bluuebunny/crossref_metadata_2025 huggingface dataset:
Distinct properties of wild‐type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type
Authors: Calero Miguel , Pawlik Monika , Soto Claudio , Castaño Eduardo M. , Sigurdsson Einar M. , Kumar Asok , Gallo Gloria , Frangione Blas , Levy Efrat
Published: March 2003

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild‐type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild‐type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild‐type protein forms amorphous aggregates. We also show by circular dichroism, steady‐state fluorescence and Fourier‐transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing α‐helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three‐dimensional structure different from that of the wild‐type protein. The structural differences between variant and wild‐type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.
UTP evokes noradrenaline release from rat sympathetic neurons by activation of protein kinase C
Authors: Vartian Nina , Moskvina Eugenia , Scholze Thomas , Unterberger Ursula , Allgaier Clemens , Boehm Stefan
Published: March 2003

The pathway involved in UTP‐evoked noradrenaline release was investigated in cultures of rat superior cervical ganglia. Northern blots revealed an age‐related increase in levels of mRNA for P2Y6 receptors in cultures obtained at postnatal days 1 and 5, respectively, but no change in transcripts for P2Y1 and P2Y2. Likewise, UTP‐evoked overflow of previously incorporated [3H]noradrenaline was six‐fold higher in neurons obtained at postanatal day 5. Various protein kinase C inhibitors diminished UTP‐, but not electrically, induced tritium overflow by > 70%, as did down‐regulation of protein kinase C by 24 h exposure to phorbol ester. β‐Phorbol‐12,13‐dibutyrate and dioctanoylglycerol caused concentration‐dependent increases in [3H] outflow of up to 6% of total radioactivity, and the secretagogue actions of these agents were reduced in the presence of protein kinase C inhibitors and in neurons pretreated with phorbol ester. Overflow evoked by dioctanoylglycerol was attenuated in the absence of extracellular Ca2+ and in the presence of tetrodotoxin or Cd2+. In addition to triggering tritium overflow, UTP reduced currents through muscarinic K+ channels which, however, were not affected by phorbol esters. This action of UTP was not altered by protein kinase C inhibitors. These results indicate that P2Y6 receptors mediate UTP‐evoked noradrenaline release from rat sympathetic neurons via activation of protein kinase C, but not inhibition of KM channels.
Effects of protracted nicotine exposure and withdrawal on the expression and phosphorylation of the CREB gene transcription factor in rat brain
Authors: Pandey Subhash C. , Roy Adip , Xu Tiejun , Mittal Navdha
Published: March 2003

Addiction to nicotine may result in molecular adaptations in the neurocircuitry of specific brain structures via changes in the cyclic AMP‐responsive element binding protein (CREB)‐dependent gene transcription program. We therefore investigated the effects of chronic nicotine exposure and its withdrawal on CREB and phosphorylated CREB (p‐CREB) protein levels in the rat brain. We report here that chronic nicotine exposure (1‐h withdrawal) had no effect on the expression of CREB and p‐CREB in the rat cortex and amygdala. On the other hand, decreases in the expression of CREB protein and phosphorylation of CREB occur in the cingulate gyrus, and in the parietal and the piriform but not in the frontal cortex during nicotine withdrawal (18 h) after nicotine exposure. It was also observed that CREB and p‐CREB protein levels were significantly decreased in the medial and basolateral, but not in the central amygdala during nicotine withdrawal (18 h) after chronic nicotine exposure. Furthermore, it was found that nicotine withdrawal (18 h) after chronic nicotine exposure leads to decreased CRE‐DNA binding without modulating cAMP‐dependent protein kinase A activity in the cortex and the amygdala of rats. In addition, chronic nicotine treatment produced anxiolytic effects whereas nicotine withdrawal (18 h) produced anxiety in rats as measured by the elevated plus‐maze test. These results provide the first evidence that decreased CREB activity and/or expression in specific cortical and amygdaloid brain structures may be involved in the underlying molecular mechanisms of nicotine dependence.
Oxyhemoglobin induces caspase‐mediated cell death in cerebral endothelial cells
Authors: Meguro Toshinari , Chen Betty , Lancon John , Zhang John H.
Published: March 2003

Damaged endothelium is one of the pathological changes of the cerebral vasospastic vessels following subarachnoid hemorrhage. Our recent study shows that oxyhemoglobin (OxyHb) induces apoptosis in vascular endothelial cells. Apoptosis generally requires the action of various classes of proteases, including a family of cysteine proteases, known collectively as the caspases. This study was undertaken to investigate the activation of caspases and the efficacy of caspase inhibitors, z‐IETD‐fmk and z‐LEHD‐fmk, for oxyhemoglobin‐induced apoptosis in vascular endothelial cells. Cultured bovine brain microvascular endothelial cells (passages 5–9) were used for this study. OxyHb (10 µmol/L) was added during the 24–72 h incubation with and without caspase‐8 or − 9 inhibitors (z‐IETD‐fmk and z‐LEHD‐fmk). Counting surviving cells, DNA laddering, western blotting of poly(ADP‐ribose) polymerase, and measurement of caspase activities were employed to confirm the cytotoxic effects of OxyHb and the protective effects of the caspase inhibitors. OxyHb produced cell detachment in a time‐dependent manner and increased caspase‐8 and ‐9 activities in the cells. z‐IETD‐fmk and z‐LEHD‐fmk (100 µmol/L) attenuated OxyHb‐induced cell loss, DNA laddering, and proteolytic cleavage of PARP, although a lower concentration (10 µmol/L) of caspase inhibitors showed partial effects. OxyHb activates caspase‐8 and ‐9 in cultured vascular endothelial cells, and blocking the action of the caspases with the inhibitors efficiently prevents loss of vascular endothelial cells from OxyHb‐induced apoptosis in vitro. These results suggest that the caspase cascade participates in OxyHb‐induced apoptosis.
Plasmalogen deficiency in early Alzheimer's disease subjects and in animal models: molecular characterization using electrospray ionization mass spectrometry
Authors: Han Xianlin , Holtzman David M. , McKeel Daniel W.
Published: March 2003

