bluuebunny/crossref_metadata_2025 huggingface dataset:
Mast Cells Contain Large Quantities of Secretagogue‐Sensitive N‐Acetylaspartate
Authors: Burlina Alessandro P. , Ferrari Vanni , Facci Laura , Skaper Stephen D. , Burlina Alberto B.
Published: July 2010

Abstract: Mast cells play a central role in both immediate allergic reactions and inflammation. A functional nerve‐mast cell interaction has been proposed, given the morphological association between mast cells and neuropeptide‐containing peripheral nerves. We now show that purified rat peritoneal mast cells contain large quantities of N‐acetylaspartate (NAA; 747.50 nmol/mg of protein). Mast cell levels of NAA were rapidly reduced, by 64.0 and 86.4%, following treatment with compound 48/80 and mastoparan, respectively. These secretagogues strongly decreased mast cell histamine content over the same time period, suggesting also that NAA is stored in secretory granules. The data are the first to show that NAA is present in an immune effector cell type. Because NAA may be involved in myelin synthesis and glutamyl peptide metabolism, NAA released from mast cells following nervous or other stimuli could participate in neuroimmune interactions. Mast cells in multiple sclerosis plaques may contribute to the reported elevations in brain NAA in this disease.
Alternative Splicing of the Human Serotonin Transporter Gene
Authors: Bradley Christopher C. , Blakely Randy D.
Published: July 2010

Abstract: To explore the structural basis for regulation of human serotonin transporter (hSERT) gene expression, we used primer extension and 5′ rapid amplification of cDNA ends (5′RACE) techniques to estimate levels of and identify 5′‐noncoding elements of hSERT mRNAs and genomic cloning to place these elements within the overall map of the hSERT gene. Primer extension on JAR cell mRNA suggested the presence of significant hSERT mRNA sequence upstream of the 5′ end of our cloned hSERT cDNA. Using 5′RACE and reverse transcription‐PCR (RT‐PCR) methodologies, we cloned these sequences from brain and placenta and found this material to be composed of alternatively spliced exons using a previously reported noncoding exon (1A) and a novel 97‐bp noncoding exon (1B). RT‐PCR of JAR cell mRNA blotted with exon‐specific oligonucleotides revealed both exons 1A and 1B to be regulated in a cholera toxin‐dependent manner. To clarify the structure of the hSERT gene including exon 1B, we isolated and characterized genomic hSERT clones from Lambda Fix II and P1 artificial chromosome libraries. In agreement with previous findings, a single hSERT gene was identified that accounts for hybridizing bands on genomic Southern blots and was found to utilize 13 exons to encode the transporter's coding sequences along with the two noncoding 5′ exons. Exon 1B was identified ∼14 kb downstream of exon 1A in the hSERT gene and 737 bp upstream of exon 2, where the initiation site for translation is located. Exon 1B is surrounded by several elements potentially suitable for regulating serotonin transporter gene expression in vivo, including consensus sites for transcription factors AP‐1, AP‐2, CREB/ATF, and NF‐κB. These data reveal additional complexity in hSERT gene structure and expression that may be relevant to regulated and compromised transporter expression in vivo.
Involvement of a Caspase‐3‐Like Cysteine Protease in 1‐Methyl‐4‐Phenylpyridinium‐Mediated Apoptosis of Cultured Cerebellar Granule Neurons
Authors: Du Yangshen , Dodel Richard C. , Bales Kelly R. , Jemmerson Ronald , Hamilton‐Byrd Elizabeth , Paul Steven M.
Published: July 2010

