bluuebunny/crossref_metadata_2025 huggingface dataset:
Apolipoprotein D mRNA expression is elevated in PDAPP transgenic mice
Authors: Thomas Elizabeth A. , Sautkulis Lauren N. , Criado Jose R. , Games Dora , Sutcliffe J. Gregor
Published: March 2003

Apolipoprotein D (apoD) expression is known to be elevated in select regions of rodent and human brain in association with different types of CNS pathology. To investigate a potential role for apoD in the neuropathology of Alzheimer's disease, we have measured apoD mRNA expression in transgenic mice expressing mutated human amyloid precursor protein under control of platelet‐derived growth factor promoter (PDAPP mice). In situ hybridization analysis revealed increased apoD mRNA expression in brains of aged (26 months) PDAPP transgenic mice compared to aged littermate controls. These increases were most prominent in the hippocampal fimbria, corpus callosum and other white matter tracts. No substantial increases in expression were observed in white matter regions in young (6 months) PDAPP transgenic mice compared to young controls. Comparison between aged and young control mice revealed increased apoD expression in similar white matter regions of the aged animals. These findings suggest that, although increases in apoD expression are a normal feature of brain aging, super‐increases may represent a glial cell compensatory response to beta‐amyloid deposition in Alzheimer's disease.
Differing effects of substrate and non‐substrate transport inhibitors on glutamate uptake reversal
Authors: Anderson, Christopher M. , Bridges Richard J. , Chamberlin A. Richard , Shimamoto Keiko , Yasuda‐Kamatani Yoshimi , Swanson Raymond A.
Published: March 2003

Na+‐dependent excitatory amino acid transporters (EAATs) normally function to remove extracellular glutamate from brain extracellular space, but EAATs can also increase extracellular glutamate by reversal of uptake. Effects of inhibitors on EAATs can be complex, depending on cell type, whether conditions favor glutamate uptake or uptake reversal and whether the inhibitor itself is a substrate for the transporters. The present study assessed EAAT inhibitors for their ability to inhibit glutamate uptake, act as transporter substrates and block uptake reversal in astrocyte and neuron cultures. lthreo‐β‐hydroxyaspartate (l‐TBHA), dlthreo‐β‐benzyloxyaspartate (dl‐TBOA), ltrans‐pyrrolidine‐2,4‐dicarboxylic acid (ltrans‐2,4‐PDC) (+/–)‐cis‐4‐methy‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (cis‐4‐methy‐trans‐2,4‐PDC) and lantiendo‐3,4‐methanopyrrolidine‐2,4‐dicarboxylic acid (lantiendo‐3,4‐MPDC) inhibited l‐[14C]glutamate uptake in astrocytes with equilibrium binding constants ranging from 17 µm (dl‐TBOA and l‐TBHA) – 43 µm (cis‐4‐methy‐trans‐2,4‐PDC). Transportability of inhibitors was assessed in astrocytes and neurons. While l‐TBHA, ltrans‐2,4‐PDC, cis‐4‐methy‐trans‐2,4‐PDC and lantiendo‐3,4‐MPDC displayed significant transporter substrate activities in neurons and astrocytes, dl‐TBOA was a substrate only in astrocytes. This effect of dl‐TBOA was concentration‐dependent, leading to complex effects on glutamate uptake reversal. At concentrations low enough to produce minimal dl‐TBOA uptake velocity (≤ 10 µm), dl‐TBOA blocked uptake reversal in ATP‐depleted astrocytes; this blockade was negated at concentrations that drove substantial dl‐TBOA uptake (> 10 µm). These findings indicate that the net effects of EAAT inhibitors can vary with cell type and exposure conditions.
Local and global cerebral blood flow and glucose utilization in the α‐galactosidase A knockout mouse model of Fabry disease
Authors: Itoh Yoshiaki , Esaki Takanori , Cook Michelle , Qasba Pankaj , Shimoji Kazuaki , Alroy Joseph , Brady Roscoe O. , Sokoloff Louis , Moore David F.
Published: March 2003

Fabry disease is an X‐linked lysosomal disorder characterized by deficient α‐galactosidase A activity and intracellular accumulations of glycosphingolipids, mainly globotriaosylceramide (Gb3). Clinically, patients occasionally present CNS dysfunction. To examine the pathophysiology underlying brain dysfunction, we examined glucose utilization (CMRglc) and cerebral blood flow (CBF) globally and locally in 18 brain structures in the α‐galactosidase A gene knockout mouse. Global CMRglc was statistically significantly reduced by 22% in Fabry mice (p < 0.01). All 18 structures showed decreases in local CMRglc ranging from 14% to 33%. The decreases in all structures of the diencephalon, caudate‐putamen, brain stem, and cerebellar cortex were statistically significant (p < 0.05). Global cerebral blood flow (CBF) and local CBF measured in the same 18 structures were lower in Fabry mice than in control mice, but none statistically significantly. Histological examination of brain revealed no cerebral infarcts but abundant Gb3 deposits in the walls of the cerebral vessels with neuronal deposits localized to the medulla oblongata. These results indicate an impairment in cerebral energy metabolism in the Fabry mice, but one not necessarily due to circulatory insufficiency.
Overexpression of α7 nicotinic acetylcholine receptor prevents G1‐arrest and DNA fragmentation in PC12 cells after hypoxia
Authors: Utsugisawa Kimiaki , Nagane Yuriko , Obara Daiji , Tohgi Hideo
Published: March 2003

