bluuebunny/crossref_metadata_2025 huggingface dataset:
The Endogenous Amine 1‐Methyl‐1,2,3,4‐ Tetrahydroisoquinoline Prevents the Inhibition of Complex I of the Respiratory Chain Produced by MPP+
Authors: Parrado Juan , Absi Elhadi , Ayala Antonio , Castaño Angélica , Cano Josefina , Machado Alberto
Published: March 2003

Abstract : The endogenous monoamine

1‐methyl‐1,2,3,4‐tetrahydroisoquinoline has been shown to prevent the

neurotoxic effect of MPP+ and other endogenous neurotoxins, which

produce a parkinsonian‐like syndrome in humans. We have tested its potential

protective effect in vivo by measuring the protection of

1‐methyl‐1,2,3,4‐tetrahydroisoquinoline in the neurotoxicity elicited by

MPP+ in rat striatum by tyrosine hydroxylase immunocytochemistry.

Because we know that cellular damage caused by MPP+ is primarily

the result of mitochondrial respiratory inhibition at the complex I level, we

have extended the study further to understand this protective mechanism. We

found that the inhibitory effect on the mitochondrial respiration rate induced

by MPP+ in isolated rat liver mitochondria and striatal

synaptosomes was prevented by addition of

1‐methyl‐1,2,3,4‐tetrahydroisoquinoline. This compound has no antioxidant

capacity ; therefore, this property is not involved in its protective effect.

Thus, we postulate that the preventive effect that

1‐methyl‐1,2,3,4‐tetrahydroisoquinoline has on mitochondrial inhibition for

MPP+ could be due to a “shielding effect,” protecting the energetic machinery, thus preventing energetic failure. These results suggest that this endogenous amine may protect against the effect of several parkinsonism‐inducing compounds that are associated with progressive impairment of the mitochondrial function.
Phospholipase C, Protein Kinase C, Ca2+/Calmodulin‐Dependent Protein Kinase II, and Tyrosine Phosphorylation Are Involved in Carbachol‐Induced Phospholipase D Activation in Chinese Hamster Ovary Cells Expressing Muscarinic Acetylcholine Receptor of Caenorhabditis elegans
Authors: Min Do Sik , Cho Nam Jeong , Yoon Shin Hee , Lee Young Han , Hahn Sang‐June , Lee Kweon‐Haeng , Kim Myung‐Suk , Jo Yang‐Hyeok
Published: March 2003

Abstract : Recently, we have isolated a cDNA encoding a muscarinic

acetylcholine receptor (mAChR) from Caenorhabditis elegans. To

investigate the regulation of phospholipase D (PLD) signaling via a muscarinic

receptor, we generated stable transfected Chinese hamster ovary (CHO) cells

that overexpress the mAChR of C. elegans (CHO‐GAR‐3). Carbachol (CCh)

induced inositol phosphate formation and a significantly higher

Ca2+ elevation and stimulated PLD activity through the mAChR ; this

was insensitive to pertussis toxin, but its activity was abolished by the

phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several

apparent tyrosine‐phosphorylated protein bands after CCh treatment. The

CCh‐induced PLD activation and tyrosine phosphorylation were significantly

reduced by the protein kinase C (PKC) inhibitor calphostin C and

down‐regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover,

the Ca2+‐calmodulin‐dependent protein kinase II (CaM kinase II)

inhibitor KN62, in addition to chelation of extracellular or intracellular

Ca2+ by EGTA and BAPTA/AM, abolished CCh‐induced PLD activation and

protein tyrosine phosphorylation. Taken together, these results suggest that

the PLC/PKC‐PLD pathway and the CaM kinase II/tyrosine kinase‐PLD pathway are

involved in the activation of PLD through mAChRs of C. elegans.
Ionotropic Glutamate Receptors Trigger Microvesicle‐Mediated Exocytosis of L‐Glutamate in Rat Pinealocytes
Authors: Yatsushiro Shouki , Yamada Hiroshi , Hayashi Mitsuko , Yamamoto Akitsugu , Moriyama Yoshinori
Published: March 2003

Abstract : Rat pinealocytes receive noradrenergic innervation that

stimulates melatonin synthesis. Besides melatonin, we showed previously that

pinealocytes accumulate L‐glutamate in microvesicles and secrete it through an

exocytic mechanism. The secreted glutamate binds to the class II metabotropic

glutamate receptor and inhibits norepinephrine‐stimulated melatonin synthesis

in neighboring pinealocytes through an inhibitory cyclic AMP cascade. In this

study, it was found that, in addition to metabotropic receptors, pinealocytes

express functional ionotropic receptors. RT‐PCR and northern analyses

indicated the expression of mRNA for GluR1, KA2, and NR2C in pineal gland. The

presence of GluR1 protein was confirmed by immunological techniques, but

neither KA2 nor NR2C was detected. Consistent with this observation, the

presence of

(RS)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid or

kainate, non‐N‐methyl‐D‐aspartate receptor agonists, transiently

stimulated increased the intracellular Ca2+ concentration of

cultured pinealocytes, whereas N‐methyl‐D‐aspartate did not. These

responses were prevented by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, a selective

antagonist for non‐N‐methyl‐D‐aspartate receptors, by L‐type

Ca2+ channel blockers such as nifedipine, or by omitting

Ca2+ or Na+ in the medium. In the presence of

Ca2+ and Na+,

(RS)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid or

kainate evoked glutamate secretion from the cultured cells, which was

prevented by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, L‐type Ca2+

channel blockers, type E or B botulinum neurotoxin, or incubation at

<20°C. These results strongly suggest that GluR1 is functionally

expressed in pinealocytes and triggers microvesicle‐mediated exocytosis of

L‐glutamate via activation of L‐type Ca2+ channels. It is possible that GluR1 participates in a signaling cascade that enhances and expands the L‐glutamate signal throughout the pineal gland.
Impaired Proteasome Function in Alzheimer's Disease
Authors: Keller Jeffrey N. , Hanni Keith B. , Markesbery William R.
Published: March 2003

