bluuebunny/crossref_metadata_2025 huggingface dataset:
Subcellular Localization of Chromogranins, Calcium ChanneAmine Carriers, and Proteins of the Exocytotic Machinery in Bovine Splenic Nerve
Authors: Leitner Bernd , Lovisetti‐Scamihorn Paola , Heilmann Jutta , Striessnig Jörg , Blakely Randy D. , Eiden Lee E. , Winkler Hans
Published: March 2003

Abstract : Subcellular fractionation of bovine splenic nerves, which
consist mainly of sympathetic nerve fibers, has been useful for characterizing
cellular organelles en route to the terminal. In the present study we have
characterized the subcellular distribution of both secretory and membrane
proteins. A newly discovered chromogranin‐like protein, NESP55, was found in
large dense‐core vesicles. The endogenous processing of NESP55 was comparable
to that of chromogranins but more limited than that of secretogranin II and
chromogranin B. For membrane proteins three major types of distribution were
found. The amine carrier VMAT2 was confined to large dense‐core vesicles. VAMP
or synaptobrevin was present both in large dense‐core vesicles and in lighter
vesicles, whereas SNAP‐25, syntaxin, and two types (N and L) of
Ca2+ channels were found in a special population of lighter
vesicles but were not present in large dense‐core vesicles or at the most in
very low concentrations. The plasma membrane norepinephrine transporter was
apparently present in a separate type of vesicle, but this requires further
study. These results further characterize vesicles en route to the terminal
and establish for the first time that peptides involved in exocytosis
(syntaxin, SNAP‐25, and N‐ and L‐type Ca2+ channels) are apparently transported to the terminal in a special type of vesicle. The exclusive presence of the amine carrier in large dense‐core vesicles indicates that the formation of small dense‐core vesicles in the terminals requires a reuse of membrane components of large dense‐core vesicles.
Pathways of Cadmium Influx in Mammalian Neurons
Authors: Usai Cesare , Barberis Andrea , Moccagatta Luca , Marchetti Carla
Published: March 2003

Abstract: The Influx of the toxic cation Cd2+ was studied in fura 2‐loaded rat cerebellar granule neurons. In cells depolarized with Ca2+‐free, high‐KCI solutions, the fluorescence emission ratio (R) increased in the presence of 100 γM Cd2+. This increase was fully reversed by the Cd2+ chelator tetrakis(2‐pyridylmethyl)ethylenediamine, indicating a cadmium influx into the cell. The rate of increase, dR/dt, was greatly reduced (67 ± 5%) by 1 γM nimodipine and enhanced by 1 γM Bay K 8644. Concurrent application of nimodipine and ω‐agatoxin IVA (200 nM) blocked Cd2+ permeation almost completely (88 ± 5%), whereas ω‐conotoxin MVIIC (2 γM) reduced dR/dt by 24 ± 8%. These results indicate a primary role of voltage‐dependent calcium channels in Cd2+ permeation. Stimulation with glutamate or NMDA and glycine also caused a rise of R in external Cd2+. Simultaneous application of nimodipine and ω‐agatoxin IVA moderately reduced dR/dt (25 ± 3%). NMDA‐driven Cd2+ entry was almost completely prevented by 1 mM Mg2+, 50 γM memantine, and 10 γM 5,7‐dichlorokynurenic acid, suggesting a major contribution of NMDA‐gated channels in glutamate‐stimulated Cd2+ influx. Moreover, perfusion with α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate caused a slow increase of R. These results suggest that Cd2+ permeates the cell membrane mainly through the same pathways of Ca2+ influx.
Membrane Microviscosity Modulates μ‐Opioid Receptor Conformational Transitions and Agonist Efficacy
Authors: Emmerson Paul J. , Clark Mary J. , Medzihradsky Fedor , Remmers Ann E.
Published: March 2003

Abstract: The influence of membrane microviscosity on μ‐opioid agonist and antagonist binding, as well as agonist efficacy, was examined in membranes prepared from SH‐SY5Y cells and from a C6 glioma cell line stably expressing the rat μ‐opioid receptor (C6μ). Addition of cholesteryl hemisuccinate (CHS) to cell membranes increased membrane microviscosity and reduced the inhibitory effect of sodium and guanine nucleotides on the affinity of the full agonists sufentanil and [D‐Ala2,N‐MePhe4,Gly‐ol5]enkephalin (DAMGO) for the μ‐opioid receptor. Binding of the antagonists [3H]naltrexone and [3H]diprenorphine and the partial agonist nalbuphine was unaffected by CHS. The effect of CHS on agonist binding was reversed by subsequent addition of cis‐vaccenic acid, suggesting that the effect of CHS is the result of increased membrane microviscosity and not a specific sterol—receptor interaction. CHS addition increased the potency of DAMGO to stimulate guanosine‐5′‐O‐(3‐[35S]thio)triphosphate binding by fourfold, whereas the potency of nalbuphine was unaffected. However, nalbuphine efficacy relative to that of the full agonist DAMGO was strongly increased in CHS‐treated membranes compared with that in control membranes. Membrane rigidification also resulted in an increased efficacy for the partial agonists meperidine, profadol, and butorphanol relative to that of DAMGO as measured by agonist‐stimulated GTPase activity in control and CHS‐modified membranes. These findings support a regulatory role for membrane microviscosity in receptor‐mediated G protein activation.
Evidence for an Antiapoptotic Role of Dopamine in Developing Retinal Tissue
Authors: Varella Marcia H. , De Mello Fernando G. , Linden Rafael
Published: March 2003

Abstract : Inhibition of protein synthesis leads to apoptosis in the

undifferentiated neuroblastic layer of the retina of newborn rats. We have

shown previously that an increase in the intracellular concentration of cyclic

AMP prevented apoptosis induced in the retinal neuroblastic layer by

inhibition of protein synthesis. In this study, we tested the effects of

dopamine on retinal apoptosis and related these effects to the intracellular

concentration of cyclic AMP. Both dopamine (100 μM) and the

D1‐like agonists SKF‐38393,

6‐chloro‐7,8‐dihydroxy‐1‐phenyl‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine

