bluuebunny/crossref_metadata_2025 huggingface dataset:
Amyloid β Peptide (25–35) Inhibits Na+‐Dependent Glutamate Uptake in Rat Hippocampal Astrocyte Cultures
Authors: Harris Marni E. , Wang Yaning , Pedigo Norman W. , Hensley Kenneth , Butterfield D. Allan , Carney John M.
Published: July 2010

Abstract: Large numbers of neuritic plaques surrounded by reactive astrocytes are characteristic of Alzheimer's disease (AD). There is a large body of research supporting a causal role for the amyloid β peptide (Aβ), a main constituent of these plaques, in the neuropathology of AD. Several hypotheses have been proposed to explain the toxicity of Aβ including free radical injury and excitotoxicity. It has been reported that treatment of neuronal/astrocytic cultures with Aβ increases the vulnerability of neurons to glutamate‐induced cell death. One mechanism that may explain this finding is inhibition of the astrocyte glutamate transporter by Aβ. The aim of the current study was to determine if Aβs inhibit astrocyte glutamate uptake and if this inhibition involves free radical damage to the transporter/astrocytes. We have previously reported that Aβ can generate free radicals, and this radical production was correlated with the oxidation of neurons in culture and inhibition of astrocyte glutamate uptake. In the present study, Aβ (25–35) significantly inhibited l‐glutamate uptake in rat hippocampal astrocyte cultures and this inhibition was prevented by the antioxidant Trolox. Decreases in astrocyte function, in particular l‐glutamate uptake, may contribute to neuronal degeneration such as that seen in AD. These results lead to a revised excitotoxicity/free radical hypothesis of Aβ toxicity involving astrocytes.
High‐Resolution Separation of Amyloid β‐Peptides: Structural Variants Present in Alzheimer's Disease Amyloid
Authors: Näslund Jan , Karlström Anders R. , Tjernberg Lars O. , Schierhorn Angelika , Terenius Lars , Nordstedt Christer
Published: July 2010

Abstract: In Alzheimer's disease (AD), one of the cardinal neuropathological signs is deposition of amyloid, primarily consisting of the amyloid β‐peptide (Aβ). Structural variants of AD‐associated Aβ peptides have been difficult to purify by high‐resolution chromatographic techniques. We therefore developed a novel chromatographic protocol, enabling high‐resolution reverse‐phase liquid chromatography (RPLC) purification of Aβ variants displaying very small structural differences. By using a combination of size‐exclusion chromatography and the novel RPLC protocol, Aβ peptides extracted from AD amyloid were purified and subsequently characterized. Structural analysis by microsequencing and electrospray‐ionization mass spectrometry revealed that the RPLC system resolved a complex mixture of Aβ variants terminating at either residue 40 or 42. Aβ variants differing by as little as one amino acid residue could be purified rapidly to apparent homogeneity. The resolution of the system was further illustrated by its ability to separate structural isomers of Aβ1–40. The present chromatography system might provide further insight into the role of N‐terminally and posttranslationally modified Aβ variants, because each variant can now be studied individually.
Interaction Between A1 Adenosine and Class II Metabotropic Glutamate Receptors in the Regulation of Purine and Glutamate Release from Rat Hippocampal Slices
Authors: Di Iorio P. , Battaglia G. , Ciccarelli R. , Ballerini P. , Giuliani P. , Poli A. , Nicoletti F. , Caciagli F.
Published: July 2010

Abstract: Electrical stimulation of rat hippocampal slices evoked the release of excitatory amino acids and purines, as reflected by a time‐dependent increase in the extracellular levels of glutamate and adenosine, as well as by the increased efflux of radioactivity in slices preloaded with both [14C]glutamate and [3H]adenosine. The evoked release of excitatory amino acids and purines was amplified when slices were exposed to 8‐cyclopentyl‐1,3‐dipropylxanthine (a selective A1 adenosine receptor antagonist), (+)‐α‐methyl‐4‐carboxyphenylglycine [a mixed antagonist of metabotropic glutamate receptors (mGluRs)], or (2S,3S,4S)‐2‐methyl‐2‐(carboxycyclopropyl)glycine (a selective antagonist of class II mGluRs). In contrast, 2‐chloro‐N6‐cyclopentyladenosine (CCPA; a selective A1 receptor agonist) or (2S,1R,2R,3R)‐(2,3‐dicarboxycyclopropyl)glycine (DCG‐IV; a selective agonist of class II mGluRs) reduced the evoked release of excitatory amino acids and purines. CCPA and DCG‐IV also reduced the increase in cyclic AMP formation induced by either forskolin or electrical stimulation in hippocampal slices. The inhibitory effect of CCPA and DCG‐IV on release or cyclic AMP formation was less than additive. We conclude that the evoked release of excitatory amino acids and purines is under an inhibitory control by A1 receptors and class II mGluRs, i.e., mGluR2 or 3, which appear to operate through a common transduction pathway. In addition, although these receptors are activated by endogenous adenosine and glutamate, they can still respond to pharmacological agonists. This provides a rationale for the use of A1 or class II mGluR agonists as neuroprotective agents in experimental models of excitotoxic neuronal degeneration.
Septal and Hippocampal Glutamate Receptors Modulate the Output of Acetylcholine in Hippocampus: A Microdialysis Study
Authors: Moor Eytan , Auth Ferenc , DeBoer Peter , Westerink Ben H. C.
Published: July 2010