We investigated the neuroprotective function of α7 nicotinic acetylcholine receptor (α7nAChR) after transient hypoxia (12 h) and reoxygenation (0–72 h), comparing rat pheochromocytoma (PC12) cells overexpressing FLAG‐tagged α7nAChR (α7pCMV cells) and control PC12 cells (non‐transfected or transfected with vector only) in medium with and without nicotine. Plasma membrane degradation in the early phase after hypoxia was inhibited in PC12 cells with nicotine, and more profoundly in α7pCMV cells with nicotine. Inhibition of DNA fragmentation in the late phase after hypoxia was most remarkable in α7pCMV cells with nicotine, but, surprisingly, it was more remarkable in α7pCMV cells without nicotine than in PC12 cells with nicotine. G1‐arrest of the cell cycle, observed in control PC12 cells at 12 h after hypoxia, preceding DNA fragmentation, was not evident in α7pCMV cells, with or without nicotine. Furthermore, in α7pCMV cells with and without nicotine, the basal expression levels of total Akt were approximately 1.5‐fold higher, and the up‐regulation of Akt phosphorylated at Ser473 after hypoxia was strikingly enhanced, compared with control PC12 cells. These findings suggest that α7nAChR functions constitutively in PC12 cells, that its overexpression raises tolerance against G1‐arrest and DNA fragmentation after hypoxia, and that it can be considered a candidate target for treatment against hypoxia‐induced acute membrane degradation and delayed DNA fragmentation in neurons.
Adenosine inhibits calcium channel currents via A1 receptors on salamander retinal ganglion cells in a mini‐slice preparation
Authors: Sun Xiaolu , Barnes Steven , Baldridge William H.
Published: March 2003

The effects of adenosine on high‐voltage‐activated calcium channel currents in tiger salamander retinal ganglion cells were investigated in a mini‐slice preparation. Adenosine produced a concentration‐dependent decrease in the amplitude of calcium channel current with a maximum inhibition of 26%. The effects of adenosine on calcium channel current were both time‐ and voltage‐dependent. In cells dialyzed with GTP‐γ‐s, adenosine caused a sustained and irreversible inhibition of calcium channel current, suggesting involvement of a GTP‐binding protein. The inhibitory effect of adenosine on calcium channel current was blocked by the A1 antagonist 8‐cyclopentyltheophylline (DPCPX, 1–10 µm), but not by the A2 antagonist 3‐7‐dimethyl‐1‐propargylxanthine (DMPX, 10 µm), and was mimicked by the A1 agonist N 6‐cyclohexyladenosine (CHA, 1 µm) but not by the A2 agonist 5′‐(N‐cyclopropyl) carbox‐amidoadenosine (CPCA, 1 µm). Adenosine's inhibition of calcium channel current was not affected by the L‐type calcium channel blocker nifedipine (5 µm). However, adenosine's inhibition of calcium channel current was reduced to approximately 10% after application of ω‐conotoxin GVIA (1 µm), suggesting that adenosine inhibits N‐type calcium channels. These results show that adenosine acts on an A1 adenosine receptor subtype via a G protein‐coupled pathway to inhibit the component of calcium channel current carried in N‐type calcium channels.
Albumin promotes neuronal survival by increasing the synthesis and release of glutamate
Authors: Tabernero Arantxa , Granda Begoña , Medina Ana , Sánchez‐Abarca Luis Ignacio , Lavado Eva , Medina José M.
Published: March 2003

It is well known that the presence of albumin within the brain and the CSF is developmentally regulated. However, the physiological relevance of this phenomenon is not well established. We have previously shown that albumin specifically increases the flux of glucose and lactate through the pyruvate dehydrogenase reaction in astrocytes. Here we show that, in neurones, albumin also increases the oxidation of glucose and lactate through the pyruvate dehydrogenase‐catalysed reaction, the final purpose of this being the synthesis of glutamate. Thus, in neurones, the presence of albumin strongly increased the synthesis and release of glutamate to the extracellular medium. Our results also suggest that glutamate release caused by albumin is designed to promote neuronal survival. Thus, under culture conditions in which neurones die by apoptosis, the presence of albumin promoted neuronal survival and maintained the differentiation programme of these cells, as judged by the expression of the axonal protein, GAP‐43. The effect of albumin on neuronal survival was counteracted by the presence of DNQX, an antagonist of non‐NMDA‐glutamate receptors, suggesting that the glutamate synthesized and released due to the presence of albumin is responsible for neuronal survival. In addition, the effect of albumin seemed to depend on the activity of the NGF receptor, TrkA, suggesting that the glutamate synthesized and released due to the presence of albumin promotes neuronal survival through the activity of TrkA.
A perforated patch‐clamp study of calcium currents and exocytosis in chromaffin cells of wild‐type and α1A knockout mice
Authors: Aldea Marcos , Jun Kisun , Shin Hee‐Sup , Andrés‐Mateos Eva , Solís‐Garrido Luisa M. , Montiel Carmen , García Antonio G. , Albillos Almudena
Published: March 2003

Simultaneous recordings of inward whole‐cell Ca2+ channel currents (ICa) and increments of capacitance as an indication of exocytosis (ΔCm), were performed in voltage‐clamped single adrenal chromaffin cells from wild‐type and α1A subunit deficient mice, using the perforated‐patch configuration of the patch‐clamp technique. Using protocol #1 (one single Ca2+ channel blocker per cell), to dissect the components of ICa, L channels contributed 43%, N channels 35% and P/Q channels 30% to the total ICa of wild‐type cells. Using protocol #2 (cumulative sequential addition of 3 µm nifedipine, 1 µmω‐conotoxin GVIA, and 1 µmω‐agatoxin IVA), L, N and P/Q channels contributed 40%, 34% and 14%, respectively, to ICa; an R component of around 11% remained. In wild‐type mice the changes of ΔCm paralleled those of ICa. In α1A deficient mice the L component of ICa rose to 53% while the P/Q disappeared; the N and R components were similar. In these mice, ΔCm associated to N and R channels did not vary; however, the P/Q component was abolished while the L component increased by 20%. In conclusion, exocytosis was proportional to the relative density of each Ca2+ channel subtype, L, N, P/Q, R. Ablation of the α1A gene led to a loss of P/Q channel current and to a compensatory increase of L channel‐associated secretion; however, this compensation was not sufficient to maintain the overall exocytotic response, that was diminished by 35% in α1A‐deficient mice. This may be due to altered Ca2+ homeostasis in these mice, as compared to wild mouse chromaffin cells.
The C‐terminal C1 cassette of the N‐methyl‐d‐aspartate receptor 1 subunit contains a bi‐partite nuclear localization sequence
Authors: Holmes K. D. , Mattar P. , Marsh D. R. , Jordan V. , Weaver L. C. , Dekaban G. A.
Published: March 2003

