bluuebunny/crossref_metadata_2025 huggingface dataset:
Kennedy's Disease
Authors: Ellerby Lisa M. , Hackam Abigail S. , Propp Stephanie S. , Ellerby H. Michael , Rabizadeh Shahrooz , Cashman Neil R. , Trifiro Mark A. , Pinsky Leonard , Wellington Cheryl L. , Salvesen Guy S. , Hayden Michael R. , Bredesen Dale E.
Published: March 2003

Abstract : X‐linked spinal and bulbar muscular atrophy (SBMA),
Kennedy's disease, is a degenerative disease of the motor neurons that is
associated with an increase in the number of CAG repeats encoding a
polyglutamine stretch within the androgen receptor (AR). Recent work has
demonstrated that the gene products associated with open reading frame triplet
repeat expansions may be substrates for the cysteine protease cell death
executioners, the caspases. However, the role that caspase cleavage plays in
the cytotoxicity associated with expression of the disease‐associated alleles
is unknown. Here, we report the first conclusive evidence that caspase
cleavage is a critical step in cytotoxicity ; the expression of the AR with an
expanded polyglutamine stretch enhances its ability to induce apoptosis when
compared with the normal AR. The AR is cleaved by a caspase‐3 subfamily
protease at Asp146, and this cleavage is increased during
apoptosis. Cleavage of the AR at Asp146 is critical for the
induction of apoptosis by AR, as mutation of the cleavage site blocks the
ability of the AR to induce cell death. Further, mutation of the caspase
cleavage site at Asp146 blocks the ability of the SBMA AR to form perinuclear aggregates. These studies define a fundamental role for caspase cleavage in the induction of neural cell death by proteins displaying expanded polyglutamine tracts, and therefore suggest a strategy that may be useful to treat neurodegenrative diseases associated with polyglutamine repeat expansions.
Up‐Regulation of Immunolabeled α2A‐Adrenoceptors,Gi Coupling Proteins, and Regulatory Receptor Kinases in the Prefrontal Cortex of Depressed Suicides
Authors: García‐Sevilla Jesús A. , Escribá Pablo V. , Ozaita Andrés , La Harpe Romano , Walzer Claude , Eytan Ariel , Guimón José
Published: March 2003

Abstract : Suicide and depression are associated with an increased
density of α2‐adrenoceptors (radioligand receptor binding) in
specific regions of the human brain. The function of these inhibitory
receptors involves various regulatory proteins (Gi coupling
proteins and G protein‐coupled receptor kinases, GRKs), which work in concert
with the receptors. In this study we quantitated in parallel the levels of
immunolabeled α2A‐adrenoceptors and associated regulatory
proteins in brains of suicide and depressed suicide victims. Specimens of the
prefrontal cortex (Brodmann area 9) were collected from 51 suicide victims and
31 control subjects. Levels of α2A‐adrenoceptors,
Gα1/2 proteins, and GRK 2/3 were assessed by immunoblotting
techniques by using specific polyclonal antisera and the immunoreactive
proteins were quantitated by densitometry. Increased levels of
α2A‐adrenoceptors (31‐40%), Gα1/2 proteins
(42‐63%), and membrane‐associated GRK 2/3 (24‐32%) were found in the
prefrontal cortex of suicide victims and antidepressantfree depressed suicide
victims. There were significant correlations between the levels of GRK 2/3
(dependent variable) and those of α2A‐adrenoceptors and
Gα1/2 proteins (independent variables) in the same brain
samples of suicide victims (r = 0.56, p = 0.008) and
depressed suicide victims (r = 0.54, p = 0.041). Antemortem
antidepressant treatment was associated with a significant reduction in the
levels of Gα1/2 proteins (32%), but with modest decreases in
the levels of α2A‐adrenoceptors (6%) and GRK 2/3 (18%) in
brains of depressed suicide victims. The increased levels in concert of
α2A‐adrenoceptors, Gα1/2 proteins, and GRK
2/3 in brains of depressed suicide victims support the existence of
supersensitive α2A‐adrenoceptors in subjects with major depression.
Characterization of 8‐Epiprostaglandin F2α as aMarker of Amyloid β‐Peptide‐Induced Oxidative Damage
Authors: Mark Robert J. , Fuson Kimberly S. , May Patrick C.
Published: March 2003

Abstract : The amyloid β‐peptide (Aβ) is a major component
of the neuritic plaques that are a defining histological characteristic of
Alzheimer's disease. Aβ can be directly toxic and pro‐inflammatory to
cells in vitro. Numerous reports have shown that oxidative damage and reactive
oxygen species play a role in Aβ‐mediated neurotoxicity.
8‐Epiprostaglandin F2α (8‐isoprostane) is a well characterized product of lipid peroxidation that is formed nonenzymatically in cell membranes following an oxidative insult. We report a time‐ and concentration‐dependent increase in 8‐isoprostane levels in rat hippocampal cultures treated with Aβ(1‐40) or hydrogen peroxide. As evidence that 8‐isoprostane production is part of an Aβ toxic pathway, alkaline‐treated peptide, which shows minimal toxic activity, resulted in greatly attenuated 8‐isoprostane production. Although the increase in 8‐isoprostane levels preceded cell death, exogenously added 8‐isoprostane had no cytotoxic effects. The antioxidants vitamin E and propyl gallate attenuated Aβ‐induced 8‐isoprostane formation yet had no effect on Aβ‐induced lactate dehydrogenase release. Neither vitamin E nor propyl gallate had any effect on Aβ's ability to adopt a β‐pleated sheet structure and deposit on cells as determined by thioflavine S fluorescence. We conclude that 8‐isoprostane is an indicator of Aβ‐induced damage but not necessarily a mediator of Aβ‐induced neurotoxicity. Also, 8‐isoprostane could be a useful marker for assessing oxidative damage in the CNS.
In Situ Detection of Neuronal DNA Strand Breaks Using the Klenow Fragment of DNA Polymerase I Reveals Different Mechanisims of Neuron Death After Global Cerebral Ischemia
Authors: Jin Kunlin , Chen Jun , Nagayama Tetsuya , Chen Minzhi , Sinclair Jennifer , Graham Steven H. , Simon Roger P.
Published: March 2003

