bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the AT4 receptor in a human neuroblastoma cell line (SK‐N‐MC)
Authors: Mustafa Tomris , Chai Siew Yeen , Mendelsohn Frederick A. O. , Moeller Ingrid , Albiston Anthony L.
Published: March 2003

Angiotensin IV (Ang IV), the 3–8 fragment of angiotensin II (Ang II), binds to a distinct receptor designated the AT4 receptor. The peptide elicits a range of vascular and central actions including facilitation of memory retention and retrieval in several learning paradigms. The aim of this study was to characterize the AT4 receptor in a human cell line of neural origin. Receptor binding studies indicate that the human neuroblastoma cell line SK‐N‐MC cells express a high‐affinity Ang IV binding site with a pharmacological profile similar to the AT4 receptor: 125I]‐Ang IV and 125I]‐Nle1‐Ang IV bind specifically to the SK‐N‐MC cell membranes (Kd = 0.6 and 0.1 nm) in a saturable manner (Bmax= 1.2 pmol/mg of protein). AT4 receptor ligands, Nle1‐Ang IV, Ang IV and LVV‐haemorphin 7 (LVV‐H7), compete for the binding of [125I]‐Ang IV or [125I]‐Nle1‐Ang IV to the SK‐N‐MC cell membranes with rank order potencies of Nle1‐Ang IV > Ang IV > LVV‐H7 with IC50 values of 1.4, 8.7 and 59 nm ([125I]‐Ang IV) and 1.8, 20 and 168 nm ([125I]‐Nle1‐Ang IV), respectively. The binding of [125I]‐Ang IV or [125I]‐Nle1‐Ang IV to SK‐N‐MC cell membranes was not affected by the presence of GTPγS. Both Ang IV and LVV‐H7 stimulated DNA synthesis in this cell line up to 72 and 81% above control levels, respectively. The AT4 receptor in the SK‐N‐MC cells is a 180‐kDa glycoprotein; under non‐reducing conditions a 250‐kDa band was also observed. In summary, the human neuroblastoma cell line, SK‐N‐MC, expresses functional AT4 receptors that are responsive to Ang IV and LVV‐H7, as indicated by an increase in DNA synthesis. This is the first human cell line of neural origin shown to express the AT4 receptor.
Differentiation induces up‐regulation of plasma membrane Ca2+‐ATPase and concomitant increase in Ca2+ efflux in human neuroblastoma cell line IMR‐32
Authors: Usachev Yuriy M. , Toutenhoofd Sonja L. , Goellner Geoffrey M. , Strehler Emanuel E. , Thayer Stanley A.
Published: March 2003

Precise regulation of intracellular Ca2+ concentration ([Ca2+]i) is achieved by the coordinated function of Ca2+ channels and Ca2+ buffers. Neuronal differentiation induces up‐regulation of Ca2+ channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca2+‐ATPase (PMCA), the principal Ca2+ extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR‐32. [Ca2+]i was monitored in single cells using indo‐1 microfluorimetry. When the Ca2+‐ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca2+]i recovery after small depolarization‐induced Ca2+ loads was governed primarily by PMCAs. [Ca2+]i returned to baseline by a process described by a monoexponential function in undifferentiated cells (τ = 52 ± 4 s; n = 25). After differentiation for 12–16 days, the [Ca2+]i recovery rate increased by more than threefold (τ = 17 ± 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR‐32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up‐regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca2+ influx and efflux pathways may enable differentiated neurons to precisely localize Ca2+ signals in time and space.
Growth/differentiation factor 7 is preferentially expressed in the primary motor area of the monkey neocortex
Authors: Watakabe Akiya , Fujita Hiroshi , Hayashi Motoharu , Yamamori Tetsuo
Published: March 2003

We applied a differential display PCR technique to isolate molecules that are area‐specific in expression in the primate neocortex, and found that growth/differentiation factor 7 (GDF7), a member of the bone morphogenetic protein (BMP)/transforming growth factor (TGF) beta super‐family, is preferentially expressed in the primary motor area of African green monkeys (Cercopithecus aethiops). We proved that GDF7 is 10 times more abundant in the motor cortex than in the visual cortex by northern blotting and quantitative RT‐PCR. When we examined the neocortex of closely related rhesus monkeys (Macaca mulatta), GDF7 was also most abundant in the motor cortex, although the regional difference was reduced to 3‐fold. This differential expression pattern was observed in both newborn and infant rhesus monkeys. We found that several type I/II receptors of BMP, candidates of the receptors for GDF7, are uniformly expressed in the mature neocortex. The unique expression pattern of GDF7 suggests that it may play an active role in the motor area of the primate neocortex.
Prion protein with an E200K mutation displays properties similar to those of the cellular isoform PrPC
Authors: Rosenmann H. , Talmor G. , Halimi M. , Yanai A. , Gabizon R. , Meiner Z.
Published: March 2003

Creutzfeldt–Jakob disease (CJD) in Libyan Jews, linked to the E200K mutation in PRNP (E200KCJD), is the most prevalent of the inherited prion diseases. As other prion diseases, E200KCJD is characterized by the brain accumulation of PrPSc, a pathologic conformational isoform of a normal glycoprotein denominated PrPC. To investigate whether the E200K mutation is enough to de novo confer PrPSc properties to mutant PrP, as suggested by experiments in Chinese hamster ovary cells, we examined the biochemical behavior of E200KPrP in brains and fibroblasts from sporadic as well as homozygous and heterozygous E200KCJD patients, asymptomatic transgenic mice carrying the E200K mutation, as well as in normal and scrapie‐infected mouse neuroblastoma cells expressing E200KPrP. E200KPrP was examined for protease sensitivity, solubility in detergents, releasibility by phosphoinositol phospholypase‐C and localization in cholesterol enriched membrane microdomains (rafts). In all tissues except in brains of CJD patients and ScN2a cells, E200KPrP displayed properties similar to those of PrPC. Our results indicate that the E200K mutation does not automatically convey the properties of PrPSc to new PrP molecules. A conversion process occurs mainly in the prion disease affected brain, suggesting the presence of a tissue‐specific or age‐dependent factor, in accord with the late onset nature of inherited CJD.
Tamoxifen inhibits nitrotyrosine formation after reversible middle cerebral artery occlusion in the rat
Authors: Osuka Koji , Feustel Paul J. , Mongin Alexander A. , Tranmer Bruce I. , Kimelberg Harold K.
Published: March 2003