Abstract : Inhibition of proteasome activity is sufficient to induce neuron degeneration and death ; however, altered proteasome activity in a neurodegenerative disorder has not been demonstrated. In the present study, we analyzed proteasome activity in short‐postmortem‐interval autopsied brains from 16 Alzheimer's disease (AD) and nine age‐ and sex‐matched controls. A significant decrease in proteasome activity was observed in the hippocampus and parahippocampal gyrus (48%), superior and middle temporal gyri (38%), and inferior parietal lobule (28%) of AD patients compared with controls. In contrast, no significant decrease in proteasome activity was observed in either the occipital lobe or the cerebellum. The loss of proteasome activity was not associated with a decrease in proteasome expression, suggesting that the proteasome may become inhibited in AD by a posttranslational modification. Together, these data indicate a possible role for proteasome inhibition in the neurodegeneration associated with AD.
Oncostatin M‐Mediated Growth Inhibition of Human Glioblastoma Cells Does Not Depend on Stat3 or on Mitogen‐Activated Protein Kinase Activation
Authors: Halfter Hartmut , Stögbauer Florian , Friedrich Matthias , Serve Susanne , Serve Hubert , Ringelstein E. Bernd
Published: March 2003

Abstract: Oncostatin M (OSM) and other members of the interleukin‐6 cytokines, like ciliary neurotrophic factor and leukemia inhibitory factor, can induce differentiation of glial cells. We have recently described that OSM inhibited the growth of human glioma cells in vitro and induced a cell morphology resembling that of mature astrocytes. Using the glioblastoma cell line 86HG39, we demonstrated that treatment of the glioma cells with OSM also leads to a differentiation of the malignant glioma cells as judged by a strong increase in glial fibrillary acidic protein expression. The differentiation and the growth inhibition were not significantly blocked by expression of a dominant‐negative (dn) signal transducer and activator of transcription (Stat) 3 protein. OSM exerted a reduction in DNA synthesis even in the presence of a high expression level of dnStat3. Moreover, inhibition of the ras‐raf‐mitogen‐activated protein kinase (MAPK) pathway by the MAPK kinase 1 inhibitor PD98059 resulted in a synergistic enhancement of the OSM effect, indicating that the activation of this pathway counteracts the activity of the cytokine.
Different Subtypes of Tachykinin NK1 Receptor Binding Sites Are Present in the Rat Brain
Authors: Beaujouan Jean‐Claude , Saffroy Monique , Torrens Yvette , Glowinski Jacques
Published: March 2003

Abstract: (2‐[125I]iodohistidyl1)Neurokinin A

([125I]NKA), which labels “septide‐sensitive” but not

classic NK1 binding sites in peripheral tissues, was used to

determine whether septide‐sensitive binding sites are also present in the rat

brain. Binding studies were performed in the presence of SR 48968

(NK2 antagonist) and senktide (NK3 agonist) because

[125I]NKA also labels peripheral NK2 binding sites and,

as shown in this study, central NK3 binding sites.

[125I]NKA was found to label not only septide‐sensitive binding

sites but also a new subtype of NK1 binding site distinct from

classic NK1 binding sites. Both subtypes of [125I]NKA

binding sites were sensitive to tachykinin NK1 antagonists and

agonists but also to the endogenous tachykinins NKA, neuropeptide K (NPK), and

neuropeptide γ (NPγ). However, compounds of the septide family

such as substance P(6‐11) [SP(6‐11)] and

propionyl‐[Met(O2)11]SP(7‐11) and some NK1

antagonists, GR 82334, RP 67580, and CP 96345, had a much lower affinity for

the new NK1‐sensitive sites than for the septide‐sensitive sites.

The hypothalamus and colliculi possess only this new subtype of NK1

site, whereas both types of [125I]NKA binding sites were found in

the amygdala and some other brain structures. These results not only explain

the central effects of septide or SP(6‐11), but also those of NKA, NPK, and

NPγ, which can be selectively blocked by NK1 receptor antagonists.
Neuron‐Specific Phosphorylation of Alzheimer's β‐Amyloid Precursor Protein by Cyclin‐Dependent Kinase 5
Authors: Iijima Ko‐ichi , Ando Kanae , Takeda Shizu , Satoh Yasushi , Seki Tatsunori , Itohara Shigeyoshi , Greengard Paul , Kirino Yutaka , Nairn Angus C. , Suzuki Toshiharu
Published: March 2003

Abstract: The mature form of Alzheimer's β‐amyloid precursor

protein (APP) is phosphorylated specifically at Thr668 in neurons.

In mature neurons, phosphorylated APP is detected in neurites, with

dephosphorylated APP being found mostly in the cell body. In vitro, active

cyclin‐dependent kinase 5 (Cdk5) phosphorylated the cytoplasmic domain of APP

at Thr668. Treatment of mature neurons with an antisense oligonucleotide to Cdk5 suppressed Cdk5 expression and significantly diminished the level of phosphorylated APP. The expression of APP was unaffected in antisense‐treated neurons. These results indicate that in neurons APP is phosphorylated by Cdk5, and that this may play a role in its localization.
Role of the Peripheral Myelin Protein 22 N‐Linked Glycan in Oligomer Stability
Authors: Ryan Mary C. , Notterpek Lucia , Tobler Andreas R. , Liu Ning , Shooter Eric M.
Published: March 2003