The N‐methyl‐d‐aspartate receptor (NMDAR) is a multimeric transmembrane protein composed of at least two subunits. One subunit, NR1, is derived from a single gene and can be subdivided into three regions: the N‐terminal extracellular domain, the transmembrane regions, and the C‐terminal intracellular domain. The N‐terminal domain is responsible for Mg2+ metal ion binding and channel activity, while the transmembrane domains are important for ion channel formation. The intracellular C‐terminal domain is involved in regulating receptor activity and subcellular localization. Our recent experiments indicated that the intracellular C‐terminal domain, when expressed independently, localizes almost exclusively in the nucleus. An examination of the amino acid sequence reveals the presence of a putative nuclear localization sequence (NLS) in the C1 cassette of the NR1 intracellular C‐terminus. Using an expression vector designed to test whether a putative NLS sequence is a valid, functional NLS, we have demonstrated that a bi‐partite NLS does in fact exist within the NR1‐1 C‐terminus. Computer algorithms identified a putative helix–loop–helix motif that spanned the C0C1 cassettes of the C‐terminus. These data suggest that the NR1 subunit may represent another member of a family of transmembrane proteins that undergo intramembrane proteolysis, releasing a cytosolic peptide that is actively translocated to the nucleus leading to alterations in gene regulation.
Oxidation of DOPAC by nitric oxide: effect of superoxide dismutase
Authors: Laranjinha João , Cadenas Enrique
Published: March 2003

This study aimed to characterize the redox interaction between 3,4‐dihydroxyphenylacetic acid (DOPAC) and nitric oxide (·NO), and to assess the reductive and oxidative decay pathways of the DOPAC semiquinone originating from this interaction. The reaction between DOPAC and ·NO led to the formation of the DOPAC semiquinone radical, detected by electron paramagnetic resonance (EPR) and stabilized by Mg2+, and the nitrosyl anion detected as nitrosylmyoglobin. The EPR signal corresponding to the DOPAC semiquinone was modulated as follows: (i) it was suppressed by glutathione and ascorbic acid with the formation of new EPR spectra corresponding to the glutathionyl and ascorbyl radical, respectively; (ii) it was enhanced by Cu,Zn‐superoxide dismutase; the enzyme also accelerated the decay of the semiquinone species to DOPAC quinone. These results are interpreted as a one‐electron oxidation of DOPAC by ·NO; the reductive decay of the semiquinone back to DOPAC was facilitated by reducing agents, such as glutathione and ascorbate, whereas the oxidative decay to DOPAC quinone was facilitated by superoxide dismutase. The latter effect is understood in terms of a reversible conversion of nitrosyl anion to ·NO by the enzyme. The biological relevance of these reactions is also discussed in terms of the reactivity of peroxynitrite towards DOPAC as a model with implications for aerobic conditions.
Activation of the antioxidant response element in primary cortical neuronal cultures derived from transgenic reporter mice
Authors: Johnson D. A. , Andrews G. K. , Xu W. , Johnson J. A.
Published: March 2003

Many phase II protective genes contain a cis‐acting enhancer region known as the antioxidant response element (ARE). Increased expression of these genes contributes to the protection of cells from oxidative stress. Transgenic reporter mice were created that carry in their genome the core ARE coupled to the human placental alkaline phosphatase (hPAP) reporter gene. Primary cortical cultures derived from these mice were treated with tBHQ resulting in a dose‐dependent increase in hPAP activity. Histochemical staining for hPAP activity was observed in both glia and neurons from tBHQ‐treated cultures. The tBHQ‐mediated increase in hPAP was not affected by the antioxidant glutathione monoethyl ester (GSHEE), whereas the increase in hPAP following DEM treatment was completely blocked by GSHEE. Pre‐treatment of cultures with the PI3‐kinase inhibitor LY 294002 demonstrated a dose‐dependent decrease in tBHQ‐induced hPAP activity. In addition, the tBHQ‐mediated expression of ARE‐driven genes in primary cortical cultures was blocked by LY 294002. Interestingly, basal expression of Nrf2 was also inhibited by LY 294002. We theorize that increased levels of genes controlled by the ARE are important for cellular protection against oxidative stress. These ARE‐hPAP transgenic mice will be an important in vivo model for testing our hypothesis.
Specificity of resistance to oxidative stress
Authors: Dargusch Richard , Schubert David
Published: March 2003