(6‐Cl‐PB), and (±)‐2‐amino‐6,7‐dihydroxy‐1,2,3,4‐tetrahydronapthalene

(100 μM) blocked apoptosis induced in the neuroblastic layer by

the protein synthesis inhibitor anisomycin. The antiapoptotic effects of the

D1‐like agonists were not reversed by the D1‐like antagonist SCH‐23390 (5‐100

μM). Both dopamine and D1‐like angonists induced a five‐to

sevenfold increase in the intracellular concentration of cyclic AMP in the

retina of newborn rats. The concentration of cyclic AMP induced by the D1‐like

agonists in the presence of 100 μM SCH‐23390 was still at least

two‐ to threefold as high as control values, showing that the activation of

adenylyl cyclase by D1‐like agonists was reversed only partially by the

specific antagonist. The isoquinolinesulfonamide H‐89 (20 μM), an inhibitor of cyclic AMP‐dependent protein kinase, partially prevented the antiapoptotic effect of 6‐Cl‐PB. The data show that an early effect of dopamine in the developing retina is the control of programmed cell death. The antiapoptotic effect of dopamine is mediated, at least in part, through an atypical D1‐like receptor coupled to stimulation of adenylyl cyclase, followed by activation of cyclic AMP‐dependent protein kinase.
Regulation of Neuropeptide Y Release by Neuropeptide Y Receptor Ligands and Calcium Channel Antagonists in Hypothalamic Slices
Authors: King Peter J. , Widdowson Peter S. , Doods Henri N. , Williams Gareth
Published: March 2003

Abstract : Neuropeptide Y (NPY) is an important regulator of energy

balance in mammals through its orexigenic, antithermogenic, and insulin

secretagogue actions. We investigated the regulation of endogenous NPY release

from rat hypothalamic slices by NPY receptor ligands and calcium channel

antagonists. High‐potassium stimulation (60 mM) of the slices

produced a calcium‐dependent threefold increase in NPY release above basal

release. The Y2 receptor agonists NPY(13‐36) and

N‐acetyl[Leu28, Leu31]NPY(24‐36), the Y4

agonist rat pancreatic polypeptide (rPP), and the Y4/Y5 agonist human

pancreatic polypeptide (hPP) significantly reduced both basal and stimulated

NPY release. NPY(13‐36)‐induced reduction of NPY release could be partially

prevented in the presence of the weak Y2 antagonist

T4‐[NPY(33‐36)]4, whereas the hPP‐ and rPP‐induced inhibition of release was not affected by the Y5 antagonist CGP71683A or the Y1 antagonist BIBP3226. The selective Y1, Y2, and Y5 antagonists had no effect on either basal or potassium‐stimulated release when administered alone. The calcium channel inhibitors ω‐conotoxin GVIA (N‐type), ω‐agatoxin TK (P/Q‐type), and ω‐conotoxin MVIIC (Q‐type) all significantly inhibited potassium‐stimulated NPY release, without any effect on basal release, whereas nifedipine had no effect on either basal or stimulated release. Addition of both ω‐conotoxin GVIA and ω‐agatoxin TK together completely inhibited the potassium‐stimulated release. In conclusion, we have demonstrated that NPY release from hypothalamic slices is calcium‐dependent, involving N‐, P‐, and Q‐type calcium channels. NPY release is also inhibited by Y2 agonists and rPP/hPP, suggesting that Y2 and Y4 receptors may act as autoreceptors on NPY‐containing nerve terminals.
Relationship Between Casein Kinase I Isoforms and a Neurofilament‐Associated Kinase
Authors: Fu Zheng , Green Calvert L , Bennett Gudrun S.
Published: March 2003

Abstract : Purified neurofilaments (NFs) contain an associated kinase (NFAK) activity that phosphorylates selectively a subset of sites in the tail of NF‐M and has properties consistent with casein kinase I (CKI). Because CKI consists of a family of as many as seven genes (α, β, γ1‐3, δ, and ε), we investigated the extent to which different CKI isoforms contribute to NFAK activity. Using an NF‐M‐derived substrate, we determined that NFAK activity copurified with casein kinase activity through two purification steps. In an in‐gel kinase assay, NFAK activity occurred at 36‐40 kDa, corresponding to the size of CKIα isoforms. Chicken neurons express transcripts encoding four alternatively spliced variants of CKIα (CKIα, CKIαS, CKIαL, and CKIαLS) differing in the presence or absence of two inserts, L and S. Using antibodies against different isoforms or with broad CKI specificity, we determined that all four CKIα variants, as well as other CKI family members, are present in chicken brain. However, only CKIα and CKIαS could be detected in purified NFAK. Also, immunoprecipitation studies showed that CKIα and CKIαS together account for NFAK activity. These findings raise the possibility that only a subset of CKI isoforms may be able to associate with and/or phosphorylate NFs.
Semaphorins as Mediators of Neuronal Apoptosis
Authors: Shirvan Anat , Ziv Ilan , Fleminger Gideon , Shina Ronit , He Zhigang , Brudo Irene , Melamed Eldad , Barzilai Ari
Published: March 2003