Abstract: Peripheral myelin protein 22 (PMP22) is a 22‐kDa
glycoprotein containing a single N‐linked carbohydrate moiety. This
posttranslational modification is conserved in PMP22 across species and within
members of the PMP22 gene family; however, the function of the oligosaccharide is not known. To study the role of the PMP22 carbohydrate, site‐directed mutagenesis was used to alter the glycosylation consensus sequence and produce a glycosylation‐deficient mutant protein. This modified PMP22 was expressed in primary Schwann cells (SCs), and the effect of the N‐glycan on the turnover rate, oligomerization, and intracellular trafficking of PMP22 was determined. Our data show a slight decrease in turnover rate from a half‐life of ∼70 min for the wild‐type (wt) protein to 100 min for the glycosylation mutant. Although the presence of glycosylation‐deficient PMP22 oligomers could be detected in SCs, we observed a decrease in oligomer stability compared with the wt oligomers. Both wt and mutant proteins showed similar localization in the endoplasmic reticulum and Golgi compartments and were transported to the SC surface. These results suggest that the N‐glycan of PMP22 facilitates, in part, the stability of the PMP22 oligomer; however, the implications of PMP22 oligomerization remain unknown.
Three Distinct Ca2+Influx Pathways Couple Acetylcholine Receptor Activation to Catecholamine Secretion from PC12 Cells
Authors: Taylor Shafeena C. , Peers Chris
Published: March 2003

Abstract:Amperometry and microfluorimetry were employed to investigate the Ca2+‐dependence of catecholamine release induced from PC12 cells by cholinergic agonists. Nicotine‐evoked exocytosis was entirely dependent on extracellular Ca2+but was only partly blocked by Cd2+, a nonselective blocker of voltagegated Ca2+channels. Secretion and rises of [Ca2+]iobserved in response to nicotine could be almost completely blocked by methyllycaconitine and α‐bungarotoxin, indicating that such release was mediated by receptors composed of α7 nicotinic acetylcholine receptor subunits. Secretion and [Ca2+]irises could also be fully blocked by co‐application of Cd2+and Zn2+. Release evoked by muscarine was also fully dependent on extracellular Ca2+. Muscarinic receptor activation stimulated release of Ca2+from a caffeine‐sensitive intracellular store, and release from this store induced capacitative Ca2+entry that could be blocked by La3+and Zn2+. This Ca2+entry pathway mediated all secretion evoked by muscarine. Thus, activation of acetylcholine receptors stimulated rises of [Ca2+]iand exocytosis via Ca2+influx through voltage‐gated Ca2+channels, α7 subunit‐containing nicotinic acetylcholine receptors, and channels underlying capacitative Ca2+entry.
Enhancement of Hippocampal Neurogenesis by Lithium
Authors: Chen Guang , Rajkowska Grazyna , Du Fu , Seraji‐Bozorgzad Navid , Manji Husseini K.
Published: March 2003

Abstract: Increasing evidence suggests that mood disorders are associated with a reduction in regional CNS volume and neuronal and glial cell atrophy or loss. Lithium, a mainstay in the treatment of mood disorders, has recently been demonstrated to robustly increase the levels of the cytoprotective B‐cell lymphoma protein‐2 (bcl‐2) in areas of rodent brain and in cultured cells. In view of bcl‐2's antiapoptotic and neurotrophic effects, the present study was undertaken to determine if lithium affects neurogenesis in the adult rodent hippocampus. Mice were chronically treated with lithium, and 5‐bromo‐2‐deoxyuridine (BrdU) labeling of dividing cells was conducted over 12 days. Immunohistochemical analysis was undertaken 1 day after the last injection, and three‐dimensional stereological cell counting revealed that lithium produced a significant 25% increase in the BrdU‐labeled cells in the dentate gyrus. Double‐labeling immunofluorescence studies were undertaken to co‐localize BrdU‐positive cells with neuron‐specific nuclear protein and showed that ∼65% of the cells were double‐labeled. These results add to the growing body of evidence suggesting that mood stabilizers and antidepressants exert neurotrophic effects and may therefore be of use in the long‐term treatment of other neuropsychiatric disorders.
Coexpression of Several Types of Metabotropic Nucleotide Receptors in Single Cerebellar Astrocytes
Authors: Jiménez Ana I. , Castro Enrique , Communi Didier , Boeynaems Jean‐Marie , Delicado Esmerilda G. , Miras‐Portugal M. Teresa
Published: March 2003

Abstract: We have examined the expression of mRNA for several P2Y
nucleotide receptors by northern blot analysis in purified type 1 cerebellar
astrocyte cultures. These results suggest that different P2Y subtypes could be
responsible for ATP metabotropic calcium responses in single type 1
astrocytes. To identify these subtypes we have studied the pharmacological
profile of ATP calcium responses using fura‐2 microfluorimetry. All tested
astrocytes responded to ATP and UTP stimulations evoking similar calcium
transients. Most astrocytes also responded to 2‐methylthioATP and ADP
challenges. The agonist potency order was 2‐methylthioATP > ADP > ATP =
UTP. Cross‐desensitization experiments carried out with ATP, UTP, and
2‐methylthioATP showed that 2‐methylthioATP and UTP interact with different
receptors, P2Y1 and P2Y2 or P2Y4. In a
subpopulation of type 1 astrocytes, ATP prestimulation did not block UTP
responses, and UDP elicited clear intracellular Ca2+ concentration
responses at very low concentrations. 2‐MethylthioATP and UTP calcium
responses exhibited different sensitivity to pertussis toxin and different
inhibition patterns in response to P2 antagonists. The
P2Y1‐specific antagonist
N6‐methyl‐2′‐deoxyadenosine
3′,5′‐bisphosphate (MRS 2179) specifically blocked the
2‐methylthio‐ATP responses. We can conclude that all single astrocytes
coexpressed at least two types of P2Y metabotropic receptors: P2Y1
and either P2Y2 or P2Y4 receptors. Moreover, 30‐40% of
astrocytes also coexpressed specific pyrimidine receptors of the
P2Y6 subtype, highly selective for UDP coupled to pertussis‐toxin insensitive G protein.
Changes in the Cannabinoid Receptor Binding, G Protein Coupling, and Cyclic AMP Cascade in the CNS of Rats Tolerant to and Dependent on the Synthetic Cannabinoid Compound CP55,940
Authors: Rubino T. , Viganò D. , Massi P. , Parolaro D.
Published: March 2003