Two clonal nerve‐like cell lines derived from HT22 and PC12 have been selected for resistance to glutamate toxicity and amyloid toxicity, respectively. In the following experiments it was asked if these cell lines show cross‐resistance toward amyloid beta peptide (Aβ) and glutamate as well as toward a variety of additional neurotoxins. Conversely, it was determined if inhibitors of oxytosis, a well‐defined oxidative stress pathway, also protect cells from the neurotoxins. It is shown that both glutamate and amyloid resistant cells are cross resistant to most of the other toxins or toxic conditions, while inhibitors of oxytosis protect from glutathione and cystine depletion and H2O2 toxicity, but not from the toxic effects of nitric oxide, rotenone, arsenite or cisplatin. It is concluded that while there is a great deal of cross‐resistance to neurotoxins, the components of the cell death pathway which has been defined for oxytosis are not used by many of the neurotoxins.
Dynamics of tyrosine hydroxylase promoter activity during midbrain dopaminergic neuron development
Authors: Matsushita Natsuki , Okada Hideki , Yasoshima Yasunobu , Takahashi Kazuaki , Kiuchi Kazutoshi , Kobayashi Kazuto
Published: March 2003

Dopamine (DA)‐producing neurons in the ventral midbrain are generated from a specified neuronal lineage and form selective axonal pathways that mediate multiple CNS functions. Expression of the gene encoding tyrosine hydroxylase (TH), which is a key enzyme of catecholamine biosynthesis, is regulated during the development of midbrain DA neurons. In the present study, we report the developmental regulation and cell type specificity of TH gene promoter in the ventral midbrain by using a green fluorescent protein (GFP) reporter system. Transgenic mice were generated that express GFP in the majority of midbrain DA neurons under the control of the 9‐kb upstream region of the rat TH gene. At an early embryonic stage, GFP expression was induced in the developing DA neurons, and the expression was then markedly down‐regulated at later embryonic stages. However, the expression was reactivated and approached the adult levels during early post‐natal development. These developmental changes in GFP expression patterns suggest the presence of multistep regulatory mechanisms for TH gene expression during DA neuron development. The TH promoter appears to possess transcriptional elements at least necessary for the induction of TH expression at the early embryonic stage and its reactivation during the post‐natal development.
Convergent evidence from microdialysis and presynaptic immunolabeling for the regulation of γ‐aminobutyric acid release in the globus pallidus following acute clozapine or haloperidol administration in rats
Authors: See Ronald E. , Berglind William J. , Krentz Lisa , Meshul Charles K.
Published: March 2003

Antipsychotic drugs (APDs) have been primarily characterized for their effects on dopaminergic terminal regions in the brain, especially within the corpus striatum. Efferent GABA pathways are the primary outflow of striatal processing via their projections to the substantia nigra and the globus pallidus (GP). In the current study, we analyzed changes in pallidal GABA function following acute APD administration by means of in vivo microdialysis, followed by immunolabeling of presynaptic GABA terminal density in the contralateral hemisphere of the same animals. Acute administration of the atypical APD, clozapine (10 or 30 mg/kg, s.c.), produced a dose‐dependent decrease in extracellular GABA. A corresponding dose‐dependent increase in the density of presynaptic terminal GABA immunolabeling in the GP was found. In contrast, the typical APD, haloperidol (1 or 3 mg/kg, s.c.), had no significant effects on either measure, although a non‐significant increase in extracellular GABA and decrease in the density of GABA terminal immunolabeling was noted. Paw retraction tests conducted during the time of microdialysis showed that haloperidol produced a typical pattern of highly pronounced motor impairment, while clozapine showed an atypical profile of minimal catalepsy. These complementary results obtained from in vivo neurochemistry and presynaptic neurotransmitter labeling suggest that systemic clozapine suppresses neuronal GABA release within the GP. This decrease in released pallidal GABA may play a role in the low motor side‐effect liability of atypical APDs.
Stimulatory effect of vitamin C on autophagy in glial cells
Authors: Martin Antonio , Joseph James A. , Cuervo Ana Maria
Published: March 2003

Intracellular accumulation of damaged or abnormal proteins is a common event associated with numerous neurodegenerative diseases and other age‐related pathologies. Increasing the activity of the intracellular proteolytic systems normally responsible for the removal of these abnormal proteins might be beneficial in lessening the severity or development of those pathologies. In this study we have used human astrocyte glial cells to investigate the effect of vitamin C (ascorbate) on the intracellular turnover of proteins. Supplementation of the culture medium with physiological concentrations of vitamin C did not affect protein synthesis, but did increase the rate of protein degradation by lysosomes. Vitamin C accelerated the degradation of intra‐ and extracellular proteins targeted to the lysosomal lumen by autophagic and heterophagic pathways. At the doses analyzed, vitamin C lowered and stabilized the acidic intralysosomal pH at values that result in maximum activation of the lysosomal hydrolases.
The NADH oxidase activity of the plasma membrane of synaptosomes is a major source of superoxide anion and is inhibited by peroxynitrite
Authors: Martín‐Romero Francisco Javier , Gutiérrez‐Martín Yolanda , Henao Fernando , Gutiérrez‐Merino Carlos
Published: March 2003