Abstract : Ischemic cell injury in the brain may involve a cascade of programmed cell death. DNA damage may be either a catalyst or a consequence of this cascade. Therefore, the induction of DNA strand breaks in the rat brain following transient global ischemia was examined using (a) the Klenow labeling assay, identifying DNA single‐strand breaks (SSBs) or double‐strand breaks (DSBs) with protruding 5' termini, and (b) terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL), detecting DNA DSBs with protruding 3' termini or blunt ends. Klenow‐positive staining occurred within 2 h of reperfusion and increased with increasing durations of reperfusion. DNA damage detected with the Klenow labeling assay preceded that of TUNEL expression in the caudate putamen, reticular thalamus, thalamus, and cortex. However, in CA1, DNA SSBs were not detected until 72 h of reperfusion and occurred simultaneously with DSBs. Thus, the time course and fragmentation characteristics of DNA damage differ between the hippocampal CA1 and other selectively vulnerable brain regions. This distinct pattern suggests that the delayed neuronal death in CA1 following transient global ischemia may occur via an apoptotic mechanism different from that of other brain regions.
Scrapie‐Infected Mice and PrP Knockout Mice Share Abnormal Localization and Activity of Neuronal Nitric Oxide Synthase
Authors: Keshet Gilmor I. , Ovadia Haim , Taraboulos Albert , Gabizon Ruth
Published: March 2003

Abstract : PrPSc, the only identified component of the
scrapie prion, is a conformational isoform of PrPc. The
physiological role of PrPc, a glycolipid‐anchored glycoprotein, is
still unknown. We have shown previously that neuronal nitric oxide synthase
(nNOS) activity is impaired in the brains of mice sick with experimental
scrapie as well as in scrapie‐infected neuroblastoma cells. In this work we
investigated the cell localization of nNOS in brains of wild‐type and
scrapie‐infected mice as well as in mice in which the PrP gene was ablated. We
now report that whereas in wild‐type mice, nNOS, like PrPc, is
associated with detergent‐insoluble cholesterol‐rich membranous microdomains
(rafts), this is not the case in brains of scrapie‐infected or in those of
adult PrP% mice. Also, adult PrP%, like scrapie‐infected
mice, show reduced nNOS activity. We suggest that PrPc may play a
role in the targeting of nNOS to its proper subcellular localization. The
similarities of nNOS properties in PrP% as compared with
scrapie‐infected mice suggest that at least this role of PrPc may be impaired in scrapie‐infected brains.
Effects of Methylphenidate Analogues on Phenethylamine Substrates for the Striatal Dopamine Transporter
Authors: Wayment Hollie K , Deutsch Howard , Schweri Margaret , Schenk James O
Published: March 2003

Abstract : Methylphenidate (MPD) was found to inhibit competitively
the striatal dopamine transporter (DAT) and bind at sites on the DAT in common
with both cocaine (a non‐substrate site ligand) and amphetamine (a substrate
site ligand). Some methylphenidate analogues modified on the aromatic ring
and/or at the nitrogen were tested to determine whether the profile of
inhibition could be altered. None was found to stimulate the release of
dopamine in the time frame (≤60 s) of the experiments conducted, and each
of the analogues tested was found to noncompetitively inhibit the
transport of dopamine. It was found that halogenating the aromatic ring with
chlorine (threo‐3,4‐dichloromethylphenidate hydrochloride ; compound
1) increased the affinity of MPD to inhibit the transport of dopamine. A
derivative of MPD with simultaneous, single methyl group substitutions on the
phenyl ring and at the nitrogen
(threo‐N‐methyl‐4‐methylphenidate hydrochloride ; compound
2) bound at a site in common with MPD. A benzyl group positioned at the
nitrogen (threo‐N‐benzylmethylphenidate hydrochloride ;
compound 3) imparted properties to the inhibitor in which binding at substrate
and non‐substrate sites could be distinguished. This analogue bound at a
mutually interacting site with that of methylphenidate and had a
Kint value of 4.29 μM. Furthermore, the N‐substituted analogues (compounds 2 and 3), although clearly inhibitors of dopamine transport, were found to attenuate dramatically the inhibition of dopamine transport by amphetamine, suggesting that the development of an antagonist for substrate analogue drugs of abuse may be possible.
Complete Sequence of a Novel Protein Containing a Femtomolar‐Activity‐Dependent Neuroprotective Peptide
Authors: Bassan Merav , Zamostiano Rachel , Davidson Ariane , Pinhasov Albert , Giladi Eliezer , Perl Orly , Bassan Haim , Blat Cila , Gibney Gretchen , Glazner Gordon , Brenneman Douglas E. , Gozes Illana
Published: March 2003

Abstract : The vulnerability of neurons and the irreversibility of
loss make discoveries of neuroprotective compounds fundamentally important.
Here, the complete coding sequence of a novel protein (828 amino acids, pl
5.99), derived from mouse neuroglial cells, is revealed. The sequence
contained (1) a neuroprotective peptide, NAPVSIPQ, sharing structural and
immunological homologies with the previously reported, activity‐dependent
neurotrophic factor ; (2) a glutaredoxin active site ; and (3) a zinc binding
domain. Gene expression was enriched in the mouse hippocampus and cerebellum
and augmented in the presence of the neuropeptide vasoactive intestinal
peptide, in cerebral cortical astrocytes. In mixed neuron—astrocyte
cultures, NAPVSIPQ provided neuroprotection at subfemtomolar concentrations
against toxicity associated with tetrodotoxin (electrical blockade), the
β‐amyloid peptide (the Alzheimer's disease neurotoxin),
N‐methyl‐D‐aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. Daily NAPVSIPQ injections to newborn apolipoprotein E‐deficient mice accelerated the acquisition of developmental reflexes and prevented short‐term memory deficits. Comparative studies suggested that NAPVSIPQ was more efficacious than other neuroprotective peptides in the apolipoprotein E‐deficiency model. A potential basis for rational drug design against neurodegeneration is suggested with NAPVSIPQ as a lead compound. The relative enrichment of the novel mRNA transcripts in the brain and the increases found in the presence of vasoactive intestinal peptide, an established neuroprotective substance, imply a role for the cloned protein in neuronal function.
Hypoxia/Ischemia Induces Dephosphorylation of Rat Brain Neuromodulin/GAP‐43 In Vivo
Authors: Huang Kuo‐Ping , Huang Freesia L. , Chen Hao‐Chia
Published: March 2003