Tamoxifen (TAM), a widely used non‐steroidal anti‐estrogen, has recently been shown to be neuroprotective in a rat model of reversible middle cerebral artery occlusion (rMCAo). Tamoxifen has several potential mechanisms of action including inhibition of the release of excitatory amino acids (EAA) and nitric oxide synthase (NOS) activity. The question addressed in this study was whether TAM reduces ischemia‐induced production of nitrotyrosine, considered as a footprint of the product of nitric oxide and superoxide, peroxynitrite. In rat brain, 2 h rMCAo produced a time‐dependent increase in nitrotyrosine content in the cerebral cortex, as measured by Western blot analysis. Compared with vehicle, TAM significantly reduced nitrotyrosine levels in the ischemic cortex at 24 h. The neuronal (n)NOS inhibitor, 7‐nitroindazole also tended to reduce nitrotyrosine, but this reduction was not statistically significant. Immunostaining for nitrotyrosine was seen in cortical neurons in the MCA territory and this immunostaining was reduced by TAM. In vitro, TAM and the calmodulin inhibitor trifluoperazine inhibited, with similar EC50 values, the activity of recombinant nNOS as well as NOS activity in brain homogenates, measured by conversion of [3H]arginine to [3H]citrulline. There was marginal inhibition of recombinant inducible (i)NOS activity up to 100 µm TAM. These data suggest that TAM is an effective inhibitor of Ca2+/calmodulin‐dependent NOS and the derived peroxynitrite production in transient focal cerebral ischemia and this may be one mechanism for its neuroprotective effect following rMCAo.
Cellular defenses against excitotoxic insults
Authors: Sapolsky Robert M.
Published: March 2003

The cellular events mediating necrotic neuron death are now reasonably well understood, and involve excessive extracellular accumulation of glutamate and free cytosolic calcium. When such necrotic neurological insults occur, neurons are not passively buffeted, but instead mobilize a variety of defenses in an attempt to decrease the likelihood of neuron death, or to decrease the harm to neighboring neurons (by decreasing the likelihood of inflammation). This review considers some of these defenses, organizing them along the lines of those which decrease neuronal excitability, decrease extracellular glutamate accumulation, decrease cytosolic calcium mobilization, decrease calcium‐dependent degenerative events, enhance neuronal energetics, and bias a neuron towards apoptotic, rather than necrotic, death. Although these are currently perceived as a disparate array of cellular adaptations, some experimental approaches are suggested that may help form a more unified subdiscipline of cellular defenses against neurological insults. Such an advance would help pave the way for the rational design of therapeutic interventions against necrotic insults.
Direct binding of β‐arrestins to two distinct intracellular domains of the δ opioid receptor
Authors: Cen Bo , Yu Qingming , Guo Jun , Wu Yalan , Ling Kun , Cheng Zhijie , Ma Lan , Pei Gang
Published: March 2003

β‐Arrestins regulate opioid receptor‐mediated signal transduction and play an important role in opiate‐induced analgesia and tolerance/dependence. This study was carried out to measure the direct interaction between β‐arrestins and opioid receptor. Immunoprecipitation experiments demonstrated that β‐arrestin 1 physically interacts with delta opioid receptor (DOR) co‐expressed in human embryonic kidney 293 cells in an agonist‐enhanced manner and truncation of the carboxyl terminus of DOR partially impairs the interaction. In vitro data from glutathione‐S‐transferase pull‐down assay showed that the carboxyl terminus (CT) and the third intracellular loop (I3L) of DOR are both capable of and either domain is sufficient for binding to β‐arrestin 1 and 2. Surface plasmon resonance determination further revealed that binding of CT and I3L of DOR to β‐arrestin is additive, suggesting these two domains bind at distinctly different sites on β‐arrestin without considerable spatial hindrance. This study demonstrated for the first time the direct binding of β‐arrestins to the two distinct domains, the carboxyl terminus and the third intracellular loop, of DOR.
Activation of nuclear transcription factor kappa B (NF‐κB) is essential for dopamine‐induced apoptosis in PC12 cells
Authors: Panet Hana , Barzilai Ari , Daily Dvora , Melamed Eldad , Offen Daniel
Published: March 2003

