bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Myelin Basic Protein Phosphorylation by Mitogen‐Activated Protein Kinase During Increased Action Potential Firing in the Hippocampus
Authors: Atkins Coleen M. , Yon M. , Groome N. P. , Sweatt J. David
Published: March 2003

Abstract : Myelin basic protein (MBP) phosphorylation is a complex regulatory process that modulates the contribution of MBP to the stability of the myelin sheath. Recent research has demonstrated the modulation of MBP phosphorylation by mitogen‐activated protein kinase (MAPK) during myelinogenesis and in the demyelinating disease multiple sclerosis. Here we investigated the physiological regulation of MBP phosphorylation by MAPK during neuronal activity in the alveus, the myelinated output fibers of the hippocampus. Using a phosphospecific antibody that recognizes the predominant MAPK phosphorylation site in MBP, Thr95, we found that MBP phosphorylation is regulated by high‐frequency stimulation but not low‐frequency stimulation of the alveus. This change was blocked by application of tetrodotoxin, indicating that action potential propagation in axons is required. It is interesting that the change in MBP phosphorylation was attenuated by the reactive oxygen species scavengers superoxide dismutase and catalase and the nitric oxide synthase inhibitor N‐nitro‐L‐arginine. Removal of extracellular calcium also blocked the changes in MBP phosphorylation. Thus, we propose that during periods of increased neuronal activity, calcium activates axonal nitric oxide synthase, which generates the intercellular messengers nitric oxide and superoxide and regulates the phosphorylation state of MBP by MAPK
The GABA Paradox
Authors: Waagepetersen Helle S. , Sonnewald Ursula , Schousboe Arne
Published: March 2003

Abstract : GABA, which is present in the brain in large amounts, is distributed among distinctly different cellular pools, possibly reflecting its multiple functions as metabolite, neurotransmitter, and neurotrophin. Its metabolic enzymes also exhibit heterogeneity, because glutamate decarboxylase exists in two isoforms with different subcellular distribution and regulatory properties. Moreover, recent evidence points to a more pronounced regulatory role of the tricarboxylic acid cycle than hitherto anticipated in the biosynthetic machinery responsible for formation of GABA from glutamine. Additionally, GABAergic neurons may contain distinct populations of mitochondria having different turnover rates of the tricarboxylic acid cycle with different levels of association with GABA synthesis from 2‐oxoglutarate via glutamate. These aspects are discussed in relation to the different functional roles of GABA and its prominent involvement in epileptogenic activity.
Involvement of Inositol 1,4,5‐Trisphosphate‐Regulated Stores of Intracellular Calcium in Calcium Dysregulation and Neuron Cell Death Caused by HIV‐1 Protein Tat
Authors: Haughey N. J. , Holden C.P. , Nath A. , Geiger J.D.
Published: March 2003

Abstract : HIV‐1 infection commonly leads to neuronal cell death and

a debilitating syndrome known as AIDS‐related dementia complex. The HIV‐1

protein Tat is neurotoxic, and because cell survival is affected by the

intracellular calcium concentration ([Ca2+]i), we

determined mechanisms by which Tat increased [Ca2+]i and

the involvement of these mechanisms in Tat‐induced neurotoxicity. Tat

increased [Ca2+]i dose‐dependently in cultured human

fetal neurons and astrocytes. In neurons, but not astrocytes, we observed

biphasic increases of [Ca2+]i. Initial transient

increases were larger in astrocytes than in neurons and in both cell types

were significantly attenuated by antagonists of inositol 1,4,5‐trisphosphate

(IP3)‐mediated intracellular calcium release

[8‐(diethylamino)octyl‐3,4,5‐trimethoxybenzoate HCl (TMB‐8) and xestospongin],

an inhibitor of receptor‐Gi protein coupling (pertussis toxin), and

a phospholipase C inhibitor (neomycin). Tat significantly increased levels of

IP3 threefold. Secondary increases of neuronal

[Ca2+]i in neurons were delayed and progressive as a

result of excessive calcium influx and were inhibited by the glutamate

receptor antagonists ketamine, MK‐801, (±)‐2‐amino‐5‐phosphonopentanoic

acid, and 6,7‐dinitroquinoxaline‐2,3‐dione. Secondary increases of

[Ca2+]i did not occur when initial increases of

[Ca2+]i were prevented with TMB‐8, xestospongin,

pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin

inhibited Tat‐induced neurotoxicity. These results suggest that Tat, via

pertussis toxin‐sensitive phospholipase C activity, induces calcium release

from IP3‐sensitive intracellular stores, which leads to glutamate

receptor‐mediated calcium influx, dysregulation of

[Ca2+]i, and Tat‐induced neurotoxicity.
The Up‐Regulation of Endosomal‐Lysosomal Components in Amyloid β‐Resistant Cells
Authors: Yonghong Li  , Xu Chunhui , Schubert David
Published: March 2003

Abstract : The abundance of amyloid β peptide

(Aβ) and the selective loss of neurons are characteristics of

Alzheimer's disease. However, subpopulations of brain cells survive, including

neurons near Aβ‐rich plaques. The surviving neurons may have

gene expression profiles that allow them to be resistant to Aβ

toxicity. Here we use the differential display technique to compare the

profiles of gene expression in an Aβ‐resistant cell line with

its parental cells. Prominent among the changes are two components of the

endosomal‐lysosomal system, insulin growth factor II

receptor/mannose‐6‐phosphate receptor and arylsulfatase B. Both are more

highly expressed in the Aβ‐resistant clone, and arylsulfatase is

inducible by Aβ and hydrogen peroxide. Another lysosomal enzyme,

βglucuronidase, is also up‐regulated in

Aβ‐resistant cells. These results are consistent with the

observation that the endosomal‐lysosomal system is highly activated in

Alzheimer's disease brains, and they raise the possibility that the high

expression of endosomal‐lysosomal components is important for neuronal

survival in the presence of Aβ.
Regulation of Na+, K+ ‐ATPase Isoforms in Rat Neostriatum by Dopamine and Protein Kinase C
Authors: Nishi Akinori , Fisone Gilberto , Snyder Gretchen L. , Dulubova Irina , Aperia Anita , Nairn Angus C. , Greengard Paul
Published: March 2003

