bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional and Molecular Characterization of Individual Olfactory Neurons
Authors: Noé Johannes , Breer Heinz
Published: July 2010

Abstract: To gain an understanding of the olfactory signal transduction process, individual chemosensory neurons have been assessed for odor‐induced Ca2+ responses and the molecular elements of transduction cascades using Ca2+ imaging technique in combination with single‐cell RT‐PCR approaches. It has been demonstrated that responsiveness of cells to cyclic AMP or inositol trisphosphate odorants was blocked by specific adenylyl cyclase inhibitors or phospholipase C inhibitors, respectively. Using specific primers in single‐cell RT‐PCR analysis, olfactory marker protein, two G protein subtypes (Golf and Go), and adenylyl cyclase (subtype III) and a phospholipase C (phospholipase Cβ2‐related subtype) were identified. For a subpopulation of sensory neurons it was demonstrated that both transduction cascades coexist and are active in the same cell. These data support the notion that two second messenger pathways are active in olfactory sensory neurons and emphasize the concept of dual transduction cascades in olfaction.
GABA Synthesis in Astrocytes After Infection with Defective Herpes Simplex Virus Vectors Expressing Glutamic Acid Decarboxylase 65 or 67
Authors: New Kent C. , Rabkin Samuel D.
Published: July 2010

Abstract: Defective herpes simplex virus (HSV) vectors containing glutamic acid decarboxylase (GAD) cDNAs, either GAD65 or GAD67, were used to examine GAD function and GABA synthesis in rat cortical astrocytes, CNS cells that do not endogenously synthesize GABA. GAD vector infection resulted in isoform‐specific expression of GAD as determined by western blotting and immunohistochemistry. Astrocytes infected with a β‐galactosidase vector or uninfected expressed no GAD and contained no detectable GABA. GABA was detected in glial fibrillary acid protein‐expressing cells after GAD65 vector infection. Significant amounts of GABA, as determined by HPLC, were synthesized in cultures infected with either GAD vector. The levels of GABA in GAD67 vector‐infected cells were almost twofold higher than in GAD65 vector‐infected cells. Vector infection did not alter levels of other intracellular amino acids. GABA was tonically released from astrocytes infected with the GAD67 vector, but no increase in release could be detected after treatment of the cells with K+, veratridine, glutamate, or bradykinin. The ability to transduce astrocytes so that they express GAD and thereby increase GABA levels provides a potential strategy for the treatment of neurologic disorders associated with hyperexcitable or diminished inhibitory activity.
Heterogeneous Expression of the Polysialyltransferases ST8Sia II and ST8Sia IV During Postnatal Rat Brain Development
Authors: Hildebrandt Herbert , Becker Christoph , Mürau Marianne , Gerardy‐Schahn Rita , Rahmann Hinrich
Published: July 2010

Abstract: Polysialic acid on the neural cell adhesion molecule is developmentally regulated and has been implicated in the plasticity of cell‐cell interactions. The sialyltransferases ST8Sia II and ST8Sia IV are able to catalyze the synthesis of polysialic acid. This study compares the expression of ST8Sia II and ST8Sia IV mRNA during postnatal rat brain development. Northern blot analysis indicated a substantial down‐regulation of ST8Sia II from high expression at postnatal day 2 to almost undetectable levels at the age of 6 months. In contrast, the decline of ST8Sia IV content was moderate. In the mature brain, ST8Sia IV is the predominant polysialyltransferase. In situ hybridization of selected brain regions at postnatal days 2, 11, and 21 confirmed the decline of ST8Sia II level in isocortex, hippocampus, and cerebellum. ST8Sia II was not detectable at any time point in the subependymal layer and the layers of the olfactory bulb. Persistent ST8Sia IV expression was localized in the subependymal layer, the glomerular layer of the olfactory bulb, and the granule cell layer of the dentate gyrus and in some widely dispersed cells of the isocortex. The distinct expression patterns of ST8Sia II and ST8Sia IV suggest their differential regulation. As discussed with regard to the persistent polysialic acid expression, ST8Sia IV should receive particular attention in the mature brain.
Characterization of Ethanol‐Sensitive and Insensitive Fibroblast Cell Lines Expressing Human L1
Authors: Wilkemeyer Michael F. , Charness Michael E.
Published: July 2010

Abstract: Ethanol inhibits L1‐mediated cell‐cell adhesion in fibroblast cell lines stably transfected with human L1. Here we show that this action of ethanol is present in only a subset of transfected NIH/3T3 and L cell clonal cell lines. All L1‐expressing cell lines had higher levels of cell adhesion than cell lines transfected with empty vector. In all ethanol‐sensitive cell lines, L1‐mediated adhesion was inhibited by ethanol (IC50 5–10 mM), 2 mM butanol, but not 5 mM pentanol. In contrast, ethanol‐insensitive cell lines were not inhibited by up to 200 mM ethanol, 2 mM butanol, or 5 mM pentanol. Ethanol sensitivity or insensitivity was a stable property of each cell line and was not associated with differences in electrophoretic mobility, abundance, or cell surface localization of L1. Fab fragments prepared from anti‐L1 polyclonal antisera inhibited cell adhesion only in the ethanol‐sensitive cell lines. These data suggest that L1 may exist in an alcohol‐sensitive or an alcohol‐insensitive state that may be governed by host cell factors.
Effects of Oxidants and Glutamate Receptor Activation on Mitochondrial Membrane Potential in Rat Forebrain Neurons
Authors: Scanlon Joelle M. , Reynolds Ian J.
Published: July 2010