Abstract: Chronic exposure to CP55,940 produced a significant
down‐regulation of cannabinoid receptors in the striatum, cortex, hippocampus,
and cerebellum of rat brain. At 24 h after SR141716‐precipitated withdrawal,
we observed a tendency to return to basal levels in the striatum and cortex,
whereas the specific binding remained lower in the hippocampus and cerebellum.
When we surveyed cannabinoid receptor‐activated G proteins, in chronic
CP55,940‐treated rats the guanosine
5′‐O‐(3‐[35S]thiotriphosphate)
([35S]GTPγS) binding assay revealed a decrease of activated G
proteins in the striatum, cortex, and hippocampus, whereas no significant
changes were seen in the cerebellum. At 24 h after the SR141716‐precipitated
withdrawal, [35S]GTPγS binding increased compared with that of rats chronically exposed to CP55,940, attaining the control level except for cerebellum, where we observed a trend to overcome the control amounts. Concerning the cyclic AMP (cAMP) cascade, which represents the major intracellular signaling pathway activated by cannabinoid receptors, in the cerebral areas from rats chronically exposed to CP55,940 we found alteration in neither cAMP levels nor protein kinase A activity. In the brain regions taken from CP55,940‐withdrawn rats, we only observed a significant up‐regulation in the cerebellum. Our findings suggest that receptor desensitization and down‐regulation are strictly involved in the development of cannabinoid tolerance, whereas alterations in the cAMP cascade in the cerebellum could be relevant in the mediation of the motor component of cannabinoid abstinence.
Complex Regulation of Tau Exon 10, Whose Missplicing Causes Frontotemporal Dementia
Authors: Gao Qing‐Sheng , Memmott John , Lafyatis Robert , Stamm Stefan , Screaton Gavin , Andreadis Athena
Published: March 2003

Abstract: Tau is a microtubule‐associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 10 of the gene is an alternatively spliced cassette that is adult‐specific and that codes for a microtubule binding domain. Recently, mutations that affect splicing of exon 10 have been shown to cause inherited frontotemporal dementia (FTDP). In this study, we establish the endogenous expression patterns of exon 10 in human tissue; by reconstituting naturally occurring FTDP mutants in the homologous context of exon 10, we show that the cis determinants of exon 10 splicing regulation include an exonic silencer within the exon, its 5′ splice site, and the relative affinities of its flanking exons to it. By cotransfections in vivo, we demonstrate that several splicing regulators affect the ratio of tau isoforms by inhibiting exon 10 inclusion.
Identification of an Amino Acid Sequence Motif in the Cytoplasmic Domain of the NCAM‐140 kDa Isoform Essential for Its Neuritogenic Activity
Authors: Kolkova Kateryna , Pedersen Nina , Berezin Vladimir , Bock Elisabeth
Published: March 2003

Abstract: The functions of the extracellular domains of neural cell adhesion molecule (NCAM) have been studied extensively, whereas the roles of the cytoplasmic domains of the transmembrane forms of NCAM are less elucidated. We investigated the importance of the cytoplasmic domain of the 140‐kDa NCAM isoform (cytNCAM‐140) and of the 180‐kDa NCAM isoform (cytNCAM‐180) in NCAM‐induced neurite extension by estimating NCAM‐dependent neurite outgrowth from PC12‐E2 cells grown in coculture with NCAM‐negative or NCAM‐positive fibroblasts. PC12‐E2 cells were transiently transfected with expression plasmids encoding cytNCAM‐140, cytNCAM‐180, the constitutively active form of the mitogen‐activated protein (MAP) kinase/extracellular signal‐regulated kinase kinase (MEK2), and the enhanced variant of the green fluorescent protein (EGFP). EGFP expression was used for identification of transfected cells. We found that expression of cytNCAM‐180 had no effect on NCAM‐stimulated neuritogenesis, whereas expression of cytNCAM‐140 strongly inhibited this process. However, if MEK2 was expressed concomitantly with cytNCAM‐140, neurite outgrowth was rescued, indicating that cytNCAM‐140 is involved in signaling via the Ras‐MAP kinase pathway. PC12‐E2 cells were subsequently transiently transfected with constructs encoding a series of fragments of cytNCAM‐140 and various full‐length cytNCAM‐140 mutants, and the residues Thr‐Glu‐Val‐Lys‐Thr (839‐843) were identified as essential in NCAM‐stimulated neuritogenesis. The combined substitution of Glu840 and Lys842 with Ala abrogated the effect of the construct, assigning a critical role to these two residues.
Abundant Tissue Butyrylcholinesterase and Its Possible Function in the Acetylcholinesterase Knockout Mouse
Authors: Li Bin , Stribley Judith A. , Ticu Andreea , Xie Weihua , Schopfer Lawrence M. , Hammond Pamela , Brimijoin Stephen , Hinrichs Steven H. , Lockridge Oksana
Published: March 2003

Abstract: We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X‐100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15‐fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X‐100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/‐ mice had 50% of normal AChE activity and AChE ‐/‐ mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X‐100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl‐binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X‐100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild‐type mice as well.
Peroxynitrite plays a role in methamphetamine‐induced dopaminergic neurotoxicity: evidence from mice lacking neuronal nitric oxide synthase gene or overexpressing copper–zinc superoxide dismutase
Authors: Imam Syed Z. , Newport Glenn D. , Itzhak Yossef , Cadet Jean L. , Islam Fakhrul , Slikker William , Ali Syed F.
Published: March 2003