Abstract : Our previous studies showed that dopamine inhibits

Na+, K+ ‐ATPase activity in acutely dissociated neurons

from striatum. In the present study, we have found that in this preparation,

dopamine inhibited significantly (by ~25%) the activity of the α3 and/or

α2 isoforms, but not the α1 isoform, of Na+,

K+ ‐ATPase. Dopamine, via D1 receptors, activates cyclic

AMP‐dependent protein kinase (PKA) in striatal neurons. Dopamine is also known

to activate the calcium‐ and phospholipid‐dependent protein kinase (PKC) in a

number of different cell types. The PKC activator phorbol 12,13‐dibutyrate

reduced the activity of Na+, K+ ‐ATPase α3 and/or

α2 isoforms (by ~30%) as well as the α1 isoform (by ~15%).

However, dopamine‐mediated inhibition of Na+, K+ ‐ATPase

activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not

affect the phosphorylation of Na+, K+ ‐ATPase isoforms

at the PKA‐dependent phosphorylation site. Phorbol ester treatment did not

alter the phosphorylation of α2 or α3 isoforms of Na+,

K+ ‐ATPase in neostriatal neurons but did increase the

phosphorylation of the α1 isoform. Thus, in rat neostriatal neurons,

treatment with either dopamine or PKC activators results in inhibition of the

activity of specific (α3 and/or α2) isoforms of Na+,

K+ ‐ATPase, but this is not apparently mediated through direct

phosphorylation of the enzyme. In addition, PKC is unlikely to mediate

inhibition of rat Na+, K+ ‐ATPase activity by dopamine in neostriatal neurons.
Acidosis Potentiates Oxidative Neuronal Death by Multiple Mechanisms
Authors: Ying Weihai , Han Shan‐Kuo , Miller Johann W. , Swanson Raymond A.
Published: March 2003

Abstract : Both acidosis and oxidative stress contribute to ischemic

brain injury. The present study examines interactions between acidosis and

oxidative stress in murine cortical cultures. Acidosis (pH 6.2) was found to

potentiate markedly neuronal death induced by H2O2

exposure. To determine if this effect was mediated by decreased antioxidant

capacity at low pH, the activities of several antioxidant enzymes were

measured. Acidosis was found to reduce the activities of glutathione

peroxidase and glutathione S‐transferase by 50‐60% (p <

0.001) and the activity of glutathione reductase by 20% (p < 0.01)

in lysates of the cortical cultures. Like acidosis, direct inhibition of

glutathione peroxidase with mercaptosuccinate also potentiated

H2O2 toxicity. Because acidosis may accelerate hydroxyl

radical production by the Fenton reaction, the effect of iron chelators was

also examined. Both desferrioxamine and

N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine, two

structurally different iron chelators, significantly reduced

H2O2‐induced neuronal death under both pH 7.2 and pH 6.2 conditions. These results suggest that the increased cell death produced by severe acidosis during cerebral ischemia may result in part from excerbation of oxidative injury. This exacerbation may result from both impaired antioxidant enzyme functions and increased intracellular free iron levels.
Mechanisms of Synaptic Vesicle Recycling Illuminated by Fluorescent Dyes
Authors: Cousin M. A. , Robinson P. J.
Published: March 2003

Abstract : The recycling of synaptic vesicles in nerve terminals

involves multiple steps, underlies all aspects of synaptic transmission, and

is a key to understanding the basis of synaptic plasticity. The development of

styryl dyes as fluorescent molecules that label recycling synaptic vesicles

has revolutionized the way in which synaptic vesicle recycling can be

investigated, by allowing an examination of processes in neurons that have

long been inaccessible. In this review, we evaluate the major aspects of

synaptic vesicle recycling that have been revealed and advanced by studies

with styryl dyes, focussing upon synaptic vesicle fusion, retrieval, and

trafficking. The greatest impact of styryl dyes has been to allow the routine

visualization of endocytosis in central nerve terminals for the first time.

This has revealed the kinetics of endocytosis, its underlying sequential

steps, and its regulation by Ca2+. In studies of exocytosis, styryl dyes have helped distinguish between different modes of vesicle fusion, provided direct support for the quantal nature of exocytosis and endocytosis, and revealed how the probability of exocytosis varies enormously from one nerve terminal to another. Synaptic vesicle labelling with styryl dyes has helped our understanding of vesicle trafficking by allowing better understanding of different synaptic vesicle pools within the nerve terminal, vesicle intermixing, and vesicle clustering at release sites. Finally, the dyes are now being used in innovative ways to reveal further insights into synaptic plasticity.
Monoamine Oxidase and Mitochondrial Respiration
Authors: Cohen Gerald , Kesler Natasa
Published: March 2003

Abstract : Mitochondrial defects encompassing complexes I‐IV of the electron transport chain characterize a relatively large number of neurodegenerative diseases. The relationships between mitochondrial lesions and recently described genetic alterations have not yet been defined. We describe a general mechanism whereby the enzymatic metabolism of neurotransmitters by monoamine oxidase (MAO) damages mitochondria, altering their protein thiol status and suppressing respiration. In these experiments, incubation of rat brain mitochondria with tyramine (a mixed MAO‐A/MAO‐B substrate) for 15 min at 27°C suppressed state 3 respiration by 32.8% and state 5 respiration by 40.1%. These changes were accompanied by a 10‐fold rise in protein‐glutathione mixed disulfides. Direct comparison of effects on respiration and MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] dye reduction during electron flow gave similar results. It is suggested that certain mitochondrial lesions may derive from the natural turnover of monoamine neurotransmitters in susceptible individuals.
Dopamine Neuronal Transport Kinetics and Effects of Amphetamine
Authors: Jones Sara R. , Joseph Joshua D. , Barak Larry S. , Caron Marc G. , Wightman R. Mark
Published: March 2003

