bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the 5′ flanking region of the rat D3 dopamine receptor gene
Authors: D'Souza Ursula M. , Wang Wei , Gao Da‐Qing , Kanda Seiji , Lee Gwang , Junn Eunsung , Hwang Cheol Kyu , Jose Pedro A. , Mouradian M. Maral
Published: March 2003

The D3 dopamine receptor has a restricted regional distribution in brain and is regulated by dopaminergic agents. Additionally, the D3 gene is implicated in the pathogenesis of several neuropsychiatric disorders or in their response to pharmacological agents. Elucidating its transcription control mechanisms is therefore of interest in order to explain these biological features of the D3 gene. In this study, the 5′ flanking region of the rat D3 gene was characterized by isolating the 5′ end of its cDNA as well as 4.6 kb of genomic sequence. Analysis of this region revealed the presence of two new exons 196‐bp and 120‐bp long, separated by an 855‐bp intron, located several kilobases upstream of the previously published coding exons. Thus, current evidence indicates that the rat D3 gene is organized into eight exons. Transcription initiation site was determined by primer extension analysis and repeated rounds of 5′ RACE and was found to localize at a pyrimidine‐rich consensus ‘initiator’ sequence, similar to the rat D2 gene. The D3 promoter lacks TATA or CAAT boxes but unlike that of other dopamine receptor genes has only 52% GC content. Functional analysis of D3 promoter deletion mutants fused to a reporter gene in TE671 cells, which endogenously express this gene, revealed strong transcriptional activity localized within 36 nucleotides upstream of transcription start site, and a potent silencer between bases −37 and −537. The D3 promoter is inactive in C6 and COS7 cells. We conclude that the D3 gene, similar to the closely related D2 gene, is transcribed from a tissue specific promoter which is under intense negative control.
Complement anaphylatoxin C5a neuroprotects through mitogen‐activated protein kinase‐dependent inhibition of caspase 3
Authors: Mukherjee Piali , Pasinetti Giulio Maria
Published: March 2003

We previously reported that pretreatment of murine cortico‐hippocampal neuronal cultures with the complement‐derived anaphylatoxin C5a, protects against glutamate neurotoxicity. In this study we explored the potential mechanisms involved in C5a‐mediated neuroprotection. We found that C5a neuroprotectsin vitrothrough inhibition of apoptotic death because pretreatment with human recombinant (hr)C5a prevented nuclear DNA fragmentation coincidental to inhibition of the pro‐apoptotic caspase 3 activity mediated by glutamate treatment. Also, hrC5a‐mediated responses appeared to be receptor‐mediated because pretreatment of cultures with the specific C5a receptor antagonist C177, prevented hrC5a‐mediated neuroprotection. Based on this evidence, we further explored possible signaling pathways involved in hrC5a inhibition of caspase 3 activation and apoptotic neuronal death. We found that treatment of cultures with the mitogen‐activated protein kinase (MAPK) pathway inhibitor PD98059 prevented hrC5a‐mediated inhibition of caspase 3 and apoptotic neuron death. MAPK pathways, whose activation by hrC5a is inhibited by PD98059 and C177, include the extracellular signal‐regulated kinase (ERK)2 and, to a lesser extent, ERK1. The study suggests that C5a may protect against glutamate‐induced apoptosis in neurons through MAPK‐mediated regulation of caspase cascades.
Differences in signal transduction pathways by which platelet‐derived and fibroblast growth factors activate extracellular signal‐regulated kinase in differentiating oligodendrocytes
Authors: Yim Sung Hye , Hammer Jeffrey A. , Quarles Richard H.
Published: March 2003

Treatment of cultured rat oligodendroglial progenitors with either platelet‐derived growth factor (PDGF) or fibroblast growth factor‐2 (FGF‐2) activated extracellular signal regulated kinase 2 (ERK2). Activation was transient in response to PDGF, whereas it was greater and more prolonged in response to FGF‐2. ERK2 activation by PDGF was preceded by a very rapid, robust and transient tyrosine phosphorylation of the PDGF receptor. Although there was consistently more activation of ERK2 in response to FGF‐2 than to PDGF, immunostaining of FGF receptors 1 (FGFR1) and 2 (FGFR2) and their tyrosine phosphorylation in progenitors was very weak, and both receptors were up‐regulated during differentiation to oligodendrocytes. Tyrosine phosphorylation of the FGF receptors was maximal from 15 to 60 min of treatment and was sustained for many hours. Binding of radioiodinated FGF‐2 to FGFR1 was predominant in progenitors, whereas binding to FGFR2 was predominant in oligodendrocytes. ERK2 activation by PDGF was more sensitive to inhibition of tyrosine kinases, whereas ERK2 activation by FGF‐2 was relatively more sensitive to inhibitors of protein kinase C. These differences in signal transduction pathways probably contribute to the different cellular responses of oligodendroglial lineage cells to PDGF and FGF‐2, respectively.
Overexpression of BETA2/NeuroD induces neurite outgrowth in F11 neuroblastoma cells
Authors: Cho Jang‐Hyeon , Kwon Il‐Sun , Kim Soyeon , Ghil Sung‐Ho , Tsai Ming‐Jer , Kim Yeon‐Soo , Lee Young‐Don , Suh‐Kim Haeyoung
Published: March 2003