To explore the hypothesis that alterations in ethanolamine plasmalogen may be directly related to the severity of dementia in Alzheimer's disease (AD), we performed a systematic examination of plasmalogen content in cellular membranes of gray and white matter from different regions of human subjects with a spectrum of AD clinical dementia ratings (CDR) using electrospray ionization mass spectrometry (ESI/MS). The results demonstrate: (1) a dramatic decrease in plasmalogen content (up to 40 mol% of total plasmalogen) in white matter at a very early stage of AD (i.e. CDR 0.5); (2) a correlation of the deficiency in gray matter plasmalogen content with the AD CDR (i.e. ∼10 mol% of deficiency at CDR 0.5 (very mild dementia) to ∼30 mol% of deficiency at CDR 3 (severe dementia); (3) an absence of alterations of plasmalogen content and molecular species in cerebellar gray matter at any CDR despite dramatic alterations of plasmalogen content in cerebellar white matter. Alterations of ethanolamine plasmalogen content in two mouse models of AD, APPV717F and APPsw, were also examined by ESI/MS. A plasmalogen deficiency was present (up to 10 mol% of total plasmalogen at the age of 18 months) in cerebral cortices, but was absent in cerebella from both animal models. These results suggest plasmalogen deficiency may play an important role in the AD pathogenesis, particularly in the white matter, and suggest that altered plasmalogen content may contribute to neurodegeneration, synapse loss and synaptic dysfunction in AD.
The transcription factor E2F1 promotes dopamine‐evoked neuronal apoptosis by a mechanism independent of transcriptional activation
Authors: Hou Sheng T. , Cowan Emily , Walker Teena , Ohan Nick , Dove Mike , Rasqinha Ingrid , MacManus John P.
Published: March 2003

The E2F1 transcription factor plays an important role in promoting neuronal apoptosis; however, it is not clear how E2F1 does this. Here we show that E2F1 is involved in dopamine (DA)‐evoked apoptosis in cerebellar granule neurons (CGNs). E2F1 –/– CGNs and CGNs expressing an antisense E2F1 cDNA were significantly protected from DA‐toxicity relative to controls. The neuronal protection was accompanied by significantly reduced caspase 3 activity. E2F1‐mediated neuronal apoptosis did not require activation of gene transcription because: (1) ectopic expression of E2F1 or its mutants lacking the transactivation domain induced neuronal apoptosis, whereas an E2F1 mutant lacking the DNA‐binding domain did not; (2) under all of these conditions, known E2F1 target genes includingcyclin A,cdc2andp19ARFwere not induced; and (3) DA‐evoked neuronal apoptosis was associated with up‐regulated E2F1, but not transcription of its target genes. Finally, E2F1‐mediated neuronal apoptosis was associated with reduced nuclear factor (NF)‐κB DNA‐binding activity. Taken together, these data suggest that E2F1 promotes DA‐evoked caspase 3‐dependent neuronal apoptosis by a mechanism independent of gene transactivation, and this may possibly occur through inhibition of anti‐apoptotic genes including NF‐κB.
Enhanced inducible mGlu1α receptor expression in Chinese hamster ovary cells
Authors: Nash Mark S. , Selkirk Julie V. , Gaymer Caroline E. , Challiss R. A. John , Nahorski Stefan R.
Published: March 2003

Inducible expression of the group‐I metabotropic glutamate receptor (mGlu1α) in Chinese hamster ovary cells allows for the study of receptor density dependent effects. However, expression levels attainable with this system are lower than those reported for various brain regions and achieved by conventional (constitutive) transfection. Thus, direct comparison of mGlu1α receptor‐mediated responses in this inducible expression system with those for receptors expressed heterologously or in vivo is compounded. We show here that inducible expression can be selectively augmented by butyrate pretreatment to levels approaching those reported for cerebral tissue. Enhanced mGlu1α receptor protein levels, agonist‐induced inositol phosphate accumulation, as well as single‐cell inositol 1,4,5‐trisphosphate production and intracellular Ca2+ mobilization occurred following co‐induction with butyrate. In contrast, endogenous purinoceptor function was unaffected. Importantly, the ability to titrate receptor expression by varying isopropyl β‐thiogalactoside concentration was retained. Sodium butyrate thus offers a simple and convenient method to enhance inducible gene expression to levels found in vivo.
Expression and role of phosphodiesterase 6 in the chicken pineal gland
Authors: Morin Fabrice , Lugnier Claire , Kameni Jacques , Voisin Pierre
Published: March 2003