Abstract : The dopamine (DA) transporter (DAT) regulates DA neurotransmission by recycling DA back into neurons. Drugs that interfere with DAT function, e.g., cocaine and amphetamine, can have profound behavioral effects. The kinetics of DA transport by DAT in isolated synaptosomal or single cell preparations have been previously studied. To investigate how DA transport is regulated in intact tissue and to examine how amphetamine affects the DAT, the kinetics of DA uptake by the DAT were examined in tissue slices of the mouse caudate‐putamen with fastscan cyclic voltammetry. The data demonstrate that inward DA transport is saturable and sodium‐dependent. Elevated levels of cytoplasmic DA resulting from disruption of vesicular storage by incubation with 10 μM Ro 4‐1284 did not generate DA efflux or decrease its uptake rate. However, incubation with 10 μM amphetamine reduced the net DA uptake rate and increased extracellular DA levels due to DA efflux through the DAT. In addition, a new, elevated steady‐state level of extracellular DA was established after electrically stimulated DA release in the presence of amphetamine, norepinephrine, and exogenous DA. These results from intact tissue are consistent with a kinetic model of the DAT established in more purified preparations in which amphetamine and other transported substances make the inwardly facing DAT available for outward transport of intracellular DA.
Ethanol Inhibits Astroglial Cell Proliferation by Disruption of Phospholipase D‐Mediated Signaling
Authors: Kötter Katja , Klein Jochen
Published: March 2003

Abstract : The activation of phospholipase D (PLD) is a common

response to mitogenic stimuli in various cell types. As PLD‐mediated signaling

is known to be disrupted in the presence of ethanol, we tested whether PLD is

involved in the ethanol‐induced inhibition of cell proliferation in rat

cortical primary astrocytes. Readdition of fetal calf serum (FCS) to

serum‐deprived astroglial cultures caused a rapid, threefold increase of PLD

activity and a strong mitogenic response ; both effects were dependent on

tyrosine kinases but not on protein kinase C. Ethanol (0.1‐2%) suppressed the

FCS‐induced, PLD‐mediated formation of phosphatidic acid (PA) as well as

astroglial cell proliferation in a concentration‐dependent manner. Moreover,

exogenous bacterial PLD increased astroglial proliferation in an

ethanol‐sensitive manner, whereas exogenous PA or lysophosphatidic acid was

less effective. Formation of PA and astroglial proliferation were strongly

inhibited by 1‐butanol (0.1‐1%), a substrate of PLD, but were unaffected by

t‐butanol, a nonsubstrate ; 2‐butanol had intermediate effects.

Platelet‐derived growth factor and endothelin‐1 mimicked the mitogenic effect

of FCS ; their effects were also inhibited by the butanols in the potency

order 1‐butanol > 2‐butanol > tert‐butanol. Our results, in

particular, the differential effects of 1‐, 2‐, and tert‐butanol with respect to PA formation and astroglial proliferation, strongly suggest that the antiproliferative effects of ethanol in glial cells are due to the disruption of the PLD signaling pathway. This mechanism may also contribute to the inhibition of astroglial growth and brain development observed in alcoholic embryopathy.
Molecular Cloning of Multiple Splicing Variants of JIP‐1 Preferentially Expressed in Brain
Authors: Kim In‐Jung , Lee Ko‐Woon , Park Byung Young , Lee Ja‐Kyeong , Park Jihyun , Choi In Young , Eom Soo‐Jung , Chang Tong‐Shin , Kim Myung Jin , Yeom Young Il , Chang Sung Key , Lee Young‐Don , Choi Eui‐Ju , Han Pyung‐Lim
Published: March 2003

Abstract: Stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) is activated by a variety of cellular or environmental stresses. Proper regulation of the SAPK/JNK pathway may be critical for cell survival or death under various conditions. In this study, we report the molecular cloning of novel isoforms of JIP‐1, which harbor a putative phosphotyrosine interaction domain and a helix‐loop‐helix domain, as well as an SH3 homologous region in the C terminus. Northern analysis indicates that transcription variant jip‐1 is expressed in brain and kidney and transcription variants jip‐2 and jip‐3 are specifically expressed in brain. In situ hybridization data showed that the hybridized jip messages were heavily concentrated in adult brain, and were particularly enriched in the cerebral cortex and hippocampus, the brain regions vulnerable to pathological states such as hypoxia‐ischemia, epilepsy, and Alzheimer’s disease. All the deduced protein products of the jip transcription variants appear to have a similar property in that they inhibit the SAPK/JNK stimulation when overexpressed. Inhibition of SAPK activation by overexpression of the novel isoform JIP‐2a resulted in suppression of etoposide‐induced cell death in a neuroglioma cell line, N18TG. These findings suggest that JIP may play an important role in regulation of the SAPK pathway that is involved in stress‐induced cellular responses.
Extended Ceramide Exposure Activates the trkA Receptor by Increasing Receptor Homodimer Formation
Authors: MacPhee Ian , Barker Philip A.
Published: March 2003