Abstract: In the present study, glutamate receptor agonists and antagonists were administered by retrograde microdialysis into either the medial septum/vertical limb of the diagonal band (MS/vDB), or hippocampus, and the output of acetylcholine (ACh) was measured in the hippocampus by using intracerebral microdialysis. Perfusion with N‐methyl‐d‐aspartate (NMDA) and (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) in the MS/vDB caused an increase in ACh output in the hippocampus. This increase was completely blocked by coadministration of their respective antagonists d(−)‐2‐amino‐5‐phosphonopentanoic acid (d‐AP5) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). Perfusion in the MS/vDB with kainic acid also caused an increase in ACh output, but coadministration of CNQX attenuated the increase only partially. Perfusion with d‐AP5 or CNQX alone in the septal probe did not affect ACh output from the hippocampus. In contrast to the results of septal administration of NMDA and AMPA, local perfusion with the same drugs in the hippocampus caused a decrease in ACh output. Whereas the results of septal administration of drugs indicate that septal cholinergic neurons probably receive excitatory glutamatergic innervation, the decrease in ACh output caused by administration of NMDA and AMPA in the hippocampus is poorly understood.
Developmental Expression of the Glycine Transporters GLYT1 and GLYT2 in Mouse Brain
Authors: Jursky Frantisek , Nelson Nathan
Published: July 2010

Abstract: Using immunocytochemical localization, the distribution of the glycine transporters GLYT1 and GLYT2 in the developing mouse brain was studied. GLYT1 and GLYT2 immunoreactivity begins during the period of fiber outgrow and synaptogenesis. GLYT2 is first expressed in spinal and spinothalamic white matter and is followed by the expression of synaptophysin. In the postnatal stages, GLYT2 staining in the white matter disappears, and a punctuated pattern in the gray matter emerges. In contrast, in the fetal brain GLYT1 immunoreactivity coincides with gray matter neuropil and processes of radial glia. GLYT1 is distributed over a much wider area of the brain than GLYT2. However, the distribution of these two GLYTs implies that GLYT1 and GLYT2 operate in concert within the area where both are present. At the day 12 embryo stage, GLYT1 antibodies stain the liver, and later they also react with the pancreas and the gastroduodenal junction. No other organs exhibit significant GLYT1 immunoreactivity. We additionally observed the presence of GLYT1 in rat fetal cerebral cortex and hippocampus, which was not detected in fetal mouse brain. Moreover, GLYT1 immunoreactivity was found in the mouse floor plate and the ventral commissure but was not present in the same regions in rats. These findings suggest possible differences in the expression of GLYT1 between these two species.
Acute and Repeated Systemic Amphetamine Administration: Effects on Extracellular Glutamate, Aspartate, and Serine Levels in Rat Ventral Tegmental Area and Nucleus Accumbens
Authors: Xue Chang‐Jiang , Ng Jeffrey P. , Li Yong , Wolf Marina E.
Published: July 2010

Abstract: Recent work indicates an important role for excitatory amino acids in behavioral sensitization to amphetamine. We therefore examined, using in vivo microdialysis in awake rats, the effects of amphetamine on efflux of glutamate, aspartate, and serine in the ventral tegmental area and nucleus accumbens, brain regions important for the initiation and expression of amphetamine sensitization, respectively. Water‐pretreated and amphetamine‐pretreated rats were compared to determine if sensitization altered such effects. In both brain regions, Ca2+‐dependent efflux of glutamate accounted for ∼20% of basal glutamate efflux. A challenge injection of water or 2.5 mg/kg of amphetamine did not significantly alter glutamate, aspartate, or serine efflux in the ventral tegmental area or nucleus accumbens of water‐ or amphetamine‐pretreated rats. However, 5 mg/kg of amphetamine produced a gradual increase in glutamate efflux in both regions that did not reverse, was observed in both water‐ and amphetamine‐pretreated rats, and was prevented by haloperidol. Although increased glutamate efflux occurred with too great a delay to mediate acute behavioral responses to amphetamine, it is possible that repeated augmentation of glutamate efflux during repeated amphetamine administration results in compensatory changes in levels of excitatory amino acid receptors in the ventral tegmental area and nucleus accumbens that contribute to development or expression of amphetamine sensitization.
Dual Signalling by the Adenosine A2a Receptor Involves Activation of Both N‐ and P‐Type Calcium Channels by Different G Proteins and Protein Kinases in the Same Striatal Nerve Terminals
Authors: Gubitz Amelie K. , Widdowson Leon , Kurokawa Masako , Kirkpatrick Karen A. , Richardson Peter J.
Published: July 2010

Abstract: Many Gs‐linked receptors have been reported to use multiple signalling pathways in transfected cells but few in their normal cell environment. We show that the adenosine A2a receptor uses two signalling pathways to increase the release of acetylcholine from striatal nerve terminals. One pathway involves activation of Gs, adenylyl cyclase, protein kinase A, and P‐type calcium channels; the other is mediated by a cholera toxin‐insensitive G protein, protein kinase C, and N‐type calcium channels. The effects of these two pathways are not additive, the second pathway being inhibited by the first; but they are equally sensitive to the A2a receptor antagonist KF17837. This demonstrates that the A2a receptor activates two signalling systems in striatal cholinergic neurons.
In Vivo and In Vitro Evidence for the Biosynthesis of Testosterone in the Telencephalon of the Female Frog
Authors: Mensah‐Nyagan Ayikoe G. , Do‐Rego Jean‐Luc , Feuilloley Marc , Marcual Albert , Lange Catherine , Pelletier Georges , Vaudry Hubert
Published: July 2010