The chicken pineal gland is directly photosensitive, with light causing an inhibition of melatonin synthesis. A possible role of phosphodiesterase 6 (PDE6, the primary effector of retinal phototransduction) in mediating this response was investigated. RT‐PCR, DNA sequencing and northern blots revealed the presence of RNA encoding both catalytic and regulatory subunits of PDE6 in the chicken pineal gland. Both rod and cone forms of PDE6 subunits mRNA were detected. The concentration of the transcripts encoding PDE6 catalytic subunits peaked at night. Western blot analysis of chicken pineal proteins with an antibody directed against the catalytic subunits of bovine rod PDE6 identified a single immunoreactive protein of 97 kDa. Anion exchange chromatography of chicken pineal soluble proteins revealed a peak of PDE6 activity that accounted for about 30% of cyclic GMP‐hydrolysis. In cultured chick pineal glands, arylalkylamine N‐acetyltransferase (AA‐NAT), the rate‐limiting enzyme of melatonin synthesis, was protected from inhibition by light when selective PDE5/6 inhibitors (zaprinast, DMPPO) were added to the culture medium. PDE5/6 inhibitors did not affect AA‐NAT activity in the dark. In contrast, a general PDE inhibitor (IBMX) increased AA‐NAT in a light‐independent manner. Together, the data indicate that rod and cone forms of PDE6 are expressed in chick pineal cells and that this enzyme plays a role in the inhibition of melatonin synthesis by light.
Lithium inhibits caspase 3 activation and dephosphorylation of PKB and GSK3 induced by K+deprivation in cerebellar granule cells
Authors: Mora Alfonso , Sabio Guadalupe , González‐Polo Rosa A. , Cuenda Ana , Alessi Dario R. , Alonso Juan C. , Fuentes José M. , Soler Germán , Centeno Francisco
Published: March 2003

Lithium protects cerebellar granule cells from apoptosis induced by low potassium, and also from other apoptotic stimuli. However, the precise mechanism by which this occurs is not understood. When cerebellar granule cells were switched to low potassium medium, the activation of caspase 3 was detected within 6 h, suggesting a role of caspase 3 in mediating apoptosis under conditions of low potassium. In the same conditions, lithium (5 mm) inhibited the activation of caspase 3 induced by low potassium. As lithium did not inhibit caspase 3 activityin vitro, these results suggest that this ion inhibits an upstream component that is required for caspase 3 activation. Lithium is known to inhibit a kinase termed glycogen sythase kinase 3 (GSK3), which is implicated in the survival pathway of phosphatidylinositol 3‐kinase/protein kinase B (PI3K/PKB). Here we demonstrate that low potassium in the absence of lithium induces the dephosphorylation, and therefore the activation, of GSK3. However, when lithium was present, GSK3 remained phosphorylated at the same level as observed under conditions of high potassium. Low potassium induced the dephosphorylation and inactivation of PKB, whereas when lithium was present PKB was not dephosphorylated. Our results allow us to propose a new hypothesis about the action mechanism of lithium, this ion could inhibit a serine‐threonine phosphatase induced by potassium deprivation.
Effect of proteasome inhibition on cellular oxidative damage, antioxidant defences and nitric oxide production
Authors: Lee MoonHee , Hyun Dong‐Hoon , Jenner Peter , Halliwell Barry
Published: March 2003

The ubiquitin/proteasome pathway plays an essential role in protein turnover in vivo, and contributes to removal of oxidatively damaged proteins. We examined the effects of proteasome inhibition on viability, oxidative damage and antioxidant defences in NT‐2 and SK‐N‐MC cell lines. The selective proteasome inhibitor, lactacystin (1 µm) caused little loss of viability, but led to significant increases in levels of oxidative protein damage (measured as protein carbonyls), ubiquitinated proteins, lipid peroxidation and 3‐nitrotyrosine, a biomarker of the attack of reactive nitrogen species (such as peroxynitrite, ONOO−) upon proteins. Higher levels (25 µm) of lactacystin did not further increase the levels of carbonyls, lipid peroxidation, 3‐nitrotyrosine, or ubiquitinated proteins, but produced increases in the levels of 8‐hydroxyguanine (a biomarker of oxidative DNA damage) and falls in levels of GSH. Lactacystin (25 µm) caused loss of viability, apparently by apoptosis, and also increased production of nitric oxide (NO·) (measured as levels of NO2− plus NO3−) by the cells; this was inhibited by N‐nitro‐l‐arginine methyl ester (l‐NAME), which also decreased cell death induced by 25 µm lactacystin and decreased levels of 3‐nitrotyrosine. The NO· production appeared to involve nNOS; iNOS or eNOS were not detectable in either cell type. Another proteasome inhibitor, epoxomicin, had similar effects.
Effect of overexpression of wild‐type and mutant Cu/Zn‐superoxide dismutases on oxidative stress and cell death induced by hydrogen peroxide, 4‐hydroxynonenal or serum deprivation: potentiation of injury by ALS‐related mutant superoxide dismutases and protection by Bcl‐2
Authors: Lee MoonHee , Hyun Dong‐Hoon , Halliwell Barry , Jenner Peter
Published: March 2003