Abstract: Binding of nerve growth factor (NGF) to the trkA tyrosine kinase receptor results in receptor homodimer formation, transphosphorylation, and kinase activation that supports neuronal differentiation and survival. We have shown previously that short‐term exposure of PC12 cells to brain‐derived neurotrophic factor or to C2‐ceramide activates a signaling pathway that results in serine phosphorylation of the trkA intracellular domain and reduces the ability of trkA to respond to NGF. Here we demonstrate that extended C2‐ceramide exposure dramatically increases NGF‐induced trkA activation and further show that C2‐ceramide augments trkA tyrosine phosphorylation even in the absence of neurotrophin. This increase in trkA receptor phosphotyrosine is reflected in increased activation of both Erk1 and Erk2 and of the catalytic subunit of phosphatidylinositol 3‐kinase. The C2‐ceramide‐mediated increase in tyrosine phosphorylation is blocked completely by the trk kinase inhibitor K252A, indicating that this increase results from an effect of C2‐ceramide on trkA kinase activity. Consistent with this, crosslinking studies show that C2‐ceramide promotes the formation of active trkA receptor homodimers. Together, these data suggest that chronic C2‐ceramide treatment increases trkA activation by altering properties of the plasma membrane, which favors the formation of trkA homodimers.
Inhibition of Excessive Neuronal Apoptosis by the Calcium Antagonist Amlodipine and Antioxidants in Cerebellar Granule Cells
Authors: Mason R. Preston , Leeds Peter R. , Jacob Robert F. , Hough Christopher J. , Zhang Kai‐Gao , Mason Pamela E. , Chuang De‐Maw
Published: March 2003

Abstract: Neuronal cell death as a result of apoptosis is associated with cerebrovascular stroke and various neurodegenerative disorders. Pharmacological agents that maintain normal intracellular Ca2+ levels and inhibit cellular oxidative stress may be effective in blocking abnormal neuronal apoptosis. In this study, a spontaneous (also referred to as age‐induced) model of apoptosis consisting of rat cerebellar granule cells was used to evaluate the antiapoptotic activities of voltage‐sensitive Ca2+ channel blockers and various antioxidants. The results of these experiments demonstrated that the charged, dihydropyridine Ca2+ channel blocker amlodipine had very potent neuroprotective activity in this system, compared with antioxidants and neutral Ca2+ channel blockers (nifedipine and nimodipine). Within its effective pharmacological range (10‐100 nM), amlodipine attenuated intracellular neuronal Ca2+ increases elicited by KCl depolarization but did not affect Ca2+ changes triggered by N‐methyl‐D‐aspartate receptor activation. Amlodipine also inhibited free radical‐induced damage to lipid constituents of the membrane in a dose‐dependent manner, independent of Ca2+ channel modulation. In parallel experiments, spontaneous neuronal apoptosis was inhibited in dose‐ and time‐dependent manners by antioxidants (U‐78439G, α‐tocopherol, and melatonin), nitric oxide synthase inhibitors (N‐nitro‐L‐arginine and N‐nitro‐D‐arginine), and a nitric oxide chelator (hemoglobin) in the micromolar range. These results suggest that spontaneous neuronal apoptosis is associated with excessive Ca2+ influx, leading to further intracellular Ca2+ increases and the generation of reactive oxygen species. Agents such as amlodipine that block voltage‐sensitive Ca2+ channels and inhibit cellular oxidative stress may be effective in the treatment of cerebrovascular stroke and neurodegenerative diseases associated with excessive apoptosis.
Novel Effects of FCCP [Carbonyl Cyanide p‐(Trifluoromethoxy)phenylhydrazone] on Amyloid Precursor Protein Processing
Authors: Connop Bruce P. , Thies Robert L. , Beyreuther Konrad , Ida Nobuo , Reiner Peter B.
Published: March 2003

Abstract: Amyloidogenic processing of the β‐amyloid precursor protein (APP) has been implicated in the pathology of Alzheimer’s disease. Because it has been suggested that catabolic processing of the APP holoprotein occurs in acidic intracellular compartments, we studied the effects of the protonophore carbonyl cyanide p‐(trifluoromethoxy)phenylhydrazone (FCCP) and the H+‐ATPase inhibitor bafilomycin A1 on APP catabolism in human embryonic kidney 293 cells expressing either wild‐type or “Swedish” mutant APP. Unlike bafilomycin A1, which inhibits β‐amyloid production in cells expressing mutant but not wild‐type APP, FCCP inhibited β‐amyloid production in both cell types. Moreover, the effects of FCCP were independent of alterations in total cellular APP levels or APP maturation, and the concentrations used did not alter either cellular ATP levels or cell viability. Bafilomycin A1, which had no effect on β‐amyloid production in wild‐type cells, inhibited endocytosis of fluorescent transferrin, whereas concentrations of FCCP that inhibited β‐amyloid production in these cells had no effect on endosomal function. Thus, in wild‐type‐expressing cells it appears that the β‐amyloid peptide is not produced in the classically defined endosome. Although bafilomycin A1 decreased β‐amyloid release from cells expressing mutant APP but not wild‐type APP, it altered lysosomal function in both cell types, suggesting that in normal cells β‐amyloid is not produced in the lysosome. Although inhibition of β‐amyloid production by bafilomycin A1 in mutant cells may occur via changes in endosomal/lysosomal pH, our data suggest that FCCP inhibits wild‐type β‐amyloid production by acting on a bafilomycin A1‐insensitive acidic compartment that is distinct from either the endosome or the lysosome.
Carboxyfullerene Prevents Iron‐Induced Oxidative Stress in Rat Brain
Authors: Lin Anya M. Y. , Chyi B. Y. , Wang S. D. , Yu H.‐H. , Kanakamma P. P. , Luh T.‐Y. , Chou C. K. , Ho L. T.
Published: March 2003

