebellum of 9.5‐ and 30‐day‐old rats, respectively. Immunocytochemical examination showed higher amounts of neurotrophin‐3 protein in the central lobe than in the anterior lobe. Our results provide evidence that neurotrophin‐3 regulates the proliferation of granule precursors and supports the survival of mature granule neurons in restricted lobules, suggesting an involvement in limited regions at a specific stage in development of the rat cerebellum.
Protective Effect of the Energy Precursor Creatine Against Toxicity of Glutamate and β‐Amyloid in Rat Hippocampal Neurons
Authors: Brewer Gregory J. , Wallimann Theo W.
Published: March 2003

Abstract: The loss of ATP, which is needed for ionic homeostasis, is an early event in the neurotoxicity of glutamate and β‐amyloid (Aβ). We hypothesize that cells supplemented with the precursor creatine make more phosphocreatine (PCr) and create large energy reserves with consequent neuroprotection against stressors. In serum‐free cultures, glutamate at 0.5‐1 mM was toxic to embryonic hippocampal neurons. Creatine at >0.1 mM greatly reduced glutamate toxicity. Creatine (1 mM) could be added as late as 2 h after glutamate to achieve protection at 24 h. In association with neurotoxic protection by creatine during the first 4 h, PCr levels remained constant, and PCr/ATP ratios increased. Morphologically, creatine protected against glutamate‐induced dendritic pruning. Toxicity in embryonic neurons exposed to Aβ (25‐35) for 48 h was partially prevented by creatine as well. During the first 6 h of treatment with Aβ plus creatine, the molar ratio of PCr/ATP in neurons increased from 15 to 60. Neurons from adult rats were also partially protected from a 24‐h exposure to Aβ (25‐35) by creatine, but protection was reduced in neurons from old animals. These results suggest that fortified energy reserves are able to protect neurons against important cytotoxic agents. The oral availability of creatine may benefit patients with neurodegenerative diseases.
The Activity‐Regulated Cytoskeletal‐Associated Protein Arc Is Expressed in Different Striosome‐Matrix Patterns Following Exposure to Amphetamine and Cocaine
Authors: Tan Andrew , Moratalla Rosario , Lyford Gregory L. , Worley Paul , Graybiel Ann M.
Published: March 2003

Abstract: The activity‐regulated, cytoskeletal‐associated gene, arc, is a brain‐enriched immediate‐early gene whose expression is rapidly induced in the striatum by dopamine receptor agonists. This rapid induction of arc in the striatum is similar to that of other early response genes such as c‐fos, junB, ▵fosB, fra, and NGFI‐A, which code for transcription factors. Unlike these proteins, however, Arc is a cytoskeletal protein expressed not only in the nucleus of neurons but also in their dendrites. We investigated the patterns of Arc expression evoked in the rat striatum by acute exposures to two psychomotor stimulants, cocaine and amphetamine. Cocaine induced arc in striatal neurons that were broadly distributed within both striosome and matrix compartments of the caudoputamen. Amphetamine also evoked Arc expression in striatal projection neurons, but these were heavily concentrated in the striosomal compartment and only sparsely in the matrix compartment in the rostral striatum. The contrasting patterns of Arc expression evoked by cocaine and amphetamine parallel those of c‐Fos, JunB, FRA, and NGFI‐A expression induced by these two psychomotor stimulants. This difference in the action of cocaine and amphetamine at the level of protein expression may be linked to the different effects of these psychomotor stimulants on behavior.
The Human Nucleus Accumbens Is Highly Susceptible to G Protein Down‐Regulation by Methamphetamine and Heroin
Authors: McLeman Elizabeth R. , Warsh Jerry J. , Ang Lee , Li Peter P. , Kalasinsky Kathleen S. , Ross Brian M. , Tong Junchao , Schmunk Gregory , Adams Vernard , Kish Stephen J.
Published: March 2003