Abstract: Exposure of various neuronal cells or cell lines to high concentrations of 1‐methyl‐4‐phenylpyridinium (MPP+), the active metabolite of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), results in cell death. Recently, it has been reported that low concentrations of MPP+ induce apoptosis in susceptible neurons. We have further characterized MPP+‐mediated toxicity of cultured cerebellar granule neurons (CGNs) and found that exposure of CGNs to relatively low concentrations of MPP+ results in apoptosis, whereas higher concentrations result in necrosis. Cotreatment of CGNs with MPP+ and the tetrapeptide inhibitor of caspase‐3‐like proteases, acetyl‐DEVD‐CHO, markedly attenuates apoptotic but not necrotic death of these neurons. The more specific inhibitor of caspase‐1‐like proteases, acetyl‐YVAD‐CHO, however, was ineffective against MPP+ neurotoxicity. Moreover, cytoplasmic extracts prepared from MPP+‐treated CGNs contain markedly increased protease activity that cleaves the caspase‐3 substrate acetyl‐DEVD‐p‐nitroaniline. Finally, the cytoplasmic concentration of the apoptogenic protein cytochrome c was increased in a time‐dependent fashion in MPP+‐treated CGNs before the onset of apoptosis. Our data confirm that the neurotoxicity of MPP+ is due to both necrosis and apoptosis and suggest that the latter is mediated by activation of a caspase‐3‐like protease.
Neurotrophic Effects of l‐DOPA in Postnatal Midbrain Dopamine Neuron/Cortical Astrocyte Cocultures
Authors: Mena Maria Angeles , Davila Viviana , Sulzer David
Published: July 2010

Abstract: l‐DOPA is toxic to catecholamine neurons in culture, but the toxicity is reduced by exposure to astrocytes. We tested the effect of l‐DOPA on dopamine neurons using postnatal ventral midbrain neuron/cortical astrocyte cocultures in serum‐free, glia‐conditioned medium. l‐DOPA (50 µM) protected against dopamine neuronal cell death and increased the number and branching of dopamine processes. In contrast to embryonically derived glia‐free cultures, where l‐DOPA is toxic, postnatal midbrain cultures did not show toxicity at 200 µMl‐DOPA. The stereoisomer d‐DOPA (50–400 µM) was not neurotrophic. The aromatic amino acid decarboxylase inhibitor carbidopa (25 µM) did not block the neurotrophic effect. These data suggest that the neurotrophic effect of l‐DOPA is stereospecific but independent of the production of dopamine. However, l‐DOPA increased the level of glutathione. Inhibition of glutathione peroxidase by l‐buthionine sulfoximine (3 µM for 24 h) blocked the neurotrophic action of L‐DOPA. N‐Acetyl‐l‐cysteine (250 µM for 48 h), which promotes glutathione synthesis, had a neurotrophic effect similar to that of l‐DOPA. These data suggest that the neurotrophic effect of l‐DOPA may be mediated, at least in part, by elevation of glutathione content.
The Serotonin 5‐HT2C Receptor Is a Prominent Serotonin Receptor in Basal Ganglia: Evidence from Functional Studies on Serotonin‐Mediated Phosphoinositide Hydrolysis
Authors: Wolf William A. , Schutz Laurence J.
Published: July 2010

Abstract: Serotonin (5‐hydroxytryptamine; 5‐HT) 5‐HT2A and 5‐HT2C receptors belong to the class of phosphoinositide‐specific phospholipase C (PLC)‐linked receptors. Conditions were established for measuring 5‐HT2A‐linked and 5‐HT2C‐linked PLC activity in membranes prepared from previously frozen rat frontal cortex and caudate. In the presence of Ca2+ (300 nM) and GTPγS (1 µM), 5‐HT increased PLC activity in caudate membranes. Pharmacological analysis using the selective 5‐HT2A antagonist, spiperone, and the nonselective 5‐HT2A/2C antagonist, mianserin, demonstrated that over half of the 5‐HT‐stimulated PLC activity was due to stimulation of 5‐HT2C receptors as opposed to 5‐HT2A receptors. Radioligand binding assays with [3H]RP 62203 and [3H]‐mesulergine were used to quantify 5‐HT2A and 5‐HT2C sites, respectively, in caudate. From these data, the Bmax for caudate 5‐HT2A sites and 5‐HT2C sites was 165.4 ± 9.7 fmol/mg of protein and 49.7 ± 3.3 fmol/mg of protein, respectively. In contrast to that in caudate, PLC activity in frontal cortex was stimulated by 5‐HT in a manner that was inhibited by the 5‐HT2A‐selective antagonists, spiperone and ketanserin. Taken together, the results indicate that 5‐HT2A‐ and 5‐HT2C‐linked PLC activity can be discerned in brain regions possessing both receptor subtypes using membranes prepared from previously frozen tissue. More importantly, significant 5‐HT2C‐mediated phosphoinositide hydrolysis was observed in caudate, despite the relatively low density of 5‐HT2C sites. The significance of these observations with respect to the physiological function of 5‐HT2C receptors is discussed.
Metabolism of Catecholamines by Catechol‐O‐Methyltransferase in Cells Expressing Recombinant Catecholamine Transporters
Authors: Eshleman Amy J. , Stewart Emilie , Evenson Anna K. , Mason John N. , Blakely Randy D. , Janowsky Aaron , Neve Kim A.
Published: July 2010