The use of methamphetamine (METH) leads to neurotoxic effects in mammals. These neurotoxic effects appear to be related to the production of free radicals. To assess the role of peroxynitrite in METH‐induced dopaminergic, we investigated the production of 3‐nitrotyrosine (3‐NT) in the mouse striatum. The levels of 3‐NT increased in the striatum of wild‐type mice treated with multiple doses of METH (4 × 10 mg/kg, 2 h interval) as compared with the controls. However, no significant production of 3‐NT was observed either in the striata of neuronal nitric oxide synthase knockout mice (nNOS –/–) or copper–zinc superoxide dismutase overexpressed transgenic mice (SOD‐Tg) treated with similar doses of METH. The dopaminergic damage induced by METH treatment was also attenuated in nNOS–/– or SOD‐Tg mice. These data further confirm that METH causes its neurotoxic effects via the production of peroxynitrite.
Peroxynitrite affects Ca2+ influx through voltage‐dependent calcium channels
Authors: Ohkuma Seitaro , Katsura Masashi , Higo Atsuko , Shirotani Kenji , Hara Atsuo , Tarumi Chihaya , Ohgi Tadaaki
Published: March 2003

The effect of peroxynitrite (OONO−) on voltage‐dependent Ca2+ channels (VDCCs) was examined by measuring [45Ca2+] influx into mouse cerebral cortical neurones. OONO− time‐ and dose‐dependently increased [45Ca2+] influx and this increase was abolished by manganese (III) tetrakis (4‐benzoic acid) porphyrin, a scavenger for OONO−. Inhibition of cyclic GMP (cGMP) formation did not alter the OONO−‐induced [45Ca2+] influx. OONO−, as well as 30 mm KCl, significantly increased fluorescence intensity of cell‐associated bis‐(1,3‐dibutylbarbituric acid) trimethine oxonol (bis‐oxonol). Tetrodotoxin and membrane stabilizers such as lidocaine dose‐dependently suppressed OONO−‐induced [45Ca2+] influx. Although each of 1 µm nifedipine and 1 µmω‐agatoxin VIA (ω‐ATX) significantly inhibited the OONO−‐induced [45Ca2+] influx and the concomitant presence of these agents completely abolished the influx, 1 µmω‐conotoxin GVIA (ω‐CTX) showed no effect on the influx. On the other hand, OONO− itself reduced 30 mm KCl‐induced [45Ca2+] influx to the level of [45Ca2+] influx induced by OONO− alone, and the magnitude of this reduction was as same as that of KCl‐induced [45Ca2+] influx by ω‐CTX. These results indicate that OONO− increases [45Ca2+] influx into the neurones through opening P/Q‐ and L‐type VDCCs subsequent to depolarization, and inhibits the influx through N‐type VDCCs.
Activation and redistribution of c‐Jun N‐terminal kinase/stress activated protein kinase in degenerating neurons in Alzheimer's disease
Authors: Zhu Xiongwei , Raina Arun K. , Rottkamp Catherine A. , Aliev Gjumrakch , Perry George , Boux Heather , Smith Mark A.
Published: March 2003

Cellular responses to increased oxidative stress appear to be a mechanism that contributes to the varied cytopathology of Alzheimer's disease (AD). In this regard, we suspect that c‐Jun N‐terminal kinase/Stress activated protein kinase (JNK/SAPK), a major cellular stress response protein induced by oxidative stress, plays an important role in Alzheimer disease in susceptible neurons facing the dilemma of proliferation or death. We found that JNK2/SAPK‐α and JNK3/SAPK‐β were related to neurofibrillary pathology and JNK1/SAP‐Kγ related to Hirano bodies in cases of AD but were only weakly diffuse in the cytoplasm in all neurons in control cases and in non‐involved neurons in diseased brain. In this regard, in hippocampal and cortical regions of individuals with severe AD, the activated phospho‐JNK/SAPK was localized exclusively in association with neurofibrillar alterations including neurofibrillary tangles, senile plaque neurites, neuropil threads and granulovacuolar degeneration structures (GVD), completely overlapping with τ‐positive neurofibrillary pathology, but was virtually absent in these brain regions in younger and age‐matched controls without pathology. However, in control patients with some pathology, as well as in mild AD cases, there was nuclear phospho‐JNK/SAPK and translocation of phospho‐JNK/SAPK from nuclei to cytoplasm, respectively, indicating that the activation and re‐distribution of JNK/SAPK correlates with the progress of the disease. By immunoblot analysis, phospho‐JNK/SAPK is significantly increased in AD over control cases. Together, these findings suggest that JNK/SAPK dysregulation, probably resulting from oxidative stress, plays an important role in the increased phosphorylation of cytoskeletal proteins found in AD.
Identification of genes up‐regulated by retinoic‐acid‐induced differentiation of the human neuronal precursor cell line NTERA‐2 cl.D1
Authors: Leypoldt Frank , Lewerenz Jan , Methner Axel
Published: March 2003

The human teratocarcinoma cell line NTERA‐2 cl.D1 (NT2 cells) can be induced with retinoic acid and cell aggregation to yield postmitotic neurones. This seems to model the in vivo situation, as high concentrations of retinoic acid, retinoic acid binding proteins, and receptors have been detected in the embryonic CNS and the developing spinal cord suggesting a role for retinoic acid in neurogenesis. Suppression subtractive hybridization was used to detect genes up‐regulated by this paradigm of neuronal differentiation. Microfibril‐associated glycoprotein 2 was found to be drastically up‐regulated and has not been implicated in neuronal differentiation before. Suppression subtractive hybridization also identified DYRK4, a homologue of the Drosophila gene minibrain. Minibrain mutations result in specific defects in the development of the fly central nervous system. In adult rats, DYRK4 is only expressed in testis, but our results suggest an additional role for DYRK4 in neuronal differentiation. We have shown that suppression subtractive hybridization in conjunction with an efficient screening procedure is a valuable tool to produce a repertoire of differentially expressed genes and propose a new physiological role for several identified genes and expressed sequence tags.
Neurons reduce glial responses to lipopolysaccharide (LPS) and prevent injury of microglial cells from over‐activation by LPS
Authors: Chang Raymond C. C. , Chen Wei , Hudson Pearlie , Wilson Belinda , Han David S. K. , Hong Jau‐Shyong
Published: March 2003