Abstract: Although the nucleus accumbens is assumed to be a critical brain “pleasure center,” its function in humans is unknown. As animal data suggest that a unique feature of this small brain area is its high sensitivity to down‐regulation of an inhibitory G protein by drugs of abuse, we compared G protein levels in postmortem nucleus accumbens with those in seven other brain regions of chronic users of cocaine, methamphetamine, and heroin, and of matched controls. Biochemical changes were restricted to the nucleus accumbens in which concentrations of Gαi1 and/or Gαi2 were reduced by 32‐49% in the methamphetamine and heroin users. This selective responsiveness to these abused drugs implies a special role for the human nucleus accumbens in mechanisms of drug reinforcement and suggests that some features of the drug‐dependent state (e.g., tolerance) might be related to inhibition of Gαi‐linked receptor activity.
Commercial Recombinant Human β‐Nerve Growth Factor and Adult Rat Dorsal Root Ganglia Contain an Identical Molecular Species of Nerve Growth Factor Prohormone
Authors: Reinshagen Max , Geerling Irmlind , Eysselein Viktor E. , Adler Guido , Huff Kenneth R. , Moore Geoffrey Philip , Lakshmanan Jayaraman
Published: March 2003

Abstract: Examination of commercial recombinant human β‐nerve growth factor (rh‐β‐NGF) preparations with polyclonal antibodies specific to 13‐kDa NGF and pro‐NGF‐specific domains revealed the presence of high‐molecular‐mass immunoreactive proteins, including a 60‐kDa NGF prohormone. On incubation with a mixture of N‐ and O‐specific glycosidases, the 60‐kDa NGF prohormone generated a 32‐kDa protein corresponding to the molecular size of NGF precursor predicted by the cloned human NGF cDNA. Highly sensitive chemiluminescence immunoblot analysis of adult rat dorsal root ganglia, spinal cord, and colon tissues with NGF‐ and pro‐NGF domain‐specific antibodies also revealed the presence of high‐molecular‐mass proteins, including the 60‐kDa NGF prohormone. Based on the presence of the 60‐kDa NGF prohormone in dorsal root ganglia and its efferent tissues, we suggest that proteolytically unprocessed, glycosylated NGF prohormone may mediate interactions between neurons and the tissues they innervate.
Modulation of GABAA Receptor Subunit mRNA Levels in Olivocerebellar Neurons of Purkinje Cell Degeneration and Weaver Mutant Mice
Authors: Rotter A. , Rath S. , Evans J. E. , Frostholm A.
Published: March 2003

Abstract: In olivocerebellar circuits, changes in the subunit composition of GABAA receptors occur at a time of extensive synaptic remodeling. In the deep cerebellar nuclei, GABAA receptor α1, β2, and γ2 subunit mRNA expression increases throughout neonatal development, whereas in the inferior olivary complex, the perinatal combination of α3, α5, β3, and γ2 mRNAs switches to the adult combination of α2, α4, β3, and γ1 during postnatal week 2. In situ hybridization was used to examine changes in subunit expression in the olivocerebellar nuclei of Purkinje cell degeneration and weaver mutant mice. In Purkinje cell degeneration, subunit transcripts decreased below control levels in olivary neurons; however, α1, β2, and γ2 transcript levels were slightly increased in the medial nucleus of the deep cerebellar nuclei. In weaver olivary neurons, although the switch from early‐ to late‐onset subunit mRNAs occurred as in normal mice, transcript levels were differentially modulated by the mutation. Our studies indicate that major alterations in synaptic connectivity do not prevent developmentally programmed switches in GABAA receptor gene expression but can modulate the timing and level of transcript expression in afferent and efferent neurons.
Evolutionarily Distinct Classes of S27 Ribosomal Proteins with Differential mRNA Expression in Rat Hypothalamus
Authors: Thomas Elizabeth A. , Alvarez Carlos E. , Sutcliffe J. Gregor
Published: March 2003