Abstract: Neurons and glial cells are capable of synthesizing various steroid hormones, but biosynthesis of testosterone in the CNS has never been reported. The aim of the present study was to demonstrate the synthesis of testosterone in the frog brain. The presence of 17β‐hydroxysteroid dehydrogenase (17β‐HSD)‐like immunoreactivity was detected in a population of glial cells located in the telencephalon. Reversed‐phase HPLC analysis of brain tissue extracts combined with radioimmunoassay detection revealed the presence of substantial amounts of testosterone and 5α‐dihydrotestosterone (5α‐DHT) in the telencephalon where 17β‐HSD‐positive cells were visualized. In male frogs, castration totally suppressed testosterone and 5α‐DHT in the blood and in the rhombencephalon but did not affect the concentration of these two steroids in the telencephalon. Chemical characterization of testosterone in female frog telencephalon extracts was performed by coupling HPLC analysis with gas chromatography‐mass spectrometry. Using the pulse‐chase technique with [3H]pregnenolone as a precursor, the formation of a series of metabolites was observed, including dehydroepiandrosterone, androstenedione, testosterone, 5α‐DHT, and estradiol. These data demonstrate the existence of an active form of 17β‐HSD in the frog telencephalon, which is likely involved in testosterone biosynthesis within the brain.
Striatal Malonate Lesions Are Attenuated in Neuronal Nitric Oxide Synthase Knockout Mice
Authors: Schulz Jörg B. , Huang Paul L. , Matthews Russell T. , Passov David , Fishman Mark C. , Beal M. Flint
Published: July 2010

Abstract: Intrastriatal administration of the reversible succinate dehydrogenase inhibitor malonate produces both energy depletion and striatal lesions by a secondary excitotoxic mechanism. To investigate the role of nitric oxide (NO•) in the pathogenesis of the lesions we examined malonate toxicity in mice in which the genes for neuronal nitric oxide synthase (nNOS) or endothelial nitric oxide synthase (eNOS) were disrupted. Malonate striatal lesions were significantly attenuated in the nNOS mutant mice, and they were significantly increased in the eNOS mutant mice. Malonate‐induced increases in levels of 2,3‐ and 2,5‐dihydroxybenzoic acid/salicylate, markers of hydroxyl radical generation, were significantly attenuated in the nNOS knockout mice. Malonate‐induced increases in 3‐nitrotyrosine, a marker for peroxynitrite‐mediated damage, were blocked in the nNOS mice, whereas a significant increase occurred in the eNOS mice. These findings show that NO• produced by nNOS results in generation of peroxynitrite, which plays a role in malonate neurotoxicity.
Intricate Regulation of Tyrosine Hydroxylase Activity and Gene Expression
Authors: Kumer Sean C. , Vrana Kent E.
Published: July 2010

Abstract: Tyrosine hydroxylase catalyzes the rate‐limiting step in the biosynthesis of the catecholamines dopamine, norepinephrine, and epinephrine. Therefore, the regulation of tyrosine hydroxylase enzyme number and intrinsic enzyme activity represents the central means for controlling the synthesis of these important biogenic amines. An intricate scheme has evolved whereby tyrosine hydroxylase activity is modulated by nearly every documented form of regulation. Beginning with the genomic DNA, evidence exists for the transcriptional regulation of tyrosine hydroxylase mRNA levels, alternative RNA processing, and the regulation of RNA stability. There is also experimental support for the role of both translational control and enzyme stability in establishing steady‐state levels of active tyrosine hydroxylase protein. Finally, mechanisms have been proposed for feedback inhibition of the enzyme by catecholamine products, allosteric modulation of enzyme activity, and phosphorylation‐dependent activation of the enzyme by various different kinase systems. Given the growing literature suggesting that different tissues regulate tyrosine hydroxylase mRNA levels and activity in different ways, regulatory mechanisms provide not only redundancy but also diversity in the control of catecholamine biosynthesis.
Excitatory Amino Acid Receptors in the Ventral Tegmental Area Regulate Dopamine Release in the Ventral Striatum
Authors: Karreman Mignon , Westerink Ben H. C. , Moghaddam Bita
Published: July 2010

Abstract: The role of excitatory amino acid (EAA) receptors located in the ventral tegmental area (VTA) in tonic and phasic regulation of dopamine release in the ventral striatum was investigated. Microdialysis in conscious rats was used to assess dopamine release primarily from the nucleus accumbens shell region of the ventral striatum while applying EAA antagonists or agonists to the VTA. Infusion of the AMPA/kainate receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (25 and 100 µM) into the VTA did not affect dopamine release in the ventral striatum. In contrast, intra‐VTA infusion of the NMDA receptor antagonist 2‐amino‐5‐phosphopentanoic acid (100 and 500 µM) dose‐dependently decreased the striatal release of dopamine. Intra‐VTA application of the ionotropic EAA receptor agonists NMDA and AMPA dose‐dependently (10 and 100 µM) increased dopamine efflux in the ventral striatum. However, infusion of 50 or 500 µM trans‐(±)‐1‐amino‐1,3‐cyclopentanedicarboxylic acid (ACPD), a metabotropic EAA receptor agonist, did not significantly affect these levels. These data suggest that NMDA receptors in the VTA exert a tonic excitatory influence on dopamine release in the ventral striatum. Furthermore, dopamine neurotransmission in this region may be enhanced by activation of NMDA and AMPA receptors, but not ACPD‐sensitive metabotropic receptors, located in the VTA. These data further suggest that EAA regulation of dopamine release primarily occurs in the VTA as opposed to presynaptically at the terminal level.
Melatonin Effect on [3H]Glutamate Uptake and Release in the Golden Hamster Retina
Authors: Faillace María P. , Keller Sarmiento María I. , Rosenstein Ruth E.
Published: July 2010