Abstract: To determine if catechol‐O‐methyltransferase (COMT) metabolizes catecholamines within cell lines used for heterologous expression of plasmalemmal transporters and alters the measured characteristics of 3H‐substrate transport, the uptake of monoamine transporter substrates was assessed in three cell lines (C6 glioma, L‐M fibroblast, and HEK293 cells) that had been transfected with the recombinant human transporters. Uptake and cellular retention of 3H‐catecholamines was increased by up to fourfold by two COMT inhibitors, tropolone and Ro 41‐0960, with potencies similar to those for inhibition of COMT activity, whereas the uptake of two transporter substrates that are not substrates for COMT, [3H]serotonin and [3H]MPP+, was unaffected. Direct measurement of monoamine substrates by HPLC confirmed that tropolone (1 mM) increased the retention of the catecholamines dopamine and norepinephrine, but not the retention of serotonin in HEK293 cells. Saturation analysis of the uptake of [3H]dopamine by C6 cells expressing the dopamine transporter demonstrated that tropolone (1 mM) decreased the apparent Km of transport from 0.61 µM to 0.34 µM without significantly altering the maximal velocity of transport. These data suggest that endogenous COMT activity in mammalian cells may alter neurotransmitter deposition and thus the apparent kinetic characteristics of transport.
Endogenous Glutamate Increases Extracellular Concentrations of Dopamine, GABA, and Taurine Through NMDA and AMPA/Kainate Receptors in Striatum of the Freely Moving Rat: A Microdialysis Study
Authors: Segovia Gregorio , Del Arco Alberto , Mora Francisco
Published: July 2010

Abstract: Interactions between glutamate (Glu), dopamine (DA), GABA, and taurine (Tau) were investigated in striatum of the freely moving rat by using microdialysis. Intrastriatal infusions of the selective Glu uptake inhibitor l‐trans‐pyrrolidine‐3,4‐dicarboxylic acid (PDC) were used to increase the endogenous extracellular [Glu]. Correlations between extracellular [Glu] and extracellular [DA], [GABA], and [Tau], and the effects of a selective blockade of ionotropic Glu receptors, were studied. PDC (1, 2, and 4 mM) produced a dose‐related increase in extracellular [Glu]. At the highest dose of PDC, [Glu] increased from 1.55 ± 0.35 to 6.11 ± 0.88 µM. PDC also increased extracellular [DA], [GABA], and [Tau]. The increasing [Glu] was correlated significantly with increasing [DA], [GABA], and [Tau]. PDC also decreased extracellular concentrations of DA metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and 4‐hydroxy‐3‐methoxyphenylacetic acid (HVA). Perfusion with the NMDA‐receptor antagonist 3‐[(R)‐2‐carboxypiperazin‐4‐yl]‐propyl‐1‐phosphonic acid (1 mM) or the AMPA/kainate‐receptor antagonist 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX) (1 mM) attenuated the increases produced by PDC (4 mM) on [DA], [GABA], and [Tau], and decreases in [DOPAC] and [HVA]. DNQX also attenuated the increases in [Glu] induced by PDC. These data show that endogenous Glu plays a role in modulating the extracellular concentrations of DA, GABA, and Tau in striatum of the freely moving rat.
A Temporary Local Energy Pool Coupled to Neuronal Activity: Fluctuations of Extracellular Lactate Levels in Rat Brain Monitored with Rapid‐Response Enzyme‐Based Sensor
Authors: Hu Yibai , Wilson George S.
Published: July 2010