BETA2/NeuroD, a basic helix‐loop‐helix (bHLH) transcription factor, has been shown to play important roles in the development of the nervous system and the maintenance and formation of pancreatic and enteroendocrine cells. The gain of function of BETA2/NeuroD in neurogenesis has been shown in Xenopus embryos. In this study, we investigated the neurogenic potential of BETA2/NeuroD using neuroblastoma cell line, F11, which could be induced to differentiate into neurons in the presence of cAMP. To induce or block the expression of BETA2/NeuroD, expression vectors for the full‐length and a C‐terminal deletion mutant of BETA2 were constructed and their transactivation potential was verified using reporter genes containing the insulin promoter sequences. Overexpression of BETA2 with full‐length construct induced neurite outgrowth in F11 cells in the absence of cAMP. In contrast, the C‐terminal deletion mutant, BETA2(1–233), which has dominant negative activity, inhibited neurite outgrowth induced by cAMP in F11 cells. These results indicate that BETA2/NeuroD plays an important role in terminal differentiation of neuroblastoma cells. They also imply that BETA2/NeuroD or related bHLH factors plays an essential role for differentiation of F11 neuroblastoma cells.
Up‐regulation of cyclooxygenase‐1 in neuroblastoma cell lines by retinoic acid and corticosteroids
Authors: Schneider Norbert , Lanz Stefan , Ramer Robert , Schaefer Dirk , Goppelt‐Struebe Margarete
Published: March 2003

Cyclooxygenases‐1 and ‐2 are both expressed in neuronal cells in vivo. In the neuroblastoma cell lines NG108 and N2a, however, only cyclooxygenase‐1 was detectable. Differentiation of the cells with retinoic acid increased cyclooxygenase‐1 mRNA and protein expression within 24 and 48 h, respectively. A further increase was observed when the cells were concomitantly treated with the glucocorticoid dexamethasone (a 2–3‐fold increase compared with retinoic acid alone). In the absence of retinoic acid, dexamethasone only slightly up‐regulated cyclooxygenase‐1 expression. The inhibitor of protein synthesis cycloheximide abrogated the effect of dexamethasone, indicating the involvement of newly synthesised proteins. Retinoic acid increased the transcription of cyclooxygenase‐1 mRNA, determined with a luciferase‐coupled promoter construct. Dexamethasone only slightly augmented cyclooxygenase‐1‐promoter activity but increased cyclooxygenase‐1 mRNA stability. Other corticosteroids, hydrocortisone and aldosterone, also up‐regulated cyclooxygenase‐1 whereas neurosteroids or oestrogen were ineffective. Up‐regulation was mediated primarily by the glucocorticoid receptor, because the receptor antagonist RU486 strongly reduced the effects of all corticosteroids. This indicated that in NG108 cells, the mineralocorticoid aldosterone may bind to the glucocorticoid receptor. Treatment of NG108 or N2a cells with corticosteroids did not alter the morphological phenotype obtained during differentiation. We thus show that corticosteroids, which down‐regulate cyclooxygenase expression in most cell types, up‐regulate cyclooxygenase‐1 during neuronal differentiation.
Visualizing differences in ligand‐induced β‐arrestin–GFP interactions and trafficking between three recently characterized G protein‐coupled receptors
Authors: Evans Nicholas A. , Groarke D. Alex , Warrack John , Greenwood Catherine J. , Dodgson Kathryn , Milligan Graeme , Wilson Shelagh
Published: March 2003

β‐Arrestin 1‐GFP or β‐arrestin 2‐GFP were coexpressed transiently with G protein‐coupled receptor kinase 2 within cells stably expressing the orexin‐1, apelin or melanin‐concentrating hormone (MCH), receptors. In response to agonist ligands both the orexin‐1 and apelin receptors were able to rapidly translocate both β‐arrestin 1‐GFP and β‐arrestin 2‐GFP from cytoplasm to the plasma membrane. For the MCH receptor this was only observed for β‐arrestin 2‐GFP. β‐Arrestin 1‐GFP translocated by the apelin receptor remained at the plasma membrane during prolonged exposure to ligand even though the receptor became internalized. By contrast, for the orexin‐1 receptor, internalization of β‐arrestin 1‐GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Co‐internalization of the orexin‐1 receptor was observed by monitoring the binding and trafficking of TAMRA‐(5‐ and 6‐carboxytetramethylrhodamine) labelled orexin‐A. Subsequent addition of an orexin‐1 receptor antagonist resulted in cessation of incorporation of β‐arrestin 1‐GFP into vesicles at the plasma membrane and a gradual clearance of β‐arrestin 1‐GFP from intracellular vesicles. For the melanin‐concentrating hormone receptor the bulk of translocated β‐arrestin 2‐GFP was maintained at concentrated foci close to, or at, the plasma membrane. These results demonstrate very distinct features of β‐arrestin–GFP interactions and trafficking for three G protein‐coupled receptors for which the natural ligands have only recently been identified and which were thus previously considered as orphan receptors.
Nerve growth factor induces P2X3 expression in sensory neurons
Authors: Ramer Matt S. , Bradbury Elizabeth J. , McMahon Stephen B.
Published: March 2003

Glial cell line‐derived neurotrophic factor (GDNF) and nerve growth factor (NGF) are neuroprotective for subpopulations of sensory neurons and thus are candidates for pain treatment. However, delivering these factors to damaged neurons will invariably result in undamaged systems also being treated, with possible consequences for sensory processing. In sensory neurons the purinergic receptor P2X3 is found predominantly in GDNF‐sensitive nociceptors. ATP signalling via the P2X3 receptor may contribute to pathological pain, suggesting an important role for this receptor in regulating nociceptive function. We therefore investigated the effects of intrathecal GDNF or NGF on P2X3 expression in adult rat spinal cord and dorsal root ganglia (DRG). In control spinal cords, P2X3 expression was restricted to a narrow band of primary afferent terminals within inner lamina II (IIi). Glial cell line‐derived neurotrophic factor treatment increased P2X3 immunoreactivity within lamina IIi but not elsewhere in the cord. Nerve growth factor treatment, however, induced novel P2X3 expression, with intense immunoreactivity in axons projecting to lamina I and outer lamina II and to the ventro‐medial afferent bundle beneath the central canal. In the normal DRG, we found a greater proportion of P2X3‐positive neurons at cervical levels, many of which were large‐diameter and calcitonin gene‐related peptide‐positive. In both cervical and lumbar DRG, the number of P2X3‐positive cells increased following GDNF or NGF treatment. De novo expression of P2X3 in NGF‐sensitive nociceptors may contribute to chronic inflammatory pain.
Effects of insulin‐like growth factor‐1 on retinal endothelial cell glucose transport and proliferation
Authors: DeBosch Brian J. , Baur Elisabeth , Deo Baljit K. , Hiraoka Miki , Kumagai Arno K.
Published: March 2003

