bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐1β up‐regulates expression of neurofilament light in human neuronal cells
Authors: Rempel Hans , Kusdra Leonard , Pulliam Lynn
Published: March 2003

Elevated expression of interleukin‐1 (IL‐1β), a pro‐inflammatory cytokine secreted by activated microglia, is a pathogenic marker of numerous neurodegenerative processes including Alzheimer's disease (AD). We have characterized a link between IL‐1β and the 68‐kDa neurofilament light (NF‐L) protein, which is a major component of the neuronal cytoskeleton. Using human brain aggregate cultures, we found that IL‐1β treatment significantly increased NF‐L expression in primary neurons. Analysis of mRNA levels demonstrated elevated NF‐L expression within 72 h while imaging of neurons by immunofluorescent staining for NF‐L confirmed IL‐1β‐induced NF‐L protein expression. These observations suggest a potential inflammatory‐induced mechanism for deregulation of an important cytoskeletal protein, NF‐L, possibly leading to neuronal dysfunction.
l‐DOPA and glia‐conditioned medium have additive effects on tyrosine hydroxylase expression in human catecholamine‐rich neuroblastoma NB69 cells
Authors: Rodríguez‐Martín E. , Canals S. , Casarejos M. J. , de Bernardo S. , Handler A. , Mena M. A.
Published: March 2003

The aim of this study was to investigate the effect of l‐DOPA and glia‐conditioned medium (GCM) on cell viability, tyrosine hydroxylase (TH) expression, dopamine (DA) metabolism and glutathione (GSH) levels of NB69 cells. l‐DOPA (200 µm) induced differentiation of NB69 cells of more than 4 weeks in vitro, as shown by phase‐contrast microscopy and TH immunocytochemistry, and decreased replication, as shown by 5‐bromodeoxyuridine immunostaining. l‐DOPA did not increase the number of necrotic or apoptotic cells, as shown by morphological features, Trypan Blue, lactate dehydrogenase activity, bis‐benzimide staining and TUNEL assay. Furthermore, l‐DOPA (200 µm) increased Bcl‐xL protein expression. Incubation of cells with l‐DOPA (50, 100, 200 µm) for 24 h resulted in an increase in TH protein levels (174, 196 and 212% versus control). Neither carbidopa, an inhibitor of l‐aromatic amino acid decarboxylase enzyme, nor l‐buthionine sulfoximine, which inhibits GSH synthesis, or ascorbic acid, an antioxidant, blocked the l‐DOPA‐induced effect on TH protein expression. l‐DOPA (0, 50, 100 and 200 µm) plus GCM further increased the amount of TH protein (346, 446, 472 and 424%). l‐DOPA (200 µm) increased TH protein levels to 132, 191 and 245% of controls after incubation for 24, 48 and 72 h. DA metabolism in NB69 cells was increased in cultures treated with either l‐DOPA (200–300 µm) or GCM and these two agents had a synergistic effect on DA metabolism. In addition, l‐DOPA (200 µm) or/and GCM‐treated cells increased their GSH extracellular levels (223, 257, 300% of controls) after 48 h of treatment. The l‐DOPA‐induced increase of TH protein expression in NB69 cells was independent of DA production, free radicals and GSH up‐regulation.
Semaphorin4F interacts with the synapse‐associated protein SAP90/PSD‐95
Authors: Schultze Wiebke , Eulenburg Volker , Lessmann Volkmar , Herrmann Lutz , Dittmar Thomas , Gundelfinger Eckart D. , Heumann Rolf , Erdmann Kai S.
Published: March 2003

Semaphorins are a family of secreted and membrane‐associated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post‐synaptic density protein SAP90/PSD‐95. Using the yeast two‐hybrid system and coprecipitation assays we were able to show an interaction between the extreme C‐terminus of Sema4F and the PDZ domains of SAP90/PSD‐95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD‐95 in COS cells leads to translocation of SAP90/PSD‐95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C‐terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post‐synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD‐95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses.
Activity of cytisine and its brominated isosteres on recombinant human α7, α4β2 and α4β4 nicotinic acetylcholine receptors
Authors: Houlihan Lee M. , Slater Yvonne , Guerra Doris L. , Peng Jian‐Hong , Kuo Yen‐Ping , Lukas Ronald J. , Cassels Bruce K. , Bermudez Isabel
Published: March 2003

Effects of cytisine (cy), 3‐bromocytisine (3‐Br‐cy), 5‐bromocytisine (5‐Br‐cy) and 3,5‐dibromocytisine (3,5‐diBr‐cy) on human (h) α7‐, α4β2‐ and α4β4 nicotinic acetylcholine (nACh) receptors, expressed in Xenopus oocytes and cell lines, have been investigated. Cy and its bromo‐isosteres fully inhibited binding of both [α‐125I]bungarotoxin ([α‐125I]BgTx) to hα7‐ and [3H]cy to hα4β2‐ or hα4β4‐nACh receptors. 3‐Br‐cy was the most potent inhibitor of both [α‐125I]BgTx and [3H]cy binding. Cy was less potent than 3‐Br‐cy, but 5‐Br‐cy and 3,5‐diBr‐cy were the least potent inhibitors. Cy and 3‐Br‐cy were potent full agonists at hα7‐nACh receptors but behaved as partial agonists at hα4β2‐ and hα4β4‐nACh receptors. 5‐Br‐cy and 3,5‐diBr‐cy had low potency and were partial agonists at hα7‐ and hα4β4‐nACh receptors, but they elicited no responses on hα4β2‐nACh receptors. Cy and 3‐Br‐cy produced dual dose–response curves (DRC) at both hα4β2‐ and hα4β4‐nACh receptors, but ACh produced dual DRC only at hα4β2‐nACh receptors. Low concentrations of cy, 3‐Br‐cy and 5‐Br‐cy enhanced ACh responses of oocytes expressing hα4β2‐nACh receptors, but at high concentrations they inhibited the responses. In contrast, 3,5‐diBr‐cy only inhibited, in a competitive manner, ACh responses of hα4β2‐nACh receptors. It is concluded that bromination of the pyridone ring of cy produces marked changes in effects of cy that are manifest as nACh receptor subtype‐specific differences in binding affinities and in functional potencies and efficacies.
Inhibition of neurite extension by overexpression of individual domains of LIM kinase 1
Authors: Birkenfeld Jörg , Betz Heinrich , Roth Dagmar
Published: March 2003