Abstract: Both glutamate and reactive oxygen species have been implicated in excitotoxic neuronal injury, and mitochondria may play a key role in the mediation of this process. In this study, we examined whether glutamate‐receptor stimulation and oxidative stress interact to affect the mitochondrial membrane potential (ΔΨ). We measured ΔΨ in rat forebrain neurons with the ratiometric fluorescent dye JC‐1 by using laser scanning confocal imaging. Intracellular oxidant levels were measured by using the oxidation‐sensitive dyes 2′,7′‐dichlorodihydrofluorescein (DCFH2) and dihydroethidium (DHE). Application of hydrogen peroxide (0.3–3 mM) or 1 mM xanthine/0.06 U/ml xanthine oxidase decreased ΔΨ in a way that was independent of the presence of extracellular Ca2+ and was not affected by the addition of cyclosporin A, suggesting the presence of either a cyclosporin A‐insensitive form of permeability transition, or a separate mechanism. tert‐Butylhydroperoxide (730 µM) had less of an effect on ΔΨ than hydrogen peroxide despite similar effects on intracellular DCFH2 or DHE oxidation. Hydrogen peroxide‐, tert‐butylhydroperoxide‐, and superoxide‐enhanced glutamate, but not kainate, induced decreases in ΔΨ. The combined effect of peroxide or superoxide plus glutamate was Ca2+ dependent and was partially inhibited by cyclosporin A. These results suggest that oxidants and glutamate depolarize mitochondria by different mechanisms, and that oxidative stress may enhance glutamate‐mediated mitochondrial depolarization.
Presynaptic Inhibition by Concanavalin A: Are α‐Latrotoxin Receptors Involved in Action Potential‐Dependent Transmitter Release?
Authors: Boehm Stefan , Huck Sigismund
Published: July 2010

Abstract: Effects of concanavalin A on transmitter release were investigated in primary cultures of chick sympathetic neurons. The lectin reduced electrically evoked [3H]noradrenaline release by up to 30% with half‐maximal inhibition at 0.16 µM. Concanavalin A also reduced the release triggered by extracellular Ca2+ in neurons depolarized by 25 mM K+ or rendered Ca2+‐permeable by the ionophore A23187. The inhibitory action of concanavalin A on electrically evoked release was additive to that of the α2‐adrenergic agonist UK 14,304. Inactivation of Gs and Gi/Go type G proteins by either cholera or pertussis toxin did not alter the inhibitory effect of the lectin. Concanavalin A failed to affect the resting membrane potential, action potential waveforms, or voltage‐dependent K+ and Ca2+ currents. In contrast, the lectin efficiently blocked both the Ca2+‐dependent and ‐independent α‐latrotoxin‐induced transmitter release, but only when applied before the toxin. The reduction of electrically evoked, as well as α‐latrotoxin‐evoked, release by concanavalin A was attenuated in the presence of glucose and abolished by methyl α‐d‐mannopyranoside. The dimeric derivative, succinyl‐concanavalin A, was significantly less active than tetrameric concanavalin A. In bovine adrenal chromaffin cells, which displayed only weak secretory responses to α‐latrotoxin, concanavalin A failed to alter K+‐evoked catecholamine secretion. These results show that concanavalin A causes presynaptic inhibition in sympathetic neurons and indicate that cross‐linking of α‐latrotoxin receptors may reduce action potential‐dependent transmitter release.
Traumatic Brain Injury Increases β‐Amyloid Peptide 1‐42 in Cerebrospinal Fluid
Authors: Raby Charlotte A. , Morganti‐Kossmann Maria C. , Kossmann Thomas , Stahel Philip F. , Watson M. Desiree , Evans Lori M. , Mehta Pankaj D. , Spiegel Katharyn , Kuo Yu‐Min , Roher Alex E. , Emmerling Mark R.
Published: July 2010

Abstract: The β‐amyloid peptides, Aβ1‐42 and Aβ1‐40, were quantified in ventricular CSF taken daily for up to 3 weeks from six individuals with severe traumatic brain injury (TBI). There was considerable interindividual variability in the levels of Aβ peptides, but in general Aβ1‐42 levels equalled or exceeded those of Aβ1‐40. Averaging the daily totals of our trauma cohort revealed that the levels of Aβ1‐42 and Aβ1‐40 rose after injury, peaking in the first week and then declining toward control levels over the next 2 weeks. Aβ1‐42 levels were on average two to three times higher in the trauma cohort than in CSF from nontrauma samples. Compared with nontrauma samples, the Aβ1‐40/Aβ1‐42 ratio decreased about fivefold in the trauma patients, further indicative of increased Aβ1‐42 levels. The ratio remained low at all time points studied. No change was measured in the levels of β‐amyloid precursor protein during the same interval. These results suggest that Aβ1‐42 becomes elevated in the CSF after severe brain trauma.
Glycosylation of a Vesicular Monoamine Transporter: A Mutation in a Conserved Proline Residue Affects the Activity, Glycosylation, and Localization of the Transporter
Authors: Yelin Rodrigo , Steiner‐Mordoch Sonia , Aroeti Benjamin , Schuldiner Shimon
Published: July 2010