Mutations in Cu/Zn‐superoxide dismutase (SOD1) are associated with some cases of familial amyotrophic lateral sclerosis (ALS). We overexpressed Bcl‐2, wild‐type SOD1 or mutant SOD1s (G37R and G85R) in NT‐2 and SK‐N‐MC cells. Overexpression of Bcl‐2 rendered cells more resistant to apoptosis induced by serum withdrawal, H2O2 or 4‐hydroxy‐2‐trans‐nonenal (HNE). Overexpression of Bcl‐2 had little effect on levels of protein carbonyls, lipid peroxidation, 8‐hydroxyguanine (8‐OHG) or 3‐nitrotyrosine. Serum withdrawal or H2O2 raised levels of protein carbonyls, lipid peroxidation, 8‐OHG and 3‐nitrotyrosine, changes that were attenuated in cells overexpressing Bcl‐2. Overexpression of either SOD1 mutant tended to increase levels of lipid peroxidation, protein carbonyls, and 3‐nitrotyrosine and accelerated viability loss induced by serum withdrawal, H2O2 or HNE, accompanied by greater rises in oxidative damage parameters. The effects of mutant SOD1s were attenuated by Bcl‐2. By contrast, expression of wild‐type SOD1 rendered cells more resistant to loss of viability induced by serum deprivation, HNE or H2O2. The levels of lipid peroxidation in wild‐type SOD1 transfectants were elevated. Overexpression of mutant SOD1s makes cells more predisposed to undergo apoptosis in response to several insults. Our cellular systems appear to mimic events in patients with ALS or transgenic mice overexpressing mutant SOD1.
Regional changes in density of serotonin transporter in the brain of 5‐HT1A and 5‐HT1B knockout mice, and of serotonin innervation in the 5‐HT1B knockout
Authors: Ase Ariel R. , Reader Tomás A. , Hen René , Riad Mustapha , Descarries Laurent
Published: March 2003

5‐HT1A knockout (KO) mice display an anxious‐like phenotype, whereas 5‐HT1B KOs are over‐aggressive. To identify serotoninergic correlates of these altered behaviors, autoradiographic measurements of 5‐HT1A and 5‐HT1B serotonin (5‐HT) receptors and transporter (5‐HTT) were obtained using the radioligands [3H]8‐OH‐DPAT, [125I]cyanopindolol and [3H]citalopram, respectively. By comparison to wild‐type, density of 5‐HT1B receptors was unchanged throughout brain in 5‐HT1A KOs, and that of 5‐HT1A receptors in 5‐HT1B KOs. In contrast, decreases in density of 5‐HTT binding were measured in several brain regions of both genotypes. Moreover, 5‐HTT binding density was significantly increased in the amygdalo‐hippocampal nucleus and ventral hippocampus of the 5‐HT1B KOs. Measurements of 5‐HT axon length and number of axon varicosities by quantitative 5‐HT immunocytochemistry revealed proportional increases in the density of 5‐HT innervation in these two regions of 5‐HT1B KOs, whereas none of the decreases in 5‐HTT binding sites were associated with any such changes. Several conclusions could be drawn from these results: (i) 5‐HT1B receptors do not adapt in 5‐HT1A KOs, nor do 5‐HT1A receptors in 5‐HT1B KOs. (ii) 5‐HTT is down‐regulated in several brain regions of 5‐HT1A and 5‐HT1B KO mice. (iii) This down‐regulation could contribute to the anxious‐like phenotype of the 5‐HT1A KOs, by reducing 5‐HT clearance in several territories of 5‐HT innervation. (iv) The 5‐HT hyperinnervation in the amygdalo‐hippocampal nucleus and ventral hippocampus of 5‐HT1B KOs could play a role in their increased aggressiveness, and might also explain their better performance in some cognitive tests. (v) These increases in density of 5‐HT innervation provide the first evidence for a negative control of 5‐HT neuron growth mediated by 5‐HT1B receptors.
Calcium and pituitary adenylate cyclase‐activating polypeptide induced expression of circadian clock gene mPer1 in the mouse cerebellar granule cell culture
Authors: Akiyama Masashi , Minami Youichi , Nakajima Tatsuki , Moriya Takahiro , Shibata Shigenobu
Published: March 2003

Mammalian circadian clock genes Per1 and Per2 are rhythmically expressed not only in the suprachiasmatic nucleus where the mammalian circadian clock exists, but also in other brain regions and peripheral tissues. The induced circadian oscillation of Per genes after treatment with high concentrations of serum or various drugs in cultured cells suggests the ubiquitous existence of the oscillatory mechanism. These treatments also result in a rapid surge of expression of Per1. It has been shown that multiple signaling pathways are involved in Per1 gene induction in culture cells. We used a dispersed primary cell culture made up of mouse cerebellar granule cells to examine the stimuli inducing the mPer genes and their signaling pathways in neuronal tissues expressing mPer genes. We demonstrated that mPer1, but not mPer2, mRNA expression was dependent on the depolarization state controlled by extracellular KCl concentration in the granule cell culture. Nifedipine treatment reduced mPer1 induction, suggesting that mPer1 mRNA expression depends on intracellular calcium concentration regulated through a voltage‐dependent Ca2+ channel. Transient mPer1 mRNA induction was observed after elevating KCl concentration in the medium from 5 mm to 25 mm. This increased expression was suppressed by a calmodulin antagonist, or CaMKII/IV inhibitor, but not by MEK inhibitors. Addition of pituitary adenylate cyclase‐activating polypeptide‐38 to the medium also induced transient Per1 gene expression. This induction was mimicked by dibutyryl‐cAMP and suppressed by a protein kinase A (PKA) inhibitor, but not by MEK inhibitors. These results suggest that Ca2+/calmodulin‐dependent protein kinase II/IV‐ and PKA‐dependent pathways are involved in high‐KCl and PACAP‐induced mPer1 induction, respectively, and neural tissues use multiple signaling pathways for mPer1 induction similar to culture cells.
Accumulation of non‐erythroid αII‐spectrin and calpain‐cleaved αII‐spectrin breakdown products in cerebrospinal fluid after traumatic brain injury in rats
Authors: Pike Brian R. , Flint Jeremy , Dutta Satavisha , Johnson Erik , Wang Kevin K. W. , Hayes Ronald L.
Published: March 2003