Abstract: A successfully developed enzyme‐based lactate microsensor with rapid response time allows the direct and continuous in vivo measurement of lactic acid concentration with high temporal resolution in brain extracellular fluid. The fluctuations coupled to neuronal activity in extracellular lactate concentration were explored in the dentate gyrus of the hippocampus of the rat brain after electrical stimulation of the perforant pathway. Extracellular glucose and oxygen levels were also detected simultaneously by coimplantation of a fast‐response glucose sensor and an oxygen electrode, to provide novel information of trafficking of energy substances in real time related to local neuronal activity. The results first give a comprehensive picture of complementary energy supply and use of lactate and glucose in the intact brain tissue. In response to acute neuronal activation, the brain tissue shifts immediately to significant energy supply by lactate. A local temporary fuel “reservoir” is established behind the blood‐brain barrier, evidenced by increased extracellular lactate concentration. The pool can be depleted rapidly, up to 28% in 10–12 s, by massive, acute neuronal use after stimulation and can be replenished in ∼20 s. Glutamate‐stimulated astrocytic glycolysis and the increase of regional blood flow may regulate the lactate concentration of the pool in different time scales to maintain local energy homeostasis.
Functional Expression of a GLT‐1 Type Na+‐Dependent Glutamate Transporter in Rat Pinealocytes
Authors: Yamada Hiroshi , Yatsushiro Shouki , Yamamoto Akitsugu , Hayashi Mitsuko , Nishi Tsuyoshi , Futai Masamitsu , Yamaguchi Akihito , Moriyama Yoshinori
Published: July 2010

Abstract: Pinealocytes, the neuroendocrine cells that produce melatonin, accumulate glutamate in microvesicles through a specific vesicular transporter energetically coupled with vacuolar‐type proton ATPase. The glutamate is secreted into the extracellular space through microvesicle‐mediated exocytosis and then stimulates neighboring pinealocytes, resulting in inhibition of norepinephrine‐dependent melatonin synthesis. In this study, we identified and characterized the plasma membrane‐type glutamate transporter in rat pinealocytes. The [3H]‐glutamate uptake by cultured pinealocytes was driven by extracellular Na+, saturated with the [3H]glutamate concentration used, and significantly inhibited by l‐glutamate, l‐aspartate, β‐threo‐hydroxyaspartate, pyrrolidine dicarboxylate, and l‐cysteine sulfinate, substrates or inhibitors of the plasma membrane glutamate transporter. Consistently, the clearance of extracellular glutamate, as measured by HPLC, was also dependent on Na+ and inhibited by β‐threo‐hydroxyaspartate and l‐cysteine sulfinate. Immunological studies with site‐specific antibodies against three isoforms of the Na+‐dependent glutamate transporter (GLT‐1, GLAST, and EAAC1) revealed the expression of only the GLT‐1 type transporter in pineal glands. Expression of the GLT‐1 type transporter in pineal glands was further demonstrated by means of reverse transcription‐polymerase chain reaction with specific DNA probes. Immunohistochemical analysis indicated that the immunological counterpart(s) of the GLT‐1 is localized in pinealocytes. These results suggested that the GLT‐1‐type Na+‐dependent transporter is expressed and functions as a reuptake system for glutamate in rat pinealocytes. The physiological role of the transporter in the termination of the glutamate signal in the pineal gland is discussed.
Lipid Free Radical Generation and Brain Cell Membrane Alteration Following Nitric Oxide Synthase Inhibition During Cerebral Hypoxia in the Newborn Piglet
Authors: Numagami Yoshihiro , Zubrow Alan B. , Mishra Om P. , Delivoria‐Papadopoulos Maria
Published: July 2010