Abstract : Shrinkage and collapse of the neuritic network are often observed during the process of neuronal apoptosis. However, the molecular and biochemical basis for the axonal damage associated with neuronal cell death is still unclear. We present evidence for the involvement of axon guidance molecules with repulsive cues in neuronal cell death. Using the differential display approach, an up‐regulation of collapsin response mediator protein was detected in sympathetic neurons undergoing dopamine‐induced apoptosis. A synchronized induction of mRNA of the secreted collapsin‐1 and the intracellular collapsin response mediator protein that preceded commitment of neurons to apoptosis was detected. Antibodies directed against a conserved collapsin‐derived peptide provided marked and prolonged protection of several neuronal cell types from dopamine‐induced apoptosis. Moreover, neuronal apoptosis was inhibited by antibodies against neuropilin‐1, a putative component of the semaphorin III/collapsin‐1 receptor. Induction of neuronal apoptosis was also caused by exposure of neurons to semaphorin III‐alkaline phosphatase secreted from 293EBNA cells. Anti‐collapsin‐1 antibodies were effective in blocking the semaphorin III‐induced death process. We therefore suggest that, before their death, apoptosis‐destined neurons may produce and secrete destructive axon guidance molecules that can affect their neighboring cells and thus transfer a “death signal” across specific and susceptible neuronal populations.
Tyrosine Hydroxylase Is Inactivated by Catechol‐Quinones and Converted to a Redox‐Cycling Quinoprotein
Authors: Kuhn Donald M. , Arthur Robert E. , Thomas David M. , Elferink Lisa A.
Published: March 2003

Abstract Quinone derivatives of DOPA, dopamine, and N‐acetyldopamine inactivate tyrosine hydroxylase, the initial and rate‐limiting enzyme in the biosynthesis of the catecholamine neurotransmitters. The parent catechols are inert in this capacity. The effects of the catecholquinones on tyrosine hydroxylase are prevented by antioxidants and reducing reagents but not by scavengers of hydrogen peroxide, hydroxyl radicals, or superoxide radicals. Quinone modification of tyrosine hydroxylase modifies enzyme sulfhydryl groups and results in the formation of cysteinyl‐catechols within the enzyme. Catecholquinones convert tyrosine hydroxylase to a redox‐cycling quinoprotein. Quinotyrosine hydroxylase causes the reduction of the transition metals iron and copper and may therefore contribute to Fenton‐like reactions and oxidative stress in neurons. The discovery that a phenotypic marker for catecholamine neurons can be converted into a redox‐active species is highly relevant for neurodegenerative conditions such as Parkinson’s disease.
Signaling Mechanisms Involved in the Activation of Arachidonic Acid Metabolism in Human Astrocytoma Cells by Tumor Necrosis Factor‐α
Authors: Hernández Marita , Bayón Yolanda , Crespo Mariano Sánchez , Nieto María Luisa
Published: March 2003

Abstract : Tumor necrosis factor‐α (TNF‐α) is a cytokine

that elicits cell responses by activating the mitogen‐activated protein kinase

(MAP kinase) cascade and transcription factors such as nuclear

factor‐kB (NF‐kB). As these elements play a central role in

the mechanisms of signaling involved in the activation of cytosolic

phospholipase A2 (cPLA2) and cyclooxygenase‐2 (COX‐2),

the effect of TNF‐α on arachidonate (AA) metabolism in 1321N1

astrocytoma cells was assayed. TNF‐α produced a phosphorylation of

cPLA2, which was preceded by an activation of both c‐Jun N‐terminal

kinase (JNK) and p38‐MAP kinase, and this was associated with the release of

[3H]AA. In contrast, TNF‐α did not activate the extracellular

signal‐regulated kinase (MAP kinase) p42, nor did it elicit a mitogenic

response. Analysis of [3H]AA metabolites by reverse‐phase HPLC

showed that all of the [3H]AA released during the first hour after

TNF‐α addition eluted as authentic AA, whereas in samples obtained at 24

h after addition of TNF‐α, 25% of the [3H]AA had been

converted into COX products as compared with only 9% in control cells. In

keeping with these findings, TNF‐α produced an increase of COX‐2

expression, as judged from both RT‐PCR studies and immunoblot of COX‐2

protein, and a long‐lasting activation of NF‐kB. These data show that

TNF‐α produces in astrocytoma cells an early activation of both p38‐MAP

kinase and JNK, which is followed by the phosphorylation of cPLA2

and the release of AA. On the other hand, the activation of NF‐kB may explain the induction of the expression of COX‐2 and the delayed generation of prostanoids.
Dopamine D2 Receptor Isoforms Expressed in AtT20 Cells Differentially Couple to G Proteins to Acutely Inhibit High Voltage‐Activated Calcium Channels
Authors: Wolfe Shannyn E. , Morris Stephen J.
Published: March 2003

Abstract : The dopamine D2 receptor belongs to the

serpentine superfamily of receptors, which have seven transmembrane segments

and activate G proteins. D2 receptors are known to be linked,

through Gαo‐ and Gαi‐containing G proteins,

to several signaling pathways in neuronal and secretory cells, including

inhibition of adenylyl cyclase and high voltage‐activated Ca2+

channels (HVA‐CCs). The dopamine D2 receptor exists in two

alternatively spliced isoforms, “long” and “short”

(D2L and D2S, respectively), which have identical ligand

binding sites but differ by 29 amino acids in the third intracellular loop,

the proposed site for G protein interaction. This has led to the speculation

that the two isoforms may interact with different G proteins. We have

transfected the AtT20 cell line with either D2L (KCL line) or

D2S (KCS line) to facilitate experimentation on the individual

isoforms. Both lines show dopamine agonist‐dependent inhibition of Q‐type

HVA‐CCs. We combined G protein antisense knock‐down studies with

multiwavelength fluorescence video microscopy to measure changes in HVA‐CC

inhibition to investigate the possibility of differential G protein coupling

to this inhibition. The initial, rapid, K+ depolarization‐induced

increase in intracellular Ca2+ concentration is due to influx

through HVA‐CCs. Our studies reveal that both D2 isoforms couple to

Gαo to partially inhibit this influx. However, D2L

also couples to Gα13, whereas D2S couples to

Gα12. These data support the hypothesis of differential

coupling of D2 receptor isoforms to G proteins.
In Vitro Evidence for Increased Facilitation of Striatal Acetylcholine Release via Pre‐ and Postsynaptic NMDA Receptotors in Hemiparkinsonian Rats
Authors: Marti M. , Sbrenna S. , Fuxe K. , Bianchi C. , Beani L. , Morari M.
Published: March 2003