Abstract : The in vivo state of phosphorylation and the modification
of two Cys residues of neuromodulin/GAP‐43 (Nm) were analyzed by electrospray
ionization‐mass spectrometry (ES‐MS). The protein was purified from rat brain
with homogenization buffer containing 1% Nonidet P‐40, protease inhibitors,
protein phosphatase inhibitors, and sulfhydryl reagent, 4‐vinylpyridine. Nm
was purified by HPLC and ion‐exchange chromatography, and the various
fractions were identified by ES‐MS as unphosphorylated and mono‐, di‐, tri‐,
and tetraphosphorylated species. All of these Nm species contained 2 mol of
added 4‐vinylpyridine per mol of Nm, suggesting that that the two Cys residues
are in the reduced form in the brain. In vivo, the majority of Nm is in the
phosphorylated form (~80%), of which the levels of the mono‐ and diphospho
forms are higher than those of the tri‐ and tetraphospho species. Four in vivo
phosphorylation sites, Ser41, Thr95, Ser142,
and Thr172, were identified by amino acid sequencing and tandem
ES‐MS of the peptides derived from Lys‐C endoproteinase digestion. Among these
sites, only Ser41 is a known target of PKC, whereas the kinases
responsible for the phosphorylation of the other three novel sites are
unknown. Hypoxia/ischemia caused a preferential dephosphorylation of
Ser41 and Thr172, whereas Thr95 is the least susceptible to dephosphorylation.
Inhibitors of V‐Type ATPases, Bafilomycin A1 and Concanamycin A, Protect Against β‐Amyloid‐Mediated Effects on 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) Reduction
Authors: Kane Michael D. , Schwarz Roy D. , Pierre Linda St. , Watson M. Desiree , Emmerling Mark R. , Boxer Peter A. , Walker Glenn K.
Published: March 2003

Abstract: The functional viability of cells can be evaluated using a number of different assay determinants. One common assay involves exposing cells to 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), which is converted intracellularly to a colored formazan precipitate and often used to assess amyloid peptide‐induced cytotoxic effects. The MTT assay was employed to evaluate the role of endosomal uptake and lysbsomal acidification in amyloid peptide‐treated differentiated PC12 cell cultures using selective vacuolar‐type (N‐type) ATPase inhibitors. The macrolides bafilomycin A1 (BAF) and concanamycin A (CON) block lysosomal acidification through selective inhibition of the V‐type ATPase. Treating nerve growth factor‐differentiated PC12 cells with nanomolar concentrations of BAF or CON provides complete protection against the effects of β‐amyloid peptides Aβ(1‐42), Aβ(1‐40), and Aβ(25‐35) and of amylin on MTT dye conversion. These macrolides do not inhibit peptide aggregation, act as antioxidants, or inhibit Aβ uptake by cells. Measurements of lysosomal acidification reveal that the concentrations of BAF and CON effective in reversing Aβ‐mediated MTT dye conversion also reverse lysosomal pH. These results suggest that lysosomal acidification is necessary for Aβ effects on MTT dye conversion.
Adenosine Stimulates Cone Photoreceptor Myoid Elongation via an Adenosine A2‐Like Receptor
Authors: Rey Homero L. , Burnside Beth
Published: March 2003

Abstract: In several parts of the nervous system, adenosine has been shown to function as an extracellular neuromodulator binding to surface receptors on target cells. This study examines the possible role of adenosine in mediating light and circadian regulation of retinomotor movements in teleost cone photoreceptors. Teleost cones elongate in the dark and contract in the light. In continuous darkness, the cones continue to elongate and contract at subjective dusk and dawn in response to circadian signals. We report here that exogenous adenosine triggers elongation (the dark/night movement) in isolated cone inner segment‐cone outer segment preparations (CIS‐COS) in vitro. Agonist/antagonist potency profiles indicate that adenosine's effect on cone movement is mediated by an A2‐like adenosine receptor, which like other A2 receptors enhances adenylate cyclase activity. Although closest to that expected for A2 receptors, the antagonist potency profile for CIS‐COS does not correspond exactly to any known A2 receptor subtype, suggesting that the cone receptor may be a novel A2 subtype. Our findings are consistent with previous reports that retinal adenosine levels are higher in the dark, and further suggest that adenosine could act as a neuromodulatory “dark signal” influencing photoreceptor metabolism and function in the fish retina.
Differential Generation of High‐Density Lipoprotein by Endogenous and Exogenous Apolipoproteins in Cultured Fetal Rat Astrocytes
Authors: Ito Jin‐ichi , Zhang Li‐Yong , Asai Michiyo , Yokoyama Shinji
Published: March 2003

Abstract: Most peripheral cells generate cholesterol‐rich high‐density lipoprotein (HDL) with exogenous apolipoprotein as one of the mechanisms for the maintenance of cellular cholesterol homeostasis. Astrocytes isolated from fetal rat brain showed a unique behavior in this reaction. Consistent with previous findings, the astrocytes synthesized apolipoprotein (apo) E and generated cholesterol‐rich pre‐β‐HDL‐like lipoprotein with this apoE, and cellular cholesterol and phospholipids. When exogenous apoA‐I and E were added to the medium, they caused generation of additional HDL with cellular phospholipid. It is interesting that this additional part was very poor in cholesterol except for the generation of relatively cholesterol‐rich HDL only in the initial few hours of the incubation. The mobilization of intracellular cholesterol for this reaction was also very limited, reflecting the poor cholesterol incorporation into the HDL. Thus, the results demonstrated a unique profile of HDL generation and cholesterol efflux by apolipoproteins in rat astrocytes, with endogenous apoE producing cholesterol‐rich HDL and exogenous apolipoproteins producing cholesterol‐poor HDL. These lipoproteins may play differential roles in cholesterol transport in the CNS.
Activity of α‐Secretase as the Common Final Effector of Protein Kinase C‐Dependent and ‐Independent Modulation of Amyloid Precursor Protein Metabolism
Authors: Racchi Marco , Solano Daniela C. , Sironi Marita , Govoni Stefano
Published: March 2003