Insulin‐like growth factor‐1 (IGF‐1) plays important roles in the developing and mature retina and in pathological states characterized by retinal neovascularization, such as diabetic retinopathy. The effects of IGF‐1 on glucose transport and proliferation and the signal transduction pathways underlying these effects were studied in a primary bovine retinal endothelial cell (BREC) culture model. IGF‐1 stimulated uptake of the glucose analog 2‐deoxyglucose in a dose‐dependent manner, with a maximal uptake at 25 ng/mL (3.3 nm) after 24 h. Increased transport occurred in the absence of an increase in total cellular GLUT1 transcript or protein. IGF‐1 stimulated activity of both protein kinase C (PKC) and phosphatidylinositol‐3 kinase (PI3 kinase), and both pathways were required for IGF‐1‐mediated BREC glucose transport and thymidine incorporation. Use of a selective inhibitor of the β isoform of PKC, LY379196, revealed that IGF‐1 stimulation of glucose transport was mediated by PKC‐β; however, inhibition of PKC‐β had no effect on BREC proliferation. Taken together, these data suggest that the actions of IGF‐1 in retinal endothelial cells couple proliferation with delivery of glucose, an essential metabolic substrate. The present studies extend our general understanding of the effects of IGF‐1 on vital cellular activities within the retina in normal physiology and in pathological states such as diabetic retinopathy.
Selective inhibition of β2‐adrenergic receptor‐mediated cAMP generation by activation of the P2Y2 receptor in mouse pineal gland tumor cells
Authors: Suh Byung‐Chang , Kim Jong‐So , Namgung Uk , Han Sung , Kim Kyong‐Tai
Published: March 2003

Rhythmic noradrenergic signaling from the hypothalamic clock in the suprachiasmatic nucleus to the pineal gland causes an increase in intracellular cAMP which regulates the circadian fluctuation of melatonin synthesis. The activation of phospholipase C (PLC)‐coupled P2Y2 receptors upon treatment with ATP and UTP exclusively inhibited the isoproterenol‐stimulated cAMP production in mouse pineal gland tumor cells. However, the activation of other PLC‐coupled receptors including P2Y1 and bombesin receptors had little or no effect on the isoproterenol‐stimulated cAMP production. Also, ATP did not inhibit cAMP production caused by forskolin, prostaglandin E2, or the adenosine analog NECA. These results suggest a selective coupling between signalings of P2Y2 and β2‐adrenergic receptors. The binding of [3H]CGP12177 to β2‐adrenergic receptors was not effected by the presence of ATP or UTP. Ionomycin decreased the isoproterenol‐stimulated cAMP production, whereas phorbol 12‐myristate 13‐acetate slightly potentiated the isoproterenol response. Chelation of intracellular Ca2+, however, had little effect on the ATP‐induced inhibition of cAMP production, while it completely reversed the ionomycin‐induced inhibition. Treatment of cells with pertussis toxin almost completely blocked the inhibitory effect of nucleotides. Pertussis toxin also inhibited the nucleotide‐induced increase in intracellular Ca2+ and inositol 1,4,5‐trisphosphate production by 30–40%, suggesting that the ATP‐mediated inhibition of the cAMP generation and the partial activation of PLC are mediated by pertussis toxin‐sensitive Gi‐protein. We conclude that one of the functions of P2Y2 receptors on the pineal gland is the selective inhibition of β‐adrenergic receptor‐mediated signaling pathways via the inhibitory G‐proteins.
Developmental changes in the expression of parkin and UbcR7, a parkin‐interacting and ubiquitin‐conjugating enzyme, in rat brain
Authors: Wang Mei , Suzuki Toshiaki , Kitada Tohru , Asakawa Shinichi , Minoshima Shinsei , Shimizu Nobuyoshi , Tanaka Keiji , Mizuno Yoshikuni , Hattori Nobutaka
Published: March 2003

Parkin is a product of the Park2 gene the mutation of which causes autosomal recessive juvenile parkinsonism (AR‐JP) characterized by selective dopaminergic neuronal death and absence of Lewy bodies. Recently we found that parkin is directly linked to the ubiquitin (Ub)‐proteasome pathway as a Ub‐protein ligase (E3) collaborating with a Ub‐conjugating enzyme (E2) UbcH7. Here we analysed by in situ hybridization the expression of mRNAs for parkin and UbcR7 (rat orthologue of human UbcH7) in the developing rat brain. Parkin mRNA increased in parallel with neuronal maturation, but was unevenly distributed in various brain regions after four postnatal days. The expression pattern of the UbcR7 mRNA was almost identical to that of the parkin mRNA in all cases examined. Both parkin and UbcR7 mRNAs were distributed in neurones but not glial cells. Our findings indicate that parkin is expressed not only in the substantia nigra, but also uniformly in various brain regions in a development‐dependent manner. Co‐expression of UbcR7 with parkin suggests that UbcR7 may interact with parkin in vivo for ubiquitination of yet unidentified target protein(s).
Attenuation of neurotoxicity in cortical cultures and hippocampal slices from E2F1 knockout mice
Authors: Gendron Tania F. , Mealing Geoff A. R. , Paris James , Lou Allison , Edwards Andrei , Hou Sheng T. , MacManus John P. , Hakim Antoine M. , Morley Paul
Published: March 2003