Abstract : The NMDA‐evoked acetylcholine release from striatal
slices and synaptosomes was investigated in rats subjected to unilateral
injection of 6‐hydroxydopamine into the substantia nigra. In slices prepared
from the striatum contralateral to the lesion, the NMDA‐evoked endogenous
acetylcholine release was not significant at 10 μM NMDA and
maximal at 100 μM NMDA (124 ± 19%). Conversely, in slices
taken from the dopamine‐depleted striatum, NMDA was effective even at 10
μM (41 ± 4%), and at 100 μM (196 ± 24%)
efficacy was nearly doubled. In synaptosomes prepared from the contralateral
striatum, NMDA maximally stimulated 20 mM KCl‐induced endogenous
acetylcholine release at 1 μM (66 ± 5.1%), with lower
concentrations (0.01‐0.1 μM) being ineffective. Conversely, in
synaptosomes prepared from the dopamine‐depleted striatum, NMDA maximally
enhanced the K+‐evoked acetylcholine release at 0.1 μM (118 ± 12.4%). Concentration‐response curves of NMDA‐evoked acetylcholine release in sham‐operated rats could be superimposed on those observed in the contralateral striatum of the 6‐hydroxydopamine‐lesioned animals. The present data support the view of an increased glutamatergic regulation of striatal acetylcholine release via pre‐ and postsynaptic NMDA receptors during Parkinson's disease.
Expression and Signaling of Group I Metabotropic Glutamate Receptors in Astrocytes and Microglia
Authors: Biber Knut , Laurie David J. , Berthele Achim , Sommer Bernd , Tölle Thomas R. , Gebicke‐Härter Peter‐J. , Van Calker Dietrich , Boddeke Hendrikus W. G. M.
Published: March 2003

Abstract: Stimulation of astrocytes with the excitatory neurotransmitter glutamate leads to the formation of inositol 1,4,5‐trisphosphate and the subsequent increase of intracellular calcium content. Astrocytes express both ionotropic receptors and metabotropic glutamate (mGlu) receptors, of which mGlu5 receptors are probably involved in glutamate‐induced calcium signaling. The mGlu5 receptor occurs as two splice variants, mGlu5a and mGlu5b, but it was hitherto unknown which splice variant is responsible for the glutamate‐induced effects in astrocytes. We report here that both mRNAs encoding mGlu5 receptor splice variants are expressed by cultured astrocytes. The expression of mGlu5a receptor mRNA is much stronger than that of mGlu5b receptor mRNA in these cells. In situ hybridization experiments reveal neuronal expression of mGlu5b receptor mRNA in adult rat forebrain but a strong neuronal expression of mGlu5a mRNA only in olfactory bulb. Signals for mGlu5a receptor mRNA in the rest of the brain were diffuse and weak but consistently above background. Activation of mGlu5 receptors in astrocytes yields increases in inositol phosphate production and transient calcium responses. It is surprising that the rank order of agonist potency [quisqualate > (2S, 1′S,2′S)‐2‐(carboxycyclopropyl)glycine = trans‐(1S,3R)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid (1S, 3R‐ACPD) > glutamate] differs from that reported for recombinantly expressed mGlu5a receptors. The expression of mGlu5a receptor mRNA and the occurrence of 1S, 3R‐ACPD‐induced calcium signaling were found also in cultured microglia, indicating for the first time expression of mGlu5a receptors in these macrophage‐like cells.
The Lipid Peroxidation Product 4‐Hydroxy‐2,3‐Nonenal Increases AP‐1‐Binding Activity Through Caspase Activation in Neurons
Authors: Camandola Simonetta , Poli Giuseppe , Mattson Mark P.
Published: March 2003

Abstract: The transcription factor activator protein‐1 (AP‐1) is activated in response to physiological activity in neuronal circuits and in response to neuronal injury associated with various acute and chronic neurodegenerative conditions. The membrane lipid peroxidation product 4‐hydroxy‐2,3‐nonenal (HNE) is increasingly implicated in the disruption of neuronal calcium homeostasis that occurs in various paradigms of neuronal excitotoxicity and apoptosis. The possible mechanistic links between lipid peroxidation and alterations in gene transcription during neuronal apoptosis have not previously been examined. We now report that exposure of cultured rat cortical neurons to an apoptotic concentration of HNE results in a large increase in AP‐1 DNA‐binding activity. The protein synthesis inhibitor cycloheximide blocked the induction of AP‐1, consistent with a requirement for induction of expression of AP‐1 family members. The broad‐spectrum caspase inhibitor N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethyl ketone and the caspase‐3 inhibitor N‐acetyl‐Asp‐Glu‐Val‐Asp‐aldehyde blocked HNE‐induced increases in AP‐1 DNA‐binding activity, demonstrating a requirement for caspase activation in the activation of AP‐1. HNE induced phosphorylation of c‐Jun N‐terminal kinase (JNK), which was prevented by caspase inhibitors, indicating that HNE was acting at or upstream of JNK phosphorylation. The intracellular calcium chelator BAPTA‐acetoxymethyl ester completely prevented stimulation of AP‐1 DNA‐binding by HNE, indicating a requirement for calcium. Moreover, agents that suppress mitochondrial calcium uptake (ruthenium red) and membrane permeability transition (cyclosporin A) attenuated AP‐1 activation by HNE, suggesting a contribution of mitochondrial alterations to AP‐1 activation. Collectively, our data suggest a scenario in which HNE disrupts neuronal calcium homeostasis and perturbs mitochondrial function, resulting in caspase activation. Activated caspases, in turn, induce activation of JNK, resulting in stimulation of AP‐1 DNA‐binding protein production. This transcriptional pathway induced by HNE may modulate the cell death process.
Immunocytochemical Localization of β‐Methylcrotonyl‐CoA Carboxylase in Astroglial Cells and Neurons in Culture
Authors: Bixel M. Gabriele , Hamprecht Bernd
Published: March 2003