Abstract: The effect of melatonin on [3H]glutamate uptake and release in the golden hamster retina was studied. In retinas excised in the middle of the dark phase, i.e., at 2400 h, melatonin (0.1 and 10 nM) significantly increased [3H]glutamate uptake, and this effect persisted in a Ca2+‐free medium. On the other hand, melatonin significantly increased [3H]glutamate release in retinas excised at 2400 h, but this effect was Ca2+ sensitive. Melatonin significantly increased 45Ca2+ uptake by a crude synaptosomal fraction from retinas of hamsters killed at 2400 h. In retinas excised at 1200 h, melatonin had no effect on [3H]glutamate uptake, [3H]glutamate release, or 45Ca2+ uptake at any concentration tested. Cyclic GMP analogues, i.e., 8‐bromoguanosine 3′,5′‐cyclic monophosphate and 2′‐O‐dibutyrylguanosine 3′,5′‐cyclic monophosphate, significantly increased [3H]glutamate uptake, [3H]glutamate release, and 45Ca2+ uptake by tissue removed at 1200 and 2400 h, suggesting that the effects of melatonin could correlate with a previously described effect of melatonin on cyclic GMP levels in the golden hamster retina. Taking into account the key role of glutamate in visual mechanisms, the results suggest the participation of melatonin in retinal physiology.
Mivazerol, a Novel α2‐Agonist and Potential Anti‐Ischemic Drug, Inhibits KCl‐Stimulated Neurotransmitter Release in Rat Nervous Tissue Preparations
Authors: El Tamer Ahmed , Prokopenko Igor , Wülfert Ernst , Hanin Israel
Published: July 2010

Abstract: In this study, we have investigated the effect of mivazerol, [3‐(1H‐imidazol‐4‐yl)methyl‐1]‐2‐hydroxy‐benzamide hydrochloride, a new α2‐agonist lacking hypotensive properties and a potential anti‐ischemic drug, on the evoked release of norepinephrine, aspartate, and glutamate in tissue preparations from hippocampus, spinal cord T1–T5 section, rostrolateral ventricular medulla, and nucleus tractus solitarii of the brainstem of rat. A simple and efficient in vitro procedure to study pharmacologically the release of norepinephrine and glutamate is described. Tissues were chopped into (0.3 × 0.2 × 0.2 mm3) sections and the resulting minces were used for this study. Exposure to KCl (10–75 mM) for 5 min served as a stimulus for the release response. One, S (for aspartate and for glutamate release), or two such stimuli, S1 and S2 (for norepinephrine release) were conducted. The release of norepinephrine (+150% above baseline) was inhibited in a dose‐dependent manner by mivazerol in hippocampus (IC50 = 1.5 × 10−8M), spinal cord (IC50 = 5 × 10−8M), rostrolateral ventricular medulla (IC50 = 10−7M), and nucleus tractus solitarii (IC50 = 7.5 × 10−8M), and by clonidine in hippocampus (IC50 = 5 × 10−8M), spinal cord (IC50 = 4.5 × 10−8M), rostrolateral ventricular medulla (IC50 = 2.5 × 10−7M), and nucleus tractus solitarii (IC50 = 10−7M). This effect was counteracted by the selective α2‐antagonists yohimbine and rauwolscine. A significant glutamate and aspartate release response was also induced by KCl (35 mmol/L) in hippocampus (+250 and +135%, respectively) and spinal cord (+120 and +55%, respectively), in vitro. However, neither mivazerol nor clonidine, at doses up to 10 µM, had any significant effect on KCl‐induced glutamate release in spinal cord, whereas mivazerol blocked completely the release of both amino acids in hippocampus and only the release of aspartate in spinal cord. On the other hand, clonidine (1 µM) was only effective in reducing by 40% the release of aspartate in hippocampus. These data indicate that (1) inhibition of KCl‐induced norepinephrine release by mivazerol is mediated by its action on α2‐adrenergic receptors; (2) at concentrations selective for α2‐adrenergic receptors, only mivazerol was effective in blocking the KCl‐induced glutamate release in hippocampal tissue; and (3) at the same concentrations, both mivazerol and clonidine were unable to inhibit glutamate release in the spinal cord. These data suggest that prevention of hyperadrenergic activity by mivazerol in perioperative patients may be mediated through its effect on the release of norepinephrine and/or the release of glutamate and aspartate in regions of the CNS that are involved in the control of cardiovascular homeostasis.
Seizure Activity Results in a Rapid Induction of Nuclear Factor‐κB in Adult but Not Juvenile Rat Limbic Structures
Authors: Rong Yongqi , Baudry Michel
Published: July 2010

Abstract: Previous studies have indicated that increased formation of oxygen free radicals is likely to participate in the cascade of events leading to neuronal damage following kainic acid (KA)‐induced seizure activity. As reactive oxygen species are involved in signal transduction pathways leading to nuclear factor‐κB (NF‐κB) activation, we examined the effects of KA treatment on the activation of NF‐κB in adult and juvenile rat brain. For comparison, changes in two other transcription factors, activator protein‐1 (AP‐1) and Sp1, were also determined. In adult rat piriform cortex and hippocampus, significant induction of NF‐κB was observed at 4 h after KA injection, and the maximal increase was reached at 8–16 h posttreatment. NF‐κB binding activities returned to control levels by 5 days after injection. NF‐κB binding activities were slightly decreased in adult rat cerebellum at 8 and 16 h after KA treatment. In the juvenile rat, no significant changes in NF‐κB binding activity were observed in piriform cortex, hippocampus, and cerebellum after KA injection. Changes in AP‐1 binding activity were qualitatively similar to those observed with NF‐κB in adult but not juvenile rat brain, as AP‐1 was significantly induced in juvenile piriform cortex and hippocampus following KA injection. On the other hand, little or no changes in Sp1 activity were detected in adult and juvenile rat brain. Our results provide further evidence that oxidative stress participates in neuronal damage resulting from KA‐induced seizure activity.
Reduction of Serotonergic Function in Rat Brain by Tryptophan Depletion: Effects in Control and Fluvoxamine‐Treated Rats
Authors: Bel Núria , Artigas Francesc
Published: July 2010