The E2F1 transcription factor modulates neuronal apoptosis induced by staurosporine, DNA damage and β‐amyloid. We demonstrate E2F1 involvement in neuronal death induced by the more physiological oxygen‐glucose deprivation (OGD) in mouse cortical cultures and by anoxia in mouse hippocampal slices. E2F1(+/+) and (−/−) cultures were comparable, in that they contained similar neuronal densities, responded with similar increases in intracellular calcium concentration ([Ca2+]i) to glutamate receptor agonists, and showed similar NMDA receptor subunit mRNA expression levels for NR1, NR2A and NR2B. Despite these similarities, E2F1(−/−) cultures were significantly less susceptible to neuronal death than E2F1(+/+) cultures 24 and 48 h following 120–180 min of OGD. Furthermore, the absence of E2F1 significantly improved the ability of CA1 neurons in hippocampal slices to recover synaptic transmission following a transient anoxic insult in vitro. These results, along with our finding that E2F1 mRNA levels are significantly increased following OGD, support a role for E2F1 in the modulation of OGD‐ and anoxia‐induced neuronal death. These findings are consistent with studies showing that overexpression of E2F1 in postmitotic neurons causes neuronal degeneration and the absence of E2F1 decreases infarct volume following cerebral ischemia.
Apoptotic signaling in dopamine‐induced cell death: the role of oxidative stress, p38 mitogen‐activated protein kinase, cytochrome c and caspases
Authors: Junn Eunsung , Mouradian M. Maral
Published: March 2003

Oxidative stress generated by dopamine (DA) oxidation could be one of the factors underlying the selective vulnerability of nigral dopaminergic neurons in Parkinson's diseases. Here we show that DA induces apoptosis in SH‐SY5Y neuroblastoma cells demonstrated by activation of caspase‐9 and caspase‐3, cleavage of poly(ADP‐ribose) polymerase as well as nuclear condensation. We also show that p38 mitogen‐activated protein kinase is activated within 10 min of DA treatment, which precedes the onset of apoptosis because the potent p38 kinase inhibitor SB203580 protects against DA‐induced cell death as well as against caspase‐9 and caspase‐3 activation. In addition, the antioxidant N‐acetyl‐l‐cysteine (NAC) effectively blocks DA‐induced p38 kinase activation, caspase‐9 and caspase‐3 cleavage and subsequent apoptosis, indicating that DA triggers apoptosis via a signaling pathway that is initiated by the generation of reactive oxygen species (ROS). Dopamine exerts its toxicity principally intracellularly as the DA uptake inhibitor, nomifensine significantly reduces DA‐induced cell death as well as activation of p38 kinase and caspase‐3. Furthermore, DA induces mitochondrial cytochrome c release, which is dependent on p38 kinase activation and precedes the cleavage of caspases. These observations indicate that DA induces apoptosis primarily by generating ROS, p38 kinase activation, cytochrome c release followed by caspase‐9 and caspase‐3 activation.
6‐Hydroxydopamine increases the hydroxylation and nitration of phenylalanine in vivo: implication of peroxynitrite formation
Authors: Ferger Boris , Themann Claudia , Rose Sarah , Halliwell Barry , Jenner Peter
Published: March 2003

In the present study, we investigated the effect of the dopaminergic neurotoxin 6‐hydroxydopamine (6‐OHDA) on hydroxyl free radical and peroxynitrite formation in vivo using d‐phenylalanine as a novel mechanistic probe. In vivo microdialysis was carried out in the striatum of freely moving male Wistar rats. The microdialysis probes were perfused with artificial cerebrospinal fluid containing 5 mm d‐phenylalanine (flow rate 2 µL/min). After obtaining a stable baseline 6‐OHDA was delivered into the striatum via reverse microdialysis for 60 min. HPLC measurements of the effluent were performed using photodiode array detection for determination of phenylalanine derived o‐tyrosine and m‐tyrosine (as hydroxylation markers) as well as of nitrotyrosine and nitrophenylalanine (as nitration markers). The basal levels of the hydroxylation derived products of phenylalanine were approximately 100‐fold higher than those of the nitration derived products. 6‐OHDA (0.1, 1, 10 mm) significantly increased o‐ and m‐tyrosine up to nine‐ and 13‐fold, respectively, whereas levels of 3‐nitrotyrosine and 4‐nitrophenylalanine were significantly increased up to 422‐ and 358‐fold, respectively. The results demonstrate that phenylalanine is a sensitive in vivo marker for 6‐OHDA‐induced hydroxylation and nitration reactions which are clearly concentration dependent. We conclude that peroxynitrite formation is involved in 6‐OHDA‐induced neurochemical effects.
Evidence that increases of mitochondrial immunoreactive IL‐1β by HIV‐1 gp120 implicate in situ cleavage of pro‐IL‐1β in the neocortex of rat
Authors: Corasaniti M. Tiziana , Turano Paola , Bilotta Anna , Malorni Walter , Stringaro A. Rita , Nisticò Robert , Finazzi‐Agró Alessandro , Bagetta Giacinto
Published: March 2003