Lin‐11, Isl‐1 and Mec‐3 (LIM) kinases are serine/threonine kinases that phosphorylate cofilin, an actin depolymerizing protein. LIM kinases have a highly modular structure composed of two N‐terminal LIM domains (LIM 1/2), a PSD‐95, Dlg and ZO‐1 (PDZ) domain and a C‐terminal protein kinase domain. Here, we overexpressed individual domains of mouse LIM kinase 1 (LIMK1) in PC12 cells and investigated their effects on neurite outgrowth. Although none of the LIMK1 domains had an effect on spontaneous neurite outgrowth, the N‐terminal LIM 1/2 domains strongly inhibited differentiation of PC12 cells after stimulation with both nerve growth factor (NGF) and the Rho‐kinase inhibitor Y‐27632. In contrast, the overexpressed PDZ domain reduced neurite outgrowth only when differentiation had been induced by Y‐27632, but not by NGF. Our data suggest that the different non‐catalytic N‐terminal domains of LIMK1 contribute to the regulation of neurite extension by using distinct signal transduction pathways.
Pro‐inflammatory and pro‐oxidant properties of the HIV protein Tat in a microglial cell line: attenuation by 17β‐estradiol
Authors: Bruce‐Keller Annadora J. , Barger Steven W. , Moss Naeeta I. , Pham Jaqueline T. , Keller Jeffrey N. , Nath Avindra
Published: March 2003

Microglia are activated in humans following infection with human immunodeficiency virus (HIV), and brain inflammation is thought to be involved in neuronal injury and dysfunction during HIV infection. Numerous studies indicate a role for the HIV regulatory protein Tat in HIV‐related inflammatory and neurodegenerative processes, although the specific effects of Tat on microglial activation, and the signal transduction mechanisms thereof, have not been elucidated. In the present study, we document the effects of Tat on microglial activation and characterize the signal transduction pathways responsible for Tat's pro‐inflammatory effects. Application of Tat to N9 microglial cells increased multiple parameters of microglial activation, including superoxide production, phagocytosis, nitric oxide release and TNFα release. Tat also caused activation of both p42/p44 mitogen activated protein kinase (MAPK) and NFκB pathways. Inhibitor studies revealed that Tat‐induced NFκB activation was responsible for increased nitrite release, while MAPK activation mediated superoxide release, TNFα release, and phagocytosis. Lastly, pre‐treatment of microglial cells with physiological concentrations of 17β‐estradiol suppressed Tat‐mediated microglial activation by interfering with Tat‐induced MAPK activation. Together, these data elucidate specific components of the microglial response to Tat and suggest that Tat could contribute to the neuropathology associated with HIV infection through microglial promulgation of oxidative stress.
Identification and characterization of the human parkin gene promoter
Authors: West Andrew , Farrer Matt , Petrucelli Leonard , Cookson Mark , Lockhart Paul , Hardy John
Published: March 2003

Compound mutations and homozygous loss of function of the parkin gene causes juvenile and early onset, autosomal recessive parkinsonism. Pathologically, the disease is associated with loss of dopaminergic neurons in the substantia nigra pars compacta and locus ceruleus, usually without Lewy body pathology. Hemizygous families have been described that may harbor mutations outside of the open reading frame. The parkin gene promoter has yet to be characterized, and therein, mutations in hemizygous families may plausibly be identified. To identify the promoter of the parkin gene, the transcription start site was defined by a combination of primer extension and 5′ RACE. Five kilobases of DNA 5′ to the parkin start codon were directly sequenced from a BAC containing parkin exon 1 and evaluated for promoter motifs. The parkin promoter lacks TATA or CAAT boxes and appears to share homology to the α‐synuclein promoter. Deletion constructs demonstrated core promoter activity and tissue specific enhancing regions in HEK‐293T and SH‐SY5Y cells.
Nicotinic acetylcholine receptors containing α7 subunits on rat cortical neurons do not undergo long‐lasting inactivation even when up‐regulated by chronic nicotine exposure
Authors: Kawai Hideki , Berg Darwin K.
Published: March 2003

Chronic exposure to (–)nicotine has been widely reported to up‐regulate nicotinic acetylcholine receptors on neurons and induce long‐term inactivation as a possible cause. Nicotinic receptors containing α7 subunits are among the most abundant in brain and influence diverse cellular events. Whole‐cell patch clamp recording from embryonic rat cortical neurons in culture was used to identify responses from α7‐containing receptors. Immunochemical staining for glutamic acid decarboxylase (GAD) indicated that both GABAergic and non‐GABAergic neurons expressed the receptors. Exposure to micromolar concentrations of nicotine for 1–4 days caused up‐regulation of the receptors as measured by [α‐125I]‐bungarotoxin binding. Carbachol produced the same up‐regulation, and cell counts demonstrated that neuronal survival was unchanged. The up‐regulation was accompanied by an increased whole‐cell response; no evidence was found for long‐lasting inactivation. Autonomic α7‐containing receptors also avoided long‐lasting inactivation, even though the receptors were down‐regulated by nicotine. Blocking protein synthesis or protein glycosylation prevented receptor up‐regulation on cortical neurons, suggesting that new synthesis was required. No evidence was found for a pre‐existing intracellular pool that supplied receptors to the surface. The results indicate that α7‐containing receptors differ from other receptor subtypes in their regulation by nicotine and demonstrate further that long‐lasting inactivation is not an obligatory requirement for up‐regulation in this case.
The role of 5‐HT1B receptors in the regulation of serotonin cell firing and release in the rat brain
Authors: Adell Albert , Celada Pau , Artigas Francesc
Published: March 2003