Abstract: The metabolic fate of the amyloid precursor protein (APP)
is one of the key factors in the pathogenesis of Alzheimer's disease (AD). A
complex cellular mechanism regulates the rate at which the precursor is
cleaved by α‐secretase and released as soluble protein in the
extracellular space. We show here that α‐secretase constitutes the
common final effector of several independent means of stimulation of soluble
APP (sAPP) release. The release of sAPP by α‐secretase resembles that of
several other membrane‐bound proteins with soluble counter‐parts, a process
that is sensitive to matrix metalloprotease inhibitors. The hydroxamic
acid‐based compound KD‐IX‐73‐4 inhibits phorbol ester‐mediated sAPP release
from COS cells with an IC50 of 8 μM, consistent with previous data for the same compound against leukocyte L‐selectin shedding. Beyond direct protein kinase C (PKC) activation we show that KD‐IX‐73‐4 inhibits also receptor‐mediated sAPP release induced by carbachol in SH‐SY5Y cells and by bradykinin in human skin fibroblasts, with the latter being a PKC‐independent mechanism. Altogether these data suggest that all pharmacological means of stimulating sAPP release converge to a hydroxamic acid‐based inhibitor‐sensitive proteolytic enzyme. Moreover, because KD‐IX‐73‐4 was effective in the inhibition of stimulated but not constitutive sAPP release, these data suggest the existence of different enzymes regulating the two metabolic pathways leading to sAPP secretion.
Activation of Caspase‐3 in Developmental Models of Programmed Cell Death in Neurons of the Substantia Nigra
Authors: Jeon Beom S. , Kholodilov Nikolai G. , Oo Tinmarla F. , Kim Sang‐Yun , Tomaselli Kevin J. , Srinivasan Anu , Stefanis Leonidas , Burke Robert E.
Published: March 2003

Abstract: Programmed cell death has been proposed to play a role in the death of neurons in acute and chronic degenerative neurologic disease. There is now evidence that the caspases, a family of cysteine proteases, mediate programmed cell death in various cells. In neurons, caspase‐3 (CPP32/Yama/apopain), in particular, has been proposed to play a role. We examined the expression of caspase‐3 in three models of programmed cell death affecting neurons of the substantia nigra in the rat: natural developmental neuron death and induced developmental death following either striatal target injury with quinolinic acid or dopamine terminal lesion with intrastriatal injection of 6‐hydroxydopamine. Using an antibody to the large (p17) subunit of activated caspase‐3, we have found that activated enzyme is expressed in apoptotic profiles in all models. Increased p17 immunostaining correlated with increased enzyme activity. The sub‐cellular distribution of activated caspase‐3 differed among the models: In natural cell death and the target injury model, it was strictly nuclear, whereas in the toxin model, it was also cytoplasmic. We conclude that p17 immunostaining is a useful marker for programmed cell death in neurons of the substantia nigra.
Prostate Apoptosis Response‐4 Mediates Trophic Factor Withdrawal‐Induced Apoptosis of Hippocampal Neurons
Authors: Chan Sic L. , Tammariello Steven P. , Estus Steve , Mattson Mark P.
Published: March 2003

Abstract: Prostate apoptosis response‐4 (Par‐4) is the product of a gene up‐regulated in prostate cancer cells undergoing apoptosis. We now report that Par‐4 mRNA and protein levels rapidly and progressively increase 4‐24 h following trophic factor withdrawal (TFW) in cultured embryonic rat hippocampal neurons. The increased Par‐4 levels follow an increase of reactive oxygen species, and precede mitochondrial membrane depolarization, caspase activation, and nuclear chromatin condensation/fragmentation. Pretreatment of cultures with 17β‐estradiol, vitamin E, and uric acid largely prevented Par‐4 induction and cell death following TFW, demonstrating necessary roles for oxidative stress and membrane lipid peroxidation in TFW‐induced neuronal apoptosis. Par‐4 antisense oligonucleotide treatment blocked Par‐4 protein increases and attenuated mitochondrial dysfunction, caspase activation, and cell death following TFW. Collectively, our data identify Par‐4 as an early and pivotal player in neuronal apoptosis resulting from TFW and suggest that estrogen and antioxidants may prevent apoptosis, in part, by suppressing Par‐4 production.
Apoptotic Pathways Mobilized in Microglia and Neurones as a Consequence of Chromogranin A‐Induced Microglial Activation
Authors: Kingham P. J. , Cuzner M. L. , Pocock J. M.
Published: March 2003

Abstract : Senile plaques of Alzheimer's brain are characterized by

activated microglia and immunoreactivity for the peptide chromogranin A. We

have investigated the mechanisms by which chromogranin A activates microglia,

producing modulators of neuronal survival. Primary cultures of rat

brain‐derived microglia display a reactive phenotype within 24 h of exposure

to 10nM chromogranin A, culminating in microglial death via apoptotic

mechanisms mediated by interleukin‐1β converting enzyme. The signalling

cascade initiated by chromogranin A triggers nitric oxide production followed

by enhanced microglial glutamate release, inhibition of which prevents

microglial death. The plasma membrane carrier inhibitor aminoadipate and the

type II/III metabotropic glutamate receptor antagonist

(RS)‐α‐methyl‐4‐sulphonophenylglycine are equally protective. A significant amount of the released glutamate occurs from bafilomycin‐sensitive stores, suggesting a vesicular mode of release. Inhibition of this component of release affords significant microglial protection. Conditioned medium from activated microglia kills cerebellar granule cells by inducing caspase‐3‐dependent neuronal apoptosis. Brain‐derived neurotrophic factor is partially neuroprotective, as are ionotropic glutamate receptor antagonists, and, when combined with boiling of conditioned medium, full protection is achieved ; nitric oxide synthase inhibitors are ineffective.
Store‐Operated Ca2+Influx and Voltage‐Gated Ca2+Channels Coupled to Exocytosis in Pheochromocytoma (PC12) Cells
Authors: Taylor S. C. , Peers C.
Published: March 2003