The microenvironment of the CNS has been considered to tonically inhibit glial activities. It has been shown that glia become activated where neuronal death occurs in the aging brain. We have previously demonstrated that neurons tonically inhibit glial activities including their responses to the bacterial endotoxin lipopolysaccharide (LPS). It is not clear whether activation of glia, especially microglia in the aging brain, is the consequence of disinhibition due to neuronal death. This study was designed to determine if glia regain their responsiveness to LPS once the neurons have died in aged cultures. When cultured alone, glia from postnatal day one rat mesencephalons stimulated with LPS (0.1–1000 ng/mL) produced both nitric oxide (NO) and tumor necrosis factor α (TNFα), yielding a sigmoid and a bell‐shaped curve, respectively. When neuron‐containing cultures were prepared from embryonic day 14/15 mesencephalons, the shape of the dose–response curve for NO was monotonic and the bell‐shaped curve for TNFα production was shifted to the right. After 1 month of culture under conditions where neurons die, the production curves for NO and TNFα in LPS‐stimulated glia shifted back to the left compared to mixed neuron–glia cultures. Immunostaining of rat microglia for the marker CR3 (the receptor for complement component C3) demonstrated that high concentrations of LPS (1 µg/mL) reduced the number of microglia in mixed‐glial cultures. In contrast, reduction of CR3 immunostaining was not observed in LPS‐stimulated mixed neuron–glia cultures. Taken together, the results demonstrate that disinhibition of the glial response to LPS occurs after neurons die in aged cultures. Once neurons have died, the responsiveness of glia to LPS is restored. Neurons prevented injury to microglia by reducing their responsiveness to LPS. This study broadens our understanding of the ways in which the CNS microenvironment affects cerebral inflammation.
Effect of the overexpression of wild‐type or mutant α‐synuclein on cell susceptibility to insult
Authors: Lee MoonHee , Hyun Dong‐Hoon , Halliwell Barry , Jenner Peter
Published: March 2003

Mutations in α‐synuclein (A30P and A53T) are involved in some cases of familial Parkinson's disease (FPD), but it is not known how they result in nigral cell death. We examined the effect of α‐synuclein overexpression on the response of cells to various insults. Wild‐type α‐synuclein and α‐synuclein mutations associated with FPD were overexpressed in NT‐2/D1 and SK‐N‐MC cells. Overexpression of wild‐type α‐synuclein delayed cell death induced by serum withdrawal or H2O2, but did not delay cell death induced by 1‐methyl‐4‐phenylpyridinium ion (MPP+). By contrast, wild‐type α‐synuclein transfectants were sensitive to viability loss induced by staurosporine, lactacystin or 4‐hydroxy‐2‐trans‐nonenal (HNE). Decreases in glutathione (GSH) levels were attenuated by wild‐type α‐synuclein after serum deprivation, but were aggravated following lactacystin or staurosporine treatment. Mutant α‐synucleins increased levels of 8‐hydroxyguanine, protein carbonyls, lipid peroxidation and 3‐nitrotyrosine, and markedly accelerated cell death in response to all the insults examined. The decrease in GSH levels was enhanced in mutant α‐synuclein transfectants. The loss of viability induced by toxic insults was by apoptosic mechanism. The presence of abnormal α‐synucleins in substantia nigra in PD may increase neuronal vulnerability to a range of toxic agents.
Direct inhibition of c‐Jun N‐terminal kinase in sympathetic neurones preventsc‐junpromoter activation and NGF withdrawal‐induced death
Authors: Eilers Andreas , Whitfield Jonathan , Shah Bina , Spadoni Cesare , Desmond Howard , Ham Jonathan
Published: March 2003

c‐Jun N‐terminal kinases (JNKs) regulate gene expression by phosphorylating transcription factors, such as c‐Jun. Studies withJnkknockout mice suggest that JNK activity may be required for excitotoxin‐induced apoptosis in the adult hippocampus and for apoptosis in the developing embryonic neural tube. Here we investigate the role of JNKs in classical neurotrophin‐regulated developmental neuronal death by using nerve growth factor (NGF)‐dependent sympathetic neurones. In this system, NGF withdrawal leads to an increase in JNK activity, an increase in c‐Jun protein levels and c‐Jun N‐terminal phosphorylation before the cell death commitment point, and c‐Jun activity is required for cell death. To inhibit JNK activity in sympathetic neurones we have used two different JNK inhibitors that act by distinct mechanisms: the compound SB 203580 and the JNK binding domain (JBD) of JNK interacting protein 1 (JIP‐1). We demonstrate that JNK activity is required for c‐Jun phosphorylation,c‐junpromoter activation and NGF withdrawal‐induced apoptosis. We also show that ATF‐2, a c‐Jun dimerization partner that can regulatec‐jungene expression, is activated following NGF deprivation. Finally, by co‐expressing the JBD and a regulatable c‐Jun dominant negative mutant we demonstrate that JNK and AP‐1 function in the same pro‐apoptotic signalling pathway after NGF withdrawal.
Iron, neuromelanin and ferritin content in the substantia nigra of normal subjects at different ages: consequences for iron storage and neurodegenerative processes
Authors: Zecca Luigi , Gallorini Mario , Schünemann Volker , Trautwein Alfred X. , Gerlach Manfred , Riederer Peter , Vezzoni Paolo , Tampellini Davide
Published: March 2003