Abstract: The role of N‐glycosylation in the expression, ligand recognition, activity, and intracellular localization of a rat vesicular monoamine transporter (rVMAT1) was investigated. The glycosylation inhibitor tunicamycin induced a dose‐dependent decrease in the rVMAT1‐mediated uptake of [3H]serotonin. Part of this effect was due to a general toxic effect of the drug. Therefore, to assess the contribution of each of the glycosylation sites to the transporter activity, the three putative N‐glycosylation sites were mutated individually, in combination, and in toto (“triple” mutant). Mutation of each glycosylation site caused a minor and additive decrease in activity, up to the triple mutant, which retained at least 50% of the wild‐type activity. No significant differences were found either in the time dependence of uptake or the apparent affinity for ligands of the triple mutant compared with the wild‐type protein. It is interesting that in contrast to plasma‐membrane neurotransmitter transporters, the unglycosylated form of rVMAT1 distributed in the cell as the wild‐type protein. Pro43 is a highly conserved residue located at the beginning of the large loop in which all the potential glycosylation sites are found. A Pro43Leu mutant transporter was inactive. It is remarkable that despite the presence of glycosylation sites, the mutant transporter was not glycosylated. Moreover, the distribution pattern of the Pro43Leu mutant clearly differed from that of the wild type. In contrast, a Pro43Gly mutant displayed an activity practically identical to the wild‐type protein. As this replacement generated a protein with wild‐type characteristics, we suggest that the conformation conferred by the amino acid at this position is essential for activity.
Fatty Acid Composition of Human Brain Phospholipids During Normal Development
Authors: Martínez Manuela , Mougan Isabel
Published: July 2010

Abstract: The fatty acid composition of phosphatidylethanolamine (PE), ethanolamine plasmalogens (EPs), phosphatidylserine (PS), phosphatidylcholine (PC), and sphingomyelin was studied in 22 human forebrains, ranging in age from 26 prenatal weeks to 8 postnatal years. Phospholipids were separated by two‐dimensional TLC, and the fatty acid methyl esters studied by capillary column GLC. Docosahexaenoic acid (22:6n‐3) increased with age in PE and PC, whereas arachidonic acid (20:4n‐6) remained quite constant. In EP, 22:6n‐3 increased less markedly than 20:4n‐6, adrenic (22:4n‐6) and oleic (18:1n‐9) acids being the predominant fatty acids during postnatal age. In PS, 18:1n‐9 increased dramatically throughout development, and 20:4n‐6 and 22:4n‐6 increased only until ∼6 months of age. Although 22:6n‐3 kept quite constant during development in PS, its percentage decreased due to the accretion of other polyunsaturated fatty acids (PUFAs). As a characteristic myelin lipid, sphingomyelin was mainly constituted by very long chain saturated and monounsaturated fatty acids. Among them, nervonic acid (24:1n‐9) was the major very long chain fatty acid in Sp, followed by 24:0, 26:1n‐9, and 26:0, and its accretion after birth was dramatic. As myelination advanced, 18:1n‐9 increased markedly in all four glycerophospholipids, predominating in EP, PS, and PC. In contrast, 22:6n‐3 was the most important PUFA in PE in the mature forebrain.
NMR‐Based Identification of Intra‐ and Extracellular Compartments of the Brain Pi Peak
Authors: Gilboe David D. , Kintner Douglas B. , Anderson Mark E. , Fitzpatrick James H.
Published: July 2010

Abstract: The Pi peak in a 31P NMR spectrum of the brain can be deconvoluted into six separate Lorentzian peaks with the same linewidth as that of the phosphocreatine peak in the spectrum. In an earlier communication we showed that the six Pi peaks in normal brain represent two extracellular and four intracellular compartments. In that report we have identified the first of the extracellular peaks by marking plasma with infused Pi, thereby substantially increasing the amplitude of the single peak at pH 7.35. 2‐Deoxyglucose‐6‐phosphate (2‐DG‐6‐P) was placed in the brain interstitial space by microdialysis. The resulting 2‐DG‐6‐P peak was deconvoluted into three separate peaks. The chemical shift of the principle 2‐DG‐6‐P peak gave a calculated pH of 7.24 ± 0.02 for interstitial fluid pH, a value that agreed well with the pH of the second extracellular Pi peak at pH 7.25 ± 0.01. We identified the intracellular compartments by selectively stressing cellular energy metabolism in three of the four intracellular spaces. A seizure‐producing chemical, flurothyl, was used to activate the neuron, thereby causing a demand for energy that could not be completely met by oxidative phosphorylation alone. The resulting loss of high‐energy phosphate reserves caused a significant increase in intracellular Pi only in those cells associated with the Pi peak at pH 6.95 ± 0.01. This suggests that this compartment represents the neuron. Ammonia is detoxified in the astrocyte (glutamine synthetase) by incorporating it into glutamine, a process that requires large amounts of glucose and ATP. The intraarterial infusion of ammonium acetate into the brain stressed astrocyte energy metabolism resulting in an increase in the Pi of the cells at pH of 7.05 ± 0.01 and 7.15 ± 0.02. This finding, coupled with our observation that these same cells take up infused Pi probably via the astrocyte end‐foot processes, lead us to conclude that these two compartments represent two different types of astrocytes, probably protoplasmic and fibrous, respectively. As a result of this study, we now believe the brain contains four extracellular and four intracellular compartments.
Interaction of α‐Phenyl‐N‐tert‐Butyl Nitrone and Alternative Electron Acceptors with Complex I Indicates a Substrate Reduction Site Upstream from the Rotenone Binding Site
Authors: Hensley Kenneth , Pye Quentin N. , Maidt Michael L. , Stewart Charles A. , Robinson Kent A. , Jaffrey Fatima , Floyd Robert A.
Published: July 2010