Abstract: Carboxyfullerene, a water‐soluble carboxylic acid derivative of a fullerene, was investigated as a protective agent against iron‐induced oxidative stress in the nigrostriatal dopaminergic system of anesthetized rats. Intranigral infusion of exclusive carboxyfullerene did not increase lipid peroxidation in substantia nigra or deplete dopamine content in striatum. Infusion of ferrous citrate (iron II) induced degeneration of the nigrostriatal dopaminergic system. An increase in lipid peroxidation in substantia nigra as well as decreases in K+‐evoked dopamine overflow and dopamine content in striatum were observed 7 days after the infusion. Co‐infusion of carboxyfullerene prevented iron‐induced oxidative injury. Furthermore, tyrosine hydroxylase‐immunoreactive staining showed that carboxyfullerene inhibited the iron‐induced loss of the dopaminergic nerve terminals in striatum. The antioxidative action of carboxyfullerene was verified by in vitro studies. Incubation of brain homogenates increased the formation of the Schiff base fluorescent products of malonaldehyde, an indicator of lipid peroxidation. Both autooxidation (without exogenous iron) and iron‐induced elevation of lipid peroxidation of brain homogenates were suppressed by carboxyfullerene in a dose‐dependent manner. Our results suggest that intranigral infusion of carboxyfullerene appears to be nontoxic to the nigrostriatal dopaminergic system. Furthermore, the potent antioxidative action of carboxyfullerene protects the nigrostriatal dopaminergic system from iron‐induced oxidative injury.
Targeting of Embryonic and Postnatal Autonomic and Enteric Neurons with a Vasoactive Intestinal Peptide Transgene
Authors: Waschek James A. , Bravo Dawn T. , Sena Matthew , Casillas Robert , Rodriguez Williams , Nguyen Thinh , Colburn Steve
Published: March 2003

Abstract : The neuropeptide vasoactive intestinal peptide (VIP) is expressed in several distinct sites in the CNS, in cholinergic and enteric ganglia, and in a small subpopulation of neurons within sympathetic ganglia. Previous studies on the human VIP gene indicate that transcription in neural crest‐derived neuroblastoma and pheochromocytoma cell lines is controlled in part by multiple regulatory elements located along 4.5 kb of upstream 5′ flanking sequence. In the current studies, transgenic mice were created with a chimeric gene consisting of 16.5 kb of the mouse VIP gene fused to the β‐galactosidase reporter. In situ hybridization analysis in adult mice indicated that reporter gene expression was correctly targeted to neurons in the esophagus, stomach, small intestine, and colon. No expression was observed in the brain, including regions that contain abundant VIP‐expressing cells, such as the thalamus, amygdala, cerebral cortex, hippocampus, and suprachiasmatic nucleus. Analysis of transgene expression in neonatal and embryonic day 13.5 mice revealed a near perfect correlation between VIP and β‐galactosidase gene expression in cranial cholinergic ganglia and the superior cervical ganglia, and lack of transgene expression in sensory ganglia and in nonneuronal tissue. Potential ectopic transgene expression was observed in neonates, in the cerebellar external granule layer and in a small subpopulation of neurons in the olfactory epithelium. We conclude that the 16.5 kb of VIP gene used in these studies contains sequences sufficient for directing expression specifically to VIP neurons in the PNS, and that sequences located elsewhere on the gene are required for proper CNS expression. The VIP gene sequences used here should be capable of targeting other gene products to specific populations of embryonic and adult peripheral neurons without causing significant expression in the CNS.
Pituitary Adenylate Cyclase‐Activating Polypeptide Regulation of Vasoactive Intestinal Polypeptide Transcription Requires Ca2+ Influx and Activation of the Serine/Threonine Phosphatase Calcineurin
Authors: Lee Hyeon‐Woo , Hahm Sung Ho , Hsu Chang‐Mei , Eiden Lee E.
Published: March 2003

Abstract : A > 15‐fold increase in vasoactive intestinal

polypeptide (VIP) mRNA and VIP peptide levels occurred in primary chromaffin

cells following exposure to the neurotrophic neuropeptide pituitary adenylate

cyclase‐activating polypeptide (PACAP)‐27 with an EC50 of ~2

nM. PACAP induction of VIP expression was blocked by methoxyverapamil or by a combination of nimodipine and ω‐conotoxin MVIIC, indicating a requirement for PACAP‐initiated calcium entry through voltage‐dependent calcium channels for regulation of VIP biosynthesis. Ascomycin, which inhibits calcineurin through formation of an ascomycin/FKBP12/calcineurin ternary complex, abolished the PACAP‐evoked increase in VIP expression, whereas rapamycin, which also binds to FKBP12 but does not cause inhibition of calcineurin, did not. Cyclosporin A, which inhibits calcineurin through formation of a cyclosporin A/cyclophilin/calcineurin complex, also abolished PACAP‐evoked VIP biosynthesis. These data indicate that PACAP regulates the expression of VIP via a signaling pathway that requires calcium influx and activation of calcineurin.
Spermine‐Induced Toxicity in Cerebellar Granule Neurons Is Independent of Its Actions at NMDA Receptors
Authors: Segal Jeff A. , Skolnick Phil
Published: March 2003

Abstract: The neurotoxic actions of polyamines such as spermine have been linked to their modulation of NMDA receptors, resulting in an excitotoxic cell death. Here, we demonstrate that chronic exposure to the polyamine spermine and acute exposure to the combination of spermine and glutamate result in significant toxicity to primary cultures of cerebellar granule neurons (CGNs). However, in both cases this cell death (a) lacks the characteristic cell swelling associated with the necrotic cell death induced by glutamate and (b) is characterized by the widespread formation of apoptotic nuclei. Whereas dizocilpine (MK‐801) blocks the synergistic cell death resulting from acute exposure to spermine plus glutamate, neither MK‐801 nor the calcium chelator EGTA appreciably attenuates CGN death resulting from chronic exposure to spermine. Consistent with previous reports, glutamate, both acute and chronic, causes CGN death that is characterized by cell swelling, sensitivity to MK‐801 and EGTA, and only small numbers of apoptotic nuclei. Spermine‐induced toxicity is not blocked by either the protein synthesis inhibitor cycloheximide or the pancaspase inhibitor tert‐butoxycarbonyl‐Asp‐(O‐methyl) fluoromethyl ketone. However, the antioxidant butylated hydroxyanisole is an effective blocker of spermine‐induced CGN death, suggesting a free‐radical component to this cell death. The intact spermine molecule, rather than a catabolic by‐product, is required for cell death because the amine oxidase inhibitors N1,N2‐bis(2,3‐butadienyl)‐1,4‐butanediamine and aminoguanidine fail to block this toxicity. Thus, in CGNs, spermine‐induced toxicity does not occur by its modulation of NMDA receptors, although, under some circumstances, NMDA receptor activation can modulate spermine‐induced toxicity.
In Situ Oxidative Catalysis by Neurofibrillary Tangles and Senile Plaques in Alzheimer’s Disease
Authors: Sayre Lawrence M. , Perry George , Harris Peggy L. R. , Liu Yahua , Schubert Kathryn A. , Smith Mark A.
Published: March 2003