The etiology of Parkinson's disease is still unknown, though current investigations support the notion of the pivotal involvement of oxidative stress in the process of neurodegeneration in the substantia nigra (SN). In the present study, we investigated the molecular mechanisms underlying cellular response to a challenge by dopamine, one of the local oxidative stressors in the SN. Based on studies showing that nuclear factor kappa B (NF‐κB) is activated by oxidative stress, we studied the involvement of NF‐κB in the toxicity of PC12 cells following dopamine exposure. We found that dopamine (0.1–0.5 m m) treatment increased the phosphorylation of the IκB protein, the inhibitory subunit of NF‐κB in the cytoplasm. Immunoblot analysis demonstrated the presence of NF‐κB‐p65 protein in the nuclear fraction and its disappearance from the cytoplasmic fraction after 2 h of dopamine exposure. Dopamine‐induced NF‐κB activation was also evidenced by electromobility shift assay using radioactive labeled NF‐κB consensus DNA sequence. Cell‐permeable NF‐κB inhibitor SN‐50 rescued the cells from dopamine‐induced apoptosis and showed the importance of NF‐κB activation to the induction of apoptosis. Furthermore, flow cytometry assay demonstrated a higher level of translocated NF‐κB‐p65 in the apoptotic nuclei than in the unaffected nuclei. In conclusion, our findings suggest that NF‐κB activation is essential to dopamine‐induced apoptosis in PC12 cells and it may be involved in nigral neurodegeneration in patients with Parkinson's disease.
Effects of R‐ and S‐apomorphine on MPTP‐induced nigro‐striatal dopamine neuronal loss
Authors: Grünblatt Edna , Mandel Silvia , Maor Gila , Youdim Moussa B. H.
Published: March 2003

In order to establish whether the antioxidant and iron‐chelating activities of R‐apomorphine (R‐APO), a D1‐D2 receptor agonist, may contribute to its neuroprotective property, its S‐isomer, which is not a dopamine agonist, was studied. The neuroprotective property of R‐ and S‐APO has been studied in the MPTP model of Parkinson's disease (PD). Both S‐APO (0.5–1 mg/kg, subcutaneous) and R‐APO (10 mg/kg) pretreatment of C57‐BL mice, protected against MPTP (24 mg/kg, intraperitoneally) induced dopamine (DA) depletion and reduction in tyrosine hydroxylase (TH) activity. However, only R‐APO prevented nigro‐striatal neuronal cell degeneration, as indicated by the immunohistochemistry of TH positive neurones in substantia nigra and by western analysis of striatal TH content. R‐APO prevented the reduction of striatal‐GSH and the increase in the ratio of GSSG over total glutathione, caused by MPTP treatment. In vitro both R‐APO and S‐APO inhibited monoamine oxidase A and B activity at relatively high concentrations (100 and 300 µmol/L, respectively). The elevated activity of TH induced by the two enantiomers may contribute to the maintenance of normal DA levels, suggesting that one of the targets of these molecules may involve upregulation of TH activity. It is suggested that the antioxidant and iron‐chelating properties, possible monoamine oxidase inhibitory actions, together with activation of DA receptors, may participate in the mechanism of neuroprotection by APO enantiomers against MPTP.
Developmental expression of metabotropic P2Y1 and P2Y2 receptors in freshly isolated astrocytes from rat hippocampus
Authors: Zhu Yingzi , Kimelberg Harold K.
Published: March 2003

There are at least three subtypes of cloned metabotropic P2 receptors linked to intracellular Ca2+ rises in rat brain cells, namely, P2Y1, P2Y2 and P2Y4. In this study we explore the subtypes of the metabotropic P2 receptors seen in freshly isolated astrocytes (FIAs) from P8‐P25 rats. We found by single cell RT‐PCR that in process‐bearing FIAs from hippocampi of P8‐P12 rats, 31% of the glial fibrillary acidic protein (GFAP) mRNA (+) cells expressed P2Y1 mRNA while only 5% of the cells tested expressed P2Y2 mRNA. The expression of P2Y1 receptor mRNA was not changed in FIAs from the hippocampi of P18‐P25 rats, but 38% of the GFAP mRNA (+) cells in the P18‐P25 age group then showed P2Y2 mRNA. We also studied whether the mRNA was expressing functional receptor protein by measuring Ca2+ responses to specific agonists for P2Y1 and P2Y2. We found that similar proportions of GFAP mRNA (+) FIAs responded to ATP or UTP as showed mRNAs for P2Y 1 and P2Y2, respectively. Total tissue RNA from P9 and P24 rat hippocampus showed a 2.8‐fold increase in P2Y2 mRNA levels from P9 to P24 with a decrease in P2Y1 mRNA. Thus, this study shows a marked up‐regulation of mRNA for P2Y2 from 9 to 24 days in rat hippocampus, and some of this increase is likely due to the protoplasmic astrocytes which is being translated into functional receptor protein in these cells.
Protection of intracellular dopamine cytotoxicity by dopamine disposition and metabolism factors
Authors: Weingarten Paul , Zhou Qun‐Yong
Published: March 2003

Dopamine has been hypothesized as a contributing factor for the selective degeneration of dopaminergic neurons in Parkinson's disease. However, the cytotoxic mechanisms of dopamine and its metabolites remain poorly understood. Using a stable aromatic amino acid decarboxylase (AADC) expressing a fibroblast cell line, we previously demonstrated a novel, non‐oxidative cytotoxicity of intracellular dopamine. In this study, we further investigate the roles of dopamine metabolism and disposition proteins against intracellular dopamine cytotoxicity by co‐expressing these factors in AADC‐expressing cells. Our results indicate that overexpression of the vesicular monoamine transporter and monoamine oxidase A‐induced protection against intracellular dopamine toxicity, and conversely that pharmacological inhibition of these pathways potentiated l‐DOPA toxicity in catecholaminergic PC12 cells. Macrophage migration inhibitory factor and glutathione S‐transferase (GST), factors that have recently been shown to be involved in dopamine metabolism, also exhibited a strong protective role against intracellular dopamine cytotoxicity. Our results support a potential role for non‐oxidative cytoplasmic dopamine toxicity, and imply that disruption in dopamine disposition and/or metabolism could underlie the progressive degeneration of dopaminergic neurons in Parkinson's disease.
A unique spacer domain of synaptotagmin IV is essential for Golgi localization
Authors: Fukuda Mitsunori , Ibata Keiji , Mikoshiba Katsuhiko
Published: March 2003