Abstract: Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O2 to hydrogen peroxide (H2O2) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H2O2 formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI‐mediated enzyme inactivation. The O2 consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H2O2 generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate‐linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH‐coupled H2O2 flux was accelerated by rotenone. α‐Phenyl‐N‐tert‐butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex I‐stimulated H2O2 flux and NBT reduction and also protected complex I from DPI‐mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H2O2 synthesis, possessing an “electron leak” site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H2O2 production by PBN suggests a novel explanation for the broad‐spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.
Dopamine D1B Receptor Chimeras Reveal Modulation of Partial Agonist Activity by Carboxyl‐Terminal Tail Sequences
Authors: Sugamori Kim S. , Scheideler Mark A. , Vernier Philippe , Niznik Hyman B.
Published: July 2010

Abstract: NNC 01‐0012, a second‐generation benzazepine compound, pharmacologically differentiates multiple vertebrate D1 receptor subtypes (D1A, D1B, D1C, and D1D) and displays high selectivity and affinity for dopamine D1C receptors. Functionally, whereas NNC 01‐0012 acts as a full or poor antagonist at D1C and D1A receptor‐mediated cyclic AMP production, respectively, it exhibits partial agonist activity at the D1B receptor. To define some of the structural motifs that regulate the pharmacological and functional differentiation of vertebrate dopamine D1 receptors by NNC 01‐0012, a series of receptor chimeras were constructed in which the divergent carboxyl‐terminal (CT) receptor tails were replaced with the corresponding sequences of D1A, D1B, or D1C receptors. Substitution of the vertebrate D1B carboxyl‐terminal‐tail at position Tyr345 with carboxyl‐terminal‐tail sequences of the D1A receptor abolished the partial agonist activity of NNC 01‐0012 without affecting dopamine‐stimulated cyclic AMP accumulation. At vertebrate D1B/D1Cct‐tail receptor mutants, however, the intrinsic activity of the partial agonist NNC 01‐0012 (10 µM) was markedly enhanced (∼60% relative to 10 µM dopamine) with no concomitant alteration in the molecule's ligand binding affinity or constitutive activity of the chimeric receptor. Similar results were obtained with other benzazepines such as SKF‐38393 and SCH‐23390, which act as partial agonists at vertebrate D1B receptors. Substitution of D1A and D1C receptor carboxyl‐terminal tails with sequences encoded by the D1B receptor carboxyl‐terminal tail did not, however, produce receptors with functional characteristics significantly different from wild type. Taken together, these data clearly suggest that in addition to well‐characterized domains and amino acid residues in the third cytoplasmic loop, partial agonist activity at the D1B receptor is modulated by sequence‐specific motifs within the carboxyl‐terminal tail, a region that may underlie the possible structural basis for functionally divergent roles of multiple dopamine D1‐like receptors.
Structural Analysis of the Sixth Immunoglobulin‐Like Domain of Mouse Neural Cell Adhesion Molecule L1 and Its Interactions with αvβ3, αIIbβ3, and α5β1 Integrins
Authors: Blaess Sandra , Kammerer Richard A. , Hall Heike
Published: July 2010

Abstract: Previous experiments suggested that the human cell adhesion molecule L1 interacts with different integrins via its sixth immunoglobulin‐like domain in an RGD‐dependent manner. Here we have described the expression of this domain from early postnatal mouse brain, analyzed the structure of the recombinant protein by circular dichroism and fluorescence spectroscopy, and performed solid‐phase binding studies to αvβ3, αIIbβ3, and α5β1 integrins. The domain was found to have the expected β‐sheet organization, which was lost in the presence of guanidine hydrochloride. The midpoint of the single‐step transition occurred at 1.5 M guanidine hydrochloride. The sixth immunoglobulin‐like domain of mouse brain L1 contains two RGD motifs and was found to bind in a concentration‐dependent and saturable way to αvβ3, αIIbβ3, and α5β1 integrins, suggesting specific interactions with these ligands. However, only the interaction to αvβ3 could be inhibited in a concentration‐dependent manner by an RGD‐containing peptide, and the IC50 was determined to be ∼20 nM. Mutants of the domain, which lack either one or both of the RGD sites, demonstrated that the RGD site comprising residues 562–564 is involved in the interaction to αvβ3. Our findings indicate an RGD‐independent mechanism for the interactions to αIIbβ3 and α5β1, as no involvement of any RGD motif could be demonstrated.
Mice Deficient in Group IV Cytosolic Phospholipase A2 Are Resistant to MPTP Neurotoxicity
Authors: Klivenyi Peter , Beal M. Flint , Ferrante Robert J. , Andreassen Ole A. , Wermer Marieke , Chin Mi‐Reyoung , Bonventre Joseph V.
Published: July 2010

Abstract: Phospholipase A2 (PLA2) enzymes are critical regulators of prostaglandin and leukotriene synthesis, and they may also play an important role in the generation of intracellular free radicals. The group IV cytosolic form of phospholipase A2 (cPLA2) is regulated by changes in intracellular calcium concentration, and the enzyme preferentially acts to release arachidonic acid esterified at the sn‐2 position of phospholipids. We examined the susceptibility of mice carrying a targeted mutation of the cPLA2 gene to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐induced neurotoxicity. Mutant mice have no functional cPLA2 activity. Mice that were homozygous for the mutation (cPLA2−/−) were significantly resistant to MPTP‐induced dopamine depletion as compared with littermate control (cPLA2+/+) and heterozygous mice (cPLA2+/−). These findings provide evidence that cPLA2 plays a role in MPTP neurotoxicity and suggest that cPLA2 may play a role in the development of Parkinson's disease in humans.
Continuous Treatment with Morphine Increases Diazepam Binding Inhibitor mRNA in Mouse Brain
Authors: Katsura Masashi , Hara Atsuo , Higo Atsuko , Tarumi Chihaya , Hibino Yasushi , Ohkuma Seitaro
Published: July 2010