Plasma membrane vesicles from adult rat brain synaptosomes (PMV) have an ascorbate‐dependent NADH oxidase activity of 35–40 nmol/min/(mg protein) at saturation by NADH. NADPH is a much less efficient substrate of this oxidase activity, with a Vmax 10‐fold lower than that measured for NADH. Ascorbate‐dependent NADH oxidase activity accounts for more than 90% of the total NADH oxidase activity of PMV and, in the absence of NADH and in the presence of 1 mm ascorbate, PMV produce ascorbate free radical (AFR) at a rate of 4.0 ± 0.5 nmol AFR/min/(mg protein). NADH‐dependent ·O2− production by PMV occurs with a rate of 35 ± 3 nmol/min/(mg protein), and is a coreaction product of the NADH oxidase activity, because: (i) it is inhibited by more than 90% by addition of ascorbate oxidase, (ii) it is inhibited by 1 µg/mL wheat germ agglutinin (a potent inhibitor of the plasma membrane AFR reductase activity), and (iii) the KM(NADH) of the plasma membrane NADH oxidase activity and of NADH‐dependent ·O2− production are identical. Treatment of PMV with repetitive micromolar ONOO– pulses produced almost complete inhibition of the ascorbate‐dependent NADH oxidase and ·O2− production, and at 50% inhibition addition of coenzyme Q10 almost completely reverts this inhibition. Cytochrome c stimulated 2.5‐fold the plasma membrane NADH oxidase, and pretreatment of PMV with repetitive 10 µm ONOO– pulses lowers the K0.5 for cytochrome c stimulation from 6 ± 1 (control) to 1.5 ± 0.5 µm. Thus, the ascorbate‐dependent plasma membrane NADH oxidase activity can act as a source of neuronal ·O2−, which is up‐regulated by cytosolic cytochrome c and down‐regulated under chronic oxidative stress conditions producing ONOO–.
Adenosine A1 receptor down‐regulation in mothers and fetal brain after caffeine and theophylline treatments to pregnant rats
Authors: León David , Albasanz José Luis , Ruíz María Angeles , Fernández Mercedes , Martín Mairena
Published: March 2003

Pregnant rats were treated daily with 1 g/L of caffeine or theophylline in their drinking water during pregnancy and the effect of these methylxanthines on adenosine A1 receptor was assayed using binding and reverse transcription polymerase chain reaction (RT–PCR) assays in brains from both mothers and full‐term fetuses. In plasma membranes from pregnant rat brain, caffeine and theophylline caused a significant decrease in total receptor numbers, of the same order in both cases (30%), with no significant changes on receptor affinity. The effect of these adenosine receptor antagonists on plasma membranes from fetal brains was more marked, being detected at approximately 50% of the total receptors detected in control conditions. However, in this tissue, a significant increase in the receptor affinity, of the same order in both cases, was also detected after antagonist administration. No significant variation on the potency of caffeine and theophylline as antagonists was detected after treatments in mothers; however, higher affinities were detected in fetuses. A decrease in the total receptor numbers in fetal brain was associated with an increase in the mRNA coding A1 receptor, as determined by RT–PCR assays, not having detected any mRNA difference in maternal brain. No variation in the levels of mRNA coding A2A receptor was detected in any case. These results suggest that maternal caffeine or theophylline intake modulates adenosine A1 receptor, causing a down‐regulation of adenosine A1 receptor in brain in both mothers and fetuses.
Calpain activates caspase‐3 during UV‐induced neuronal death but only calpain is necessary for death
Authors: McCollum Adrian T. , Nasr Payman , Estus Steven
Published: March 2003

While caspases have been strongly implicated in delayed neuronal death in a variety of experimental paradigms, other proteases such as calpain can also contribute to neuronal death. To evaluate the relative roles of caspase and calpain, we used a model system wherein UV treatment induced moderate or severe delayed cortical neuronal death, as quantified by propidium iodide and calcein AM. UV treatment led to increases in both caspase and calpain activation. Calpain inhibitor III (MDL‐28170) reduced caspase activation, suggesting that caspase activation was mediated by calpain. Calpain contributed to neuronal death, as indicated by strong neuroprotection provided by calpain inhibitor III, calpeptin, or Ca2+‐free medium. In contrast, caspase inhibitors were not neuroprotective. These results suggest that UV neurotoxicity is mediated by a loss of Ca2+ homeostasis which leads to a calpain‐dependent, caspase‐independent cell death. That calpain, but not caspase, may mediate death in instances involving the activation of both proteases may have relevance to other neuronal death models.
Anesthetic and ethanol effects on spontaneously opening glycine receptor channels
Authors: Beckstead Michael J. , Phelan Rachel , Trudell James R. , Bianchini Michael J. , Mihic S. John
Published: March 2003

Strychnine‐sensitive glycine receptors mediate inhibitory neurotransmission occurring in the brain stem and spinal cord. Alcohols, volatile anesthetics and inhaled drugs of abuse are positive allosteric modulators of glycine receptor function, normally enhancing function only in the presence of glycine. A complication in studying allosteric actions on ligand‐gated ion channels is in the dissection of their effects on neurotransmitter binding from their effects on channel opening. Mutation of an aspartate residue at position 97 to arginine in the glycine receptor α1 subunit simulated the effects of glycine binding, producing receptors that exhibited tonic channel opening in the absence of neurotransmitter; i.e. these receptors demonstrated a dissociation of channel opening from neurotransmitter binding. In these receptors, ethanol, enflurane, chloroform, halothane, 1,1,1‐trichloroethane and toluene elicited inward currents in the absence of glycine. We previously identified mutations on ligand‐gated ion channels that eliminate ethanol, anesthetic and inhalant actions (such as S267I on α1 glycine receptors). The double mutant (D97R and S267I) receptors were both constitutively active and resistant to the enhancing effects of ethanol and enflurane. These data demonstrate that ethanol and volatile anesthetics can affect glycine receptor channel opening independently of their effects on enhancing neurotransmitter binding.
Systemic fatty acid responses to transient focal cerebral ischemia: influence of neuroprotectant therapy with human albumin
Authors: Rodriguez de Turco Elena B. , Belayev Ludmila , Liu Yitao , Busto Raul , Parkins Nilda , Bazan Nicolas G. , Ginsberg Myron D.
Published: March 2003