Synaptotagmin (Syt) family members consist of six separate domains: a short amino terminus, a single transmembrane domain, a spacer domain, a C2A domain, a C2B domain and a short carboxyl (C) terminus. Despite sharing the same domain structures, several synaptotagmin isoforms show distinct subcellular localization. Syt IV is mainly localized at the Golgi, while Syt I, a possible Ca2+‐sensor for secretory vesicles, is localized at dense‐core vesicles and synaptic‐like microvesicles in PC12 cells. In this study, we sought to identify the region responsible for the Golgi localization of Syt IV by immunocytochemical and biochemical analyses as a means of defining the distinct subcellular localization of the synaptotagmin family. We found that the unique C‐terminus of the spacer domain (amino acid residues 73–144) between the transmembrane domain and the C2A domain is essential for the Golgi localization of Syt IV. In addition, the short C‐terminus is probably involved in proper folding of the protein, especially the C2B domain. Without the C‐terminus, Syt IVΔC proteins are not targeted to the Golgi and seem to colocalize with an endoplasmic reticulum (ER) marker (i.e. induce crystalloid ER‐like structures). On the basis of these results, we propose that the divergent spacer domain among synaptotagmin isoforms may contain certain signals that determine the final destination of each isoform.
Involvement of calcium–calmodulin protein kinase but not mitogen‐activated protein kinase in light‐induced phase delays and Per gene expression in the suprachiasmatic nucleus of the hamster
Authors: Yokota Shin‐ichi , Yamamoto Masakazu , Moriya Takahiro , Akiyama Masashi , Fukunaga Kohji , Miyamoto Eishichi , Shibata Shigenobu
Published: March 2003

It is known that Ca2+‐dependent phosphorylation of cAMP response element binding protein (CREB) and the rapid induction of mPer1 and mPer2, mouse period genes in the suprachiasmatic nucleus (SCN) are associated with light‐induced phase shifting. The CREB/CRE transcriptional pathway has been shown to be activated by calcium/calmodulin dependent kinase II (CaMKII) and mitogen‐activated protein kinase (MAPK); however, there is a lack of evidence concerning whether the activation of CaMKII and/or MAPK elicited by photic stimuli are associated with the change in Per gene expression and behavioral phase shifting. In this experiment, we found there was an inhibitory effect by KN93, CaMKII inhibitor, on hamster Per1 and Per2 expression in the SCN and on phase delays in wheel running rhythm induced by light pulses. PD98059 and U0126, MAPK kinase inhibitors, however, affected neither light‐induced Per1 and Per2 expression nor behavioral phase delays, even though PD98059 attenuated the light‐induced phosphorylation of MAPK in the SCN. The present findings demonstrate that the light‐induced activation of CaMKII plays an important role in the induction of Per1 and Per2 mRNA in the hamster SCN as well as phase shifting. These results suggest that gated induction of Per1 and/or Per2 genes through CaMKII‐CREB/CRE accompanied with photic stimuli may be a critical step in phase shifting.
Inhibition of Ras extracellular‐signal‐regulated kinase (ERK) mediated signaling promotes ciliary neurotrophic factor (CNTF) expression in Schwann cells
Authors: Abe Koji , Namikawa Kazuhiko , Honma Masaru , Iwata Tatsuya , Matsuoka Ichiro , Watabe Kazuhiko , Kiyama Hiroshi
Published: March 2003

Ciliary neurotrophic factor (CNTF) can prevent injury‐induced motor neuron death. However, it is also evident that expression of CNTF in Schwann cells is suppressed during nerve regeneration. In this report, we have addressed the mechanism underlying the down‐regulation of CNTF expression in injured nerves using a mouse Schwann cell line IMS32 and mouse sciatic nerve. In IMS32 cells, activation of the Ras extracellular‐signal‐regulated kinase (ERK) pathway by adenoviral vector‐mediated expression of dominant active MEK1 did not alter a basal level of CNTF expression, whereas inhibition of the Ras‐ERK pathway by using adenoviral vectors resulted in a marked increase in CNTF expression. This inverse relation between before and after axotomy was also observed in mouse sciatic nerve. In the axotomized sciatic nerve, the phosphorylated ERK was markedly increased; in contrast, the expression of CNTF was markedly decreased. These findings suggest that an inactive state of ERK is crucial for the CNTF expression in Schwann cells, and that activation of ERK following nerve injury critically influences the expression of CNTF. This might well explain why CNTF is highly expressed in quiescent Schwann cells in the peripheral nervous system, and also why CNTF is not abundant in axotomized nerves or cultured Schwann cells in which the proliferation signal is obviously active.
Caspase‐2 activation is redundant during seizure‐induced neuronal death
Authors: Henshall David C. , Skradski Shana L. , Bonislawski David P. , Lan Jing‐Quan , Simon Roger P.
Published: March 2003

Seizure‐induced neuronal death may be under the control of the caspase family of cell death proteases. We examined the role of caspase‐2 in a model of focally evoked limbic seizures with continuous EEG recording. Seizures were elicited by microinjection of kainic acid into the amygdala of the rat and terminated after 40 min by diazepam. Caspase‐2 was constitutively present in brain, mostly within neurons, and was detected in both cytoplasm and nucleus. Cleaved caspase‐2 (12 kDa) was detected immediately following seizure termination within injured ipsilateral hippocampus, contiguous with increased Val‐Asp‐Val‐Ala‐Asp (VDVADase) activity, a putative measure of activated caspase‐2. Expression of receptor interacting protein (RIP)‐associated Ich‐1‐homologous protein with death domain (RAIDD) was increased following seizures, whereas expression of RIP and tumor necrosis factor receptor associated protein with death domain (TRADD), other components thought to be linked to the caspase‐2 activation and signaling mechanism, were unchanged. Intracerebroventricular administration of z‐VDVAD‐fluoromethyl ketone blocked seizure‐induced caspase‐2 activity but did not alter caspase‐8 activity and failed to affect DNA fragmentation or neuronal death. These data support activation of caspase‐2 following seizures but suggest that parallel caspase pathways may circumvent deficits in caspase‐2 function to complete the cell death process.
Amyloid β peptides mediate hypoxic augmentation of Ca2+ channels
Authors: Green Kim N. , Peers Chris
Published: March 2003