Abstract: The administration of tryptophan (Trp)‐free amino acid mixtures to depressed patients responding to serotonin [5‐hydroxytryptamine (5‐HT)] uptake inhibitors (SSRIs) worsens their clinical state. This procedure reduces Trp availability to brain and thus impairs 5‐HT synthesis. We have examined the influence of Trp depletion on extracellular 5‐HT and 5‐hydroxyindoleacetic acid (5‐HIAA) concentrations in the rat brain using in vivo microdialysis. The treatment with the SSRI fluvoxamine significantly increased 5‐HT content in dialysates from frontal cortex, as compared with control rats (10.2 ± 2.7 vs. 3.1 ± 0.4 fmol per fraction), whereas 5‐HIAA was unaffected. Food deprivation for 20 h reduced dialysate 5‐HT content to almost control values in fluvoxamine‐treated rats (10.2 ± 2.7 vs. 4.3 ± 0.6 fmol per fraction) but did not alter dialysate 5‐HIAA content (7.8 ± 0.4 vs. 7.2 ± 0.5 pmol per fraction). The administration of Trp‐free amino acid mixtures to fluvoxamine‐treated rats significantly attenuated the release of 5‐HT in frontal cortex (∼50%) and, to a lesser extent, in the midbrain raphe nuclei. This effect was more marked in rats not deprived from food before the experiments (67% reduction of dialysate 5‐HT content in frontal cortex) and was absent in control rats (treated with saline). In contrast, dialysate 5‐HIAA was markedly affected by Trp depletion in all groups, including controls (65–75% reductions). These data show that the administration of an amino acid mixture with the same composition and dose (in milligrams per kilogram of body weight) as those inducing a severe mood impairment in depressed patients reduces 5‐HT and 5‐HIAA concentrations in brain dialysates. The reduction of 5‐HT release, however, occurs only in animals previously treated with the antidepressant fluvoxamine for 2 weeks, which would be consistent with a marked reduction of 5‐HT‐mediated transmission in treated depressed patients but not in healthy controls.
Apoptosis as the Mechanism of Neurodegeneration in Batten's Disease
Authors: Lane Steven C. , Jolly Robert D. , Schmechel Donald E. , Alroy Joseph , Boustany Rose‐Mary
Published: July 2010

Abstract: Batten's disease is a genetic neurodegenerative disease of childhood. Its hallmarks are retinitis pigmentosa and neuronal degeneration. As some types of photoreceptor death in mice are mediated by apoptosis, we investigated whether apoptosis is responsible for retinal and neuronal degeneration in the late infantile and juvenile forms of Batten's disease. Using the terminal dUDP nick end‐labeling (TUNEL) staining method, we detected apoptotic neuronal cells in brain from patients and a canine model and in brain and retina from an ovine model for Batten's disease. We confirmed apoptosis by flow cytometry, electron microscopy, and DNA laddering. This is the first inherited neurodegenerative disease involving brain and retina in which apoptosis has been established as the mechanism of neuronal and photoreceptor cell death in both humans and animal models.
Kynurenine Disposition in Blood and Brain of Mice: Effects of Selective Inhibitors of Kynurenine Hydroxylase and of Kynureninase
Authors: Chiarugi Alberto , Carpenedo Raffaella , Moroni Flavio
Published: July 2010

Abstract: To study the regulation of the synthesis of quinolinic and kynurenic acids in vivo, we evaluated (a) the metabolism of administered kynurenine by measuring the content of its main metabolites 3‐hydroxykynurenine, anthranilic acid, and 3‐hydroxyanthranilic acid in blood and brain of mice; (b) the effects of (m‐nitrobenzoyl)alanine, a selective inhibitor of kynurenine hydroxylase and of (o‐methoxybenzoyl)alanine, a selective inhibitor of kynureninase, on this metabolism; and (c) the effects of (o‐methoxybenzoyl)alanine on liver kynureninase and 3‐hydroxykynureninase activity. The conclusions drawn from these experiments are (a) the disposition of administered kynurenine preferentially occurs through hydroxylation in brain and through hydrolysis in peripheral tissues; (b) (m‐nitrobenzoyl)alanine, the inhibitor of kynurenine hydroxylase, causes the expected changes in brain kynurenine metabolism, such as a decrease of 3‐hydroxykynurenine, and an increase of kynurenic acid; and (c) (o‐methoxybenzoyl)alanine, the kynureninase inhibitor, increases brain concentration of the cytotoxic compound 3‐hydroxykynurenine, and unexpectedly does not reduce brain concentration of 3‐hydroxyanthranilic acid, the direct precursor of quinolinic acid. Taken together, the experiments suggest that the systemic administration of a kynurenine hydroxylase inhibitor is a rational approach to increase the brain content of kynurenate and to decrease that of cytotoxic kynurenine metabolites, such as 3‐hydroxykynurenine and quinolinic acid.
In Vitro Phosphorylation of the Cytoplasmic Domain of the Amyloid Precursor Protein by Glycogen Synthase Kinase‐3β
Authors: Aplin Andrew E. , Gibb Graham M. , Jacobsen J. Steven , Gallo Jean‐Marc , Anderton Brian H.
Published: July 2010

Abstract: The two pathological lesions found in the brains of Alzheimer's disease patients, neurofibrillary tangles and neuritic plaques, are likely to be formed through a common pathway. Neurofibrillary tangles are intracellular aggregates of paired helical filaments, the main component of which is hyperphosphorylated forms of the microtubule‐associated protein τ. Extracellular neuritic plaques and diffuse and vascular amyloid deposits are aggregates of β‐amyloid protein, a 4‐kDa protein derived from the amyloid precursor protein (APP). Using conditions in vitro under which two proline‐directed protein kinases, glycogen synthase kinase‐3β (GSK‐3β) and mitogen‐activated protein kinase (MAPK), were able to hyperphosphorylate τ, GSK‐3β but not MAPK phosphorylated recombinant APPcyt. The sole site of phosphorylation in APPcyt by GSK‐3β was determined by phosphoamino acid analysis and phosphorylation of APPcyt mutant peptides to be Thr743 (numbering as for APP770). This site was confirmed by endoproteinase Glu‐C digestion of APPcyt and peptide sequencing. The ability of GSK‐3β to phosphorylate APPcyt and τ provides a putative link between the two lesions and indicates a critical role of GSK‐3β in the pathogenesis of Alzheimer's disease.
Characterization of the Glycolipid Associated with Alzheimer Paired Helical Filaments
Authors: Goux Warren J. , Rodriguez Santiago , Sparkman Dennis R.
Published: July 2010