Human albumin therapy is highly neuroprotective in focal cerebral ischemia. Because albumin is the main carrier of free fatty acids (FFA) in plasma, we investigated the content and composition of plasma FFA in jugular vein (JV), femoral artery (FA) and femoral vein (FV) of rats given intravenous human albumin (1.25 g/kg) or saline vehicle (5 mL/kg) 1 h after a 2 h middle cerebral artery occlusion (MCAo) or sham surgery. Arachidonic acid was the only FFA significantly increased by MCAo in all plasma samples prior to albumin administration, remaining at the same level regardless of subsequent treatments. Albumin treatment induced in both MCAo‐ and sham‐groups a 1.7‐fold increase in total plasma FFA (mainly 16:0, 18:1, 18:2n‐6) during 90‐min reperfusion. MCAo selectively stimulated the albumin‐mediated mobilization of n‐3 polyunsaturated fatty acids (PUFA), with an early increase in 22:5n‐3 and 22:6n‐3 in the FA prior to detectable changes in the JV. In the MCAo‐albumin group, the lower level of FFA in JV as compared with FA and FV suggests an albumin‐mediated systemic mobilization and supply of FFA to the brain, which may favor the replenishment of PUFA lost from cellular membranes during ischemia and/or to serve as an alternative source of energy, thus contributing to albumin neuroprotection.
Adrenergic inducibility of AP‐1 binding in the rat pineal gland depends on prior photoperiod
Authors: Guillaumond F. , Becquet D. , Bosler O. , François‐Bellan A. M.
Published: March 2003

The main known function of the pineal gland in mammals is the temporal synchronization of physiological rhythms to seasonal changes of day length (photoperiod). In rat, the transcription factor activating protein‐1 (AP‐1) displays a circadian rhythm in its DNA binding in the pineal gland, which results from the rhythmic expression of Fra‐2. We postulated that, if AP‐1 is an important component of pineal gland functioning, then variations in photoperiodic conditions should lead to an adaptation of the AP‐1 binding rhythm. Here we show that AP‐1 binding patterns adapt to variations in lighting conditions, in the same way as the rhythm of arylalkylamine‐N‐acetyltransferase (AA‐NAT) activity. This adaptation appeared to result from photoperiodic adaptation of the rhythmic fra‐2 gene expression and was reflected byan adapted delay between the onset of night and the acrophase of the nocturnal peak. We further showed that photoperiodic adaptation of both the AP‐1 binding and AA‐NAT activity rhythms resulted from adapted changes in adrenergic inducibility of both variables at night onset. We finally provided evidence that AP‐1 shared with the CREM gene encoding the transcriptional repressor protein inducible cAMP early repressor (ICER) the ability to be hypersensitive or subsensitive to adrenergic stimuli, depending on prior photoperiod.
Mice expressing the α1B‐adrenergic receptor induces a synucleinopathy with excessive tyrosine nitration but decreased phosphorylation
Authors: Papay Robert , Zuscik Michael J. , Ross Sean A. , Yun June , McCune Dan F. , Gonzalez‐Cabrera Pedro , Gaivin Robert , Wendy B. Macklin  , Drazba Judy , Perez Dianne M.
Published: March 2003

We had previously reported that systemic overexpression of the α1B‐adrenergic receptor (AR) in a transgenic mouse induced a neurodegenerative disease that resembled the parkinsonian‐like syndrome called multiple system atrophy (MSA). We now report that our mouse model has cytoplasmic inclusion bodies that colocalize with oligodendrocytes and neurons, are positive for α‐synuclein and ubiquitin, and therefore may be classified as a synucleinopathy. α‐Synuclein monomers as well as multimers were present in brain extracts from both normal and transgenic mice. However, similar to human MSA and other synucleinopathies, transgenic mice showed an increase in abnormal aggregated forms of α‐synuclein, which also increased its nitrated content with age. However, the same extracts displayed decreased phosphorylation of α‐synuclein. Other traits particular to MSA such as Purkinje cell loss in the cerebellum and degeneration of the intermediolateral cell columns of the spinal cord also exist in our mouse model but differences still exist between them. Interestingly, long‐term therapy with the α1‐AR antagonist, terazosin, resulted in protection against the symptomatic as well as the neurodegeneration and α‐synuclein inclusion body formation, suggesting that signaling of the α1B‐AR is the cause of the pathology. We conclude that overexpression of the α1B‐AR can cause a synucleinopathy similar to other parkinsonian syndromes.
22R‐Hydroxycholesterol protects neuronal cells from β‐amyloid‐induced cytotoxicity by binding to β‐amyloid peptide
Authors: Yao Zhi‐Xing , Brown Rachel C. , Teper Gary , Greeson Janet , Papadopoulos Vassilios
Published: March 2003

22R‐hydroxycholesterol, a steroid intermediate in the pathway of pregnenolone formation from cholesterol, was found at lower levels in Alzheimer's disease (AD) hippocampus and frontal cortex tissue specimens compared to age‐matched controls. β‐Amyloid (Aβ) peptide has been shown to be neurotoxic and its presence in brain has been linked to AD pathology. 22R‐hydroxycholesterol was found to protect, in a dose‐dependent manner, against Aβ‐induced rat sympathetic nerve pheochromocytoma (PC12) and differentiated human Ntera2/D1 teratocarcinoma (NT2N) neuron cell death. Other steroids tested were either inactive or acted on rodent neurons only. The effect of 22R‐hydroxycholesterol was found to be stereospecific because its enantiomer 22S‐hydroxycholesterol failed to protect the neurons from Aβ‐induced cell death. Moreover, the effect of 22R‐hydroxycholesterol was specific for Aβ‐induced cell death because it did not protect against glutamate‐induced neurotoxicity. The neuroprotective effect of 22R‐hydroxycholesterol was seen when using Aβ1−42 but not the Aβ25−35 peptide. To investigate the mechanism of action of 22R‐hydroxycholesterol we examined the direct binding of this steroid to Aβ using a novel cholesterol‐protein binding blot assay. Using this method the direct specific binding, under native conditions, of 22R‐hydroxycholesterol to Aβ1−42 and Aβ17−40, but not Aβ25−35, was observed. These data suggest that 22R‐hydroxycholesterol binds to Aβ and the formed 22R‐hydroxycholesterol/Aβ complex is not toxic to rodent and human neurons. We propose that 22R‐hydroxycholesterol offers a new means of neuroprotection against Aβ toxicity by inactivating the peptide.
Effect of tumor necrosis factor‐α converting enzyme (TACE) and metalloprotease inhibitor on amyloid precursor protein metabolism in human neurons
Authors: Blacker Megan , Noe Mark C. , Carty Thomas J. , Goodyer Cynthia G. , LeBlanc Andréa C.
Published: March 2003