Although a number of increased CSF proteins have been correlated with brain damage and outcome after traumatic brain injury (TBI), a major limitation of currently tested biomarkers is a lack of specificity for defining neuropathological cascades. Identification of surrogate biomarkers that are elevated in CSF in response to brain injury and that offer insight into one or more pathological neurochemical events will provide critical information for appropriate administration of therapeutic compounds for treatment of TBI patients. Non‐erythroid αII‐spectrin is a cytoskeletal protein that is a substrate of both calpain and caspase‐3 cysteine proteases. As we have previously demonstrated, cleavage of αII‐spectrin by calpain and caspase‐3 results in accumulation of protease‐specific spectrin breakdown products (SBDPs) that can be used to monitor the magnitude and temporal duration of protease activation. However, accumulation of αII‐spectrin and αII‐SBDPs in CSF after TBI has never been examined. Following a moderate level (2.0 mm) of controlled cortical impact TBI in rodents, native αII‐spectrin protein was decreased in brain tissue and increased in CSF from 24 h to 72 h after injury. In addition, calpain‐specific SBDPs were observed to increase in both brain and CSF after injury. Increases in the calpain‐specific 145 kDa SBDP in CSF were 244%, 530% and 665% of sham‐injured control animals at 24 h, 48 h and 72 h after TBI, respectively. The caspase‐3‐specific SBDP was observed to increase in CSF in some animals but to a lesser degree. Importantly, levels of these proteins were undetectable in CSF of uninjured control rats. These results indicate that detection of αII‐spectrin and αII‐SBDPs is a powerful discriminator of outcome and protease activation after TBI. In accord with our previous studies, results also indicate that calpain may be a more important effector of cell death after moderate TBI than caspase‐3.
Aberrant metal binding by prion protein in human prion disease
Authors: Wong Boon‐Seng , Chen Shu G. , Colucci Monica , Xie Zhiliang , Pan Tao , Liu Tong , Li Ruliang , Gambetti Pierluigi , Sy Man‐Sun , Brown David R.
Published: March 2003

Human prion diseases are characterized by the conversion of the normal prion protein (PrPC) into a pathogenic isomer (PrPSc). Distinct PrPSc conformers are associated with different subtypes of prion diseases. PrPC binds copper and has antioxidation activity. Changes in metal‐ion occupancy can lead to significant decline of the antioxidation activity and changes in conformation of the protein. We studied the trace element status of brains from patients with sporadic Creutzfeldt–Jakob disease (sCJD). We found a decrease of up to 50% of copper and an increase in manganese of approximately 10‐fold in the brain tissues from sCJD subjects. We have also studied the metal occupancy of PrP in sCJD patients. We observed striking elevation of manganese and, to a lesser extent, of zinc accompanied by significant reduction of copper bound to purified PrP in all sCJD variants, determined by the PrP genotype and PrPSc type, combined. Both zinc and manganese were undetectable in PrPC preparations from controls. Copper and manganese changes were pronounced in sCJD subjects homozygous for methionine at codon 129 and carrying PrPSc type‐1. Anti‐oxidation activity of purified PrP was dramatically reduced by up to 85% in the sCJD variants, and correlated with increased in oxidative stress markers in sCJD brains. These results suggest that altered metal‐ion occupancy of PrP plays a pivotal role in the pathogenesis of prion diseases. Since the metal changes differed in each sCJD variants, they may contribute to the diversity of PrPSc and disease phenotype in sCJD. Finally, this study also presented two potential approaches in the diagnosis of CJD; the significant increase in brain manganese makes it potentially detectable by MRI, and the binding of manganese by PrP in sCJD might represent a novel diagnostic marker.
Distinct roles of the Y1 and Y2 receptors on neuropeptide Y‐induced sensitization to sedation
Authors: Naveilhan Philippe , Canals Josep M. , Arenas Ernest , Ernfors Patrik
Published: March 2003

Intracranial injection of neuropeptide Y (NPY) increases the sensitivity to sodium pentobarbital and ketamin sedation and has similar properties as GABA agonists on sleep. Mice sensitive to sedation have increased levels of NPY in many brain regions and Y1−/− mice show a marked resistance to barbiturates. Here we characterized the role of the NPY Y receptors in anesthetic‐induced sedation. We show that Y1 and Y2, but not Y5, receptors participate in the modulation of sedation. Administration of a Y1 agonist increased the sodium pentobarbital‐induced sedation and Y1−/− mice were less sensitive to this anesthetic. However, Y2−/− mice display increased sensitivity, showing that Y2 modulates GABAergic induced sedation both pharmacologically and physiologically and has a functionally opposing role to the Y1 receptor. Analysis of Y1−/−/Y2−/− double mutant mice show that increased sensitivity by Y1 occurs independent of the Y2 receptor, while the decreased sensitivity mediated by Y2 depend on an intact Y1 receptor. In contrast to sodium pentobarbital, both Y1 and Y2 receptors increase the sensitivity in a collaborative fashion to NMDA antagonist‐induced sedation. These data demonstrate the physiological and pharmacological impact of the Y1 and Y2 receptors on sedation.
Developmental and regional distribution of aspartoacylase in rat brain tissue
Authors: Bhakoo Kishore K. , Craig Tim J. , Styles Peter
Published: March 2003