Abstract: In the present study, analytical techniques including gas chromatography/mass spectrometry (GC/MS)‐assisted carbohydrate linkage‐analysis, one‐ and two‐dimensional NMR, and matrix‐assisted laser desorption/ionization time of flight mass spectroscopy (MALDI‐MS) have been used to characterize the structure of the glycolipid associated with the paired helical filaments (PHF) isolated from the neurofibrillary tangles of Alzheimer's diseased brain. The 1H NMR spectrum of acid‐hydrolyzed protein‐resistant core PHF (prcPHF) displays resonances that can be assigned to fatty acid and glucose. There are no resonances present that would indicate the presence of protein, amino acids, or a sphingosine base. Using two‐dimensional homonuclear correlated spectroscopy, homonuclear Hartmann‐Hahn, and heteronuclear multiple quantum coherence experiments, resonances in the 1H and 13C NMR spectrum of native PHF were assigned to a nonreducing terminal α‐1,6‐glycosidically linked glucose, an internal α‐1,6‐linked glucose, and an α‐1,2,6‐linked glucose. The narrow line‐widths observed for these residues suggest that they arise from glucose residues undergoing rapid segmental motion. The carbohydrate portion of the PHF‐associated glycolipid was analyzed using GC/MS linkage analysis and confirmed the presence of terminal and internal α‐1,6‐linked glucose and α‐1,2,6‐linked glucose in a molar ratio of 2:1:1. Three components of the PHF‐associated glycolipid fraction having masses 2,416, 2,325, and 2,237 Da were observed using MALDI‐MS. The least abundant, heavier mass component (2,416 Da) was best fit to a structure with a tridecamer of glucose having a single esterified C20 fatty acid (Glc13 + C20 or Glc13 + C20:1), whereas the more abundant, lower mass components were best fit to noncovalently associated glycolipid dimers, each with a glucose pentamer or hexamer having two C14, C16, or C18 esterified fatty acids {D[(Glc5 + C18) + (Glc6 + C16)] or D[(Glc5 + C14) + (Glc6 + C14)]}. The ratio of glucose to fatty acid calculated from these best‐fit structures of the more abundant mass components (5.5 ± 1.1:1.0) is in reasonable agreement with the same ratio calculated from peak integrations in the NMR spectra of acid‐hydrolyzed prcPHF (6.2 ± 1.6). Structural similarities between PHF‐associated glycolipid and other glycolipid amphiphiles known to form PHF‐like filaments indirectly suggest that this unique glycolipid may be an integral component of the PHF suprastructure.
Glutamate Uptake by Squid Nerve Fiber Sheaths
Authors: García R. A. G.
Published: July 2010

Abstract: The squid giant nerve exhibits neuron‐Schwann cell interactions that appear to involve glutamate as a mediator; however, there is no information available about the possible fate of the released glutamate. In this study, it is demonstrated that the periaxonal sheaths of the extrasynaptic regions of squid giant nerves (where the glial cells are located) possess the capacity to transport glutamate. In whole intact nerves incubated with low‐glutamate concentrations for long periods of time, the majority of the glutamate incorporated into the tissue was found in the sheaths. Axoplasm‐free sheaths incubated for long periods of time with low concentrations of glutamate were able to accumulate this amino acid against a large apparent concentration gradient. Sheath glutamate uptake occurred in a sodium‐dependent fashion over a wide concentration range and displayed both high‐ and low‐affinity components. Glutamate uptake at concentrations below the Km of the high‐affinity component was independent of homoexchange and displayed a specificity that is similar to that described for high‐affinity glutamate transport in mammalian brain. It is proposed that the sheath transport systems may be involved in the regulation of glutamate levels in the intercellular clefts of the nerve fiber, as part of the glutamatergic neuron‐glial signaling mechanisms in the squid giant nerve fiber.
Preferential In Vivo Incorporation of [3H]Arachidonic Acid from Blood into Rat Brain Synaptosomal Fractions Before and After Cholinergic Stimulation
Authors: Jones Collins R. , Arai Toshanari , Bell Jane M. , Rapoport Stanley I.
Published: July 2010

Abstract: Awake adult male rats were infused intravenously with [3H]arachidonic acid for 5 min, with or without prior administration of an M1 cholinergic agonist, arecoline (15 mg/kg i.p.). Methylatropine was also administered (4 mg/kg s.c.) to control and arecoline‐treated animals. At 15 min postinfusion, the animals were killed, brains were removed and frozen, and subcellular fractions were obtained from homogenates of whole brain. Total radioactivity and radioactivity in various lipid classes were determined for each fraction following normalization for exposure by use of a unidirectional incorporation coefficient, k⋆brain. In control animals, incorporation was greatest in synaptosomal and microsomal fractions, accounting for 50 and 30% of total label incorporated into membrane lipids, respectively. Arecoline increased incorporation in these two fractions by up to 400% but did not increase incorporation into the myelin, mitochondrial, or cytosolic fractions. Of the incorporated radioactivity, 50–80% was in phospholipid in microsomal and synaptosomal fractions, indicating that phospholipid is the major lipid affected by cholinergic stimulation. These results demonstrate that plasma [3H]arachidonic acid is preferentially incorporated into phospholipids of synaptosomal and microsomal fractions of rat brain. Cholinergic stimulation increases incorporation into these fractions, likely by activation of phospholipase A2 and/or C in association with acyltransferase activity. Thus, intravenously infused radiolabeled arachidonic acid can be used to examine synapse‐mediated changes in brain phospholipid metabolism in vivo.
Type I Brain Hexokinase: Axonal Transport and Membrane Associations Within Central Nervous System Presynaptic Terminals
Authors: Garner Judy A. , Linse Klaus D. , Polk Roberta K.
Published: July 2010