The release of 5‐HT in terminal areas of the rodent brain is regulated by 5‐HT1B receptors. Here we examined the role of 5‐HT1B receptors in the control of 5‐HT output and firing in the dorsal raphe nucleus (DR), median raphe nucleus (MnR) and forebrain of the rat in vivo. The local perfusion (30–300 µm) of the selective 5‐HT1B receptor agonist CP‐93,129 to freely moving rats decreased 5‐HT release in the DR and more markedly in the MnR. Likewise, 300 µm CP‐93,129 reduced 5‐HT output in substantia nigra pars reticulata, ventral pallidum, lateral habenula and the suprachiasmatic nucleus. The effect of CP‐93,129 was prevented by SB‐224289, but not by WAY‐100635, selective 5‐HT1B and 5‐HT1A receptor antagonists, respectively. SB‐224289 did not alter dialysate 5‐HT in any raphe nuclei. The intravenous administration of the brain‐penetrant selective 5‐HT1B receptor agonist CP‐94,253 (0.5–2.0 mg/kg) to anesthetized rats decreased dialysate 5‐HT in dorsal hippocampus and globus pallidus, increased it in MnR and left it unaltered in the DR and medial prefrontal cortex. SB‐224289, at a dose known to block 5‐HT1B autoreceptor‐mediated effects (5 mg/kg), did not prevent the effect of CP‐94,253 on MnR 5‐HT. The intravenous administration of CP‐94,253 (0.05–1.6 mg/kg) to anesthetized rats increased the firing rate of MnR, but not DR‐5‐HT neurons. The local perfusion of CP‐94,253 in the MnR showed a biphasic effect, with 5‐HT reductions at 0.3–3 µm and increase at 300 µm. These results suggest that 5‐HT cell firing and release in midbrain raphe nuclei (particularly in the MnR) are under control of 5‐HT1B receptors. The activation of 5‐HT1B autoreceptors (possibly located on 5‐HT nerve endings and/or varicosities within DR and MnR) reduces 5‐HT release. The effects of higher concentrations of 5‐HT1B receptor agonists seem more compatible with the activation of 5‐HT1B heteroreceptors on inhibitory neurons.
Oxidative inactivation of calcineurin by Cu,Zn superoxide dismutase G93A, a mutant typical of familial amyotrophic lateral sclerosis
Authors: Ferri Alberto , Gabbianelli Roberta , Casciati Arianna , Celsi Fulvio , Rotilio Giuseppe , Carrì Maria Teresa
Published: March 2003

Calcineurin is a serine/threonine phosphatase involved in a wide range of cellular responses to calcium mobilizing signals. Previous evidence supports the notion of the existence of a redox regulation of this enzyme, which might be relevant for neurodegenerative processes, where an imbalance between generation and removal of reactive oxygen species could occur. In a recent work, we have observed that calcineurin activity is depressed in two models for familial amyotrophic lateral sclerosis (FALS) associated with mutations of the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1), namely in neuroblastoma cells expressing either SOD1 mutant G93A or mutant H46R and in brain areas from G93A transgenic mice.

In this work we report that while wild‐type SOD1 has a protective effect, calcineurin is oxidatively inactivated by mutant SOD1s in vitro; this inactivation is mediated by reactive oxygen species and can be reverted by addition of reducing agents. Furthermore, we show that calcineurin is sensitive to oxidation only when it is in an ‘open’, calcium‐activated conformation, and that G93A‐SOD1 must have its redox‐active copper site available to substrates in order to exert its pro‐oxidant properties on calcineurin. These findings demonstrate that both wild‐type and mutant SOD1s can interfere directly with calcineurin activity and further support the possibility of a relevant role for calcineurin‐regulated biochemical pathways in the pathogenesis of FALS.
Parvalbumin overexpression alters immune‐mediated increases in intracellular calcium, and delays disease onset in a transgenic model of familial amyotrophic lateral sclerosis
Authors: Beers David R. , Ho Bao‐Kuang , Siklós Laszlo , Alexianu Maria E. , Mosier Dennis R. , Mohamed A. Habib , Otsuka Yasushi , Kozovska Milena E. , McAlhany Robert E. , Smith R. Glenn , Appel Stanley H.
Published: March 2003

Intracellular calcium is increased in vulnerable spinal motoneurons in immune‐mediated as well as transgenic models of amyotrophic lateral sclerosis (ALS). To determine whether intracellular calcium levels are influenced by the calcium‐binding protein parvalbumin, we developed transgenic mice overexpressing parvalbumin in spinal motoneurons. ALS immunoglobulins increased intracellular calcium and spontaneous transmitter release at motoneuron terminals in control animals, but not in parvalbumin overexpressing transgenic mice. Parvalbumin transgenic mice interbred with mutant SOD1 (mSOD1) transgenic mice, an animal model of familial ALS, had significantly reduced motoneuron loss, and had delayed disease onset (17%) and prolonged survival (11%) when compared with mice with only the mSOD1 transgene. These results affirm the importance of the calcium binding protein parvalbumin in altering calcium homeostasis in motoneurons. The increased motoneuron parvalbumin can significantly attenuate the immune‐mediated increases in calcium and to a lesser extent compensate for the mSOD1‐mediated ‘toxic‐gain‐of‐function’ in transgenic mice.
BMCP1: a mitochondrial uncoupling protein in neurons which regulates mitochondrial function and oxidant production
Authors: Kim‐Han Jeong Sook , Reichert Susan A. , Quick Kevin L. , Dugan Laura L.
Published: March 2003