Clinical studies indicate that neurodegeneration caused by Alzheimer's amyloid β peptide (AβP) formation can be triggered or induced by prolonged (chronic) hypoxia. Here, we demonstrate that 24‐h culture of PC12 cells in 10% O2 leads to induction of a Cd2+‐resistant Ca2+ influx pathway and selective potentiation of l‐type Ca2+ current. Both effects were suppressed or prevented by a monoclonal antibody raised against the N′‐terminus of AβP, and were fully mimicked by AβP1−40 and AβP1−42, but not by AβP40−1. Potentiation of l‐type currents was also induced by exposure to AβP25−35. Our results indicate that hypoxia induces enhancement of Ca2+ channels, which is mediated by increased AβP formation.
Evidence against an essential role of endogenous brain dopamine in methamphetamine‐induced dopaminergic neurotoxicity
Authors: Yuan Jie , Callahan Brian T. , McCann Una D. , Ricaurte George A.
Published: March 2003

The present studies examined the role of endogenous dopamine (DA) in methamphetamine (METH)‐induced dopaminergic neurotoxicity while controlling for temperature‐related neuroprotective effects of the test compounds, reserpine and α‐methyl‐p‐tyrosine (AMPT). To determine if the vesicular pool of DA was essential for the expression of METH‐induced DA neurotoxicity, reserpine (3 mg/kg, given iintraperitoneally 24–26 h prior to METH) was given prior to a toxic dose regimen of METH. Despite severe striatal DA deficits during the period of METH exposure, mice treated with reserpine prior to METH developed long‐term reductions in striatal DA axonal markers, suggesting that vesicular DA stores were not crucial for the development of METH neurotoxicity, but leaving open the possibility that cytoplasmic DA might be involved. To evaluate this possibility, cytoplasmic DA stores were depleted with AMPT prior to METH administration. When this study was carried out at 28°C, complete neuroprotection was observed, likely due to lingering effects on core temperature because when the same study was repeated at 33°C (to eliminate AMPT's hypothermic effect in METH‐treated animals), the previously observed neuroprotection was no longer evident. In the third and final set of experiments, mice were pretreated with a combination of reserpine and AMPT, to deplete both vesicular and cytoplasmic DA pools, and to reduce striatal DA levels to negligible values during the period of METH administration (< 0.05%). When core temperature differences were eliminated by raising ambient temperature, METH‐induced DA neurotoxic changes were evident in mice pretreated with reserpine and AMPT. Collectively, these findings bring into question the view that endogenous DA plays an essential role in METH‐induced DA neurotoxicity.
Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro‐induced neurogenesis
Authors: Varju P. , Schlett K. , Eisel U. , Madarász E.
Published: March 2003

NE‐7C2 neuroectodermal cells derived from forebrain vesicles of p53‐deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all‐trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT‐PCR studies revealed that GluRε1 and GluRε4 subunit mRNAs were transcribed by both non‐induced and neuronally differentiated cells. GluRε3 subunit mRNAs were not synthesized by NE‐7C2 cells and increased numbers of messages from the GluRε2 gene were detected only after neural network formation. The presence of the GluRζ1 protein was detected throughout neural induction, whereas retinoic acid‐induced neuron formation elevated the amount of exon 21 (C1)‐ and exon 22 (C2)‐containing GluRζ1 mRNAs and resulted in the appearance of exon 5 (N1)‐containing transcripts. NMDA‐elicited Ca2+‐signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin‐I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE‐7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.
A 27‐bp region of the inducible nitric oxide synthase promoter regulates expression in glial cells
Authors: Gavrilyuk Vitaliy , Horvath Peter , Weinberg Guy , Feinstein Douglas L.
Published: March 2003

The expression of inducible nitric oxide synthase (NOS2) in glial cells is inhibited by neurotransmitters such as norepinephrine (NE) which elevate cAMP levels. We examined the molecular basis for this effect using a 2.2‐kb fragment of the rat NOS2 promoter transfected into rat C6 glioma cells. Promoter activation (up to six‐fold) by lipopolysaccharide (LPS) and interferon‐γ (IFNγ) was reduced by NE, which alone had no effect. However, a promoter construct extending to bp −130 and containing the proximal nuclear factor‐kappa B (NF‐κB) binding site was minimally activated by LPS and cytokines, but activated up to three‐fold by NE. Deletion analysis identified a 27‐bp region (bp −187 to −160) as critical for mediating this suppressive effect. This region also enhanced promoter activation by LPS and cytokines, and prevented activation by NE alone. Gel shift analysis revealed constitutive binding to this region, and induction by NE of additional complexes which could be blocked by an antibody against CREB. NE also increased levels of the IκBα protein which could contribute to its suppressive effects. These results identify a critical role for this 27‐bp region in regulation of NOS2 promoter activation and suppression by cAMP.
Intraneuronal N‐acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin‐associated aspartoacylase
Authors: Chakraborty Goutam , Mekala Praveen , Yahya Daniel , Wu Gusheng , Ledeen Robert W.
Published: March 2003