Abstract: Microamperometry was used to monitor quantal catecholamine release from individual PC12 cells in response to raised extracellular K+and caffeine. K+‐evoked exocytosis was entirely dependent on Ca2+influx through voltage‐gated Ca2+channels, and of the subtypes of such channels present in these cells, influx through N‐type was primarily responsible for triggering exocytosis. L‐type channels played a minor role in mediating K+‐evoked secretion, whereas P/Q‐type channels did not appear to be involved in secretion at all. Caffeine also evoked catecholamine release from PC12 cells, but only in the presence of extracellular Ca2+. Application of caffeine in Ca2+‐free solutions evoked large, transient rises of [Ca2+]i, but did not trigger exocytosis. When Ca2+was restored to the extracellular solution (in the absence of caffeine), store‐operated Ca2+influx was observed, which evoked exocytosis. The amount of secretion evoked by this influx pathway was far greater than release triggered by influx through L‐type Ca2+channels, but less than that caused by Ca2+influx through N‐type channels. Our results indicate that exocytosis may be regulated even in excitable cells by Ca2+influx through pathways other than voltage‐gated Ca2+channels.
Role of Serotonin2A and Serotonin2B/2C Receptor Subtypes in the Control of Accumbal and Striatal Dopamine Release Elicited In Vivo by Dorsal Raphe Nucleus Electrical Stimulation
Authors: De Deurwaerdère Philippe , Spampinato Umberto
Published: March 2003

Abstract : This study investigates, using in vivo microdialysis, the role of serotonin2A (5‐HT2A) and 5‐HT2B/2C receptors in the effect of dorsal raphe nucleus (DRN) electrical stimulation on dopamine (DA), 3,4‐dihydroxyphenylacetic acid (DOPAC), and 5‐hydroxyindoleacetic acid (5‐HIAA) extracellular levels monitored in the nucleus accumbens (NAC) and the striatum of halothane‐anesthetized rats. Following DRN stimulation (300 μA, 1 ms, 20 Hz, 15 min) DA release was enhanced in the NAC and reduced in the striatum. The 5‐HT2A antagonist SR 46349B (0.5 mg/kg) and the mixed 5‐HT2A/2B/2C antagonist ritanserin (0.63 mg/kg) significantly reduced the effect of DRN stimulation on DA release in the NAC but not in the striatum. DA responses to DRN stimulation were not affected by the 5‐HT2B/2C antagonist SB 206553 (5 mg/kg) in either region. None of these compounds was able to modify the enhancement of DOPAC and 5‐HIAA outflow induced by DRN stimulation in either the NAC or the striatum. Finally, in both brain regions basal DA release was significantly increased only by SB 206553. These results indicate that 5‐HT2A but not 5‐HT2B/2C receptors participate in the facilitatory control exerted by endogenous 5‐HT on accumbal DA release. Conversely, 5‐HT2B/2C receptors tonically inhibit basal DA release in both brain regions.
Enhancing Effect of Manganese on L‐DOPA‐Induced Apoptosis in PC12 Cells
Authors: Migheli Rossana , Godani Chiara , Sciola Luigi , Delogu Maria R , Serra Pier Andrea , Zangani Danilo , De Natale Guglielmo , Miele Egidio , Desole Maria S
Published: March 2003

Abstract : L‐DOPA and manganese both induce oxidative stress‐mediated apoptosis in catecholaminergic PC12 cells. In this study, exposure of PC12 cells to 0.2 mM MnCl2 or 10‐20 μM L‐DOPA neither affected cell viability, determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay, nor induced apoptosis, tested by flow cytometry, fluorescence microscopy, and the TUNEL technique. L‐DOPA (50 μM) induced decreases in both cell viability and apoptosis. When 0.2 mM MnCl2 was associated with 10, 20, or 50 μM L‐DOPA, a concentration‐dependent decrease in cell viability was observed. Apoptotic cell death also occurred. In addition, manganese inhibited L‐DOPA effects on dopamine (DA) metabolism (i.e., increases in DA and its acidic metabolite levels in both cell lysate and incubation medium). The antioxidant N‐acetyl‐L‐cysteine significantly inhibited decreases in cell viability, apoptosis, and changes in DA metabolism induced by the manganese association with L‐DOPA. An increase in autoxidation of L‐DOPA and of newly formed DA is suggested as a mechanism of manganese action. These data show that agents that induce oxidative stress‐mediated apoptosis in catecholaminergic cells may act synergistically.
In Vitro Autoradiographic Visualization of Guanosine‐5’‐O‐(3‐[35S]Thio)triphosphate Binding Stimulated by Sphingosine 1‐Phosphate and Lysophosphatidic Acid
Authors: Waeber Christian , Chiu Mary L.
Published: March 2003