Outside the nervous system, members of the mitochondrial uncoupling protein (UCP) family have been proposed to contribute to control of body temperature and energy metabolism, and regulation of mitochondrial production of reactive oxygen species (ROS). However, the function of brain mitochondrial carrier protein 1 (BMCP1), which is highly expressed in brain, remains to be determined. To study BMCP1 expression and function in the nervous system, a high‐affinity antibody to BMCP1 was generated and used to analyze tissue expression of BMCP1 protein in mouse. BMCP1 protein was highly expressed in heart and kidney, but not liver or lung. In the nervous system, BMCP1 was present in cortex, basal ganglia, substantia nigra, cerebellum, and spinal cord. Both BMCP1 mRNA and protein expression was almost exclusively neuronal. To study the effect of BMCP1 expression on mitochondrial function, neuronal (GT1‐1) cell lines with stable overexpression of BMCP1 were generated. Transfected cells had higher State 4 respiration and lower mitochondrial membrane potential (ψm), consistent with greater mitochondrial uncoupling. BMCP1 expression also decreased mitochondrial production of ROS. These data suggest that BMCP1 can modify mitochondrial respiratory efficiency and mitochondrial oxidant production, and raise the possibility that BMCP1 might alter the vulnerability of brain to both acute injury and to neurodegenerative conditions.
Wheat germ agglutinin‐binding glycoproteins are decreased in Alzheimer's disease cerebrospinal fluid
Authors: Fodero Lisa R. , Sáez‐Valero Javier , Barquero Maria‐Sagrario , Marcos Alberto , McLean Catriona A. , Small David H.
Published: March 2003

A number of biomarkers (e.g. Aβ, tau) has been identified in Alzheimer's disease CSF. However, none fulfils the criteria of sensitivity and specificity (> 80%) needed for the development of an accurate diagnostic test. The lack of a suitable marker has prompted the search for new CSF biomarkers. In this study, the glycosylation of CSF proteins was examined using lectin blotting. Lumbar CSF was collected ante mortem from 22 non‐Alzheimer's disease and 12 probable Alzheimer's disease cases and ventricular CSF collected post mortem from 7 non‐Alzheimer's disease and 16 Alzheimer's disease cases confirmed by pathologic examination. When CSF glycoproteins were stained with wheat germ agglutinin (WGA), the staining intensity was found to be significantly lower in the Alzheimer's disease group. No difference in staining was found using other lectins (Canavalia ensiformis agglutinin, Ricinus communis agglutinin, Lens culinaris agglutinin). The measurement of WGA‐reactive glycoproteins in CSF may be a useful biomarker for diagnosis of Alzheimer's disease.
The degenerative effect of a single intranigral injection of LPS on the dopaminergic system is prevented by dexamethasone, and not mimicked by rh‐TNF‐α, IL‐1β and IFN‐γ
Authors: Castaño A. , Herrera A. J. , Cano J. , Machado A.
Published: March 2003

It is becoming widely accepted that the inflammatory response is involved in neurodegenerative disease. In this context, we have developed an animal model of dopaminergic system degeneration by the intranigral injection of lipopolysaccharide (LPS), a potent inductor of inflammation. To address the importance of the inflammatory response in the LPS‐induced degeneration of nigral dopaminergic neurones, we carried out two different kinds of studies: (i) the possible protective effect of an anti‐inflammatory compound, and (ii) the effect of the intranigral injection of inflammatory cytokines (TNF‐α, IL‐1β and IFN‐γ) on dopaminergic neurones viability. Present results show that dexamethasone, a potent anti‐inflammatory drug that interferes with many of the features characterizing pro‐inflammatory glial activation, prevented the loss of catecholamine content, Tyrosine hydroxylase (TH) activity and TH immunostaining induced by LPS‐injection and also the bulk activation of microglia/macrophages. Surprisingly, injection of the pro‐inflammatory cytokines failed to reproduce the LPS effect. Taken together, our results suggest that inflammatory response is implicated in LPS‐induced neurodegeneration. This damage may be due, at least in part, to a cascade of events independent of that described for TNF‐α/IL‐1β/IFN‐γ.
p75 neurotrophin receptor is required for constitutive and NGF‐induced survival signalling in PC12 cells and rat hippocampal neurones
Authors: Bui Nguyen Truc , König Hans‐Georg , Culmsee Carsten , Bauerbach Elke , Poppe Monika , Krieglstein Josef , Prehn Jochen H. M.
Published: March 2003

We have previously shown that nerve growth factor (NGF)‐induced activation of nuclear factor‐κB increased neuronal expression of Bcl‐xL, an anti‐apoptotic Bcl‐2 family protein. In the present study we determined the role of the p75 neurotrophin receptor in constitutive and NGF‐induced survival signalling. Treatment of rat pheochromocytoma (PC12) cells with a blocking anti‐rat p75 antibody or inhibition of p75 expression by antisense oligonucleotides reduced constitutive and NGF‐induced bcl‐xL expression. Treatment with the blocking anti‐p75 antibody also inhibited NGF‐induced activation of the survival kinase Akt. Inhibition of phosphatidylinositol‐3‐kinase (PI3 kinase) activity or overexpression of a dominant‐negative mutant of Akt kinase inhibited NGF‐induced nuclear factor‐κB activation. Activation of Akt kinase by NGF was also observed in PC12nnr5 cells and cultured rat hippocampal neurones which both lack significant TrkA expression. Treatment of hippocampal neurones with the blocking anti‐p75 antibody inhibited constitutive and NGF‐induced Bcl‐xL expression, activation of Akt, and blocked the protective effect of NGF against excitotoxic and apoptotic injury. Our data suggest that the p75 neurotrophin receptor mediates constitutive and NGF‐induced survival signalling in PC12 cells and hippocampal neurones, and that these effects are mediated via the PI3‐kinase pathway.
Reduced number of unmyelinated sensory axons in peripherin null mice
Authors: Larivière R. C. ,   M. D. Nguyen,, Ribeiro‐da‐Silva A. , Julien J.‐P.
Published: March 2003