Abstract: While studying the delivery of cytoplasmic proteins to the presynaptic terminals of CNS neurons, we discovered unique characteristics of one protein (p118) conveyed in slow component b (SCb) of axonal transport, the large group of proteins representing the cytoplasmic matrix. Alone among the SCb group, p118 coisolated with the synaptic junctional complex on biochemical fractionation of the radiolabeled synaptic regions. Purification and amino acid sequencing of this protein revealed it is most likely the guinea pig form of type I (brain) hexokinase (ATP:d‐hexose 6‐phosphotransferase, EC 2.7.1.1). Further biochemical treatments were consistent with this identity. The majority of type I brain hexokinase has been thought to be associated primarily with membranes, in particular the mitochondrial outer membrane. We found that the majority of type I hexokinase is transported toward the terminals at a rate at least 10 times slower than that exhibited by the maximal or average rate of mitochondria. This suggests that, in the axon, the enzyme exhibits transient or dynamic interactions with mitochondria that are moving more rapidly. It is not clear whether hexokinase binds exclusively to mitochondria, or also exhibits association with nonmitochondrial membranes. The unexpected enrichment of hexokinase during synaptic junctional complex purification may result from its strong association with the presynaptic membrane portion of the synapse.
Developmental Expression of GABA Transporters GAT1 and GAT4 Suggests Involvement in Brain Maturation
Authors: Jursky Frantisek , Nelson Nathan
Published: July 2010

Abstract: cDNA clones representing four pharmacologically distinct GABA transporters (GAT1–GAT4) were previously identified in mouse brain. Two of these, GAT1 and GAT4, were found to be brain specific. We studied GAT1 and GAT4 in the developing rat brain using polyclonal antibodies against recombinant fusion proteins. Patterns of immunoreactivity were very similar in the embryonic and early postnatal stages for both transporters. However, whereas GAT1 immunoreactivity was detected in distinct patterns in gray matter and growing axons, GAT4 immunoreactivity was found in a subset of radial glial cell fascicles. These patterns usually oriented perpendicularly to the axons expressing GAT1. Our results suggest a transient relationship between GAT4‐expressing radial glial elements and GAT1‐expressing axons. The presence of GAT1 in the cortical marginal zone and the numerous GAT4‐positive fascicles observed in the fetal anterior commissure indicate that both transporters may play a role in processes of brain maturation. Because the beginning of expression for both GAT1 and GAT4 correlates with the expression of the α1 subunit of the GABA receptor, the transporters may be connected with the maturation of adult‐type GABAergic inhibitory system in the brain.
Tryptophan Hydroxylase: Cloning and Expression of the Rat Brain Enzyme in Mammalian Cells
Authors: D'Sa Carrol M. , Arthur Robert E. , States J. Christopher , Kuhn Donald M.
Published: July 2010

Abstract: A cDNA encoding full‐length tryptophan hydroxylase was produced by reverse transcriptase‐PCR from rat brain mRNA and expressed transiently in a human fibroblast cell line. Catalytic activity was low unless transfected cells were grown in the presence of FeSO4. Recombinant tryptophan hydroxylase was found almost exclusively within the soluble compartment of the cell and was dependent on tryptophan and tetrahydrobiopterin for activity. The catalytic activity of recombinant tryptophan hydroxylase was stimulated >25‐fold by Fe(II) and to a somewhat lesser extent by the polyanions heparin and phosphatidylserine. The enzyme was inhibited by desferrioxamine and dopamine, both of which complex iron. When extracts from transfected cells were subjected to sucrose gradient centrifugation and analytical gel filtration, the recombinant enzyme behaved the same as the native enzyme from brain. A monoclonal antibody against phenylalanine hydroxylase that cross‐reacts with brain tryptophan hydroxylase was capable of immunoprecipitating the recombinant hydroxylase from solution. These data indicate that recombinant tryptophan hydroxylase expressed in mammalian cells is assembled into tetramers of ∼220,000 daltons. Its catalytic and physical properties appear to be very similar to those of the native enzyme from brain.
2′,3′‐Cyclic Nucleotide 3′‐Phosphodiesterase Binds to Actin‐Based Cytoskeletal Elements in an Isoprenylation‐Independent Manner
Authors: De Angelis Dino A. , Braun Peter E.
Published: July 2010

Abstract: 2′,3′‐Cyclic nucleotide 3′‐phosphodiesterase (CNP) is an isoprenylated protein enriched in myelin and oligodendrocytes but also present in several other tissues at low levels. CNP binds avidly to membranes and in addition possesses several characteristics of cytoskeletal proteins. The role of isoprenylation in the association of CNP with the cytoskeleton was analyzed by ectopic expression in L cells of epitope‐tagged CNP1 and a non‐isoprenylated mutant CNP1. Using nonionic detergent extraction, drug‐mediated cytoskeletal disruption, and coimmunoprecipitation with an anti‐actin antibody, we show that CNP1 is associated with actin‐based cytoskeletal elements independently of its isoprenylation status. A control protein, p21c‐H‐ras, which is also modified by isoprenylation at its carboxyl‐terminus, does not bind to cytoskeletal structures as judged by the same criteria. We present a model that accounts for the association of CNP1 with membranes and the cytoskeleton.
Activity of Ionotropic Glutamate Receptors in Retinal Cells: Effect of Ascorbate/Fe2+‐Induced Oxidative Stress
Authors: Agostinho Paula , Duarte Carlos B. , Oliveira Catarina R.
Published: July 2010