Abstract: Effects of acute and chronic morphine treatment on the expression of diazepam binding inhibitor (DBI) mRNA in the mouse brain were examined. Cerebral DBI mRNA expression significantly increased in morphine‐dependent mice, and this increase is more remarkable in morphine‐withdrawn mice, whereas a single administration of morphine (50 mg/kg) produced no changes in the expression. Simultaneous administration of naloxone (3 mg/kg) with morphine completely abolished the increase in cerebral DBI mRNA expression observed in morphine‐dependent and ‐withdrawn mice. These results indicate that a chronic functional interaction between morphine and opioid receptors has a critical role in increases in DBI mRNA expression.
Peptide Fragments of β‐Amyloid Precursor Protein: Effects on Parallel Fiber‐Purkinje Cell Synaptic Transmission in Rat Cerebellum
Authors: Hartnell Nick A. , Suh Y. ‐H.
Published: July 2010

The effects of peptide fragments of the β‐amyloid precursor protein (βAPP) on parallel fiber (PF)‐Purkinje cell synaptic transmission in the rat cerebellum were examined. Transient inward currents associated with calcium influx were induced by localized applications of the 105‐amino acid carboxy‐terminal fragment (CT105) of βAPP to discrete dendritic regions of intact Purkinje cells. βAPP and the amyloid β (Aβ) peptide fragments Aβ1‐16, Aβ25‐35, and Aβ1‐42 had little or no effect. Inward currents were also observed following applications of CT105 to isolated patches of somatic Purkinje cell membrane. All five proteins/peptides induced some depression of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate (AMPA) receptor‐mediated synaptic transmission between PFs and Purkinje cells, through a combination of pre‐ and postsynaptic effects. CT105 induced the greatest depression, which spread to distant synapses following local application and which was prevented by inhibition of nitric oxide synthase. These data indicate that CT fragments of the βAPP can modulate AMPA‐mediated glutamatergic synaptic transmission in the cerebellar cortex. These fragments may therefore be considered alternative candidates for some of the neurotoxic effects of Alzheimer's disease.
Increased Generation of Alternatively Cleaved β‐Amyloid Peptides in Cells Expressing Mutants of the Amyloid Precursor Protein Defective in Endocytosis
Authors: Cescato Renzo , Dumermuth Eric , Spiess Martin , Paganetti Paolo A.
Published: July 2010

The subcellular location of the secretases processing the β‐amyloid precursor protein (APP) is not established yet. We analyzed the generation of the β‐amyloid peptide (Aβ) in human embryonic kidney 293 cell lines stably expressing wild‐type and noninternalizing mutants of human APP. APP lacking the entire cytoplasmic domain or with both tyrosine residues of the motif GYENPTY mutated to alanine showed at least fivefold reduced endocytosis. In these cell lines, the production of Aβ1‐40 was substantially reduced, but accompanied by the appearance of two prominent alternative Aβ peptides differing at the amino‐termini. Based on antibody reactivity and mobility in high‐resolution gels in comparison with defined Aβ fragments, these peptides were identified as Aβ3‐40 and Aβ5‐40. Notably, these alternative Aβ peptides were not generated when the APP mutants were retained in the early secretory pathway by treatment with brefeldin A. These results indicate that the alternative processing is the result of APP accumulation at the plasma membrane and provide evidence of distinct β‐secretase activities. Cleavage amino‐terminal to position 1 of Aβ occurs predominantly in endosomes, whereas the processing at positions 3 or 5 takes place at the plasma membrane.
Nigrostriatal Reduction of Aromatic L‐Amino Acid Decarboxylase Activity in MPTP‐Treated Squirrel Monkeys: In Vivo and In Vitro Investigations
Authors: Yee R.E. , Huang S.‐C. , Stout D.B. , Irwin I. , Shoghi‐Jadid K. , Togaski D.M. , DeLanney L.E. , Langston J.W. , Satyamurthy N. , Farahani K.F. , Phelps M.E. , Barrio J.R.
Published: July 2010

Aromatic L‐amino acid decarboxylase (AAAD) activity was examined in vivo with positron emission tomography (PET) using 6‐[18F]fluoro‐L‐DOPA (FDOPA) in squirrel monkeys lesioned with graded doses of the neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). In vitro biochemical determinations of AAAD activity in caudate, putamen, substantia nigra, and nucleus accumbens were performed in the same animals to establish a direct comparison of in vivo and in vitro measurements. In vivo and in vitro AAAD activities in caudate/putamen were substantially reduced in animals treated with the highest dose of MPTP (2.0 mg/kg). The percent change in the striatal FDOPA uptake (Ki) and decarboxylation rate constant (k3) values resulting from MPTP treatment showed highly significant correlations with in vitro‐determined AAAD activities. However, decarboxylase rates within individual animals presented as ~ 10‐fold difference between in vivo and in vitro values. Lower in vivo k3 measurements may be attributed to several possibilities, including transport restrictions limiting substrate availability to AAAD within the neuron. In addition, reductions in AAAD activity in the substantia nigra did not parallel reductions in AAAD activity within the striatum, supporting the notion of a nonlinear relationship between nigrostriatal cell degeneration and terminal losses. This work further explores the role of AAAD in Parkinson's disease, a more important factor than previously thought.
GluR1 Glutamate Receptor Subunit Is Regulated Differentially in the Primate Basal Ganglia Following Nigrostriatal Dopamine Denervation
Authors: Betarbet Ranjita , Porter Richard H. P. , Greenamyre J. Timothy
Published: July 2010