Despite its growing use as a radiological indicator of neuronal viability, the biological function of N‐acetylaspartate (NAA) has remained elusive. This is due in part to its unusual metabolic compartmentalization wherein the synthetic enzyme occurs in neuronal mitochondria whereas the principal metabolizing enzyme, N‐acetyl‐l‐aspartate amidohydrolase (aspartoacylase), is located primarily in white matter elements. This study demonstrates that within white matter, aspartoacylase is an integral component of the myelin sheath where it is ideally situated to produce acetyl groups for synthesis of myelin lipids. That it functions in this manner is suggested by the fact that myelin lipids of the rat optic system are well labeled following intraocular injection of [14C‐acetyl]NAA. This is attributed to uptake of radiolabeled NAA by retinal ganglion cells followed by axonal transport and transaxonal transfer of NAA into myelin, a membrane previously shown to contain many lipid synthesizing enzymes. This study identifies a group of myelin lipids that are so labeled by neuronal [14C]NAA, and demonstrates a different labeling pattern from that produced by neuronal [14C]acetate. High performance liquid chromatographic analysis of the deproteinated soluble materials from the optic system following intraocular injection of [14C]NAA revealed only the latter substance and no radiolabeled acetate, suggesting little or no hydrolysis of NAA within mature neurons of the optic system. These results suggest a rationale for the unusual compartmentalization of NAA metabolism and point to NAA as a neuronal constituent that is essential for the formation and/or maintenance of myelin. The relevance of these findings to Canavan disease is discussed.
Aging increases the susceptiblity to methamphetamine‐induced dopaminergic neurotoxicity in rats: correlation with peroxynitrite production and hyperthermia
Authors: Imam Syed Z. , Ali Syed F.
Published: March 2003

Methamphetamine (METH) produces dopaminergic neurotoxicity by the production of reactive oxygen (ROS) and nitrogen (RNS) species. The role of free radicals has also been implicated in the process of aging. The present study was designed to evaluate whether METH‐induced dopaminergic neurotoxicity and hyperthermia is a result of peroxynitrite production and if these effects correlate with age. One‐, six‐ and 12‐month‐old male rats (n = 8) were administered a single dose of METH (0, 5, 10, 20, and 40 mg/kg, intraperitoneally). The formation of 3‐nitrotyrosine (3‐NT) as a marker of peroxynitrite production as well as dopamine and its metabolites DOPAC and HVA were measured in the striatum 4‐h after METH‐administration. Rectal temperature was monitored every 30 min after METH administration until 4 h. At 40 mg/kg METH, a 100% mortality in 12‐month‐old animals was observed, whereas no deaths occurred in 1‐ or 6‐month‐old rats. An age‐dependent increase in hyperthermia was observed after METH‐administration. A similar pattern of dose‐dependent increase in the formation of 3‐NT and in the depletion of dopamine and its metabolites with age was observed in the striatum. Furthermore, no effect was observed at 5 mg/kg METH in 1‐month‐old animals, whereas the effect was significant in 6‐ and 12‐month‐old animals. These data suggest that aging increases the susceptibility of the animals toward METH‐induced peroxynitrite generation and striatal dopaminergic neurotoxicity.
Deficiency of presenilin‐1 increases calcium‐dependent vulnerability of neurons to oxidative stress in vitro
Authors: Nakajima Mitsunari , Miura Masami , Aosaki Toshihiko , Shirasawa Takuji
Published: March 2003

We examined the function of presenilin‐1 (PS1) on neuronal resistance to oxidative stress. CNS neurons cultured from PS1‐deficient mice exhibited increased vulnerability to H2O2 treatment compared with those from wild‐type mice. Antioxidants protected the cultured neurons against the oxidative stress. An intracellular calcium chelator, BAPTA AM, as well as an L‐type voltage‐dependent calcium channel blocker, nifedipine, rescued the neurons from H2O2‐induced death, while an N‐type voltage‐dependent calcium channel blocker, ω‐conotoxin, or calcium release blockers from ER stores, dantrolene and xestospongin C, failed to rescue them. Wild‐type and PS1‐deficient neurons showed comparable increases of cytoplasmic free calcium levels after exposure to H2O2. Taken together with the data that PS1‐deficient neurons exhibited increased vulnerability to glutamate, these findings imply that PS1 confers resistance to oxidative stress on neurons in calcium‐dependent manners.
Mechanisms underlying extracellular ATP‐evoked interleukin‐6 release in mouse microglial cell line, MG‐5
Authors: Shigemoto‐Mogami Yukari , Koizumi Schuichi , Tsuda Makoto , Ohsawa Keiko , Kohsaka Shinichi , Inoue Kazuhide
Published: March 2003

Microglia play various important roles in the CNS via the synthesis of cytokines. The ATP‐evoked production of interleukin‐6 (IL‐6) and its intracellular signals were examined using a mouse microglial cell line, MG‐5. ATP, but not its metabolites, produced IL‐6 in a concentration‐dependent manner. Although ATP activated two mitogen‐activated protein kinases, i.e. p38 and extracellular signal‐regulated protein kinase, only p38 was involved in the IL‐6 induction. However, the activation of p38 was not sufficient for the IL‐6 induction because 2′‐ and 3′‐O‐(4‐benzoylbenzoyl) ATP, an agonist to P2X7 receptors, failed to produce IL‐6 despite the fact that it activated p38. Unlike in other cytokines in microglial cells, P2Y rather than P2X7 receptors seem to have a major role in the IL‐6 production by the cells. The ATP‐evoked IL‐6 production was attenuated by Gö6976, an inhibitor of Ca2+‐dependent protein kinase C (PKC). The P2Y receptor responsible for these responses was insensitive to pertussis toxin (PTX) and was linked to phospholipase C. Taken together, ATP acting on PTX‐insensitive P2Y receptors activates p38 and Ca2+‐dependent PKC, thereby resulting in the mRNA expression and release of IL‐6 in MG‐5. This is a novel pathway for the induction of cytokines in microglia.
Restricted transport of anti‐transferrin receptor antibody (OX26) through the blood–brain barrier in the rat
Authors: Moos Torben , Morgan Evan H.
Published: March 2003