Information on the molecular distribution and ageing trend of brain iron in post‐mortem material from normal subjects is scarce. Because it is known that neuromelanin and ferritin form stable complexes with iron(III), in this study we measured the concentration of iron, ferritin and neuromelanin in substantia nigra from normal subjects, aged between 1 and 90 years, dissected post mortem. Iron levels in substantia nigra were 20 ng/mg in the first year of life, had increased to 200 ng/mg by the fourth decade and remained stable until 90 years of age. The H‐ferritin concentration was also very low (29 ng/mg) during the first year of life but increased rapidly to values of ≈ 200 ng/mg at 20 years of age, which then remained constant until the eighth decade of life. L‐Ferritin also showed an increasing trend during life although the concentrations were ≈ 50% less than that of H‐ferritin at each age point. Neuromelanin was not detectable during the first year, increased to ≈ 1000 ng/mg in the second decade and then increased continuously to 3500 ng/mg in the 80th year. A Mössbauer study revealed that the high‐spin trivalent iron is probably arranged in a ferritin‐like iron−oxyhydroxide cluster form in the substantia nigra. Based on this data and on the low H‐ and L‐ferritin content in neurones it is concluded that neuromelanin is the major iron storage in substantia nigra neurones in normal individuals.
Selective blockade of neurokinin (NK)1 receptors facilitates the activity of adrenergic pathways projecting to frontal cortex and dorsal hippocampus in rats
Authors: Millan Mark J. , Lejeune Françoise , De Nanteuil Guillaume , Gobert Alain
Published: March 2003

The selective NK1 receptor antagonist, GR205,171 (2.5–40.0 mg/kg, i.p.), dose‐dependently elevated dialysate levels of noradrenaline (NA), but not serotonin (5‐HT), in the frontal cortex of freely moving rats. This action was exerted stereospecifically inasmuch as its less active isomer, GR226,206, was ineffective. In the dorsal hippocampus, GR205,171 (but not GR226,206) also significantly increased dialysate levels of NA, whereas levels of 5‐HT were unaffected. Further, in anaesthetized rats, GR205,171 dose‐dependently (1.0–4.0 mg/kg, i.v.) increased the firing rate of adrenergic perikarya in the locus coeruleus. In contrast, their activity was not modified by GR226,206. These findings indicate that selective blockade of NK1 receptors enhances the activity of ascending adrenergic pathways in rats. Adrenergic mechanisms may, thus, be involved in the potential antidepressant and other functional properties of NK1 receptor antagonists.
5‐HT1A receptor mutant mice exhibit enhanced tonic, stress‐induced and fluoxetine‐induced serotonergic neurotransmission
Authors: Parsons, Loren H. , Kerr Tony M. , Tecott Laurence H.
Published: March 2003

Mutant mice that lack serotonin1A receptors exhibit enhanced anxiety‐related behaviors, a phenotype that is hypothesized to result from impaired autoinhibitory control of midbrain serotonergic neuronal firing. Here we examined the impact of serotonin1A receptor deletion on forebrain serotonin neurotransmission using in vivo microdialysis in the frontal cortex and ventral hippocampus of serotonin1A receptor mutant and wild‐type mice. Baseline dialysate serotonin levels were significantly elevated in mutant animals as compared with wild‐types both in frontal cortex (mutant = 0.44 ± 0.05 n

m; wild‐type = 0.28 ± 0.03 n

m) and hippocampus (mutant = 0.46 ± 0.07 n

m; wild‐type = 0.27 ± 0.04 n

m). A stressor known to elicit enhanced anxiety‐like behaviors in serotonin1A receptor mutants increased dialysate 5‐HT levels in the frontal cortex of mutant mice by 144% while producing no alteration in cortical 5‐HT in wild‐type mice. There was no phenotypic difference in the effect of this stressor on serotonin levels in the hippocampus. Fluoxetine produced significantly greater increases in dialysate 5‐HT content in serotonin1A receptor mutants as compared with wild‐types, with two‐ and three‐fold greater responses being observed in the hippocampus and frontal cortex, respectively. This phenotypic effect was mimicked in wild‐types by pretreatment with the serotonin1A antagonist 4‐iodo‐N‐[2‐[4‐(methoxyphenyl)‐1‐piperazinyl]ethyl]‐N‐2‐pyridinyl‐benzamide (p‐MPPI). These results indicate that deletion of central serotonin1A receptors results in a tonic disinhibition of central serotonin neurotransmission, with a greater dysregulation of serotonin release in the frontal cortex than ventral hippocampus under conditions of stress or increased interstitial serotonin levels.
Protein kinase C‐mediated functional regulation of dopamine transporter is not achieved by direct phosphorylation of the dopamine transporter protein
Authors: Chang Mi‐Yoon , Lee Sang‐Hun , Kim Jin‐Hyuk , Lee Ki‐Hwan , Kim Young‐Sik , Son Hyeon , Lee Yong‐Sung
Published: March 2003

Dopaminergic neurotransmission is terminated by the action of the presynaptic dopamine transporter (DAT). It mediates Na+/Cl− ‐dependent re‐uptake of extracellular dopamine (DA) into the cell, and is regarded as a major regulatory mechanism for synaptic transmission. Previous works have documented that protein kinase C (PKC) activator or inhibitor alters DA uptake by DAT, suggesting that PKC phosphorylation plays an important regulatory mechanism in DAT function. Based on the existence of consensus amino acid sequences for PKC phosphorylation, it has been postulated that PKC regulation of DAT is mediated by the direct phosphorylation of DAT protein. In this study, we try to discover whether the functional regulation of DAT by PKC is due to direct phosphorylation of DAT. The PKC null mutant hDAT, where all putative PKC phosphorylation sites are eliminated, has been constructed by the replacement of serine/threonine residues with glycines. The mutation itself showed no effect on the functional activities of DAT. The DA uptake activity of PKC null mutant was equivalent to those of wild‐type hDAT (80–110% of wild‐type). Phorbol ester activation of PKC inhibited DA uptake of wild‐type hDAT by 35%, and staurosphorine blocked the effect of phorbol ester on DA uptake. The same phenomena was observed in PKC null mutant DAT, although no significant phosphorylation was observed by PKC activation. Confocal microscopic analysis using EGFP‐fused DAT revealed that the activation of PKC by phorbol ester elicited fluorescent DAT to be internalized into the intracellular space both in wild‐type and PKC null mutant DAT in a similar way. These results suggest that PKC‐mediated regulation of DAT function is achieved in an indirect manner, such as phosphorylation of a mediator protein or activation of a clathrin‐mediated pathway.
Identification of a novel member of the T1R family of putative taste receptors
Authors: Sainz Eduardo , Korley Julius N. , Battey James F. , Sullivan Susan L.
Published: March 2003