Nigrostriatal dopaminergic denervation is associated with complex changes in the functional and neurochemical anatomy of the basal ganglia. The excitatory neurotransmitter glutamate mediates neural signaling at crucial points of this circuitry, and glutamate receptors are differentially distributed in the basal ganglia. Available evidence suggests that the glutamatergic corticostriatal and subthalamofugal pathways become overactive after nigrostriatal dopamine depletion. In this study, we have analyzed the regulation of the GluR1 subunit of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate glutamate receptor in the basal ganglia of primates following 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahy‐dropyridine‐induced dopamine denervation. The dopamine denervation resulted in distinct alterations in GluR1 distribution: (1) GluR1 protein expression was markedly increased in caudate and putamen, and this was most pronounced in the striosomes; (2) GluR1 protein was altered minimally in subthalamic nucleus; (3) expression of GluR1 was down‐regulated in the globus pallidus by 63% and in the substantia nigra by 57%. The down‐regulation of GluR1 expression in the output nuclei of the basal ganglia, the internal segment of the globus pallidus and the substantia nigra pars reticulata, may be a compensation for the overactive glutamatergic input from subthalamic nucleus, which arises after striatal dopamine denervation. Our results indicate that the glutamatergic system undergoes regulatory changes in response to altered basal ganglia activity in a primate model of Parkinson's disease. Targeted manipulation of the glutamatergic system may be a viable approach to the symptomatic treatment of Parkinson's disease.
THIS ARTICLE HAS BEEN RETRACTED: Diminished Glutathione Levels Cause Spontaneous and Mitochondria‐Mediated Cell Death in Neurons from Trisomy 16 Mice:
Authors: Schuchmann S. , Heinemann U.
Published: July 2010

It has been suggested that the increased neuronal death in cultures from trisomy 16 (Ts16) mice, a model of Down's syndrome, might result from a diminished concentration of reduced glutathione (GSH). In this study we used microfluorometric techniques to investigate the effect of GSH levels on neuronal survival in diploid and Ts16 cultures. Addition of the GSH precursors cysteine and cystine and the antioxidant tocopherol to the culture medium increased the GSH concentration up to 126.0% in diploid and up to 111.9% in Ts16 neurons. Moreover, we observed a reduced spontaneous neuronal death rate in diploid and Ts16 cultures. Following the application of 50‐100 μM glutamate to culture medium, we found a GSH increase in the presence of cysteine, cystine, tocopherol, and cyclosporin A, an inhibitor of mitochondrial permeability transition (diploid, 105.8‐110.8%; Ts16, 83.1‐96.3%). However, only tocopherol and cyclosporin A had a protective effect on glutamate‐induced neuronal death. The results suggest that reduced GSH levels affect the increase of a spontaneous and a mitochondria‐mediated, cyclosporin A‐sensitive type of neuronal cell death. Therefore, elevating intracellular GSH concentration may have neuroprotective effects in Down's syndrome and Alzheimer's disease.
Early Leptin Response to a Palatable Diet Predicts Dietary Obesity in Rats: Key Role of Melanocortin‐4 Receptors in the Ventromedial Hypothalamic Nucleus
Authors: Harrold Joanne A. , Williams Gareth , Widdowson Peter S.
Published: July 2010

We have investigated whether interactions between leptin and hypothalamic melanocortin‐4 receptors (MC4‐Rs) determine individual susceptibility to dietary obesity in rats. Animals with relatively high plasma leptin levels 1 week after presentation of palatable food, before weight increased significantly, subsequently showed lower food intake and weight gain after 8 weeks of palatable feeding than those with low early leptin levels. The rats with lesser weight gain also showed significantly greater down‐regulation of MC4‐Rs, which mediate hypophagia, in specific hypothalamic areas, namely, the arcuate, dorsomedial, and ventromedial (VMH) nuclei and the median eminence. We suggest that this reflects enhanced receptor exposure to endogenous α‐melanocyte‐stimulating hormone, an appetite‐suppressing peptide produced by hypothalamic proopiomelanocortin neurones. It is striking that plasma leptin levels at 1 week were inversely correlated with MC4‐R density in the VMH, suggesting that this is a key site of leptin action. The early leptin response to palatable feeding may therefore "program" subsequent feeding behaviour and weight gain by regulating neurones that project selectively to the VMH.
Differential Control of Tyrosine Hydroxylase Activation and Catecholamine Secretion by Voltage‐Operated Ca2+ Channels in Bovine Chromaffin Cells
Authors: O'Farrell Melanie , Marley Philip D.
Published: July 2010

Contributions of L‐, N‐, and P/Q‐type voltage‐operated Ca2+ channels to two responses of bovine adrenal chromaffin cells have been studied using the nonreceptor stimulus K+ depolarization. Tyrosine hydroxylase activity and catecholamine secretion were both increased by K+ over a similar concentration range and in a Ca2+‐dependent manner. At a submaximal concentration of 20 mM K+, tyrosine hydroxylase activation was reduced by nitrendipine but unaffected individually by (±)‐Bay K 8644, ω‐conotoxin GVIA, ω‐agatoxin IVA, and ω‐conotoxin MVIIC. It was fully blocked by combined inhibition of L‐, N‐, and P/Q‐type channels. With a maximal concentration of 50 mM K+, tyrosine hydroxylase activation was unaffected by nitrendipine as well as by each of the other drugs on its own; however, it was reduced by 71% by combined inhibition of L‐, N‐, and P/Q‐type channels. In contrast, catecholamine secretion with both 20 and 50 mM K+ was enhanced by (±)‐Bay K 8644, partially inhibited by nitrendipine and ω‐conotoxin MVIIC, and completely blocked by a combination of antagonists for L‐, N‐, and P/Q‐type channels. The results show that Ca2+ entry through voltage‐operated Ca2+ channels can differentially regulate distinct chromaffin cell responses and that this is an intrinsic property of the mechanisms by which Ca2+ entry activates these responses. It is not dependent on the parallel activation of other signaling events by receptors.
Recombinant Expression of α‐Bungarotoxin in Pichia pastoris Facilitates Identification of Mutant Toxins Engineered to Recognize Neuronal Nicotinic Acetylcholine Receptors
Authors: Levandoski Mark M. , Caffery Philip M. , Rogowski Robert S. , Lin Yingxin , Shi Qing‐Luo , Hawrot Edward
Published: July 2010