Immunoelectron microscopy analysis of brain tissue sections and rat‐specific sandwich ELISA allowed the localization of interleukin‐1β (IL‐1β) immunoreactivity in the mitochondria and cytosol of neocortical tissue preparations from the brain of naive, untreated, rats and rats receiving a single daily injection into one lateral cerebral ventricle (i.c.v.) of bovine serum albumin (BSA; 100 ng/day) for seven consecutive days. Interestingly, seven days i.c.v. treatment with the HIV‐1 coat protein gp120 (100 ng/day) enhances IL‐1β immunoreactivity in the cellular fractions studied. Elevation of mitochondrial immunoreactive IL‐1β levels seems to originate from the conversion operated by the interleukin converting enzyme (ICE) of mitochondrial pro‐IL‐1β; in fact, IL‐1β increases reported in the ELISA experiments were paralleled by a decrease of the mitochondrial pro‐IL‐1β 31‐kDa band in conjunction with enhanced expression of the p20 component of activated ICE. In conclusion, the present results demonstrate that gp120‐enhanced neocortical expression of IL‐1β originates, at least in part, from in situ cleavage of mitochondrial pro‐IL‐1β and suggest that this, together with the central role of the mitochondrion in the expression of programmed cell death, may be important for apoptosis induced by the viral coat protein in the brain of rats.
Amyloid β proteins inhibit Cl−‐ATPase activity in cultured rat hippocampal neurons
Authors: Yagyu K. , Kitagawa K. , Irie T. , Wu B. , Zeng X. T. , Hattori N. , Inagaki C.
Published: March 2003

Cl−‐ATPase in the CNS is a candidate for an outwardly directed neuronal Cl− transporter requiring phosphatidylinositol‐4‐phosphate (PI4P) for its optimal activity. To test its pathophysiological changes in a phosphatidylinositol (PI) metabolism disorder, the effects of neurotoxic factors in Alzheimer's disease (AD), amyloid β proteins (Aβs), on the Cl−‐ATPase activity were examined using primary cultured rat hippocampal neurons. Amyloid β proteins (1–40, 1–42 and 25–35) concentration‐dependently (1–100 nm) and time‐dependently (from 1 h to 6 day) decreased Cl−‐ATPase activity and elevated intracellular Cl− concentrations ([Cl−]i), Aβ25–35 being the most potent. Addition of inositol or 8‐Br‐cyclic GMP completely reversed these Aβ‐induced changes. The recoveries in enzyme activity were attenuated by an inhibitor of PI 4‐kinase, 10 µm wortmannin or 20 µm quercetin, but not by a PI 3‐kinase inhibitor, 50 nm wortmannin or 10 µm LY294002. The PI, PIP and PIP2 levels of the plasma membrane‐rich fraction were lower in the Aβ‐treated cells as compared with each control. In the Aβ‐exposed culture, but not in control, stimulation by 10 µm glutamate for 10 min significantly increased fragmentation of DNA and decreased cell viability. Addition of inositol or 8‐Br‐cyclic GMP prevented the effect of Aβ‐treatment on the neurotoxicity of glutamate. Thus, Aβs reduce neuronal Cl−‐ATPase activity, resulting in an increase in [Cl−]i probably by lowering PI4P levels, and this may reflect a pre‐apoptotic condition in early pathophysiological profiles of AD.
The inhibition of phosphatidylinositol‐3‐kinase induces neurite retraction and activates GSK3
Authors: Sanchez S. , Sayas C. L. , Lim F. , Diaz‐Nido J. , Avila J. , Wandosell F.
Published: March 2003

It has been extensively described that neuronal differentiation involves the signalling through neurotrophin receptors to a Ras‐dependent mitogen‐activated protein kinase (MAPK) cascade. However, signalling pathways from other neuritogenic factors have not been well established. It has been reported that cAMP may activate protein kinase (PKA), and it has been shown that PKA‐mediated stimulation of MAPK pathway regulates not only neuritogenesis but also survival. However, extracellular regulated kinases (ERKs) mediated pathways are not sufficient to explain all the processes which occur in neuronal differentiation. Our present data show that: in cAMP‐mediated neuritogenesis, using the SH‐SY5Y human neuroblastoma cell line, there exists a link between the activation of PKA and stimulation of phosphatidylinositol 3‐kinase (PI3K). Both kinase activities are essential to the initial elongation steps. Surprisingly, this neuritogenic process appears to be independent of ERKs. While the activity of PI3K is essential for elongation and maintenance of neurites, its inhibition causes retraction. In this neurite retraction process, GSK3 is activated. Using both a pharmacological approach and gene transfer of a dominant negative form of GSK3, we conclude that this induced retraction is a GSK3‐dependent process which in turn appears to be a common target for transduction pathways involved in lysophosphatidic acid‐mediated and PI3K‐mediated neurite retraction.
Green tea polyphenol (–)‐epigallocatechin‐3‐gallate prevents N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐induced dopaminergic neurodegeneration
Authors: Levites Yona , Weinreb Orly , Maor Gila , Youdim Moussa B. H. , Mandel Silvia
Published: March 2003

In the present study we demonstrate neuroprotective property of green tea extract and (–)‐epigallocatechin‐3‐gallate in N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine mice model of Parkinson's disease. N‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine neurotoxin caused dopamine neuron loss in substantia nigra concomitant with a depletion in striatal dopamine and tyrosine hydroxylase protein levels. Pretreatment of mice with either green tea extract (0.5 and 1 mg/kg) or (–)‐epigallocatechin‐3‐gallate (2 and 10 mg/kg) prevented these effects. In addition, the neurotoxin caused an elevation in striatal antioxidant enzymes superoxide dismutase (240%) and catalase (165%) activities, both effects being prevented by (–)‐epigallocatechin‐3‐gallate. (–)‐Epigallocatechin‐3‐gallate itself also increased the activities of both enzymes in the brain. The neuroprotective effects are not likely to be caused by inhibition of N‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine conversion to its active metabolite 1‐methyl‐4‐phenylpyridinium by monoamine oxidase‐B, as both green tea and (–)‐epigallocatechin‐3‐gallate are very poor inhibitors of this enzyme in vitro (770 µg/mL and 660 µM, respectively). Brain penetrating property of polyphenols, as well as their antioxidant and iron‐chelating properties may make such compounds an important class of drugs to be developed for treatment of neurodegenerative diseases where oxidative stress has been implicated.
Differential activation of phospholipases by mitogenic EGF and neurogenic PDGF in immortalized hippocampal stem cell lines
Authors: Sung Jee Young , Lee Sang Yoon , Min Do Sik , Eom Tae Yeon , Ahn Young Soo , Choi Myung‐Un , Kwon Yunhee Kim , Chung Kwang Chul
Published: March 2003