Anti‐transferrin receptor IgG2a (OX26) transport into the brain was studied in rats. Uptake of OX26 in brain capillary endothelial cells (BCECs) was > 10‐fold higher than isotypic, non‐immune IgG2a (Ni‐IgG2a) when expressed as % ID/g. Accumulation of OX26 in the brain was higher in 15 postnatal (P)‐day‐old rats than in P0 and adult (P70) rats. Iron‐deficiency did not increase OX26 uptake in P15 rats. Three attempts were made to investigate transport from BCECs further into the brain. (i) Using a brain capillary depletion technique, 6–9% of OX26 was identified in the post‐capillary compartment consisting of brain parenchyma minus BCECs. (ii) In cisternal CSF, the volume of distribution of OX26 was higher than for Ni‐IgG2a when corrected for plasma concentration. (iii) Immunohistochemical mapping revealed the presence of OX26 almost exclusively in BCECs; extravascular staining was observed only in neurons situated periventricularly. The data support the hypothesis of facilitated uptake of OX26 due to the presence of transferrin receptors at the blood–brain barrier (BBB). However, OX26 accumulation in the post‐capillary compartment was too small to justify a conclusion of receptor‐mediated transcytosis of OX26 occurring in BCECs. Accumulation of OX26 in the post‐capillary component may result from a diphasic transport that involves high‐affinity accumulation of OX26 by the BCECs, clearly exceeding that of Ni‐IgG2a, followed by a second transport mechanism that releases OX26 non‐specifically further into the brain. The periventricular localization suggests that OX26 probably also derives from transport across the blood–CSF barrier.
Isovolumetric regulation mechanisms in cultured cerebellar granule neurons
Authors: Tuz Karina , Ordaz Benito , Vaca Luis , Quesada Octavio , Pasantes‐Morales Herminia
Published: March 2003

Cultured cerebellar granule neurons exposed to gradual reductions in osmolarity (− 1.8 mOsm/min) maintained constant volume up to − 50% external osmolarity (πo), showing the occurrence of isovolumetric regulation (IVR). Amino acids, Cl−, and K+ contributed at different phases of IVR, with early efflux threshold for [3H]taurine, d‐[3H]aspartate (as marker for glutamate) of πo− 2% and − 19%, respectively, and more delayed thresholds of − 30% for [3H]glycine and − 25% and − 29%, respectively, for Cl− (125I) and K+ (86Rb). Taurine seems preferentially involved in IVR, showing the lowest threshold, the highest efflux rate (five‐fold over other amino acids) and the largest cell content decrease. Taurine and Cl− efflux were abolished by niflumic acid and 86Rb by 15 mm Ba2+. Niflumic acid essentially prevented IVR in all ranges of πo. Cl−‐free medium impaired IVR when πo decreased to − 24% and Ba2+ blocked it only at a late phase of − 30% πo. These results indicate that in cerebellar granule neurons: (i) IVR is an active process of volume regulation accomplished by efflux of intracellular osmolytes; (ii) the volume regulation operating at small changes of πo is fully accounted for by mechanisms sensitive to niflumic acid, with contributions of both Cl− and amino acids, particularly taurine; (iii) Cl− contribution to IVR is delayed with respect to other niflumic acid‐sensitive osmolyte fluxes (osmolarity threshold of − 25% πo); and (iv), K+ fluxes do not contribute to IVR until a late phase (< − 30% πo).
Regulated expression of an actin‐associated protein, synaptopodin, during long‐term potentiation
Authors: Yamazaki Mitsue , Matsuo Ryota , Fukazawa Yugo , Ozawa Fumiko , Inokuchi Kaoru
Published: March 2003

We report NMDA receptor‐dependent expression of synaptopodin mRNA in the dentate granule cells of the hippocampus following induction of long‐term potentiation (LTP) in vivo. Synaptopodin did not belong to immediate‐early genes, as de novo protein synthesis was required for the induction of synaptopodin gene transcription. An increased level of synaptopodin mRNA was observed at 75 min and 3.5 h after the onset of LTP. Importantly, there was correlation between the induction of mRNA expression and the persistence of LTP. Synaptopodin immunoreactivity was elevated specifically in synaptic layers, middle and outer molecular layers of dentate gyrus where LTP was induced. As synaptopodin is an actin‐associated protein present in spine neck and implicated in the modulation of cell morphology, our results suggest that synaptopodin, by regulating the dynamics of the actin cytoskeleton, contributes to the morphological change in spine shape considered to be important for the maintenance of synaptic plasticity.
Chronic benzodiazepine treatment of cells expressing recombinant GABAA receptors uncouples allosteric binding: studies on possible mechanisms
Authors: Ali Noore J. , Olsen Richard W.
Published: March 2003