A snake venom‐derived α‐neurotoxin, α‐bungarotoxin (αBgtx), is the classic competitive antagonist of nicotinic acetylcholine receptors (nAChRs). The very high specificity and essentially irreversible binding of αBgtx to various nAChRs make αBgtx the prime candidate for studying the molecular determinants of specificity for nAChR‐ligand interactions. To facilitate site‐directed mutagenesis of αBgtx for functional analysis, we have developed a recombinant expression system for αBgtx using the methylotropic yeast Pichia pastoris. A synthetic gene coding for αBgtx was subcloned into an expression vector that directs secretion of the recombinant αBgtx (rBgtx) when stably integrated into the yeast genome. Expression of rBgtx was induced by growth of yeast cultures with methanol as the sole carbon source. The activity of the rBgtx in the cell‐free medium was measured by competition with 125I‐Bgtx for binding to Torpedo nAChR‐enriched membranes. The rBgtx, purified to homogeneity by standard HPLC, has the correct predicted amino terminal sequence and molecular mass. Its circular dichroism spectrum is very similar to that of authentic venom‐derived αBgtx, and the biological activity of the rBgtx is identical to that of authentic αBgtx. We have used the Pichia expression system to study a double point mutation of αBgtx, rBgtx‐K38P/L42Q, that has a high affinity for α3β2 neuronal nAChRs. This is the first demonstration of engineering an α‐neurotoxin to recognize non‐α7 neuronal nicotinic receptors.
c‐DNA Microarray to determine molecular events in neurodegeneration and neuroprotection
Authors: Youdim M. B. H. , Levites Y. , Weinreb O. , Mandel S.
Published: July 2010

Cell death in CNS involves complex processes, many of which have not been identified biochemically. At the present biochemical techniques cannot adequately establish these. However, the advent of cDNA microarray or microchips, in which the expression of thousands of genes can be measured at once to give a global assessment in disease pathology, its progress or animal models, has simplified this. We have employed this technique to study the mechanism of neurotoxicity of MPTP and 6‐hydroxydoapmine induced in neuronally derived cells in culture, in the animal models of Parkinson's disease and neuroprotection initiated by monoamine oxidase B inhibitor, rasagiline; iron chelators, R‐apomorphine and EGCG and other neuroprotective drugs. Our studies have clearly indicated that MPTP induced early gene expression, prior to cell death (first 24 h), are prerequirement for 51 late gene expression changes implicated at the time of neuronal death. The latter genes include those involved in iron metabolism, oxidative stress, inflammatory processes, glutaminergic excitotoxicity, nitric oxide, growth factors, transcription factors, cell cycle, intermediatory metabolism and other gene previously not identified. The expressions of many of the latter genes, also identified by in situ hybridization, are prevented when the animals are pretreated with the above neuroprotective drugs. These studies have clearly shown that neurodegeneratrion is a complex cascades of ‘domino’ effect. Thus a single neuroprotective drug treatment may not be adequate to prevent it, but, that a cocktail of drugs might.
New aspects of amino acid neurotransmitter synthesis in cultured neurons
Authors: Sonnewald U. , Waagepetersen H. , Qu H. , Schousboe A.
Published: July 2010

The synthesis and degradation of GABA, glutamate and aspartate, has been studied extensively, both in vivo and in vitro. Cell culture studies have shown that metabolism of these amino acids is compartmentalized (Qu et al. 2000; Waagepetersen et al. 1998). By magnetic resonance spectroscopy (MRS) analysis of 13C incorporation into different positions in cerebral metabolites, information has been obtained giving strong support to the concept of intracellular heterogeneity in different brain cell cultures. It has been suggested that compartmentalisation of metabolism also exists at the mitochondrial level (Sonnewald et al. 1998). It was shown that metabolism and uptake of glutamate in cerebellar granule neurons was differentially affected by thiopental (Qu et al. 2000). TCA cycle activity in cortical neurons has been shown to be associated with multiple compartments as well (Waagepetersen et al. 1998). Cycling ratios calculated from MRS data obtained from cell extracts, showed a significant effect of depolarization on the ratio of glutamate but not on that of GABA. An observation that indicates multiple TCA cycles as well, in this case with regard to synthesis of glutamate and GABA. A growing amount of evidence seems to point strongly towards intracellular compartmentalisation of neurotransmitter amino acid metabolism in individual cells.

References Qu H. et al. (2000) Neurochem. Int. 37, 207–215.

Waagepetersen H. et al. (1998) JCBFM18, 109–117.