Peripherin is a type III intermediate filament (IF) abundantly expressed in developing neurons, but in the adult, it is primarily found in neurons extending to the peripheral nervous system. It has been suggested that peripherin may play a role in axonal elongation and/or cytoskeletal stabilization during development and regeneration. To further clarify the function of peripherin, we generated and characterized mice with a targeted disruption of the peripherin gene. The peripherin null mice were viable, reproduced normally and did not exhibit overt phenotypes. Microscopic analysis revealed no gross morphological defects in the ventral and dorsal roots, spinal cord, retina and gut, but protein analyses showed increased levels of the type IV IF α‐internexin in ventral roots of peripherin null mice. Whereas the number and caliber of myelinated motor and sensory axons in the L5 roots remained unchanged in peripherin knockout mice, there was a substantial reduction (∼ 34%) in the number of L5 unmyelinated sensory fibers that correlated with a decreased binding of the lectin IB4. These results demonstrate a requirement of peripherin for the proper development of a subset of sensory neurons.
NF‐κB‐mediated up‐regulation of Bcl‐XSand Bax contributes to cytochromecrelease in cyanide‐induced apoptosis
Authors: Shou Yan , Li Nianyu , Li Li , Borowitz Joseph L. , Isom Gary E.
Published: March 2003

Cyanide induces apoptosis through cytochromecactivated caspase cascade in primary cultured cortical neurons. The underlying mechanism for cytochromecrelease from mitochondria after cyanide treatment is still unclear. In this study, the roles of endogenous Bcl‐2 proteins in cyanide‐induced apoptosis were investigated. After cyanide (100–500 µm) treatment for 24 h, two pro‐apoptotic Bcl‐2 proteins, Bcl‐XSand Bax were up‐regulated as shown by western blot and RT‐PCR analysis. The expression levels of two antiapoptotic Bcl‐2 proteins, Bcl‐2 and Bcl‐XL, remained unchanged after cyanide treatment, whereas the mRNA levels of Bcl‐XSand Bax began to increase within 2 h and their protein levels increased 6 h after treatment. NF‐κB, a redox‐sensitive transcription factor activated after cyanide treatment, is responsible for the up‐regulation of Bcl‐XSand Bax. SN50, which is a synthetic peptide that blocks translocation of NF‐κB from cytosol to nucleus, inhibited the up‐regulation of Bcl‐XSand Bax. Similar results were obtained using a specific κB decoy DNA. NMDA receptor activation and reactive oxygen species (ROS) generation are upstream events of NF‐κB activation, as blockade of these two events by MK801,l‐NAME or PBN inhibited cyanide‐induced up‐regulation of Bcl‐XSand Bax. Up‐regulation of pro‐apoptotic Bcl‐XSand Bax contributed to cyanide‐induced cytochromecrelease, because SN50 and a specific Bax antisense oligodeoxynucleotide significantly reduced release of cytochromecfrom mitochondria as shown by western blot analysis. It was concluded that NF‐κB‐mediated up‐regulation of Bcl‐XSand Bax is involved in regulating cytochromecrelease in cyanide‐induced apoptosis.
PSD‐95 eliminates Src‐induced potentiation of NR1/NR2A‐subtype NMDA receptor channels and reduces high‐affinity zinc inhibition
Authors: Yamada Yasue , Iwamoto Takashi , Watanabe Yoshifumi , Sobue Kenji , Inui Makoto
Published: March 2003

The channel activity of NMDA receptors is regulated by phosphorylation by protein kinases and by interaction with other proteins. Recombinant NR1/NR2A subtype NMDA receptor channels are potentiated by the protein tyrosine kinase Src, an effect which is mediated by a reduction in the high‐affinity, voltage‐independent Zn2+ inhibition. However, it has been reported that Src‐induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn2+ inhibition. The post‐synaptic density protein PSD‐95 interacts with the C‐terminus of NR2 subunits of the NMDA receptor. Here we demonstrate that PSD‐95 eliminates the Src‐induced potentiation of NR1/NR2A channels expressed in oocytes and reduces the sensitivity of the channels to Zn2+. Our results reveal that the absence of Src‐induced potentiation of PSD‐95‐coupled NR1/NR2A channels is not to due to the reduced sensitivity of these channels to Zn2+. These results indicate that PSD‐95 functionally modulates NR1/NR2A channels and explain why Src‐induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn2+ inhibition.
Involvement of NF‐Y and Sp1 in basal and cAMP‐stimulated transcriptional activation of the tryptophan hydroxylase (TPH) gene in the pineal gland
Authors: Côté F. , Schussler N. , Boularand S. , Peirotes A. , Thévenot E. , Mallet J. , Vodjdani G.
Published: March 2003