Abstract: Astroglia‐rich primary cultures and brain slices rapidly metabolize branched‐chain amino acids (BCAAs), in particular leucine, as energy substrates. To allocate the capacity to degrade leucine oxidatively in neural cells, we have purified β‐methylcrotonyl‐CoA carboxylase (β‐MCC) from rat liver as one of the enzymes unique for the irreversible catabolic pathway of leucine. Polyclonal antibodies raised against β‐MCC specifically cross‐reacted with both enzyme subunits in liver and brain homogenates. Immunocytochemical examination of astroglia‐rich rat primary cultures demonstrated the presence of β‐MCC in astroglial cells, where the enzyme was found to be located in the mitochondria, the same organelle that the mitochondrial isoform of the BCAA aminotransferase (BCAT) is located in. This colocalization of the two enzymes supports the hypothesis that mitochondrial BCAT is the isoenzyme that in brain energy metabolism prepares the carbon skeleton of leucine for irreversible degradation in astrocytes. Analysis of neuron‐rich primary cultures revealed also that the majority of neurons contained β‐MCC. The presence of β‐MCC in most neurons demonstrates their ability to degrade the α‐ketoisocaproate that could be provided by neighboring astrocytes or could be generated locally from leucine by the action of the cytosolic isoform of BCAT that is known to occur in neurons.
Synergistic Depletion of Astrocytic Glutathione by Glucose Deprivation and Peroxynitrite
Authors: Ju Chung , Yoon Keum‐Na , Oh Yu‐Kyoung , Kim Hyoung‐Chun , Shin Chan Young , Ryu Jae Ryun , Ko Kwang Ho , Kim Won‐Ki
Published: March 2003

Abstract: Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO‐‐producing reagent 3‐morpholinosydnonimine (SIN‐1). Here we show that glucose deprivation and ONOO‐ synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A‐sensitive mitochondrial permeability transition (MPT) pore. Astrocytic GSH levels were only slightly decreased by glucose deprivation or SIN‐1 (200 μM) alone. In contrast, a rapid and large depletion of GSH was observed in glucose‐deprived/SIN‐1‐treated astrocytes. The depletion of GSH occurred before a significant release of lactate dehydrogenase (a marker of cell death). Superoxide dismutase and ONOO‐ scavengers completely blocked the augmented death, indicating that the reaction of nitric oxide with superoxide to form ONOO‐ was implicated. Furthermore, nitrotyrosine immunoreactivity (a marker of ONOO‐) was markedly enhanced in glucose‐deprived/SIN‐1‐treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose‐deprived/SIN‐1‐treated astrocytes. The glutathione synthase inhibitor L‐buthionine‐(S,R)‐sulfoximine markedly decreased the MTP and increased lactate dehydrogenase (LDH) releases in SIN‐1‐treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevented the MTP depolarization as well as the enhanced LDH releases in glucose‐deprived/SIN‐1‐treated astrocytes.
Chick Pineal Melatonin Synthesis
Authors: Zatz Martin , Gastel Jonathan A. , Heath James R. , Klein David C.
Published: March 2003

Abstract: Melatonin production in the pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N‐acetyltransferase [arylalkylamine N‐acetyltransferase (AA‐NAT); EC 2.3.1.87], the penultimate enzyme in melatonin synthesis. The rhythm is generated by an endogenous circadian clock. In the chick, a clock is located in the pinealocyte, which also contains two phototransduction systems. One controls melatonin production by adjusting the clock and the other acts distal to the clock, via cyclic AMP mechanisms, to switch melatonin synthesis on and off. Unlike the clock in these cells, cyclic AMP does not appear to regulate activity by altering AA‐NAT mRNA levels. The major changes in AA‐NAT mRNA levels induced by the clock seemed likely (but not certain) to generate comparable changes in AA‐NAT protein levels and AA‐NAT activity. Cyclic AMP might also regulate AA‐NAT activity via changes in protein levels, or it might act via other mechanisms, including posttranslational changes affecting activity. We measured AA‐NAT protein levels and enzyme activity in cultured chick pineal cells and found that they correlated well under all conditions. They rose and fell spontaneously with a circadian rhythm. They also rose in response to agents that increase cyclic AMP. They were raised by agents that increase cyclic AMP, such as forskolin, and lowered by agents that decrease cyclic AMP, such as light and norepinephrine. Thus, both the clock and cyclic AMP can control AA‐NAT activity by altering the total amount of AA‐NAT protein. Effects of proteosomal proteolysis inhibitors suggest that changes in AA‐NAT protein levels, in turn, reflect changes in the rate at which the protein is destroyed by proteosomal proteolysis. It is likely that cyclic AMP‐induced changes in AA‐NAT protein levels mediate rapid changes in chick pineal AA‐NAT activity. Our results indicate that light can rapidly regulate the abundance of a specific protein (AA‐NAT) within a photoreceptive cell.
Aspartate Mutations in Presenilin and γ‐Secretase Inhibitors Both Impair Notch1 Proteolysis and Nuclear Translocation with Relative Preservation of Notch1 Signaling
Authors: Berezovska Oksana , Jack Christine , McLean Pamela , Aster Jon C , Hicks Carol , Xia Weiming , Wolfe Michael S , Kimberly W Taylor , Weinmaster Gerry , Selkoe Dennis J , Hyman Bradley T
Published: March 2003