Abstract : Sphingosine 1‐phosphate or lysophosphatidic acid activation of guanosine‐5’‐O‐(3‐[35S]thio)‐triphosphate ([35S]GTPγS) binding to G proteins was studied by in vitro autoradiography in rat and guinea pig brain. The highest stimulation of [35S]GTPγS binding by sphingosine 1‐phosphate was observed in the molecular layer of the cerebellum. Marked stimulation was observed in most forebrain areas, including neocortex and striatum. With the exception of the substantia gelatinosa and nucleus of the solitary tract, sphingosine 1‐phosphate‐enhanced binding was weaker in the brainstem and spinal cord. Lysophosphatidic acid‐enhanced labeling was only observed in white matter areas. The G protein inhibitor 5’‐p‐fluorosulfonylbenzoyl guanosine completely inhibited lysophosphatidic acid‐enhanced [35S]GTPγS binding but only partially sphingosine 1‐phosphate‐enhanced binding. N‐Ethylmaleimide abolished binding stimulated by both agonists. Sphingosine 1‐phosphate enhanced labeling by another GTP analogue (β,γ‐imido[8‐3H]guanosine‐5’‐triphosphate) similarly to that of [35S]GTPγS. Lysophosphatidic acid stimulated [35S]GTPγS binding in the olfactory bulb, glia limitans, and cortical subventricular zone of 1‐day‐old rats, whereas enhanced labeling was not observed in the latter area of 5‐day‐old rats. Sphingosine 1‐phosphate stimulated binding in the cortical and striatal subventricular zones and olfactory bulb in 1‐ and 5‐day‐old rats. In the absence of radioligand for sphingosine 1‐phosphate and lysophosphatidic acid receptors, [35S]GTPγS autoradiography provides a unique opportunity to study the spatial distribution, ontogeny, and coupling properties of these receptors.
Cysteine 144 Is a Key Residue in the Copper Reduction by the β‐Amyloid Precursor Protein
Authors: Ruiz Francisca H. , González Mauricio , Bodini Mario , Opazo Carlos , Inestrosa Nibaldo C.
Published: March 2003

Abstract : The β‐amyloid precursor protein (β‐APP) contains a copper‐binding site localized between amino acids 135 and 156 (β‐APP135‐156). We have employed synthetic β‐APP peptides to characterize their capacities to reduce Cu(II) to Cu(I). Analogues of the wild‐type β‐APP135‐156 peptide, containing specific amino acid substitutions, were used to establish which residues are specifically involved in the reduction of copper by β‐APP135‐156. We report here that β‐APP’s copper‐binding domain reduced Cu(II) to Cu(I). The single‐mutant β‐APPHis147→Ala and the double‐mutant β‐APPHis147→Ala/His149→Ala showed a small decrease in copper reduction in relation to the wild‐type peptide and the β‐APPSys144→Ser mutation abolished it, suggesting that Cys144 is the key amino acid in the oxidoreduction reaction. Our results confirm that soluble β‐APP is involved in the reduction of Cu(II) to Cu(I).
A Novel Effect of Cyclic AMP on Capacitative Ca2+ Entry in Cultured Rat Cerebellar Astrocytes
Authors: Lin Wu Mei , Chen Wei‐Hao , Liu I.‐Hsiu , Tseng Chuen‐Den , Wang Seu‐Mei
Published: March 2003

Abstract : One of the most important intracellular Ca2+ regulatory mechanisms in nonexcitable cells, “capacitative Ca2+ entry” (CCE), has not been adequately studied in astrocytes. We therefore investigated whether CCE exists in cultured rat cerebellar astrocytes and studied the roles of cyclic AMP (cAMP) and protein kinase C (PKC) in CCE. We found that (1) at least two different intracellular Ca2+ stores, the endoplasmic reticulum and mitochondria, are present in cerebellar astrocytes ; (2) CCE does exist in these cells and can be inhibited by Ni2+, miconazole, and SKF 96365 ; (3) CCE can be directly enhanced by an increase in intracellular cAMP, as 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐brcAMP), forskolin, and isobutylmethylxanthine have stimulatory effects on CCE ; and (4) neither of the two potent protein kinase A (PKA) inhibitors, H8 and H89, nor a specific PKA agonist, Sp‐adenosine 3′,5′‐cyclic monophosphothioate, had a significant effect on cAMP‐enhanced Ca2+ entry. The [Ca2+]i increase was not due to a release from calcium stores, hyperpolarization of the membrane potential, inhibition of calcium extrusion, or a change in pHi, suggesting that cAMP itself probably acts as a novel messenger to modulate CCE. We also conclude that activation of PKC results in an increase in CCE. cAMP and PKC seem to modulate CCE by different pathways.
Extracellular‐Regulated Kinases and Phosphatidylinositol 3‐Kinase Are Involved in Brain‐Derived Neurotrophic Factor‐Mediated Survival and neuritogenesis of the Neuroblastoma Cell Line SH‐SY5Y
Authors: Encinas M. , Iglesias M. , Llecha N. , Comella J. X.
Published: March 2003

Abstract : Retinoic acid (RA) induces the differentiation of many cell lines, including those derived from neuroblastoma. RA treatment of SH‐SY5Y cells induces the appearance of functional Trk B and Trk C receptors. Acute stimulation of RA‐predifferentiated SH‐SY5Y cells with brain‐derived neurotrophic factor (BDNF), neurotrophin 3 (NT‐3), or neurotrophin 4/5 (NT‐4/5), but not nerve growth factor (NGF), induces Trk autophosphorylation, followed by phosphorylation of Akt and the extracellular signal‐regulated kinases (ERKs) 1 and 2. In addition, BDNF, NT‐3, or NT‐4/5, but not NGF, promotes cell survival and neurite outgrowth in serum‐free medium. The mitogen‐activated protein kinase and ERK kinase (MEK) inhibitor PD98059 blocks BDNF‐induced neurite outgrowth and growth‐associated protein‐43 expression but has no effects on cell survival. On the other hand, the phosphatidylinositol 3‐kinase inhibitor LY249002 reverses the survival response elicited by BDNF, leading to a cell death with morphological features of apoptosis.
CTCF Is Essential for Up‐Regulating Expression from the Amyloid Precursor Protein Promoter During Differentiation of Primary Hippocampal Neurons
Authors: Yang Yaxiong , Quitschke Wolfgang , Vostrov Alexander A. , Brewer Gregory J.
Published: March 2003