Abstract: Using an in situ hybridization screen for cDNA clones of brain region‐specific mRNAs, we isolated a rat transcript that encodes a ribosomal protein S27. Searching GenBank DNA databases, we found two S27 protein isoforms. One isoform, encoded by multiple genes, is extant in archaea and eukarya, but not bacteria. The second isoform appears to be recently evolved because it has been identified only in mammals. Multiple transcripts encode each isoform and exhibit different tissue expression patterns throughout rat brain and periphery, with abundant expression in the hypothalamus. In situ hybridization studies revealed predominant expression of S271 in distinct hypothalamic nuclei, such as the paraventricular, supraoptic, suprachiasmatic, arcuate, and circularis nuclei, whereas expression of S272 mRNA was discretely expressed in select neurons of the periventricular and supraoptic nuclei. Combined with the genetic evidence that S27 has extraribosomal functions in plants, the complexity of S27 biology observed here may suggest auxiliary functions for S27 proteins in the mammalian nervous system.
Oncostatin M Regulation of Interleukin‐6 Expression in Astrocytes
Authors: Van Wagoner Nicholas J , Choi Chulhee , Repovic Pavle , Benveniste Etty N
Published: March 2003

Oncostatin M (OSM) is a member of the interleukin (IL)‐6 family of cytokines and has both pro‐ and anti‐inflammatory properties. Of interest, OSM has functional effects within the CNS. We have shown recently that OSM can modulate expression of the cytokine IL‐6 in astrocytes. Herein we characterize the molecular mechanisms and signaling cascades involved in this response. OSM induces IL‐6 protein expression in a dose‐ and time‐dependent manner in astrocytes. In addition, OSM can synergize with the cytokines tumor necrosis factor‐α, IL‐1β, and transforming growth factor‐β for enhanced IL‐6 expression. Using neutralizing antibodies to gp 130, the OSM receptor (OSMR), and the leukemia inhibitory factor receptor (LIFR), we document that OSM exclusively uses the OSMR/gp 130 heterodimer in signaling events, rather than the LIFR/gp 130 heterodimer. Kinetic analysis of OSM‐induced IL‐6 mRNA reveals two up‐regulatory events. The first, peaking at 1 h, is transient, does not require protein synthesis, and is regulated at the transcriptional level. The second, peaking between 6 and 8 h, is prolonged and sensitive to puromycin, suggesting a requirement for de novo protein synthesis, and also is transcriptionally regulated. OSM‐induced IL‐6 mRNA and protein expression is inhibited by the mitogen‐activated protein kinase (MAPK) inhibitors U0126 and SB202190, suggesting a requirement for the MAPKs ERK1/2 and p38 in this response. Finally, we show that the MAPKs ERK1/2 and p38 are activated by OSM in astrocytes and that this activation is reduced by the MAPK inhibitors. These data demonstrate that OSM induces IL‐6 expression in astrocytes and that the MAPKs ERK1/2 and p38 participate in this response.
The Inhibitory Effects of Interleukin‐6 on Synaptic Plasticity in the Rat Hippocampus Are Associated with an Inhibition of Mitogen‐Activated Protein Kinase ERK
Authors: Tancredi Virginia , D'Antuono Margherita , Cafè Carla , Giovedì Silvia , Buè Maria Cristina , D'Arcangelo Giovanna , Onofri Franco , Benfenati Fabio
Published: March 2003

Several cytokines have short‐term effects on synaptic transmission and plasticity that are thought to be mediated by the activation of intracellular protein kinases. We have studied the effects of interleukin‐6 (IL‐6) on the expression of paired pulse facilitation (PPF), posttetanic potentiation (PTP), and long‐term potentiation (LTP) in the CA1 region of the hippocampus as well as on the activation of the signal transducer and activator of transcription‐3 (STAT3), the mitogen‐activated protein kinase ERK (MAPK/ERK), and the stress‐activated protein kinase/c‐Jun NH2‐terminal kinase (SAPK/JNK). IL‐6 induced a marked and dose‐dependent decrease in the expression of PTP and LTP that could be counteracted by the simultaneous treatment with the tyrosine kinase inhibitor lavendustin A (LavA) but did not significantly affect PPF. The IL‐6‐induced inhibition of PTP and LTP was accompanied by a simulation of STAT3 tyrosine phosphorylation and an inhibition of MAPK/ERK dual phosphorylation, in the absence of changes in the state of activation of SAPK/JNK. Both effects of IL‐6 on STAT3 and MAPK/ERK activation were effectively counteracted by LavA treatment. The results indicate the tyrosine kinases and MAPK/ERK are involved in hippocampal synaptic plasticity and may represent preferential intracellular targets for the actions of IL‐6 in the adult nervous system.