Abstract: The effect of oxidative stress induced by the oxidant pair ascorbate/Fe2+ on the activity of ionotropic glutamate receptors was studied in cultured chick retina cells. The release of [3H]GABA and the increase of the intracellular free Na+ concentration ([Na+]i), evoked by glutamate receptor agonists, were used as functional assays for the activity of the receptors. The results show that the maximal release of [3H]GABA evoked by kainate (KA; ∼20% of the total) or AMPA (∼11% of the total) was not different in control and peroxidized cells, whereas the EC50 values determined for peroxidized cells (33.6 ± 1.7 and 8.0 ± 2.0 µM for KA and AMPA, respectively) were significantly lower than those determined under control conditions (54.1 ± 6.6 and 13.0 ± 2.2 µM for KA and AMPA, respectively). The maximal release of [3H]GABA evoked by NMDA under K+ depolarization was significantly higher in peroxidized cells (7.5 ± 0.5% of the total) as compared with control cells (4.0 ± 0.2% of the total), and the effect of oxidative stress was significantly reduced by a phospholipase A2 inhibitor or by fatty acid‐free bovine serum albumin. The change in the intracellular [Na+]i evoked by saturating concentrations of NMDA under depolarizing conditions was significantly higher in peroxidized cells (8.9 ± 0.6 mM) than in control cells (5.9 ± 1.0 mM). KA, used at a subsaturating concentration (35 µM), evoked significantly greater increases of the [Na+]i in peroxidized cells (11.8 ± 1.7 mM) than in control cells (7.1 ± 0.8 mM). A saturating concentration (150 µM) of this agonist triggered similar increases of the [Na+]i in control and peroxidized cells. Accordingly, the maximal number of binding sites for (+)‐5‐[3H]methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate ([3H]MK‐801) was increased after peroxidation, whereas the maximal number of binding sites for [3H]KA was not affected by oxidative stress. These data suggest that under oxidative stress the activity of the ionotropic glutamate receptors is increased, with the NMDA receptor being the most affected by peroxidation.
τ Is Widely Expressed in Rat Tissues
Authors: Gu Yongjun , Oyama Fumitaka , Ihara Yasuo
Published: July 2010

Abstract: The microtubule‐associated protein τ, a major component of paired helical filaments in Alzheimer's disease, had been thought to be a neuron‐specific protein. We investigated various rat tissues using both reverse transcriptase‐coupled polymerase chain reaction and immunoblotting. τ was found to be widely expressed in many tissues besides the nervous system: at relatively high levels in the heart, skeletal muscle, lung, kidney, and testis and at low levels in the adrenal gland, stomach, and liver. In terms of the τ isoform expression, tissues fall into three classes: those expressing predominantly small τ, those expressing predominantly big τ, and those expressing both at comparable levels. The phosphorylation state of τ varied among the tissues, as shown by differences in the extents of changes in the reactivities with Tau 1 and electrophoretic mobilities after dephosphorylation. It is notable that τ in many nonneural tissues was highly phosphorylated at Ser396 (according to the numbering of the 441‐residue human τ isoform). Thus, τ is widely expressed in rat tissues.
Carbachol Inhibits Insulin‐Stimulated Phosphatidylinositol 3‐Kinase Activity in SH‐SY5Y Neuroblastoma Cells
Authors: Lavie Yaakov , Agranoff Bernard W.
Published: July 2010

Abstract: SH‐SY5Y human neuroblastoma cells express muscarinic M3 receptors as well as insulin receptors, thus offering the opportunity to investigate possible cross‐talk following activation of two distinct intracellular signal transduction pathways that convert the precursor phosphatidylinositol (PI) to its 3′ phosphate or its 4′ phosphate, respectively. In this study, the effect of carbachol on insulin‐stimulated PI 3‐kinase (PI3K) activity was examined in SH‐SY5Y cells. Insulin addition to the cell medium induced a 10–26‐fold increase in anti‐phosphotyrosine‐immunoprecipitable PI3K activity. Preincubation with 1 mM carbachol inhibited the insulin‐stimulated PI3K activity in a time‐dependent manner, with half‐maximal and maximal inhibition times of 4 and 15 min, respectively. Atropine blocked the inhibitory effect of carbachol. Although carbachol did not change the amount of 85‐kDa subunit protein regulatory unit associated with tyrosine‐phosphorylated proteins, either in control or in insulin‐stimulated cells, it appears to decrease the amount of associated 110‐kDa catalytic subunit protein in the latter instance. Because PI3K activity from SH‐SY5Y cells has been shown to be inhibited in vitro in the presence of cytidine diphosphodiacylglycerol (CDP‐DAG) or phosphatidate (PA), we examined the presence of these lipids in SH‐SY5Y cells that had been treated with carbachol. Formation of both lipids was increased in a time‐dependent manner following carbachol addition, and their increased levels are proposed to account for the observed in vivo inhibition of PI3K. Addition of the cell‐permeable homologue didecanoyl‐CDP‐DAG to intact cells inhibited insulin‐stimulated PI3K activity up to 75%, with an IC50 of 0.5 µM, a result that further supports a proposed lipid‐mediated inhibition of PI3K. Exogenously added didecanoyl‐PA, however, did not affect PI3K activity. The possibility that stimulation of the PI 4‐kinase‐mediated signal transduction pathway leads to down‐regulation of the PI3K‐mediated signal transduction pathway in vivo, via inhibition of PI3K by CDP‐DAG or by other consequences of phosphoinositidase C‐linked receptor activation, is discussed.