It has been hypothesized that a presenilin 1 (PS1)‐related enzymatic activity is responsible for proteolytic cleavage of the C‐terminal intracellular protein of Notch1, in addition to its role in β‐amyloid protein (Aβ) formation from the amyloid precursor protein (APP). We developed an assay to monitor ligand‐induced Notch1 proteolysis and nuclear translocation in individual cells : Treatment of full‐length Notch1‐enhanced green fluorescent protein‐transfected Chinese hamster ovary (CHO) cells with a soluble preclustered form of the physiologic ligand Delta leads to rapid accumulation of the C terminus of Notch1 in the nucleus and to transcriptional activation of a C‐promoter binding factor 1 (CBF1) reporter construct. Nuclear translocation was blocked by cotransfection with Notch's physiologic inhibitor Numb. Using this assay, we now confirm and extend the observation that PS1 is involved in Notch1 nuclear translocation and signaling in mammalian cells. We demonstrate that the D257A and the D385A PS1 mutations, which had been shown previously to block APP γ‐secretase activity, also prevent Notch1 cleavage and translocation to the nucleus but do not alter Notch1 trafficking to the cell surface. We also show that two APP γ‐secretase inhibitors block Notch1 nuclear translocation with an IC50 similar to that reported for APP γ‐secretase. Notch1 signaling, assessed by measuring the activity of CBF1, a downstream transcription factor, was impaired but not abolished by the PS1 aspartate mutations or γ‐secretase inhibitors. Our results support the hypotheses that (a) PS1‐dependent APP γ‐secretase‐like enzymatic activity is critical for both APP and Notch processing and (b) the Notch1 signaling pathway remains partially activated even when Notch1 proteolytic processing and nuclear translocation are markedly inhibited. The latter is an important finding from the perspective of therapeutic treatment of Alzheimer's disease by targeting γ‐secretase processing of APP to reduce Aβ production.
Amyloid Peptide Aβ1‐42 Binds Selectively and with Picomolar Affinity to α7 Nicotinic Acetylcholine Receptors
Authors: Wang Hoau‐Yan , Lee Daniel H. S. , Davis Coralie B. , Shank Richard P.
Published: March 2003

Abstract: We have recently reported evidence that a very high

affinity interaction between the β‐amyloid peptide Aβ1‐42

and the α7 nicotinic acetylcholine receptor (α7nAChR) may be a

precipitating event in the formation of amyloid plaques in Alzheimer's

disease. In the present study, the kinetics for the binding of

Aβ1‐42 to α7nAChR and α4β2nAChR were

determined using the subtype‐selective nicotinic receptor ligands

[3H]methyllycaconitine and [3H]cytisine. Synaptic

membranes prepared from rat and guinea pig cerebral cortex and hippocampus

were used as the source of receptors. Aβ1‐42 bound to the

α7nAChR with exceptionally high affinity, as indicated by

Ki values of 4.1 and 5.0 pM for rat and guinea

pig receptors, respectively. When compared with the α7nAChR, the

affinity of Aβ1‐42 for the α4β2nAChR was

∼5,000‐fold lower, as indicated by corresponding Ki

values of 30 and 23nM. The results of this study support the concept

that an exceptionally high affinity interaction between Aβ1‐42 and α7nAChR could serve as a precipitating factor in the formation of amyloid plaques and thereby contribute to the selective degeneration of cholinergic neurons that originate in the basal forebrain and project to the cortex and hippocampus.
Proteasome Inhibitor‐Induced Apoptosis of Glioma Cells Involves the Processing of Multiple Caspases and Cytochrome c Release
Authors: Wagenknecht Bettina , Hermisson Mirjam , Groscurth Peter , Liston Peter , Krammer Peter H. , Weller Michael
Published: March 2003

Abstract: The proteasome is a multiprotein complex that is involvedin the intracellular protein degradation in eukaryotes. Here, we show thathuman malignant glioma cells are susceptible to apoptotic cell death inducedby the proteasome inhibitors, MG132 and lactacystin. The execution of theapoptotic death program involves the processing of caspases 2, 3, 7, 8, and 9.Apoptosis is inhibited by ectopic expression of X‐linked inhibitor ofapoptosis (XIAP) and by coexposure to the broad‐spectrum caspase inhibitor,benzoyl‐VAD‐fluoromethyl ketone (zVAD‐fmk), but not by the preferentialcaspase 8 inhibitor, crm‐A. It is interesting that specific morphologicalalterations induced by proteasome inhibition, such as dilated roughendoplasmic reticulum and the formation of cytoplasmic vacuoles and densemitochondrial deposits, are unaffected by zVAD‐fmk. Apoptosis is alsoinhibited by ectopic expression of Bcl‐2 or by an inhibitor of proteinsynthesis, cycloheximide. Further, cytochrome c release anddisruption of mitochondrial membrane potential are prominent features ofapoptosis triggered by proteasome inhibition. Bcl‐2 is a stronger inhibitor ofcytochrome c release than zVAD‐fmk. XIAP and crm‐A fail to modulatecytochrome c release. These data place cytochrome c releasedownstream of Bcl‐2 activity but upstream of XIAP‐ and crm‐A‐sensitivecaspases. The partial inhibition of cytochrome c release by zVAD‐fmkindicates a positive feedback loop that may involve cytochrome crelease and zVAD‐fmk‐sensitive caspases. Finally, death ligand/receptorinteractions, including the CD95/CD95 ligand system, do not mediate apoptosisinduced by proteasome inhibition in human malignant glioma cells.
Chronic Hypoxia Enhances Adenosine Release in Rat PC12 Cells by Altering Adenosine Metabolism and Membrane Transport
Authors: Kobayashi Shuichi , Zimmermann Herbert , Millhorn David E.
Published: March 2003