Tumor necrosis factor‐α (TNF‐α) is implicated in inflammatory processes and much effort is being directed at inhibiting the release of TNF‐α for treatment of inflammatory conditions. In this context, the drug CP‐661,631 has been developed to inhibit the TNF‐α converting enzyme (TACE). However, TACE is also implicated in amyloid precursor protein secretion. Amyloid precursor protein (APP) undergoes constitutive and regulated secretion by α‐secretase endoproteolytic cleavage within the amyloid β peptide (Aβ) domain. Alternative cleavage at the N‐ and C‐terminus of the Aβ domain by β‐ and γ‐secretases results in the production of Aβ. In many cellular andin vivoanimal models, increased secretion of APP results in a concomitant decrease in the production of Aβ suggesting that the two pathways are intricately linked. However, in human primary neuron cultures, increased APP secretion is not associated with a decrease in total Aβ production. To determine if the use of CP‐661,631 may enhance amyloidogenic processing in human brain, we have assessed the effect of CP‐661,631 on APP metabolism in primary cultures of human neurons. Our results show that CP‐661,631 effectively prevents regulated APP secretion but does not increase total Aβ levels in human primary neuron cultures.
Induction of heat shock proteins (HSPs) by sodium arsenite in cultured astrocytes and reduction of hydrogen peroxide‐induced cell death
Authors: Fauconneau Bernard , Petegnief Valérie , Sanfeliu Coral , Piriou Alain , Planas Anna M.
Published: March 2003

Induction of heat shock proteins (HSPs) protects cells from oxidative injury. Here Hsp72, Hsp27 and heme oxygenase‐1 (HO‐1) were induced in cultured rat astrocytes, and protection against oxidative stress was investigated. Astrocytes were treated with sodium arsenite (20–50 µm) for 1 h, which was non‐toxic to cells, 24 h later they were exposed to 400 µmH2O2for 1 h, and cell death was evaluated at different time points. Arsenite triggered strong induction of HSPs, which was prevented by 1 µg/mL cycloheximide (CXH). H2O2caused cell loss and increased cell death with features of apoptosis, i.e. TdT‐mediated dUTP nick‐end labelling (TUNEL) reaction and caspase‐3 activation. These features were abrogated by pre‐treatment with arsenite, which prevented cell loss and significantly reduced the number of dead cells. The protective effect of arsenite was not detected in the presence of CHX. Pre‐treatment with arsenite increased protein kinase B (Akt) and extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation after H2O2. However, while Akt phosphorylation was prevented by CHX, Erk1/2 phosphorylation was further enhanced by CHX. The results show that transient arsenite pre‐treatment induces Hsp72, HO‐1 and, to a lesser extent, Hsp27; it reduces H2O2‐induced astrocyte death; and it causes selective activation of Akt following H2O2. It is suggested that HSP expression at the time of H2O2exposure protects astrocytes from oxidative injury and apoptotic cell death by means of pro‐survival Akt.
Overexpression of PrPc triggers caspase 3 activation: potentiation by proteasome inhibitors and blockade by anti‐PrP antibodies
Authors: Paitel E. , Alves da Costa C. , Vilette D. , Grassi J. , Checler F.
Published: March 2003

We examined the influence of cellular prion protein (PrPc) in the control of cell death in stably transfected HEK293 cell line and in the PrPc‐inducible Rov9 cells. PrPc expression in stably transfected HEK293 human cells did not modify basal apoptotic tonus but drastically potentiated staurosporine‐stimulated cellular toxicity and DNA fragmentation as well as caspase 3‐like activity and immunoreactivity. An identical staurosporine‐induced caspase 3 activation was observed after doxycycline in the PrPc‐inducible Rov9 cell line. Interestingly, proteasome inhibitors increase PrPc‐like immunoreactivity and unmasked a basal caspase 3 activation. Conversely, we show that anti‐PrPc antibodies sequestrate PrPc at the cell surface and drastically lower PrPc‐dependent caspase activation. We suggest that intracellular PrPc could sensitize human cells to pro‐apoptotic phenotype and that blockade of PrPc internalization could be a track to prevent intracellular toxicity associated with PrPc overexpression.
Spatial learning deficit in transgenic mice that conditionally over‐express GSK‐3β in the brain but do not form tau filaments
Authors: Hernández Félix , Borrell José , Guaza Carmen , Avila Jesús , Lucas José J.
Published: March 2003