The function of N‐acetyl‐aspartate (NAA), a predominant molecule in the brain, has not yet been determined. However, NAA is commonly used as a putative marker of viable neurones. To investigate the possible function of NAA, we determined the anatomical, developmental and cellular distribution of aspartoacylase, which catalyses the hydrolysis of NAA. Levels of aspartoacylase activity were measured during postnatal development in several brain regions. The differential distribution of aspartoacylase activity in purified populations of cells derived from the rat CNS was also investigated. The developmental and anatomical distribution of aspartoacylase correlated with the maturation of white matter tracts in the rat brain. Activity increased markedly after 7 days and coincided with the time course for the onset of myelination in the rat brain. Gray matter showed little activity or developmental trend. There was a 60‐fold excess in optic nerve (a white matter tract) when compared with cortex at 21 days of development. In the adult brain there was a 18‐fold difference in corpus callosum compared with cortex (stripped of corpus callosum). Cellular studies demonstrated that purified cortical neurons and cerebellar granular neurones have no activity. Primary O‐2A progenitor cells had moderate activity, with three‐fold higher activity in immature oligodendrocyte and 13‐fold increase in mature oligodendrocytes (myelinating cells of the CNS). The highest activity was seen in type‐2 astrocytes (20‐fold difference compared with O‐2A progenitors) derived from the same source. Aspartoacylase activity increased with time in freshly isolated astrocytes, with significantly higher activity after 15 days in culture. We conclude that aspartoacylase activity in the developing postnatal brain corresponds with maturation of myelination, and that the cellular distribution is limited to glial cells.
Induction of nuclear factor‐κB in nucleus accumbens by chronic cocaine administration
Authors: Ang Eugenius , Chen Jingshan , Zagouras Panos , Magna Holly , Holland Janice , Schaeffer Eric , Nestler Eric J.
Published: March 2003

ΔFosB is a Fos family transcription factor that is induced by chronic exposure to cocaine and other drugs of abuse in the nucleus accumbens and related striatal regions, brain regions that are important for the behavioral effects of these drugs. To better understand the mechanisms by which ΔFosB contributes to the effects of chronic drug treatment, we used DNA microarray analysis to identify genes that are regulated in the nucleus accumbens upon ΔFosB expression in inducible bitransgenic mice. One of the most highly regulated genes was that encoding a subunit of another transcription factor, nuclear factor‐κB (NF‐κB). Subsequent experiments confirmed the induction of NF‐κB in the nucleus accumbens of mice overexpressing ΔFosB as well as in wild‐type mice treated chronically, but not acutely, with cocaine. These results establish NF‐κB as a putative target for ΔFosB and implicate NF‐κB signaling pathways in the long‐term adaptations of nucleus accumbens neurons to cocaine.
Adenosine in the central nervous system: release mechanisms and extracellular concentrations
Authors: Latini Serena , Pedata Felicita
Published: March 2003

Adenosine has several functions within the CNS that involve an inhibitory tone of neurotransmission and neuroprotective actions in pathological conditions. The understanding of adenosine production and release in the brain is therefore of fundamental importance and has been extensively studied. Conflicting results are often obtained regarding the cellular source of adenosine, the stimulus that induces release and the mechanism for release, in relation to different experimental approaches used to study adenosine production and release. A neuronal origin of adenosine has been demonstrated through electrophysiological approaches showing that neurones can release significant quantities of adenosine, sufficient to activate adenosine receptors and to modulate synaptic functions. Specific actions of adenosine are mediated by different receptor subtypes (A1, A2A, A2B and A3), which are activated by various ranges of adenosine concentrations. Another important issue is the measurement of adenosine concentrations in the extracellular fluid under different conditions in order to know the degree of receptor stimulation and understand adenosine central actions. For this purpose, several experimental approaches have been used both in vivo and in vitro, which provide an estimation of basal adenosine levels in the range of 50–200 nm. The purpose of this review is to describe pathways of adenosine production and metabolism, and to summarize characteristics of adenosine release in the brain in response to different stimuli. Finally, studies performed to evaluate adenosine concentrations under physiological and hypoxic/ischemic conditions will be described to evaluate the degree of adenosine receptor activation.
Involvement of metabotropic glutamate receptors in excitatory amino acid and GABA release following spinal cord injury in rat
Authors: Mills Charles D. , Xu Guo‐Ying , McAdoo David J. , Hulsebosch Claire E.
Published: March 2003