Abstract: Acute exposure to hypoxia causes a release of adenosine (ADO) that is inversely related to the O2 levels in oxygen‐sensitive pheochromocytoma (PC12) cells. In the current study, chronic exposure (48 h) of PC12 cells to moderate hypoxia (5% O2) significantly enhanced the release of ADO during severe, acute hypoxia (1% O2). Investigation into the intra‐ and extracellular mechanisms underpinning the secretion of ADO in PC12 cells chronically exposed to hypoxia revealed changes in gene expression and activities of several key enzymes associated with ADO production and metabolism, as well as the down‐regulation of a nucleoside transporter. Decreases in the enzymatic activities of ADO kinase and ADO deaminase accompanied by an increase in those of cytoplasmic and ecto‐5′‐nucleotidases bring about an increased capacity to produce intra‐ and extracellular ADO. This increased potential to generate ADO and decreased capacity to metabolize ADO indicate that PC12 cells shift toward an ADO producer phenotype during hypoxia. The reduced function of the rat equilibrative nucleoside transporter rENT1 also plays a role in controlling extracellular ADO levels. The hypoxia‐induced alterations in the ADO metabolic enzymes and the rENT1 transporter seem to increase the extracellular concentration of ADO. The biological significance of this regulation is unclear but is likely to be associated with modulating cellular activity during hypoxia.
Antioxidant activity related to copper binding of native prion protein
Authors: Brown David R. , Clive Christine , Haswell Stephen J.
Published: March 2003

We have developed a method to affinity‐purify mouse prion protein (PrPc) from mouse brain and cultured cells. PrPc from mouse brain bound three copper atoms; PrPc from cultured cells bound between one and four copper atoms depending on the availability of copper in the culture medium. Purified PrPc exhibited antioxidant activity, as determined by spectrophotometric assay. Incubation of PrPc with the neurotoxic peptide, PrP106‐126, inactivated the superoxide dismutase‐like activity. Culture experiments showed that PrPc protects cells against oxidative stress relative to the amount of copper it binds. These results suggest that PrPc is a copper‐binding protein which can incorporate varying amounts of copper and exhibit protective antioxidant activity.
Neurotrophic and neurotoxic effects of nitric oxide on fetal midbrain cultures
Authors: Canals S. , Casarejos M. J. , Rodríguez‐Martín E. , De Bernardo S. , Mena M. A.
Published: March 2003

There is evidence suggesting that nitric oxide (NO) may play an important role in dopamine (DA) cell death. Thus, the aim of this study was to investigate the effects of NO on apoptosis and functionality of DA neurones and glial cells. The experiments were carried out in neuronal‐enriched midbrain cultures treated with the NO donor diethylamine–nitric oxide complexed sodium (DEA–NO). DEA–NO, at doses of 25 and 50 µm, exerted neurotrophic effects on dopamine cells, increasing the number of tyrosine hydroxylase positive (TH+) cells, TH+neurite processes, DA levels and [3H]DA uptake. A dose of 25 µmDEA–NO protected DA cells from apoptosis. In addition, it inducedde novoTH synthesis and increased intracellular reduced glutathione (GSH) levels, indicating a possible neuroprotective role for GSH. However, in doses ranging from 200 to 400 µm, DEA–NO decreased TH+cells, DA levels, [3H]DA uptake and the number of mature oligodendrocytes (O1+cells). No changes in either the amount or morphology of astrocytes and glial progenitors were detected. A dose‐ and time‐dependent increase in apoptotic cells in the DEA–NO‐treated culture was also observed, with a concomitant increase in the proapoptotic Bax protein levels and a reduction in the ratio between Bcl‐xL and Bcl‐xS proteins. In addition, DEA–NO induced a dose‐ and time‐dependent increase in necrotic cells. 1H‐[1,2,4]oxadiazolo[4,3a]quinoxaline‐1‐one (ODQ, 0.5 µm), a selective guanylate cyclase inhibitor, did not revert the NO‐induced effect on [3H]DA uptake. Glia‐conditioned medium, obtained from fetal midbrain astrocyte cultures, totally protected neuronal‐enriched midbrain cultures from NO‐induced apoptosis and rescued [3H]DA uptake and TH+cell number. In conclusion, our results show that low NO concentrations have neurotrophic effects on DA cells via a cGMP‐independent mechanism that may implicate up‐regulation of GSH. On the other hand, higher levels of NO induce cell death in both dopamine neurones and mature oligodendrocytes that is totally reverted by soluble factors released from glia.
Increased levels of oxidative stress markers detected in the brains of mice devoid of prion protein
Authors: Wong Boon‐Seng , Liu Tong , Li Ruliang , Pan Tao , Petersen Robert B. , Smith Mark A. , Gambetti Pierluigi , Perry George , Manson Jean C. , Brown David R. , Sy Man‐Sun
Published: March 2003

Although minor abnormalities have been reported in prion protein (PrP) knock‐out (Prnp−/–) mice, the normal physiological function of PrP, the causative agent implicated in transmissible spongiform encephalopathies (TSE), remains unresolved. Since there are increasing correlations between oxidative stress and amyloidoses, we decided to investigate whether PrP plays a role in oxidative modulation. We found higher levels of oxidative damage to proteins and lipids in the brain lysates of Prnp−/– as compared to wild‐type (WT) mice of the same genetic background. These two indicators, protein oxidation and lipid peroxidation, are hallmarks of cellular oxidative damage. Elevated levels of ubiquitin‐protein conjugates were also observed in Prnp−/– mice, a probable consequence of cellular attempts to remove the damaged proteins as indicated by increased proteasome activity. Taken together, these findings are indicative of a role for PrP in oxidative homeostasis in vivo.
The role of Bax in glutamate‐induced nerve cell death
Authors: Dargusch Richard , Piasecki Dana , Tan Shirlee , Liu Yuanbin , Schubert David
Published: March 2003