The expression of the tryptophan hydroxylase (TPH) gene, encoding the rate‐limiting enzyme of serotonin biosynthesis, is tightly regulated both at the transcriptional and at the post‐transcriptional levels. In the pineal gland, transcription of the gene is activated in response to an intracellular circadian increase of the cAMP concentration. We have previously shown that transcription of a 2.1‐kb fragment of the human TPH promoter is induced by cAMP, although it lacks the canonical cAMP responsive element, CRE. The minimal promoter (−73/+29) has only weak transcriptional activity but is responsive to cAMP. It contains an inverted CCAAT box, which was demonstrated to be involved in this response. Here, we have extended our investigation to the functional features of the inverted CCAAT box in the −252/+29 TPH promoter, which has a higher basal activity. We show that an additional cis‐acting sequence, the adjacent GC‐rich region, cooperates with the inverted CCAAT box for the full activation of basal transcription, and that both elements are essential for the full cAMP response. We also show that in pinealocytes, NF‐Y and Sp1 transactivators bind the inverted CCAAT box and GC‐rich‐region, respectively. These factors participate in a novel pathway for the cAMP‐mediated response of the TPH promoter, which is independent of the canonical CRE‐mediated response.
Cocaine treatment increases extracellular cholecystokinin (CCK) in the nucleus accumbens shell of awake, freely moving rats, an effect that is enhanced in rats that are behaviorally sensitized to cocaine
Authors: Beinfeld Margery C. , Connolly Kelly J. , Pierce R. Christopher
Published: March 2003

Cholecystokinin (CCK) is co‐localized with dopamine, is known to modulate dopamine neurotransmission and is involved in behavioral sensitization to psychostimulants. To better understand its role, CCK was measured by microdialysis in the nucleus accumbens (NAC) shell in response to cocaine in drug‐naive rats and in rats that are behaviorally sensitized to cocaine. Basal extracellular levels of CCK in drug‐naive rats were 0.17 pg/20 min fraction, while in cocaine‐sensitized rats, they were significantly higher (0.56 pg). Treating drug‐naive rats with cocaine caused a significant increase in CCK to 0.58 pg. Cocaine treatment of cocaine‐sensitized rats increased CCK to 0.98. When analyzed as a function of time after cocaine treatment, these increases were sustained and were significantly different from CCK levels of saline‐treated rats. In cocaine‐sensitized rats, CCK levels following cocaine treatment were also significantly higher than levels in drug‐naive animals receiving a single injection of cocaine. These results provide evidence for an activation of the mesolimbic and/or cerebral cortical CCK system in response to repeated cocaine administration. These results provide a neurochemical basis for an important role of CCK (via modulation of dopamine neurotransmission) in expression of cocaine sensitization.
Dibasic cleavage site is required for sorting to the regulated secretory pathway for both pro‐ and neuropeptide Y
Authors: Brakch Noureddine , Allemandou Flore , Cavadas Claudia , Grouzmann Eric , Brunner Hans R.
Published: March 2003

To investigate the signals governing routing of biologically active peptides to the regulated secretory pathway, we have expressed mutated and non‐mutated proneuropeptide Y (ProNPY) in pituitary‐derived AtT20 cells. The mutations were carried out on dibasic cleavage site and or ProNPY C‐terminal sequence. Targeting to the regulated secretory pathway was studied using protein kinase A (8‐BrcAMP), protein kinase C (phorbol myristate acetate) specific activators and protein synthesis inhibitor cycloheximide, and by pulse chase. The analysis of expressed peptides in cells and culture media indicated that: neuropeptide Y (NPY) and ProNPY were differently secreted, whilst NPY was exclusively secreted via regulatory pathway; ProNPY was secreted via regulated and constitutive‐like secretory pathways. ProNPY secretion behaviour was not Proteolytic cleavage efficiency‐dependent. The dibasic cleavage was essential for ProNPY and NPY cAMP‐dependent regulated secretion and may have function as a retention signal.
Time‐dependence of the contribution of pyruvate carboxylase versus pyruvate dehydrogenase to rat brain glutamine labelling from [1–13C]glucose metabolism
Authors: Merle Michel , Bouzier‐Sore Anne‐Karine , Canioni Paul
Published: March 2003

[1–13C]glucose metabolism in the rat brain was investigated after intravenous infusion of the labelled substrate. Incorporation of the label into metabolites was analysed by NMR spectroscopy as a function of the infusion time: 10, 20, 30 or 60 min. Specific enrichments in purified mono‐ and dicarboxylic amino acids were determined from 1H‐observed/13C‐edited and 13C‐NMR spectroscopy. The relative contribution of pyruvate carboxylase versus pyruvate dehydrogenase (PC/PDH) to amino acid labelling was evaluated from the enrichment difference between either C2 and C3 for Glu and Gln, or C4 and C3 for GABA, respectively. No contribution of pyruvate carboxylase to aspartate, glutamate or GABA labelling was evidenced. The pyruvate carboxylase contribution to glutamine labelling varied with time. PC/PDH decreased from around 80% after 10 min to less than 30% between 20 and 60 min. This was interpreted as reflecting different labelling kinetics of the two glutamine precursor glutamate pools: the astrocytic glutamate and the neuronal glutamate taken up by astrocytes through the glutamate‐glutamine cycle. The results are discussed in the light of the possible occurrence of neuronal pyruvate carboxylation. The methods previously used to determine PC/PDH in brain were re‐evaluated as regards their capacity to discriminate between astrocytic (via pyruvate carboxylase) and neuronal (via malic enzyme) pyruvate carboxylation.
Interleukin‐4 and interleukin‐10 modulate nuclear factor κB activity and nitric oxide synthase‐2 expression in Theiler's virus‐infected brain astrocytes
Authors: Molina‐Holgado Eduardo , Arévalo‐Martín Angel , Castrillo Antonio , Boscá Lisardo , Vela José M. , Guaza Carmen
Published: March 2003