Abstract: There is a great deal of evidence to support a pathogenic role of oxidative stress in Alzheimer’s disease (AD), but the sources of reactive oxygen species have not been directly demonstrated. In this study, using a novel in situ detection system, we show that neurofibrillary tangles and senile plaques are major sites for catalytic redox reactivity. Pretreatment with deferoxamine or diethylenetriaminepentaacetic acid abolishes the ability of the lesions to catalyze the H2O2‐dependent oxidation of 3,3′‐diaminobenzidine (DAB), strongly suggesting the involvement of associated transition metal ions. Indeed, following chelated removal of metals, incubation with iron or copper salts reestablished lesion‐dependent catalytic redox reactivity. Although DAB oxidation can also detect peroxidase activity, this was inactivated by H2O2 pretreatment before use of DAB, as shown by a specific peroxidase detection method. Model studies confirmed the ability of certain copper and iron coordination complexes to catalyze the H2O2‐dependent oxidation of DAB. Also, the microtubule‐associated protein τ, as an in vitro model for proteins relevant to AD pathology, was found capable of adventitious binding of copper and iron in a redox‐competent manner. Our findings suggest that neurofibrillary tangles and senile plaques contain redox‐active transition metals and may thereby exert prooxidant or possibly antioxidant activities, depending on the balance among cellular reductants and oxidants in the local microenvironment.
p27Kip 1 Expression by Contact Inhibition as a Prognostic Index of Human Glioma
Authors: Fuse Takahisa , Tanikawa Motoki , Nakanishi Makoto , Ikeda Kyoji , Tada Toyohiro , Inagaki Hiroshi , Asai Kiyofumi , Kato Taiji , Yamada Kazuo
Published: March 2003

Abstract: The clinical manifestations of human glioma are known to
be diverse, ranging from aggressive growth and invasion to apparent dormancy;
however, the molecular mechanism underlying this diversity has been largely
unexplored. In the present study, we characterized four human glioma cell
lines, T98G, A172, U251, and NAC6, each of which has distinct growth
properties. A172 and U251 cells continue to grow after confluency, whereas the
growth of T98G and NAC6 cells is contact inhibited. Northern and western blot
analyses revealed that at high cell density, the expression of p27Kip
1 cyclin‐dependent kinase inhibitor was dramatically enhanced at both
the RNA and the protein levels in T98G and NAC6 cells but not in A172 or U251.
These facts together with the finding that overexpression of p27Kip
1 caused G1 arrest in A172 and T98G cells suggest that the induction of
p27Kip 1 represents an important determinant of growth at high cell density. Immunohistochemical analyses of 42 primary gliomas revealed an inverse correlation between the level of p27 protein and the Ki‐67 proliferative index. Kaplan‐Meier plots demonstrated that a low level of p27 in tumors is associated with decreased overall survival. Thus, disrupted regulation of p27 expression at high cell density may play an important role in determining the clinical behavior of human gliomas as well as the prognosis for glioma patients.
Pretreatment with Calpain Inhibitor CEP‐4143 Inhibits Calpain I Activation and Cytoskeletal Degradation, Improves Neurological Function, and Enhances Axonal Survival After Traumatic Spinal Cord Injury
Authors: Schumacher P. A. , Siman R. G. , Fehlings M. G.
Published: March 2003

Abstract: The pathophysiology of traumatic spinal cord injury (SCI)
involves abnormal activation of the neutral cysteine protease calpain I (EC
3.4.22.17). In the present study we examined the effect of the calpain
inhibitor CEP‐4143 on cytoskeletal protection and neurological recovery after
SCI in adult rats. Microinjection of 50 mM CEP‐4143 into the T7
vertebral segment 10 min before a 35‐g clip compression injury resulted in
inhibition of calpain activation at 2 and 4 h postinjury, as determined by
western blotting for calpain I‐mediated spectrin degradation, and
significantly attenuated the degradation of dephosphorylated NF200
neurofilament protein at 4 and 8 h postinjury. To examine the in vivo chronic
neuroprotective effects of CEP‐4143, animals underwent microinjection with
saline or 50 mM CEP‐4143 10 min before injury, followed by weekly
blinded behavioral assessments for 6 weeks. Animals receiving CEP‐4143
treatment showed significant improvement over saline‐treated controls on the
Basso Beattie Bresnahan locomotor rating scale (p < 0.02) and
inclined plane test (p < 0.05). Counts of neurons in the red nucleus retrogradely labeled by fluorogold after introduction distal to the injury site were significantly higher in CEP‐4143‐treated animals. Finally, morphometric assessment of the injury site by computer‐assisted image analysis revealed significant tissue preservation in CEP‐4143‐treated animals. We conclude that the calpain antagonist CEP‐4143 exhibits biochemical, behavioral, and anatomical neuroprotection following traumatic SCI.
Nonlinear Decrease over Time in N‐Acetyl Aspartate Levels in the Absence of Neuronal Loss and Increases in Glutamine and Glucose in Transgenic Huntington's Disease Mice
Authors: Jenkins Bruce G. , Klivenyi Peter , Kustermann Ekkehard , Andreassen Ole A. , Ferrante Robert J. , Rosen Bruce R. , Beal M. Flint
Published: March 2003