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations resulting in glutamate receptor‐mediated excitotoxic events. The glutamate receptors include ionotropic (iGluRs) and metabotropic (mGluR) receptors. Of the three groups of mGluRs, group‐I activation can initiate intracellular pathways that lead to further transmitter release. Groups II and III mGluRs function mainly as autoreceptors to regulate neurotransmitter release. In an effort to examine the role of mGluRs in the increase in EAAs following SCI, we administered AIDA, a potent group‐I mGluR antagonist immediately after injury. To determine subtype specific roles of the group‐I mGluRs, we evaluated EAA release following LY 367385 (mGluR1 antagonist) and MPEP (mGluR5 antagonist) administration. To evaluate group‐II and ‐III mGluRs we administered APDC (group‐II agonist) and l‐AP4 (group‐III agonist) immediately following injury; additionally, we initiated treatment with CPPG (group‐II/‐III antagonist) and LY 341495 (group‐II antagonist) 5 min prior to injury. Subjects were adult male Sprague–Dawley rats (225–250 g), impact injured at T10 with an NYU impactor (12.5 mm drop). Agents were injected into the epicenter of injury, amino acids where collected by microdialysis fibers inserted 0.5 mm caudal from the edge of the impact region and quantified by HPLC. Treatment with AIDA significantly decreased extracellular EAA and GABA concentrations. MPEP reduced EAA concentrations without affecting GABA. Combining LY 367385 and MPEP resulted in a decrease in EAA and GABA concentrations greater than either agent alone. l‐AP4 decreased EAA levels, while treatment with LY 341495 increased EAA levels. These results suggest that mGluRs play an important role in EAA toxicity following SCI.
Cytosolic O‐glycosylation is abundant in nerve terminals
Authors: Cole Robert N. , Hart Gerald W.
Published: March 2003

Phosphorylation plays a key role in regulating growth cone migration and protein trafficking in nerve terminals. Here we show that nerve terminal proteins contain another abundant post‐translational modification: β‐N‐acetylglucosamine linked to hydroxyls of serines or threonines (O‐GlcNAc1). O‐GlcNAc modifications are essential for embryogenesis and mounting evidence suggests that O‐GlcNAc is a regulatory modification that affects many phosphorylated proteins. We show that the activity and expression of O‐GlcNAc transferase (OGT) and N‐acetyl‐β‐d‐glucosaminidase (O‐GlcNAcase), the two enzymes regulating O‐GlcNAc modifications, are present in nerve terminal structures (synaptosomes) and are particularily abundant in the cytosol of synaptosomes. Numerous synaptosome proteins are highly modified with O‐GlcNAc. Although most of these proteins are present in low abundance, we identified by proteomic analysis three neuron‐specific O‐GlcNAc modified proteins: collapsin response mediator protein‐2 (CRMP‐2), ubiquitin carboxyl hydrolase‐L1 (UCH‐L1) and β‐synuclein. CRMP‐2, which is involved in growth cone collapse, is a major O‐GlcNAc modified protein in synaptosomes. All three proteins are implicated in regulatory cascades that mediate intracellular signaling or neurodegenerative diseases. We propose that O‐GlcNAc modifications in the nerve terminal help regulate the functions of these and other synaptosome proteins, and that O‐GlcNAc may play a role in neurodegenerative disease.
Protein trafficking mechanisms associated with neurite outgrowth and polarized sorting in neurons
Authors: Tang Bor Luen
Published: March 2003

Neuronal differentiation in vitro and in vivo involves coordinated changes in the cellular cytoskeleton and protein trafficking processes. I review here recent progress in our understanding of the membrane trafficking aspects of neurite outgrowth of neurons in culture and selective microtubule‐based polarized sorting in fully polarized neurons, focusing on the involvement of some key molecules. Early neurite outgrowth appears to involve the protein trafficking machineries that are responsible for constitutive trans‐Golgi network (TGN) to plasma membrane exocytosis, utilizing transport carrier generation mechanisms, SNARE proteins, Rab proteins and tethering mechanisms that are also found in non‐neuronal cells. This vectorial TGN‐plasma membrane traffic is directed towards several neurites, but can be switch to concentrate on the growth of a single axon. In a mature neuron, polarized targeting to the specific axonal and dendritic domains appears to involve selective microtubule‐based mechanisms, utilizing motor proteins capable of distinguishing microtubule tracks to different destinations. The apparent gaps in our knowledge of these related protein transport processes will be highlighted.
Implications of CAD and DNase II in ischemic neuronal necrosis specific for the primate hippocampus
Authors: Tsukada Toshiyuki , Watanabe Masahiko , Yamashima Tetsumori
Published: March 2003

The exact molecular mechanism of ischemic neuronal death still remains unclear from rodents to primates. A number of studies using lower species animals have suggested implication of apoptosis cascade, while using monkeys the authors recently claimed necrosis cascade by calpain‐induced leakage of lysosomal cathepsins (calpain‐cathepsin hypothesis). This paper is to study implications of apoptotic versus necrotic cascades for the development of hippocampal CA1 neuronal death in the primate brain undergoing complete global ischemia. Here, we focused on two terminal cell death effectors; caspase‐activated DNase (CAD) and lysosomal enzyme DNase II, in the monkey CA1 sector undergoing 18 min ischemia. The expressions of their mRNA and proteins, and the subcellular localizations as well as ultrastructure and specific DNA gel electrophoresis were examined. Expression of CAD was much less in the normal brain, compared with the lymph node or heart tissues. On day 1 after ischemia, however, CAD mRNA and protein were significantly increased in the CA1 sector, and then CAD protein immunohistochemically showed a translocation from the perikarya into the nucleus. Activated DNase II protein was significantly increased on days 2 and 3 after ischemia, and also showed a similar translocation indicating lysosomal leakage. Although the post‐ischemic CA1 neurons showed positive terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin nick‐end labeling (TUNEL) staining on days 3–5, they showed eosinophilic coagulation necrosis on light microscopy, and frank membrane disruption and mild chromatin condensation on electron microscopy. Furthermore, DNA smear pattern typical for necrosis was observed instead of DNA laddering. These data altogether suggest that the post‐ischemic CA1 neuronal death of the monkey occurs not by apoptosis but by necrosis with participations of lysosomal enzymes DNase II and cathepsins as well as CAD. The interactions between apoptotic (caspase‐3 and CAD) and necrotic (calpain, cathepsin and DNase II) cascades should be studied further.
Quantitative dual‐probe microdialysis: mathematical model and analysis
Authors: Chen Kevin C. , Höistad Malin , Kehr Jan , Fuxe Kjell , Nicholson Charles
Published: March 2003