In brain astrocytes, nuclear factor κB (NF‐κB) is activated by stimuli that produce cellular stress causing the expression of genes involved in defence, including the inducible nitric oxide synthase (NOS‐2). Theiler's murine encephalomyelitis virus (TMEV) induces a persistent CNS infection and chronic immune‐mediated demyelination, similar to human multiple sclerosis. The cytokines interleukin (IL)‐4 and IL‐10 inhibit the expression of proinflammatory cytokines, counteracting the inflammatory process. Our study reports that infection of cultured astrocytes with TMEV resulted in a time‐dependent phosphorylation of IκBα, degradation of IκBα and IκBβ, activation of NF‐κB and expression of NOS‐2. The proteasome inhibitor MG‐132 blocked TMEV‐induced nitrite accumulation, NOS‐2 mRNA expression and phospho‐IκBα degradation, suggesting NF‐κB‐dependent NOS‐2 expression. Pretreatment of astrocytes with IL‐4 or IL‐10 decreased p65 nuclear translocation, NF‐κB binding activity and NOS‐2 transcription. IL‐4 and IL‐10 caused an accumulation of IκBα in TMEV‐infected astrocytes without affecting IκBβ levels. The IκB kinase activity and the degradation rate of both IκBs were not modified by either cytokine, suggesting de novo synthesis of IκBα. Indeed, IL‐4 or IL‐10 up‐regulated IκBα mRNA levels after TMEV infection. Therefore, the accumulation of IκBα might impair the translocation of the NF‐κB to the nucleus, mediating the inhibition of NF‐κB activity. Overall, these data suggest a novel mechanism of action of IL‐4 and IL‐10, which abrogates NOS‐2 expression in viral‐infected glial cells.
Glycine transporter isoforms show differential subcellular localization in PC12 cells
Authors: Geerlings Arjan , Núñez Enrique , Rodenstein Lara , López‐Corcuera Beatriz , Aragón Carmen
Published: March 2003

The subcellular localization of glycine transporters one (GLYT1) and two (GLYT2) stably expressed in PC12 cells has been studied. To facilitate visualization, enhanced green fluorescent protein (GFP) was fused to the amino terminus of both glycine transporters. Functional analysis of the GFP–GLYT1 and GFP–GLYT2 stable cell lines demonstrated that they exhibited high affinity for glycine and the characteristic properties of both glycine transporter subtypes. The GFP‐coupled transporters were differently distributed throughout the cell. GFP–GLYT1 was mainly localized on the plasma membrane, whereas most of GFP–GLYT2 was present on large dense‐core vesicles and endosomes. Both transporters were absent from the synaptic vesicle population in PC12 cells.
One melanocortin 4 and two melanocortin 5 receptors from zebrafish show remarkable conservation in structure and pharmacology
Authors: Ringholm Aneta , Fredriksson Robert , Poliakova Natalia , Yan Yi‐Lin , Postlethwait John H. , Larhammar Dan , Schiöth Helgi B.
Published: March 2003

We report the cloning, genome mapping, functional expression, pharmacology and anatomical distribution of three melanocortin (MC) receptors from zebrafish (z). Phylogenetic analysis showed with high bootstrap support that these genes represent one MC4 receptor and two MC5 receptors. Chromosomal mapping showed conserved synteny between regions containing zMC4 and human (h) MC4 receptors, whereas the two zMC5 receptor genes map on chromosome segments in which the zebrafish has several genes with two orthologues of a single mammalian gene. It is likely that the two copies of zMC5 receptors arose through a separate duplication in the teleost lineage. The zMC4, zMC5a, and zMC5b receptors share 70–71% overall amino acid identity with the respective human orthologues and over 90% in three TM regions believed to be most important for ligand binding. All three zebrafish receptors also show pharmacological properties remarkably similar to their human orthologues, with similar affinities and the same potency order, when expressed and characterized in radioligand binding assay for the natural melanocyte stimulating hormone (MSH) peptides α‐, β‐, and γ‐MSH. Stimulation of transfected mammalian cells with α‐MSH caused a dose‐dependent increase in intracellular cAMP levels for all three zebrafish receptors. All three genes were expressed in the brain, eye, ovaries and gastrointestinal tract, whereas the zMC5b receptor was also found in the heart, as determined by RT‐PCR. Our studies, which represent the first characterization of MC receptors in a nonamniote species, indicate that the MC receptor subtypes arose very early in vertebrate evolution. Important pharmacological and functional properties, as well as gene structure and syntenic relationships have been highly conserved over a period of more than 400 million years implying that these receptors participate in vital physiological functions.
Analysis of genes induced in peripheral nerve after axotomy using cDNA microarrays
Authors: Kubo Tateki , Yamashita Toshihide , Yamaguchi Atsushi , Hosokawa Ko , Tohyama Masaya
Published: March 2003

One of the most striking features of neurons in the mature peripheral nervous system is their ability to survive and to regenerate their axons following axonal injury. To perform a comprehensive survey of the molecular mechanisms that underlie peripheral nerve regeneration, we analyzed a cDNA library derived from the distal stumps of post‐injured sciatic nerve which was enriched in non‐myelinating Schwann cells using cDNA microarrays. The number of up‐ and down‐regulated genes in the transected sciatic nerve was 370 and 157, respectively, of the 9596 spotted genes. In the up‐regulated group, the number of known genes was 216 and the number of expressed sequence tag (EST) sequences was 154. In the down‐regulated group, the number of known genes was 103 and that of EST sequences was 54. We obtained several genes that were previously reported to be involved in regeneration of the injured neurons, such as cathepsin D, ninjurin 1, tenascin C, and co‐receptor for glial cell line‐derived neurotrophic factor family of trophic factors. In addition to unknown genes, there seemed to be a lot of annotated genes whose role in nerve regeneration remains unknown.
Antiproliferative action of valproate is associated with aberrant expression and nuclear translocation of cyclin D3 during the C6 glioma G1 phase
Authors: Bacon Christopher L. , Gallagher Helen C. , Haughey John C. , Regan Ciaran M.
Published: March 2003