Abstract: Mice transgenic for exon I of mutant huntingtin, with 141 CAG repeats, exhibit a profound symptomatology characterized by weight loss, motor disorders, and early death. We performed longitudinal analysis of metabolite levels in these mice using NMR spectroscopy in vivo and in vitro. These mice exhibited a large (53%), nonlinear drop in in vivo N‐acetyl aspartate (NAA) levels over time, commencing at ∼6 weeks of age, coincident with onset of symptoms. These drops in NAA levels occurred in the absence of neuronal death as measured by postmortem Nissl staining and neuronal counting but in the presence of nuclear inclusion bodies. In addition to decreased NAA, these mice showed a large elevation of glucose in the brain (600%) consistent with a diabetic profile and elevations in blood glucose levels both before and after glucose loading. In vitro NMR analysis revealed significant increases in glutamine (100%), taurine (95%) cholines (200%), and scyllo‐inositol (333%) and decreases in glutamate (24%) and succinate (47%). These results lead to two conclusions. NAA is reflective of the health of neurons and thus is a noninvasive marker, with a temporal progression similar to nuclear inclusion bodies and symptoms, of neuronal dysfunction in transgenic mice. Second, the presence of elevated glutamine is evidence of a profound metabolic defect. We present arguments that the elevated glutamine results from a decrease in neuronal‐glial glutamate‐glutamine cycling and a decrease in glutaminase activity.
Characterization of a Receptor for Insect Tachykinin‐Like Peptide Agonists by Functional Expression in a Stable Drosophila Schneider 2 Cell Line
Authors: Torfs Herbert , Shariatmadari Ramin , Guerrero Felix , Parmentier Marc , Poels Jeroen , Van Poyer Wendy , Swinnen Elfriede , De Loof Arnold , Åkerman Karl , Vanden Broeck Jozef
Published: March 2003

Abstract: STKR is an insect G protein‐coupled receptor, cloned from the stable fly Stomoxys calcitrans. It displays sequence similarity to vertebrate tachykinin [or neurokinin (NK)] receptors. Functional expression of the cloned STKR cDNA was obtained in cultured Drosophila melanogaster Schneider 2 (S2) cells. Insect tachykinin‐like peptides or “insectatachykinins,” such as Locusta tachykinin (Lom‐TK) III, produced dose‐dependent calcium responses in stably transfected S2‐STKR cells. Vertebrate tachykinins (or neurokinins) did not evoke any effect at concentrations up to 10‐5M, but an antagonist of mammalian neurokinin receptors, spantide II, inhibited the Lom‐TK III‐induced calcium response. Further analysis showed that the agonist‐induced intracellular release of calcium ions was not affected by pretreatment of the cells with pertussis toxin. The calcium rise was blocked by the phospholipase C inhibitor U73122. In addition, Lom‐TK III was shown to have a stimulatory effect on the accumulation of both inositol 1,4,5‐trisphosphate and cyclic AMP. These are the same second messengers that are induced in mammalian neurokinin‐dependent signaling processes.
Endogenous and Exogenous Fibroblast Growth Factor 2 Support Survival of Chick Retinal Neurons by Control of Neuronal Neuronal bcl‐xL and bcl‐2 Expression Through a Fibroblast Berowth Factor Receptor 1‐ and Erk‐Dependent Pathway
Authors: Désiré Laurent , Courtois Yves , Jeanny Jean‐Claude
Published: March 2003

Abstract : Fibroblast growth factor (FGF) 2 is a survival factor for

various cell types, including retinal neurons. However, little is understood

about the molecular bases of the neuroprotective role of FGF2 in the retina.

In this report, FGF2 survival activity was studied in chick retinal neurons

subjected to apoptosis by serum deprivation. Exogenous FGF2 supported neuronal

survival after serum deprivation and increased neuronal

bcl‐xL and bcl‐2 expression, through binding to

its receptor R1 (FGF‐R1), and subsequent extracellular signal‐regulated kinase

(ERK) activation. Endogenous FGF2 was transiently overexpressed after serum

deprivation. Its down‐regulation by antisense oligonucleotides and blockade of

its signaling pathway (binding to FGF‐R1, tyrosine phosphorylation, and ERK

inhibition) decreased bcl‐xL and bcl‐2 levels and

and enhanced apoptosis, suggesting that endogenous FGF2 supported neuronal

survival through a pathway similar to that of exogenous FGF2. This pathway may

serve to up‐regulate, or maintain, bcl‐xL and

bcl‐2 levels that normally decrease during the onset of apoptosis.

Indeed, long‐term ERK activation and high bcl‐xL levels

are necessary for the survival activity of both exogenous and endogenous FGF2.

Because FGF2 is upregulated following retinal injury in vivo, we suggest that

an injury‐stimulated autocrine/paracrine FGF2 loop may serve to maintain high

levels of survival proteins, such as Bcl‐xL, through ERK activation in retinal neurons.
HIV‐1 Coat Glycoprotein gp120 Induces Apoptosis in Rat Brain Neocortex by Deranging the Arachidonate Cascade in Favor of Prostanoids
Authors: Maccarrone Mauro , Bari Monica , Corasaniti M. Tiziana , Nisticó Robert , Bagetta Giacinto , Finazzi‐Agrò Alessandro
Published: March 2003

Abstract : Human immunodeficiency virus type‐1 coat glycoprotein gp

120 causes delayed programmed cell death (apoptosis) in rat brain neocortex.

Here, we investigated the possible role of the arachidonate cascade and

membrane peroxidation in this process. It is shown that gp 120 causes a rapid

increase in the activity and expression of the arachidonate‐metabolizing

enzyme prostaglandin H synthase, paralleled by increased prostaglandin

E2 levels. The selective inhibitor of prostaglandin H synthase

indomethacin inhibited enzyme activity, reduced prostaglandin E2

content, and partially protected neocortex against gp 120‐induced apoptosis.