Steady‐state microdialysis is a widely used technique to monitor the concentration changes and distributions of substances in tissues. To obtain more information about brain tissue properties from microdialysis, a dual‐probe approach was applied to infuse and sample the radiotracer, [3H]mannitol, simultaneously both in agar gel and in the rat striatum. Because the molecules released by one probe and collected by the other must diffuse through the interstitial space, the concentration profile exhibits dynamic behavior that permits the assessment of the diffusion characteristics in the brain extracellular space and the clearance characteristics. In this paper a mathematical model for dual‐probe microdialysis was developed to study brain interstitial diffusion and clearance processes. Theoretical expressions for the spatial distribution of the infused tracer in the brain extracellular space and the temporal concentration at the probe outlet were derived. A fitting program was developed using the simplex algorithm, which finds local minima of the standard deviations between experiments and theory by adjusting the relevant parameters. The theoretical curves accurately fitted the experimental data and generated realistic diffusion parameters, implying that the mathematical model is capable of predicting the interstitial diffusion behavior of [3H]mannitol and that it will be a valuable quantitative tool in dual‐probe microdialysis.
The effects of focal ischemia and reperfusion on the glutathione content of mitochondria from rat brain subregions
Authors: Anderson Michelle F. , Sims Neil R.
Published: March 2003

Glutathione is a key cellular antioxidant that is contained in both cytoplasmic and mitochondrial compartments. Previous investigations indicate that depletion of the mitochondrial pool of glutathione can greatly reduce cell viability. In the present investigation, the effect of focal cerebral ischemia on total (reduced plus oxidized) glutathione in mitochondria was assessed using a rat model of middle cerebral artery occlusion. Total glutathione was substantially decreased in mitochondria prepared from severely ischemic focal tissue in both the cerebral cortex and striatum at 2 h of vessel occlusion and persisted for at least the first 3 h of reperfusion. The loss of mitochondrial glutathione was not associated with decreases of the total tissue glutathione content and was not due to the formation of mixed disulfides with mitochondrial proteins. Thus, an imbalance between uptake and release from the mitochondria in the ischemic tissue provides the most likely explanation for the loss. Decreases in glutathione also developed in mitochondria from the moderately ischemic perifocal tissue when the period of arterial occlusion was extended to 3 h. The presence of mitochondrial glutathione depletion during ischemia showed an apparent close association with the subsequent development of tissue infarction. These findings are consistent with a role for the glutathione depletion in determining the susceptibility of brain tissue to focal ischemia.
Lack of regulation of aromatic l‐amino acid decarboxylase in intact bovine chromaffin cells
Authors: Waymire Jack C. , Haycock John W.
Published: March 2003

Aromatic l‐amino acid decarboxylase (AADC) is the second enzyme in the catecholamine biosynthetic pathway, and its activity is generally considered not to be limiting, and therefore not involved, in regulating flux through this pathway. Recent studies showing that its activity can be regulated in vivo and that the enzyme can be phosphorylated and activated in vitro have raised the possibility that AADC may play more than an obligatory role in catecholamine biosynthesis. In the present study, the phosphorylation and activity of AADC was evaluated relative to that of tyrosine hydroxylase (TH; the first and rate‐limiting enzyme in the pathway) in intact bovine chromaffin cells. Treatment of chromaffin cells with elevated potassium, acetylcholine, phorbol dibutyrate, forskolin, or okadaic acid each increased 32P incorporation into TH (after metabolic labeling of ATP pools with 32Pi) and TH activity. In contrast, as measured in matched samples, 32P incorporation into AADC was not detected and none of the treatments altered AADC activity. Thus, that AADC can be phosphorylated and activated in vitro has questionable physiological significance.
Species differences in the expression of multiple tyrosine hydroxylase protein isoforms
Authors: Haycock John W.
Published: March 2003

In subprimates, a single form of tyrosine hydroxylase (TH) is expressed, whereas two TH protein isoforms have been identified in monkeys and four isoforms have been demonstrated in humans. In order to establish the evolutionary pattern/emergence of these multiple TH isoforms, adrenal medullae from different mammalian species were analyzed by blot immunolabeling using pan‐specific TH antibodies and antibodies specific to each of the four human TH isoforms. The expression of multiple TH isoforms was primate specific and restricted to anthropoids: only a single TH isoform was detected in adrenal medullae from several subprimate and prosimian species (six species from four families), while two TH isoforms were found in all of the anthropoid species studied. The presence of four TH isoforms could only be demonstrated in human specimens. Contrary to previous suggestions, only one TH protein isoform was found in rats and only four TH protein isoforms were found in humans.