Abstract : The transcriptional mechanism underlying amyloid precursor protein (APP) regulation in primary neurons during development was investigated. We observed an approximately threefold elevation of APP mRNA levels in differentiating rat hippocampal neurons between day 1 and day 7 in culture and in rat brain hippocampi between embryonic day 18 and postnatal day 3. When an APP promoter construct extending to position ‐2,832 upstream from the main transcriptional start site was transfected into primary rat hippocampal neurons, promoter activity increased from day 1 until reaching a maximum on day 7 in culture. This increase in APP promoter activity was correlated more closely with the time course of expression of the synaptic vesicle protein synaptophysin, an indicator of synaptogenesis, than with neurofilament accumulation, an indicator of neuritogenesis. Transfection of 5′ APP promoter deletions and internal block mutations indicated that the CTCF binding domain designated APBβ was the primary contributor to the increase in APP promoter activity. Furthermore, the binding of transcription factor CTCF to the APBβ element increased approximately fivefold between day 1 and day 7, whereas the binding of USF to the APBα sequence increased only twofold. These results suggest that CTCF is pivotal for the up‐regulation of APP expression during synaptogenesis in primary neurons.
4‐Hydroxy‐2(E)‐Nonenal Inhibits CNS Mitochondrial Respiration at Multiple Sites
Authors: Picklo Matthew J. , Amarnath Venkataraman , McIntyre J. Oliver , Graham Doyle G. , Montine Thomas J.
Published: March 2003

Abstract: A destructive cycle of oxidative stress and mitochondrial dysfunction is proposed in neurodegenerative disease. Lipid peroxidation, one outcome of oxidative challenge, can lead to the formation of 4‐hydroxy‐2(E)‐nonenal (HNE), a lipophilic alkenal that forms stable adducts on mitochondrial proteins. In this study, we characterized the effects of HNE on brain mitochondrial respiration. We used whole rat brain mitochondria and concentrations of HNE comparable to those measured in patients with Alzheimer’s disease. Our results showed that HNE inhibited respiration at multiple sites. Complex I‐linked and complex II‐linked state 3 respirations were inhibited by HNE with IC50 values of ∼200 μM HNE. Respiration was apparently diminished owing to the inhibition of complex III activity. In addition, complex II activity was reduced slightly. The lipophilicity and adduction characteristics of HNE were responsible for the effects of HNE on respiration. The inhibition of respiration was not prevented by N‐acetylcysteine or aminoguanidine. Studies using mitochondria isolated from porcine cerebral cortex also demonstrated an inhibition of complex I‐ and complex II‐linked respiration. Thus, in neurodegenerative disease, oxidative stress may impair mitochondrial respiration through the production of HNE.
Effect of Congo Red on Wild‐Type and Mutated Prion Proteins in Cultured Cells
Authors: Milhavet Olliver , Mangé Alain , Casanova Danielle , Lehmann Sylvain
Published: March 2003

Abstract: Transmissible spongiform encephalopathies form a group of fatal neurodegenerative disorders that have the unique property of being infectious, sporadic, or genetic in origin. Although some doubts remain on the nature of the responsible agent of these diseases, it is clear that a protein called PrPSc (which stands for the scrapie isoform of the prion protein) has a central role in their pathology. PrPSc represents a conformational variant of a normal protein of the host: the cellular isoform of the prion protein, or PrPC. Compounds such as glycosaminoglycans and Congo red (CR) have been shown to interfere with both in vitro and in vivo PrPSc formation. It was hypothesized that CR acts by overstabilizing the conformation of PrPSc molecules or by modifying trafficking of PrPC. Using transfected cells expressing 3F4‐tagged mouse PrPs, we show here that CR does not interfere with conversion of PrP molecules carrying pathogenic mutations. On the contrary, after incubation with the drug, some of their properties, such as insolubility and protease resistance, are enhanced and are even acquired by the wild‐type molecule. This last observation suggests an alternative mechanism of action of CR and leads us to reconsider the relationship between the biochemical properties of PrP and conformational alteration of the protein.
Affinity Purification and Partial Characterization of the Zonulin/Zonula Occludens Toxin (Zot) Receptor from Human Brain
Authors: Lu R. , Wang W. , Uzzau S. , Vigorito R. , Zielke H. R. , Fasano A.
Published: March 2003

Abstract: The intercellular tight junctions (TJs) of endothelial cells represent the limiting structure for the permeability of the blood—brain barrier (BBB). Although the BBB has been recognized as being the interface between the bloodstream and the brain, little is known about its regulation. Zonulin and its prokaryotic analogue, zonula occludens toxin (Zot) elaborated by Vibrio cholerae, both modulate intercellular TJs by binding to a specific surface receptor with subsequent activation of an intracellular signaling pathway involving phospholipase C and protein kinase C activation and actin polymerization. Affinity column purification revealed that human brain plasma membrane preparations contain two Zot binding proteins of ∼55 and ∼45 kDa. Structural and kinetic studies, including saturation and competitive assays, identified the 55‐kDa protein as tubulin, whereas the 45‐kDa protein represents the zonulin/Zot receptor. Biochemical characterization provided evidence that this receptor is a glycoprotein containing multiple sialic acid residues. Comparison of the N‐terminal sequence of the zonulin/Zot receptor with other protein sequences by BLAST analysis revealed a striking similarity with MRP‐8, a 14‐kDa member of the S‐100 family of calcium binding proteins. The discovery and characterization of this receptor from human brain may significantly contribute to our knowledge on the pathophysiological regulation of the BBB.
Ligand—Receptor Interactions as Controlled by Wild‐Type and Mutant Thr370Lys α2B‐Adrenoceptor—Gα15 Fusion Proteins
Authors: Pauwels P. J. , Tardif S. , Finana F. , Wurch T. , Colpaert F. C.
Published: March 2003