Conversely, the activity and expression of the arachidonate‐metabolizing

enzyme 5‐lipoxygenase decreased upon gp 120 treatment, as well as the level of

its product, leukotriene B4. Treatment with gp 120 also reduced membrane lipid peroxidation, and this may be implicated in the execution of programmed cell death. These results suggest that early derangement of the arachidonate cascade in favor of prostanoids may be instrumental in the execution of delayed apoptosis in the brain neocortex of rats.
The Activation and Inhibition of Human Nicotinic Acetylcholine Receptor by RJR‐2403 Indicate a Selectivity for the α4β2 Receptor Subtype
Authors: Papke Roger L. , Webster J. Christopher , Lippiello Patrick M. , Bencherif Merouane , Francis Michael M.
Published: March 2003

Abstract : Human nicotinic acetylcholine (ACh) receptor subtypes

expressed in Xenopus oocytes were characterized in terms of their

activation by the experimental agonist RJR‐2403. Responses to RJR‐2403 were

compared with those evoked by ACh and nicotine. These agonists were also

characterized in terms of whether application of the drugs had the effect of

producing a residual inhibition that was manifest as a decrease in subsequent

control responses to ACh measured 5 min after the washout of the drug. For the

activation of α4β2 receptors, RJR‐2403 had an efficacy equivalent

to that of ACh and was more potent than ACh. RJR‐2403 was less efficacious

than ACh for other human receptor subtypes, suggesting that it is a partial

agonist for all these receptors. Nicotine activated peak currents in human

α4β2 and α3β2 receptors that were 85 and 50% of the

respective ACh maximum responses. Nicotine was an efficacious activator of

human α7 receptors, with a potency similar to ACh, whereas RJR‐2403 had

very low potency and efficacy for these receptors. At concentrations of <1

mM, RJR‐2403 did not produce any residual inhibition of subsequent

ACh responses for any receptor subtype. In contrast, nicotine produced

profound residual inhibition of human α4β2, α3β2, and

α7 receptors with IC50 values of 150, 200, and 150

μM, respectively. Co‐expression of the human α5 subunit with α3 and β2 subunits had the effect of producing protracted responses to ACh and increasing residual inhibition by ACh and nicotine but not RJR‐2403. In conclusion, our results, presented in the context of the complex pharmacology of nicotine for both activating and inhibiting neuronal nicotinic receptor subtypes, suggest that RJR‐2403 will be a potent and relatively selective activator of human α4β2 receptors.
Epidermal Growth Factor Induces Oxidative Neuronal Injury in Cortical Culture
Authors: Cha Yoo Kyung , Kim Yang‐Hee , Ahn Young‐Ho , Koh Jae‐Young
Published: March 2003

Abstract : Recently, we have demonstrated that certain neurotrophic factors can induce oxidative neuronal necrosis by acting at the cognate tyrosine kinase‐linked receptors. Epidermal growth factor (EGF) has neurotrophic effects via the tyrosine kinase‐linked EGF receptor (EGFR), but its neurotoxic potential has not been studied. Here, we examined this possibility in mouse cortical culture. Exposure of cortical cultures to 1‐100 ng/ml EGF induced gradually developing neuronal death, which was complete in 48‐72 h ; no injury to astrocytes was noted. Electron microscopic findings of EGF‐induced neuronal death were consistent with necrosis ; severe mitochondrial swelling and disruption of cytoplasmic membrane occurred, whereas nuclei appeared relatively intact. The EGF‐induced neuronal death was accompanied by increased free radical generation and blocked by the anti‐oxidant Trolox. Suggesting mediation by the EGFR, an EGFR tyrosine kinase‐specific inhibitor, C56, attenuated EGF‐induced neuronal death. In addition, inhibitors of extracellular signal‐regulated protein kinase 1/2 (Erk‐1/2) (PD98056), protein kinase A (H89), and protein kinase C (GF109203X) blocked EGF‐induced neuronal death. A p38 mitogen‐activated protein kinase inhibitor (SB203580) or glutamate antagonists (MK‐801 and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione) showed no protective effect. The present results suggest that prolonged activation of the EGFR may trigger oxidative neuronal injury in central neurons.
Differential Expression of the G Protein β5 Gene
Authors: Zhang Jian‐Hua , Lai Zhennan , Simonds William F.
Published: March 2003

Abstract : A neurally expressed heterotrimeric G protein β

subunit, Gβ5, has been found to exhibit functional

specialization with respect to its interactions with effector targets and

Gα subunits. A splice variant of Gβ5 that contains an

N‐terminal 42‐residue extension, Gβ5‐long, has been described

in the retina. To define better the potential range of its specialized

interactions, analysis of Gβ5 gene transcript and protein

expression in mouse brain and other tissues and cell lines was performed.

Quantification by ribonuclease protection assay of Gβ5

transcript expression in the developing brain demonstrates a fivefold increase

that occurs postnatally. Analysis of transcript expression by in situ

hybridization and ribonuclease protection assay indicates that the

Gβ5 gene is differentially expressed among multiple adult

mouse brain regions, including the motor and occipital cortex, the olfactory

bulb and associated rhinencephalic structures, hypothalamus, pontine cochlear

nuclei, and Purkinje cells in the cerebellum. Gβ5 is also

expressed in several cultured cell lines of neuroendocrine origin, including

murine αT3‐1 pituitary gonadotrophs and GT1‐7 hypothalamic cells, and

rat PC12 pheochromocytoma cells. Immunoblotting of tissue homogenates with

antibodies to two peptides common to Gβ5 and

Gβ5‐long confirmed expression of Gβ5 in

multiple brain regions and in spinal cord and expression of

Gβ5‐long in retina. Taken together, these results suggest that

the specialized molecular properties of Gβ5 have been adapted to diverse neural functions in the adult brain.