In several neuronal systems, nerve growth factor (NGF) and platelet‐derived growth factor (PDGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogenic agent. Hippocampal stem cell lines (HiB5) immortalized by the expression of a temperature‐sensitive SV40 large T antigen also respond differentially to EGF and PDGF. While EGF treatment at the permissive temperature induces proliferation, the addition of PDGF induces differentiation at the non‐permissive temperature. However, the mechanism responsible for these different cellular fates has not been clearly elucidated. In order to clarify possible critical signaling events leading to these distinct cellular outcomes, we examined whether either EGF or PDGF differentially induces the activation of phospholipases, such as phospholipase A2(PLA2), C (PLC), or D (PLD). Although EGF stimulation did not induce phospholipases, PDGF caused a rapid and transient activation of PLC and PLD, but not PLA2. When the activation of PLC or PLD was blocked, the neurite outgrowth induced by PDGF was significantly inhibited. Although the activation of PLD occurred faster than PLC, blocking of PLD activity by transient expression of lipase‐inactive mutants did not inhibit the induction of PLC activity by PDGF. These results suggest that the differential activation of phospholipases may play an important role in signal transduction by mitogenic EGF and neurotrophic PDGF in HiB5 neuronal hippocampal stem cells. In particular, the activation of phospholipase C and D may contribute to neuronal differentiation by neurogenic PDGF in the HiB5 cells.
Early ethanolamine phospholipid translocation marks stress‐induced apoptotic cell death in oligodendroglial cells
Authors: Brand Annette , Yavin Ephraim
Published: March 2003

The consequences of H2O2/Fe2+‐induced oxidative stress on translocation of ethanolamine phosphoglyceride (EPG) and serine phosphoglyceride (SPG) were studied in an oligodendroglia‐like cell line (OLN 93) following 3 days of supplementation with 0.1 mm docosahexaenoic acid (DHA) and a series of polar head group precursors, including N‐monomethyl‐ and N,N‐dimethylethanolamine at millimolar concentrations. Added DHA was predominantly esterified in EPG species and those cells enriched in DHA showed enhanced sensitivity to oxidative stress and eventually died by apoptosis. Co‐supplements with ethanolamine and DHA resulted in a rapid, but transient, EPG translocation with a maximum at 30 min following stress, as characterized by a trinitrobenzenesulfonic acid reagent. There was no significant translocation of SPG as evidenced by annexin V binding. Unlike SPG, which is usually irreversibly translocated to subserve as a tag for phagocytosis, EPG acted as a signaling molecule with biphasic kinetic characteristics. N‐Monomethyl‐ and N,N‐dimethylethanolamine supplements reduced EPG synthesis, prevented its externalization and rescued cells from apoptotic death. Following stress, the fatty acid profile of the externalized EPG showed marked losses in polyunsaturated fatty acids and aldehydes compared with the remaining intracellular EPG. Prevention of EPG species selective translocation to the outer membrane leaflet by altering phospholipid asymmetry may be important in the mechanism of rescue from cell death.
Kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection
Authors: Guillemin Gilles J. , Kerr Stephen J. , Smythe George A. , Smith Danielle G. , Kapoor Vimal , Armati Patricia J. , Croitoru Juliana , Brew Bruce J.
Published: March 2003

There is good evidence that the kynurenine pathway (KP) and one of its products, quinolinic acid (QUIN), play a role in the pathogenesis of neurological diseases, in particular AIDS dementia complex. Although QUIN has been shown to be produced in neurotoxic concentrations by macrophages and microglia, the role of astrocytes in QUIN production is controversial. Using cytokine‐stimulated cultures of human astrocytes, we assayed key enzymes and products of the KP. We found that human astrocytes lack kynurenine hydroxylase so that large amounts of kynurenine and the QUIN antagonist kynurenic acid were produced. However, the amounts of QUIN that were synthesized were subsequently completely degraded. We then showed that kynurenine in concentrations comparable with those produced by astrocytes led to significant production of QUIN by macrophages. These results suggest that astrocytes alone are neuroprotective by minimizing QUIN production and maximizing synthesis of kynurenic acid. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes become indirectly neurotoxic by the production of large concentrations of kynurenine that can be secondarily metabolized by neighbouring or infiltrating monocytic cells to form the neurotoxin QUIN.
The catalytic domain of human neuropathy target esterase mediates an organophosphate‐sensitive ionic conductance across liposome membranes
Authors: Forshaw Philip J. , Atkins Jane , Ray David E. , Glynn Paul
Published: March 2003

In humans and other vertebrates, reaction of organophosphates with a neuronal membrane protein, neuropathy target esterase (NTE), initiates events which culminate in axonal degeneration. The initiation process appears to involve modification of a property of the protein distinct from its esterase activity, subsequent to formation of a negatively charged adduct with the active site serine residue. Here, we show that membrane patches from liposomes containing NEST, a recombinant hydrophobic polypeptide comprising the esterase domain of human NTE, display a transmembrane ionic conductance with both stable and high‐frequency flickering components. An asymmetric current–voltage relationship suggested that ion flow was favoured in one direction relative to the membrane and its associated NEST molecules. Flow of anions was slightly favoured compared with cations. The flickering current formed a much larger proportion of the overall conductance in patches containing wild‐type NEST compared with the catalytically inactive S966A mutant form of the protein. The conductance across patches containing NEST, but not those with the S966A mutant, was significantly reduced after adding neuropathic organophosphates to the bathing medium. By contrast, non‐neuropathic covalent inhibitors of the catalytic activity of NEST did not reduce NEST‐mediated conductance. Future work may establish whether NTE itself mediates an organophosphate‐sensitive ion flux across intracellular membranes within intact cells.
Iodoacetate protects hippocampal neurons against excitotoxic and oxidative injury: involvement of heat‐shock proteins and Bcl‐2
Authors: Guo Zhihong , Lee Jaewon , Lane Mark , Mattson Mark P.
Published: March 2003