Deregulation of glycogen synthase kinase‐3 (GSK‐3) activity in neurones has been postulated as a key feature in Alzheimer's disease (AD) pathogenesis. This was further supported by our recent characterization of transgenic mice that conditionally over‐express GSK‐3β in hippocampal and cortical neurones. These mice, designated Tet/GSK‐3β, showed many of the biochemical and cellular aspects of AD neuropathology such as tau hyperphosphorylation and somatodendritic localization, decreased nuclear β‐catenin, neuronal death and reactive gliosis. Tet/GSK‐3β mice, however, did not show tau filament formation up to the latest tested age of 3 months at least. Here we report spatial learning deficits of Tet/GSK‐3β mice in the Morris water maze. In parallel, we also measured the increase in GSK‐3 activity while further exploring the possibility of tau filament formation in aged mice. We found a significant increase in GSK‐3 activity in the hippocampus of Tet/GSK‐3β mice whereas no tau fibrils could be found even in very old mice. These data reinforce the hypothesis of GSK‐3 deregulation in AD pathogenesis, and suggest that Tet/GSK‐3β mice can be used as an AD model and, most remarkably, can be used to test the therapeutic potential of the selective GSK‐3 inhibitors that are currently under development. Additionally, these experiments suggest that destabilization of microtubules and alteration of intracellular metabolic pathways contribute to AD pathogenesis independent of toxicity triggered by the aberrant tau deposits.
The role of thrombin and thrombin receptors in ischemic, hemorrhagic and traumatic brain injury: deleterious or protective?
Authors: Xi Guohua , Reiser Georg , Keep Richard F.
Published: March 2003

In the last two decades it has become apparent that thrombin has many extravascular effects that are mediated by a family of protease‐activated receptors (PARs). PAR‐1, ‐3 and ‐4 are activated via cleavage by thrombin. The importance of extravascular thrombin in modulating ischemic, hemorrhagic and traumatic injury in brain has recently become clear. Thus, in vitro, thrombin at low concentration protects neurons and astrocytes from cell death caused by a number of different insults. In vivo, pretreating the brain with a low dose of thrombin (thrombin preconditioning), attenuates the brain injury induced by a large dose of thrombin, an intracerebral hemorrhage or by focal cerebral ischemia. Thrombin may also be an important mediator of ischemic preconditioning. In contrast, high doses of thrombin kill neurons and astrocytes in vitro and cause disruption of the blood–brain barrier, brain edema and seizures in vivo. This review examines the role of thrombin in brain injury and the molecular mechanisms and signaling cascades involved.
GABAB receptor mRNA in the raphe nuclei: co‐expression with serotonin transporter and glutamic acid decarboxylase
Authors: Serrats Jordi , Artigas Francesc , Mengod Guadalupe , Cortés Roser
Published: March 2003

We have used double‐label in situ hybridization techniques to examine the cellular localization of GABAB receptor mRNA in relation to serotonin transporter mRNA and glutamic acid decarboxylase mRNA in the rat dorsal raphe, median raphe and raphe magnus nuclei. The degree of cellular co‐localization of these markers notably varied among the different nuclei. In the dorsal raphe, cell bodies showing GABAB receptor mRNA were very abundant, the 85% being also labelled for serotonin transporter mRNA, and a low proportion (5%) showing glutamic acid decarboxylase mRNA. In the median raphe, the level of co‐expression of GABAB receptor mRNA with serotonin transporter mRNA was significantly lower. Some cells were also identified that contained GABAB receptor mRNA in the absence of either one of the other mRNA species studied. Our results support the presence of GABAB receptors in serotonergic as well as GABAergic neurones in the dorsal and median raphe, providing the anatomical basis for the reported dual inhibitory/disinhibitory effect of the GABAB agonist baclofen on serotonergic function.
Expression and functional characterization of the extraneuronal monoamine transporter in normal human astrocytes
Authors: Inazu Masato , Takeda Hiroshi , Matsumiya Teruhiko
Published: March 2003

In this study we examined the functional expression of the extraneuronal monoamine transporter (EMT) in normal human astrocytes (NHA). RT‐PCR with EMT‐specific primers demonstrated the presence of EMT mRNA in NHA. The RT‐PCR products were subjected to restriction‐site analysis using three different enzymes (HinfI, SacI and BclI). The restriction patterns with the three enzymes were identical and were exactly as expected from the known restriction map of human EMT cDNA. DNA sequencing was performed for the RT‐PCR products from NHA. Sequence analysis demonstrated that the sequences of RT‐PCR products were identical to that of EMT. The extract of NHA was immunoblotted with anti‐EMT polyclonal antibody raised against EMT polypeptides. Western blotting indicated that anti‐EMT polyclonal antibody recognized a band of 63 kDa. Immunocytochemical staining using anti‐EMT polyclonal antibody in NHA revealed that the plasma membrane, as well as intracellular, perinuclear compartments, presumably endoplasmic reticulum or Golgi membranes, showed a considerable level of immunoreactivity. We examined the time course of temperature‐dependent [3H]MPP+ uptake in NHA for 60 min. Temperature‐dependent [3H]MPP+ uptake increased in a time‐dependent manner for the initial 45 min and almost reached a plateau level (8.70 ± 0.59 pmol/mg protein) at 60 min. In the presence of 3 µm decynium22 (D22) (the most potent EMT inhibitor), temperature‐dependent [3H]MPP+ uptake was strongly reduced by 61% (3.39 ± 0.76 pmol/mg protein at 60 min). D22‐sensitive [3H]MPP+ uptake was saturable over a MPP+ concentration of 6.25–200 µm. Km for this process was 78.01 ± 7.64 µm and Vmax was 295.4 ± 12.8 pmol/mg protein/min. D22‐sensitive [3H]MPP+ uptake was reduced when the astrocyte membrane potential was depolarized by increasing the concentration of K+ in the uptake buffer or by adding Ba2+ to the uptake buffer. These results provide evidence that the MPP+ transport activity in NHA is potential‐sensitive. Moreover, D22‐sensitive [3H]MPP+ uptake was independent of extracellular Na+. D22‐sensitive [3H]MPP+ uptake was inhibited by D22, various organic cations, steroids and monoamine neurotransmitters. Our results showed that the EMT is functionally expressed in NHA and may also play a key role in the disposition of cationic drugs, neurosteroids, the neurotoxin MPP+ and monoamine neurotransmitters in the brain.