In the gustatory system, the recognition of sugars, amino acids and bitter‐tasting compounds is the function of specialized G protein‐coupled receptors. Recently, two members of novel subfamily of G protein‐coupled receptors were proposed to function as taste receptors based on their specific expression in taste receptor cells. Here, we report the identification of a third member, T1R3, of this family of receptors. T1R3 maps near the telomere of mouse chromosome 4 rendering it a candidate for the Sac locus, a primary determinant of sweet preference in mice. Consistent with its candidacy for the Sac locus, T1R3 displays taste receptor cell‐specific expression. In addition, taster and non‐taster strains of mouse harbor different alleles of T1R3.
Dehydroepiandrosterone inhibits lipopolysaccharide‐induced nitric oxide production in BV‐2 microglia
Authors: Wang Mei‐Jen , Huang Hsueh‐Meei , Chen Huan‐Lian , Kuo Jon‐Son , Jeng Kee‐Ching G.
Published: March 2003

Levels of dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) decline during aging and reach even lower levels in Alzheimer's disease (AD). DHEA is known to exhibit a variety of functional activities in the CNS, including an increase of memory and learning, neurotrophic and neuroprotective effects, and the reduction of risk of age‐related neurodegenerative disorders. However, the influence of DHEA on the immune functions of glial cells is poorly understood. In this study, we investigated the effect of DHEA on activated glia. The production of inducible nitric oxide synthase (iNOS) was studied in lipopolysaccharide (LPS)‐stimulated BV‐2 microglia, as a model of glial activation. The results showed that DHEA but not DHEAS significantly inhibited the production of nitrite in the LPS‐stimulated BV‐2 cell cultures. Pretreatment of BV‐2 cells with DHEA reduced the LPS‐induced iNOS mRNA and protein levels in a dose‐dependent manner. The LPS‐induced iNOS activity in BV‐2 cells was decreased by the exposure of 100 µm DHEA. Moreover, DHEA suppressed iNOS gene expression in LPS‐stimulated BV‐2 cells did not require de novo synthesis of new proteins or destabilize of iNOS mRNA. Since DHEA is biosynthesized by astrocytes and neurons, our findings suggest that it might have an important regulatory function on microglia.
The transcriptional regulator Yin Yang 1 activates the myelin PLP gene
Authors: Berndt Jo Ann , Kim Jin G. , Tosic Mirjana , Kim Christopher , Hudson Lynn D.
Published: March 2003

Inauguration of the myelin program in developing oligodendrocytes requires the activation of those genes that encode the myelin proteins and the enzymes responsible for the synthesis and degradation of myelin lipids. An activator of the most abundantly expressed myelin protein, proteolipid protein (PLP), has been identified in a yeast one‐hybrid system. The ubiquitously expressed zinc finger protein Yin Yang 1 (YY1) recognizes the myelin PLP promoter in vitro and in vivo. When overexpressed in an oligodendrocyte cell line, YY1 enhances transcription of the PLP promoter. A truncated version of YY1 that includes only the four zinc finger domains has little effect. The binding site for YY1 in the PLP promoter (site 3) fits the YY1 consensus site and DNA‐protein complexes containing site 3 can be supershifted with an antibody directed against YY1 protein. Moreover, oligonucleotides with a mutated version of the PLP promoter site 3 are unable to bind to nuclear proteins or to compete for binding in a gel shift system. Finally, mutation of this site greatly reduces the activity of a 1‐kb PLP promoter region in transfected glial cells. Our results suggest that PLP is a target gene for the transcriptional regulator YY1. This versatile transcription factor and nuclear matrix protein may boost transcription of the PLP gene to meet the demands of actively myelinating oligodendrocytes.
Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain
Authors: Reaux Annabelle , De Mota Nadia , Skultetyova Ivana , Lenkei Zsolt , El Messari Said , Gallatz Katalin , Corvol Pierre , Palkovits Miklos , Llorens‐Cortès Catherine
Published: March 2003

Apelin, a peptide recently isolated from bovine stomach tissue extracts, has been identified as the endogenous ligand of the human orphan APJ receptor. We established a stable Chinese hamster ovary (CHO) cell line expressing a gene encoding the rat apelin receptor fused to the enhanced green fluorescent protein, to investigate internalization and the pharmacological profile of the apelin receptor. Stimulation of this receptor by the apelin fragments K17F (Lys1‐Phe‐Arg‐Arg‐Gln‐Arg‐Pro‐Arg‐Leu‐Ser‐His‐Lys‐Gly‐Pro‐Met‐Pro‐Phe17) and pE13F (pGlu5‐Arg‐Pro‐Arg‐Leu‐Ser‐His‐Lys‐Gly‐Pro‐Met‐Pro‐Phe17) resulted in a dose‐dependent inhibition of forskolin‐induced cAMP production and promoted its internalization. In contrast, the apelin fragments R10F (Arg8‐Leu‐Ser‐His‐Lys‐Gly‐Pro‐Met‐Pro‐Phe17) and G5F (Gly13‐Pro‐Met‐Pro‐Phe17) were inactive. The physiological role of apelin and its receptor was then investigated by showing for the first time in rodent brain: (i) detection of apelin neurons in the supraoptic and paraventricular nuclei by immunohistochemistry with a specific polyclonal anti‐apelin K17F antibody; (ii) detection of apelin receptor mRNA in supraoptic vasopressinergic neurons by in situ hybridization and immunohistochemistry; and (iii) a decrease in vasopressin release following intracerebroventricular injection of K17F, or pE13F, but not R10F. Thus, apelin locally synthesized in the supraoptic nucleus could exert a direct inhibitory action on vasopressinergic neuron activity via the apelin receptors synthesized in these cells. Furthermore, central injection of pE13F significantly decreased water intake in dehydrated normotensive rats but did not affect blood pressure. Together, these results suggest that neuronal apelin plays an important role in the central control of body fluid homeostasis.