Functional and behavioral tolerance to chronic benzodiazepine (BZ) exposure has been associated with an uncoupling of the BZ and GABA binding sites. As in rats exposed to BZ for periods of a week or longer, recombinant GABAA receptors (GABARs) expressed in Sf9 cells lose the normally observed allosteric enhancement of [3H]flunitrazepam binding by GABA agonists, which is measured in homogenized membranes after a few hours exposure to pharmacological doses of agonist BZ. Treatment of Sf9 cells expressing recombinant GABAR with various drugs that inhibit protein kinase A (PKA), but not protein kinase C (PKC), resulted in an uncoupling of the BZ and GABA binding sites; whereas promotion of phosphorylation by PKA, but not PKC, favored coupling and recoupling. However, mutation of the only PKA phosphorylation site expressed from among the subunits proved that direct phosphorylation of the GABAR was not involved in either coupling after chronic BZ exposure or reversal of uncoupling after exposure to the competitive BZ antagonist, flumazenil. Osmotic‐shock of cell membrane homogenates to lyse intracellular compartments reversed uncoupling, and uncoupling can be replicated in untreated cells by performing membrane binding assays in an acidic environment, suggesting that GABARs become internalized into an acidic intracellular environment where normal BZ binding occurs, but that potentiation by GABA is hindered. The internalization of receptors was shown by immunofluorescence after chronic exposure to either BZ or the PKA inhibitor H‐89.
Pigment epithelium‐derived factor is elevated in CSF of patients with amyotrophic lateral sclerosis
Authors: Kuncl Ralph W. , Bilak Masako M. , Bilak Stephan R. , Corse Andrea M. , Royal Walter , Becerra S. Patricia
Published: March 2003

Pigment epithelium‐derived factor (PEDF), a recently defined retinal trophic factor and anti‐angiogenic factor for the eye, is also present in the CNS and is a motor neuron protectant. We asked whether PEDF levels in CSF are altered in patients with amyotrophic lateral sclerosis (ALS). Pigment epithelium‐ derived factor protein was detected by quantitative western blot analysis with a PEDF‐specific antiserum. Levels of PEDF in CSF, expressed as a ratio to total CSF protein, were significantly elevated 3.4‐fold in 15 patients with ALS compared with neurologic disease controls (p < 0.0003). This increase does not seem likely to reflect up‐regulation of PEDF synthesis in muscle in response to denervation, as CSF PEDF was not elevated in severe denervating diseases other than ALS. Nor does the increase represent some non‐specific release in neurodegeneration, as CSF PEDF was not elevated in other neurodegenerative diseases. While the mechanism of this presumably reactive increase is not known, the distinctive, surprisingly elevated level of PEDF in the CSF may be an autoprotective reaction in ALS.
Neuronal transfer of the human Cu/Zn superoxide dismutase gene increases the resistance of dopaminergic neurons to 6‐hydroxydopamine
Authors: Barkats Martine , Millecamps Stéphanie , Bilang‐Bleuel Alicia , Mallet Jacques
Published: March 2003

Several mechanisms are thought to be involved in the progressive decline in neurons of the substantia nigra pars compacta (SNpc) that leads to Parkinson's disease (PD). Neurotoxin 6‐hydroxydopamine (6‐OHDA), which induces parkinsonian symptoms in experimental animals, is thought to be formed endogenously in patients with PD through dopamine (DA) oxidation and may cause dopaminergic cell death via a free radical mechanism. We therefore investigated protection against 6‐OHDA by inhibiting oxidative stress using a gene transfer strategy. We overexpressed the antioxidative Cu/Zn‐superoxide dismutase (SOD1) enzyme in primary culture dopaminergic cells by infection with an adenovirus carrying the human SOD1 gene (Ad‐hSOD1). Survival of the dopaminergic cells exposed to 6‐OHDA was 50% higher among the SOD1‐producing cells than the cells infected with control adenoviruses. In contrast, no significant increased survival of (6‐OHDA)‐treated dopaminergic cells was observed when they were infected with an adenovirus expressing the H2O2‐scavenging glutathione peroxidase (GPx) enzyme. These results underline the major contribution of superoxide in the dopaminergic cell death process induced by 6‐OHDA in primary cultures. Overall, this study demonstrates that the survival of the dopaminergic neurons can be highly increased by the adenoviral gene transfer of SOD1. An antioxidant gene transfer strategy using viral vectors expressing SOD1 is therefore potentially beneficial for protecting dopaminergic neurons in PD.
Expression of CD47/integrin‐associated protein induces death of cultured cerebral cortical neurons
Authors: Koshimizu Hisatsugu , Araki Toshiyuki , Takai Satomi , Yokomaku Daisaku , Ishikawa Yasuyuki , Kubota Misae , Sano Shin‐ichiro , Hatanaka Hiroshi , Yamada Masashi
Published: March 2003

The death and survival of neuronal cells are regulated by various signaling pathways during development of the brain and in neuronal diseases. Previously, we demonstrated that the neuronal adhesion molecule brain immunoglobulin‐like molecule with tyrosine‐based activation motifs/SHP substrate 1 (BIT/SHPS‐1) is involved in brain‐derived neurotrophic factor (BDNF)‐promoted neuronal cell survival. Here, we report the apoptosis‐inducing effect of CD47/integrin‐associated protein (IAP), the heterophilic binding partner of BIT/SHPS‐1, on neuronal cells. We generated a recombinant adenovirus vector expressing a neuronal form of CD47/IAP, and found that the expression of CD47/IAP by infection with CD47/IAP adenovirus induced the death of cultured cerebral cortical neurons. The numbers of TdT‐mediated biotin–dUTP nick‐end labelling (TUNEL)‐positive neurons and of cells displaying apoptotic nuclei increased by expression of CD47/IAP. Neuronal cell death was prevented by the addition of the broad‐spectrum caspase inhibitor Z‐VAD‐fmk. Furthermore, we observed that co‐expression of CD47/IAP with BIT/SHPS‐1 enhanced neuronal cell death, and that BDNF prevented it. These results suggest that CD47/IAP is involved in a novel pathway which regulates caspase‐dependent apoptosis of cultured cerebral cortical neurons. CD47/IAP‐induced death of cultured cortical neurons may be regulated by the interaction of CD47/IAP with BIT/SHPS‐1 and by BDNF.