Cell cycle progression is tightly regulated by cyclins, cyclin‐dependent kinases (cdks) and related inhibitory phophatases. Here, we employed mitotic selection to synchronize the C6 glioma cell cycle at the start of the G1 phase and mapped the temporal regulation of selected cyclins, cdks and inhibitory proteins throughout the 12 h of G1 by immunoblot analysis. The D‐type cyclins, D3 and D1, were differentially expressed during the C6 glioma G1 phase. Cyclin D1 was up‐regulated in the mid‐G1 phase (4–6 h) while cyclin D3 expression emerged only in late G1 (9–12 h). The influence of the anticonvulsant agent valproic acid (VPA) on expression of cyclins and related proteins was determined, since its teratogenic potency has been linked to cell cycle arrest in the mid‐G1 phase. Exposure of C6 glioma to VPA induced a marked up‐regulation of cyclin D3 and decreased expression of the proliferating cell nuclear antigen. In synchronized cell populations, increased expression of cyclin D3 by VPA was detected in the mid‐G1 phase (3–5 h). Immunocytochemical localization demonstrated rapid intracellular translocation of cyclin D3 to the nucleus following VPA exposure, suggesting that VPA‐induced cell cycle arrest may be mediated by precocious activation of cyclin D3 in the G1 phase.
Kynurenine 3‐mono‐oxygenase inhibitors attenuate post‐ischemic neuronal death in organotypic hippocampal slice cultures
Authors: Carpenedo Raffaella , Meli Elena , Peruginelli Fiamma , Pellegrini‐Giampietro Domenico E. , Moroni Flavio
Published: March 2003

Kynurenine 3‐mono‐oxygenase (KMO) inhibitors reduce 3‐hydroxykynurenine (3‐HK) and quinolinic acid (QUIN) neosynthesis and facilitate kynurenine metabolism towards kynurenic acid (KYNA) formation. They also reduce tissue damage in models of focal or transient global cerebral ischemia in vivo. We used organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) to investigate KMO mechanism(s) of neuroprotective activity. Exposure of the slices to 30 min of OGD caused CA1 pyramidal cell death and significantly decreased the amount of KYNA released in the incubation medium. The KMO inhibitors (m‐nitrobenzoyl)‐alanine (30–100 µm) or 3,4‐dimethoxy‐[‐N‐4‐(nitrophenyl)thiazol‐2yl]‐benzenesulfonamide (1–10 µm) reduced post‐ischemic neuronal death and increased KYNA concentrations in slice incubation media. The maximal concentration of KYNA detected in the incubation media of slices treated with KMO inhibitors was approximately 50 nm and was too low to efficiently interact with α7 nicotinic acetylcholine receptors or with the glycineb site of N‐methyl‐d‐aspartate (NMDA) receptors. On the other hand, the addition of either 3‐HK or QUIN (1–10 µm) to OGD‐exposed hippocampal slices prevented the neuroprotective activity of KMO inhibitors. Our results suggest that KMO inhibitors reduce the neuronal death found in the CA1 region of organotypic hippocampal slices exposed to 30 min of OGD by decreasing the local synthesis of 3‐HK and QUIN.
Characterization of chemokines and their receptors in the central nervous system: physiopathological implications
Authors: Bajetto Adriana , Bonavia Rudy , Barbero Simone , Schettini Gennaro
Published: March 2003

Chemokines represent key factors in the outburst of the immune response, by activating and directing the leukocyte traffic, both in lymphopoiesis and in immune surveillance. Neurobiologists took little interest in chemokines for many years, until their link to acquired immune deficiency syndrome‐associated dementia became established, and thus their importance in this field has been neglected. Nevertheless, the body of data on their expression and role in the CNS has grown in the past few years, along with a new vision of brain as an immunologically competent and active organ. A large number of chemokines and chemokine receptors are expressed in neurons, astrocytes, microglia and oligodendrocytes, either constitutively or induced by inflammatory mediators. They are involved in many neuropathological processes in which an inflammatory state persists, as well as in brain tumor progression and metastasis. Moreover, there is evidence for a crucial role of CNS chemokines under physiological conditions, similar to well known functions in the immune system, such as proliferation and developmental patterning, but also peculiar to the CNS, such as regulation of neural transmission, plasticity and survival.
Hydroxylated residues influence desensitization behaviour of recombinant α3 glycine receptor channels
Authors: Breitinger Hans‐Georg , Villmann Carmen , Rennert Janine , Ballhausen Diana , Becker Cord‐Michael
Published: March 2003

The human glycine receptor subunit α3 exists in two splice variants (α3K/L), with α3L bearing an additional segment of 15 amino acids within the cytoplasmic TM3‐4 loop. Homomeric α3K glycine receptors show faster desensitization than α3L receptors. Ion channel properties were compared of α3L, α3K, and of the triple mutant α3LΔOH = α3L(T358A/Y367F/S370A), where hydroxyl functions of the spliced insert had been removed by site‐directed mutagenesis. Upon recombinant expression in HEK 293 cells, patch‐clamp recording experiments revealed that removal of hydroxyl functions primarily affected receptor desensitization. The fraction of non‐desensitizing current was 68 ± 13% for α3L, 21 ± 13% for α3K, and 48 ± 16% for α3LΔOH. Desensitization time constants at saturating glycine concentration were 8.4 ± 2.8 s, 1.9 ± 2.3 s, and 2.8 ± 0.4 s, for α3L, α3K, and the triple mutant α3LΔOH, respectively. In contrast, single‐channel and whole‐cell properties were similar for all three constructs. Thus, ion channel activation, desensitization, and conductance properties are independently controlled by distinct structural elements. Hydroxyl functions within the M3‐4 loop of the glycine receptor α3 subunit are crucial, but not exclusive, determinants of receptor desensitization.