The role of the Bax gene product was examined in three forms of cortical nerve cell death in primary cultures. These include spontaneous cell death, oxidative glutamate toxicity, in which exogenous glutamate inhibits cystine uptake resulting in toxic oxidative stress, and ionotropic glutamate receptor‐mediated excitotoxicity following a brief exposure to 10 µm glutamate. Primary cortical and hippocampal neuron cultures were established from embryos of Bax –/+× Bax –/+ matings and the embryos genotyped and assayed for cell death in the three experimental paradigms. Cell death induced by oxidative glutamate toxicity and glutamate‐mediated excitotoxicity was not altered in the Bax –/– homozygous knockout animals. In contrast, there was an approximately 50% inhibition of spontaneous cell death. These results suggest that a classical Bax‐dependent apoptotic pathway contributes to the spontaneous cell death that takes place when nerve cells are initially exposed to cell culture conditions. A Bax‐dependent programed cell death pathway is not, however, utilized in oxidative glutamate toxicity and NMDA receptor‐mediated excitotoxicity following a brief exposure to low concentrations of glutamate.
Activation of microglia by secreted amyloid precursor protein evokes release of glutamate by cystine exchange and attenuates synaptic function
Authors: Barger Steven W. , Basile Anthony S.
Published: March 2003

Microglial activation as part of a chronic inflammatory response is a prominent component of Alzheimer's disease. Secreted forms of the β‐amyloid precursor protein (sAPP) previously were found to activate microglia, elevating their neurotoxic potential. To explore neurotoxic mechanisms, we analyzed microglia‐conditioned medium for agents that could activate glutamate receptors. Conditioned medium from primary rat microglia activated by sAPP caused a calcium elevation in hippocampal neurons, whereas medium from untreated microglia did not. This response was sensitive to the NMDA receptor antagonist, aminophosphonovaleric acid. Analysis of microglia‐conditioned by HPLC revealed dramatically higher concentrations of glutamate in cultures exposed to sAPP. Indeed, the glutamate levels in sAPP‐treated cultures were substantially higher than those in cultures treated with amyloid β‐peptide. This sAPP‐evoked glutamate release was completely blocked by inhibition of the cystine–glutamate antiporter by α‐aminoadipate or use of cystine‐free medium. Furthermore, a sublethal concentration of sAPP compromised synaptic density in microglia–neuron cocultures, as evidenced by neuronal connectivity assay. Finally, the neurotoxicity evoked by sAPP in microglia‐neuron cocultures was attenuated by inhibitors of either the neuronal nitric oxide synthase (NG‐propyl‐l‐arginine) or inducible nitric oxide synthase (1400 W). Together, these data indicate a scenario by which microglia activated by sAPP release excitotoxic levels of glutamate, probably as a consequence of autoprotective antioxidant glutathione production within the microglia, ultimately causing synaptic degeneration and neuronal death.
Phosphatidylinositol 3‐kinase activity is required for the expression of glial fibrillary acidic protein upon cAMP‐dependent induction of differentiation in rat C6 glioma
Authors: Roymans D. , Vissenberg K. , De Jonghe C. , Grobben B. , Claes P. , Verbelen J.‐P. , Van Broeckhoven C. , Slegers H.
Published: March 2003

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed upon maturation of astrocytes and upregulated during reactive astrogliosis. Its expression is modulated by several growth factors and hormones. Although an upregulation of intracellular cAMP is required for the induction of GFAP expression in astrocytes, little information is available on other downstream factors of the signal transduction pathways involved in the regulation of its expression. In this communication, we identified phosphatidylinositol 3‐kinase (PI 3‐K) as a necessary enzyme for GFAP expression in rat C6 glioma cells. Use of the specific PI 3‐K inhibitors wortmannin and LY294002 and transfection of C6 cells with a dominant negative PI 3‐K construct, resulting in a decrease of the enzymatic activity of PI 3‐K, inhibited the cAMP‐dependent expression of GFAP. Furthermore, confocal laser scanning microscopy demonstrated that inhibition of the PI 3‐K activity by LY294002 or wortmannin concomitant with induction of differentiation changes the cellular distribution leading to a pericentrosomal localization of GFAP and an altered cell shape lacking process formation. We conclude that the expression and cellular distribution of GFAP is mediated through a PI 3‐K‐dependent mechanism.
Characterization of the phosphorylation sites of the squid (Loligo pealei) high‐molecular‐weight neurofilament protein from giant axon axoplasm
Authors: Jaffe Howard , Sharma Pushkar , Grant Philip , Pant Harish C.
Published: March 2003

Axonal caliber in vertebrates is attributed, in part, to the extensive phosphorylation of NFM and NFH C‐terminal tail domain KSP repeats by proline‐directed kinases. The squid giant axon, primarily involved in rapid impulse conduction during jet propulsion motility, is enriched in squid‐specific neurofilaments, particularly the highly phosphorylated NF‐220. Of the 228 serine‐threonine candidate phosphate acceptor sites in the NF‐220 tail domain (residues 401–1220), 82 are found in numerous repeats of three different motifs SAR/K, SEK/R, K/RSP, with 62 of these tightly clustered in the C‐terminal repeat segment (residues 840–1160). Characterization of the in vivo NF‐220 phosphorylated sites should provide clues as to the relevant kinases. To characterize these sites, proteolytic digests of NF‐220 were analyzed by a combination of HPLC, electrospray tandem mass spectrometry and database searching. A total of 53 phosphorylation sites were characterized, with 47 clustered in the C‐terminal repeat segment (residues 840–1160), representing 76% (47/62) of the total acceptor sites in the region. As in mammalian NFH, approximately 64% of the K/RSP sites (14/22) in this region were found to be phosphorylated implicating proline‐directed kinases. Significantly, 78% of serines (31/40) in the KAES*EK and EKS*ARSP motifs were also phosphorylated suggesting that non proline‐directed kinases such as CKI may also be involved. This is consistent with previous studies showing that CKI is the principal kinase associated with axoplasmic NF preparations. It also suggests that phosphorylation of large macromolecules with multiple phospho‐sites requires sequential phosphorylation by several kinases.