Sonnewald U. et al. (1998) JCBFM18, 231–237.
CD36 null mice reveal a key role for CD36 in β‐amyloid induced microglial activation
Authors: El Khoury J. B. , Moore K. , Freeman M. , Luster A. D.
Published: July 2010

A major pathological feature of Alzheimer's disease (AD) is the senile plaque (SP), composed of β−amyloid fibrils (fAβ), activated microglia, astrocytes and degenerating neurons. Microglial activation may initiate and propagate neuronal degeneration. Activation of microglia involves phosphorylation of the mitogen activated protein (MAP) kinases ERK1/2, a step that may lead to chemokine and neurotoxin production. The microglial fAβ receptors involved in this process remain to be identified. We have shown that class A scavenger receptors mediate adhesion of microglia to fAβ. We investigated whether CD36, a class B scavenger receptor, is expressed on microglia in AD, and whether CD36 plays a role in signalling microglia to secrete neurotoxins and chemokines (such as MCP‐1) when they interact with fAβ. Using flow cytometry and immuno‐histochemistry, we found that CD36 is expressed on primary microglia in culture, and on microglia in AD brains. Microglia from CD36 null mice produce 80% less H2O2, and 75% less MCP‐1 than wild type (WT) microglia, when stimulated with fAβ. In addition, fAβ binding to microglia led to phosphorylation of ERK1/2 in WT but not in CD36 null cells, whereas inhibition of ERK1/2 in WT microglia blocked fAb‐induced MCP‐1 and H2O2 production. Intra‐peritoneal injection of fAβ in CD36 null mice elicited significantly less mononuclear phagocytes than in WT mice. Addition of fAβ to WT but not to CD36 null brain slices, led to migration of neighboring microglia to sites of fAβ deposition. Our data show that binding of fAb to microglial CD36 initiates an intracellular signaling cascade involving phosphorylation of ERK1/2 that lead to secretion of H2O2 and MCP‐1, and suggest that CD36 may play an important role in microglial activation in AD.
Is dopamine the answer? Other systems in the neurochemical effects of psychostimulants
Authors: Izenwasser S.
Published: July 2010

For over 20 years, the focus of studies examining the neurochemical and behavioral effects of cocaine and other psychostimulants has been on dopamine. Many behavioral studies have shown that dopamine plays an important role in the reinforcing and behavioral effects of cocaine. Cocaine binds to the dopamine transporter and inhibits dopamine uptake. While there are some effects of cocaine on dopamine receptors, dopamine levels, and the dopamine transporter, these neurochemical studies have not been able to account fully for the altered behavioral effects of cocaine following chronic cocaine administration. Recent studies by Kantak et al. have shown that the reinforcing effects of psychostimulants depend upon activation of brain nitric oxide synthase. In addition, Rocha et al. have reported that cocaine is self‐administered in animals lack dopamine transporters. This finding suggests that other neurochemical components are necessary for the reinforcing effects (and hence the abuse) of cocaine. Since cocaine binds to dopamine, norepinephrine and serotonin transporters, it is likely that a combination of effects on these systems may be responsible for the behavioral effects of cocaine. Mu‐ and kappa‐opioids regulate dopamine and serotonin and this regulation plays a role in the effects of cocaine (Izenwasser et al.). Unterwald and colleagues have shown that there are large effects of cocaine on opioid receptors and second messenger regulation. These studies show that there are interactions between multiple systems and that these interactions are important factors in the effects of abused drugs, perhaps more important than activation of dopaminergic systems alone. These findings will be discussed in terms of the implications for the development of treatments for cocaine abuse.
Iron porphyrin treatment extends survival in a transgenic animal model of amyotrophic lateral sclerosis
Authors: Wu Annie S. , Kiaei Mahmoud , Aguirre Norberto , Crow John P. , Calingasan Noel Y. , Browne Susan E. , Beal M. Flint
Published: July 2010

Oxidative damage, produced by mutant Cu/Zn superoxide dismutase (SOD1), may play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating motor neuron degenerative disease. A novel approach to antioxidant therapy is the use of metalloporphyrins that catalytically scavenge a wide range of reactive oxygen and reactive nitrogen species. In this study, we examined the therapeutic potential of iron porphyrin (FeTCPP) in the G93A mutant SOD1 transgenic mouse model of ALS. We found that intraperitoneal injection of FeTCPP significantly improved motor function and extended survival in G93A mice. Similar results were seen with a second group of mice wherein treatment with FeTCPP was initiated at the onset of hindlimb weakness—roughly equivalent to the time at which treatment would begin in human patients. FeTCPP‐treated mice also showed a significant reduction in levels of malondialdehyde (a marker of lipid peroxidation), in total content of protein carbonyls (a marker of protein oxidation), and increased neuronal survival in the spinal cord. These results therefore provide further evidence of oxidative damage in a mouse model of ALS, and suggest that FeTCPP could be beneficial for the treatment of ALS patients.
Ethanol specifically decreases peroxisome proliferator activated receptor β in B12 oligodendrocyte‐like cells
Authors: Leisewitz Andrea V. , Jung Juan E. , Perez‐Alzola Patricia , Fuenzalida Karen M. , Roth Alejandro , Inestrosa Nibaldo C. , Bronfman Miguel
Published: July 2010

Peroxisome proliferator activated receptors (PPARs) are nuclear receptors that control important genes involved in lipid metabolism. Their role in nerve cells is uncertain, although anomalous myelination of the corpus callosum has been described in the PPARβ‐null mouse, and abnormalities of this tissue have been documented in fetal alcohol syndrome in humans. We report here that ethanol treatment of B12 oligodendrocyte‐like cells induces a concentration‐ and time‐dependent decrease in the mRNA and protein levels of PPARβ, with no effect on PPARα or PPARγ. The effect on PPARβ is seen as an increase in mRNA degradation, as assessed by run‐off assays, due to a significant decrease in PPARβ mRNA half‐life, with no observed changes in intracellular localization. Our results suggest a possible link between PPARβ function and ethanol‐induced abnormal myelination in oligodendrocytes.