Abstract: Fusion proteins were constructed between either a wild‐type or mutant Thr370Lys α2B‐adrenoceptor (α2B AR) and a mouse Gα15 protein to analyze ligand‐receptor interactions at a receptor/Gα15 protein density ratio of 1. Activation of the wild‐type α2B AR‐Gα15 fusion protein in CHO‐K1 cells by (‐)‐adrenaline induced a time‐ and concentration‐dependent (pEC50 = 7.37 ± 0.13) increase in the intracellular Ca2+ concentration, which could be antagonized by RX 811059 (pKB = 7.55 ± 0.15). Whereas d‐medetomidine and oxymetazoline were as efficacious agonists as (‐)‐adrenaline, the following ligands displayed partial agonist properties: BRL 44408 < atipamezole < clonidine < UK 14304 < BHT 920. A comparison with the mutant Thr370Lys α2B AR‐Gα15 fusion protein displayed similar Ca2+ kinetics and a ligand‐mediated receptor activation profile characterized by higher potencies and greater maximal Ca2+ responses for the ligands being investigated, including the putative antagonists dexefaroxan and idazoxan. RX 811059 and RX 821002 remained silent. Similar conclusions could be made on enhancement of the ligands’ intrinsic activities by coexpression of the mutant Thr370Lys α2B AR with either a Gα15 or GαO Cys351Ile protein. The Thr370Lys α2B AR‐Gα protein interactions may modify the tertiary structure of the mutant receptor in such a way that some putative α2 AR antagonists are capable of stabilizing an active receptor conformation, thereby generating positive efficacy.
Glucocorticoids Exacerbate the Deleterious Effects of gp120 in Hippocampal and Cortical Explants
Authors: Yusim Anna , Franklin Laura , Brooke Sheila , Ajilore Olusola , Sapolsky Robert
Published: March 2003

Abstract: Glucocorticoids (GCs), the adrenal steroids secreted during stress, can compromise the ability of hippocampal neurons to survive numerous necrotic insults. We have previously observed that GCs worsen the deleterious effects of gp120, the glycoprotein of the acquired immune deficiency syndrome virus, which can indirectly damage neurons and which is thought to play a role in the neuropathological features of human immuno‐deficiency virus infection. Specifically, GCs augment gp120‐induced calcium mobilization, ATP depletion, decline in mitochondrial potential, and neurotoxicity in fetal monolayer cultures from a number of brain regions. In the present report, we demonstrate a similar gp120/GC synergy in adult hippocampal and cortical explants. We generated explants from rats that were either adrenalectomized, adrenally intact, or intact and treated with corticosterone to produce levels seen in response to major stressors. Metabolic rates in explants were then indirectly assessed with silicon microphysiometry, and cytosolic calcium concentrations were assessed with fura‐2 fluorescent microscopy. We observed that basal levels of GCs tonically augment the disruptive effects of gp120 on metabolism in the CA1 cell field of the hippocampus and in the cortex. Moreover, raising GC concentrations into the stress range exacerbated the ability of gp120 to mobilize cytosolic calcium in a number of hippocampal cell fields. Finally, we observed that the synthetic GC prednisone had similarly exacerbating effects on gp120. Thus, GCs can worsen the deleterious effects of gp120 in a system that is more physiologically relevant than the fetal monolayer culture and in a region‐specific manner.
Oxidative Modification of Creatine Kinase BB in Alzheimer’s Disease Brain
Authors: Aksenov Michael , Aksenova Marina , Butterfield D. Allan , Markesbery William R.
Published: March 2003

Abstract:Creatine kinase (CK) BB, a member of the CK gene family, is a predominantly cytosolic CK isoform in the brain and plays a key role in regulation of the ATP level in neural cells. CK BB levels are reduced in brain regions affected by neurodegeneration in Alzheimer’s disease (AD), Pick’s disease, and Lewy body dementia, and this reduction is not a result of decreased mRNA levels. This study demonstrates that posttranslational modification of CK BB plays a role in the decrease of CK activity in AD brain. The specific CK BB activity and protein carbonyl content were determined in brain extracts of six AD and six age‐matched control subjects. CK BB activity per microgram of immunoreactive CK BB protein was lower in AD than in control brain extracts, indicating the presence of inactive CK BB molecules. The analysis of specific protein carbonyl levels in CK BB, performed by two‐dimensional fingerprinting of oxidatively modified proteins, identified CK BB as one of the targets of protein oxidation in the AD brain. The increase of protein carbonyl content in CK BB provides evidence that oxidative posttranslational modification of CK BB plays a role in the loss of CK BB activity in AD.
Effect of Stress Exposure on the Activation Pattern of Enkephalin‐Containing Perikarya in the Rat Ventral Medulla
Authors: Mansi J. A. , Laforest S. , Drolet G.
Published: March 2003

Abstract: We examined the effects of acute and chronic psychogenic stress on the activation pattern of enkephalin‐containing perikarya in the rat ventrolateral medulla. Rats allocated to the chronic stress groups were subjected to 90 min of immobilization for 10 days. On the 11th day, the chronically stressed rats were exposed to homotypic (90‐min immobilization) or to heterotypic but still psychogenic (90‐min immobilization coupled to air jet stress) stress. The acute stress group was subjected once to an acute 90‐min immobilization. For each group, the rats were anesthetized either before stress (time 0) or 90, 180, and 270 min after the onset of stress. Brain sections were then processed using immunocytochemistry (Fos protein) followed by radioactive in situ hybridization histochemistry (enkephalin mRNA). Following immobilization, the acute group displayed a marked increase in the number of activated enkephalin‐containing perikarya within the paragigantocellularis and lateral reticular nuclei. This level of activation was sustained up to 180 min following the onset of the immobilization stress and had returned to baseline levels by 270 min from the initiation of the stress. However, this stress‐induced activation of enkephalin‐containing perikarya of the ventrolateral medulla was not seen following either homotypic or heterotypic stress in the chronically stressed group. These results provide evidence that enkephalin‐containing perikarya of the ventrolateral medulla may constitute a potential circuit through which they regulate some aspect of the stress responses. Conversely, this enkephalinergic influence from the ventrolateral medulla was shown to be absent following chronic stress exposure. This would suggest a decrease in enkephalin inhibitory input originating from the ventrolateral medulla, thereby allowing a neuroendocrine and/or autonomic response to chronic stress.