Mild metabolic stress may increase resistance of neurons in the brain to subsequent, more severe insults, as demonstrated by the ability of ischemic pre‐conditioning and dietary restriction to protect neurons in experimental models of stroke‐ and age‐related neurodegenerative disorders. In the present study we employed iodoacetic acid (IAA), an inhibitor of glyceraldehyde‐3‐phosphate dehydrogenase, to test the hypothesis that inhibition of glycolysis can protect neurons. Pre‐treatment of cultured hippocampal neurons with IAA can protect them against cell death induced by glutamate, iron and trophic factor withdrawal. Surprisingly, protection occurred with concentrations of IAA (2–200 nm) much lower than those required to inhibit glycolysis. Pre‐treatment with IAA results in suppression of oxyradical production and stabilization of mitochondrial function in neurons after exposure to oxidative insults. Levels of the stress heat‐shock proteins HSP70 and HSP90, and of the anti‐apoptotic protein Bcl‐2, were increased in neurons exposed to IAA. Our data demonstrate that IAA can stimulate cytoprotective mechanisms within neurons, and suggest the possible use of IAA and related compounds in the prevention and/or treatment of neurodegenerative conditions.
Ceramide inhibits the outward potassium current in rat pinealocytes
Authors: Chik C. L. , Li B. , Karpinski E. , Ho A. K.
Published: March 2003

In the present study, we investigated the effect of ceramide on the outward K+ current in rat pinealocytes using whole cell and single channel recordings. Three components of the whole cell outward K+ current were separated, an iberiotoxin (IBTX)‐sensitive K+ current (IKCa), a transient A current (IA) and a delayed rectifier current (IK). C6‐ceramide reduced all three components of the outward K+ current. C6‐ceramide (30 µm) caused a 53% inhibition of IKCa[a component that is generated by the IBTX‐sensitive K+ channel (BK channel)], a 27% inhibition of IA and a 17% inhibition of IK. Additional studies showed that the BK channel was not inhibited by dihydroC6‐ceramide, the inactive analog of C6‐ceramide, but mimicked by sphingomyelinase which increased intracellular ceramide. The ceramide inhibition of the BK channel was only partly dependent on its inhibition of the L‐type Ca2+ channel. Studies using specific kinase inhibitors showed that calphostin C (a protein kinase C inhibitor) and to a lesser degree lavendustin A (a tyrosine kinase inhibitor) were effective in reducing the ceramide inhibition of IKCa. Taken together, our results show that, in rat pinealocytes, ceramide reduces the outward K+ current predominantly by inhibiting IKCa. Moreover, protein kinase C appears to be the main kinase involved in the ceramide inhibition of IKCa.
Human immunodeficiency virus type‐1 Tat protein induces nuclear factor (NF)‐κB activation and oxidative stress in microglial cultures by independent mechanisms
Authors: Nicolini Alessia , Ajmone‐Cat Maria Antonietta , Bernardo Antonietta , Levi Giulio , Minghetti Luisa
Published: March 2003

We have extended our previous findings and shown that human immunodeficiency virus Tat protein, in addition to nitric oxide (NO), stimulated rat microglial cultures to release pro‐inflammatory cytokine interleukin‐1β and tumour necrosis factor‐α in a nuclear factor (NF)‐κB‐dependent manner. At the same time, Tat stimulated the accumulation of free radicals, as indicated by the increased levels of isoprostane 8‐epi‐prostaglandin F2α (8‐epi‐PGF2α), a reliable marker of lipid peroxidation and oxidative stress, by a mechanism unrelated to NF‐κB activation. The presence of free radical scavengers abrogated Tat‐induced 8‐epi‐PGF2α accumulation without affecting NO and cytokine production. Consistently, Tat‐induced IκBα degradation – an index of NF‐κB activation – was not affected by free radical scavengers, but was prevented by an NF‐κB‐specific inhibitor. Our observations indicate that NF‐κB plays a key role in Tat‐dependent microglial activation, and that oxidative stress and NF‐κB activation induced by Tat occur by independent mechanisms.
Cysteine and mercapturate conjugates of oxidized dopamine are in human striatum but only the cysteine conjugate impedes dopamine trafficking in vitro and in vivo
Authors: Sidell Kathrin R. , Olson Sandra J. , Ou Joyce J. , Zhang Yueli , Amarnath Ventkataraman , Montine Thomas J.
Published: March 2003

Recent results have suggested that some products of mercapturic acid pathway (MAP) metabolism of oxidized dopamine (DA) may contribute to mesostriatal dopaminergic neurodegeneration, and that at least one product, 5‐S‐cysteinyldopamine (Cys‐DA), is elevated in patients with advanced Parkinson's disease (PD) who have been treated with l‐DOPA. Here we investigated MAP enzymes and products in the midbrain and striatum of control individuals and patients with dementia with Lewy bodies (DLB) who had less severe dopaminergic degeneration than PD patients and who were not treated with l‐DOPA. We also determined the biological activity of MAP metabolites of oxidized DA using primary rat mesencephalic cultures, rat cerebral synaptosomes, and rat striatum in vivo microdialysis. Our results showed that the human mesostriatal dopaminergic pathway generates Cys‐DA but has limited enzymatic capacity for mercapturate formation, that striatal levels of MAP products of oxidized DA are not elevated in DLB patients compared with controls, and that Cys‐DA interferes with trafficking of DA in vitro and in vivo. These results indicate that while Cys‐DA is not increased in striatum of patients with mild dopaminergic neurodegeneration, it may interfere with uptake of DA in patients with advanced PD.
Species‐scanning mutagenesis of the serotonin transporter reveals residues essential in selective, high‐affinity recognition of antidepressants
Authors: Mortensen Ole V. , Kristensen Anders S. , Wiborg Ove
Published: March 2003

The serotonin transporter (SERT) is a high‐affinity sodium/chloride‐dependent neurotransmitter transporter responsible for reuptake of serotonin from the extracellular space. SERT is a selective target of several clinically important antidepressants. In a cross‐species analysis comparing human and bovine SERTs, the kinetic parameters for serotonin uptake were found to be similar, however, the pharmacological profiles of the two transporters differ. Following transient expression in COS‐1 cells, IC50 values were determined for several antidepressants and psychostimulants. The potencies of the antidepressants citalopram, fluoxetine, paroxetine and imipramine were several‐fold higher at hSERT compared with bSERT. No species selectivity was observed for the antidepressants fluvoxamine, and sertraline or for the psychostimulants cocaine, the cocaine analogue β‐carbomethoxy‐3β‐(4‐iodophenyl)tropane, or for 3,4‐methylenedioxymethamphetamine (MDMA). Analysis of six hSERT/bSERT chimeras and subsequent species‐scanning mutagenesis of each isoform revealed methionine‐180, tyrosine‐495, and phenylalanine‐513 to be responsible for the increase in citalopram and paroxetine potencies at hSERT and methionine‐180 and phenylalanine‐513 to confer species selectivity at hSERT for fluoxetine and imipramine. Results were obtained by doing the forward, bovine to human, mutations and confirmed by doing the reverse mutations. Citalopram analogues were used to define the roles of methionine‐180, tyrosine‐495, and phenylalanine‐513 and to reveal molecular interactions with individual functional groups of citalopram. We suggest that methionine‐180 interacts with the heterocyclic nucleus of citalopram or stabilizes the binding pocket and phenylalanine‐513 to be a steric blocker of antidepressant recognition.
Glutathione depletion switches nitric oxide neurotrophic effects to cell death in midbrain cultures: implications for Parkinson's disease
Authors: Canals S. , Casarejos M. J. , De Bernardo S. , Rodríguez‐Martín E. , Mena M. A.
Published: March 2003

Nitric oxide (NO) exerts neurotrophic and neurotoxic effects on dopamine (DA) function in primary midbrain cultures. We investigate herein the role of glutathione (GSH) homeostasis in the neurotrophic effects of NO. Fetal midbrain cultures were pretreated with GSH synthesis inhibitor, l‐buthionine‐(S,R)‐sulfoximine (BSO), 24 h before the addition of NO donors (diethylamine/nitric oxide‐complexed sodium and S‐nitroso‐N‐acetylpenicillamine) at doses tested previously as neurotrophic. Under these conditions, the neurotrophic effects of NO disappeared and turned on highly toxic. Reduction of GSH levels to 50% of baseline induced cell death in response to neurotrophic doses of NO. Soluble guanylate cyclase (sGC) and cyclic GMP‐dependent protein kinase (PKG) inhibitors protected from cell death for up to 10 h after NO addition; the antioxidant ascorbic acid also protected from cell death but its efficacy decreased when it was added after NO treatment (40% protection 2 h after NO addition). The pattern of cell death was characterized by an increase in chromatin condensed cells with no DNA fragmentation and with breakdown of plasmatic membrane. The inhibition of RNA and protein synthesis and of caspase activity also protected from cell death. This study shows that alterations in GSH levels change the neurotrophic effects of NO in midbrain cultures into neurotoxic. Under these conditions, NO triggers a programmed cell death with markers of both apoptosis and necrosis characterized by an early step of free radicals production followed by a late requirement for signalling on the sGC/cGMP/PKG pathway.
Pro‐apoptotic effect of presenilin 2 (PS2) overexpression is associated with down‐regulation of Bcl‐2 in cultured neurons
Authors: Araki Wataru , Yuasa Katsutoshi , Takeda Shin'ichi , Takeda Kazuya , Shirotani Keiro , Takahashi Keikichi , Tabira Takeshi
Published: March 2003

Presenilin 2 (PS2) is a polytopic membrane protein that is mutated in some cases of familial Alzheimer's disease (AD). The normal functions of PS2 and its pathogenic role in AD remain unclear. We investigated the biological role of this protein in neurons, using adenovirus‐mediated transduction of the PS2 gene into rat primary cortical neurons. Immunocytochemical analyses demonstrated increased PS2 immunoreactivity in most neurons infected with recombinant adenoviruses expressing PS2. Neurons infected with wild‐type or mutant (N141I) PS2‐expressing adenoviruses showed a significant increase in basal cell death, compared with those infected with control β‐galactosidase‐expressing adenovirus. Moreover, PS2 overexpression markedly increased neuronal susceptibility to staurosporine‐induced apoptosis. Mutant PS2 was more effective in enhancing apoptosis than its wild‐type counterpart. Staurosporine‐induced death was significantly inhibited by a specific caspase 3 inhibitor. Western analyses revealed that Bcl‐2 protein expression was specifically down‐regulated in neurons overexpressing PS2, which temporally corresponded to the accumulation of C‐ and N‐terminal fragments of PS2. Additionally, expression of mutant, but not wild‐type PS2, increased the production of β‐amyloid protein (Aβ) 42. These data collectively suggest that the pro‐apoptotic effect of PS2 is mediated by down‐regulation of Bcl‐2. PS2 mutations may increase the susceptibility of neurons to apoptotic stimuli by perturbing the regulation of cell death.