Abstract: Nitric oxide (NO) is reported to cause neuronal damage through various mechanisms. The present study tests the hypothesis that NO synthase inhibition by Nω‐nitro‐l‐arginine (NNLA) will result in decreased oxygen‐derived free radical production leading to the preservation of cell membrane structure and function during cerebral hypoxia. Ten newborn piglets were pretreated with NNLA (40 mg/kg); five were subjected to hypoxia, whereas the other five were maintained with normoxia. An additional 10 piglets without NNLA treatment underwent the same conditions. Hypoxia was induced with a lowered FiO2 and documented biochemically by decreased cerebral ATP and phosphocreatine levels. Free radicals were detected by using electron spin resonance spectroscopy with a spin trapping technique. Results demonstrated that free radicals, corresponding to alkoxyl radicals, were induced by hypoxia but were inhibited by pretreatment with NNLA before inducing hypoxia. NNLA also inhibited hypoxia‐induced generation of conjugated dienes, products of lipid peroxidation. Na+,K+‐ATPase activity, an index of cellular membrane function, decreased following hypoxia but was preserved by pretreatment with NNLA. These data demonstrate that during hypoxia NO generates free radicals via peroxynitrite production, presumably causing lipid peroxidation and membrane dysfunction. These results suggest that NO is a potentially limiting factor in the peroxynitrite‐mediated lipid peroxidation resulting in membrane injury.
The Potassium Channel Opener (−)‐Cromakalim Prevents Glutamate‐Induced Cell Death in Hippocampal Neurons
Authors: Lauritzen Inger , De Weille Jan R. , Lazdunski Michel
Published: July 2010

Abstract: (−)‐Cromakalim, a typical K+‐channel opener, prevents neuronal death induced by either glucose and oxygen privation or by high (100 µM) extracellular glutamate in primary cultures of hippocampus. (−)‐Cromakalim has no effect on the earliest events associated with exposure to glutamate. It does not prevent the rapid rise of intracellular Ca2+, the initial swelling of neurons, or the induction of c‐fos mRNA transcription. (−)‐Cromakalim inhibits all delayed effects associated with the excitotoxic effect of glutamate: (a) (−)‐cromakalim inhibits the late and major phase of intracellular Ca2+ increase occurring up to hours after glutamate application; and (b) although (−)‐cromakalim cannot prevent the initial cellular swelling induced by glutamate, cells that have been pretreated with (−)‐cromakalim return to their original size in a few hours, whereas non‐(−)‐cromakalim‐treated cells remain swollen for more prolonged periods. Many neurons surviving the initial necrotic phase of glutamate‐induced cell death undergo progressive DNA cleavage leading to apoptosis. This apoptotic process is prevented completely by (−)‐cromakalim. Glibenclamide, a potent blocker of the ATP‐sensitive K+ channel, abolishes all the beneficial effects of (−)‐cromakalim. These findings strongly suggest that (−)‐cromakalim has postsynaptic effects that are closely related to the regulation of Ca2+ homeostasis and cell volume.
β‐Amyloid Neurotoxicity In Vitro: Evidence of Oxidative Stress but Not Protection by Antioxidants
Authors: Pike Christian J. , Ramezan‐Arab Nima , Cotman Carl W.
Published: July 2010

Abstract: Recent data from several groups suggest that the primary mechanism of β‐amyloid neurotoxicity may be mediated by reactive oxygen species. To evaluate this hypothesis, we first compared the efficacy of antioxidant agents in preventing toxicity caused by oxidative insults (iron, hydrogen peroxide, and tert‐butyl hydroperoxide) and β‐amyloid peptides in cultured rat hippocampal neurons. Tested antioxidants (propyl gallate, Trolox, probucol, and promethazine) generally provided significant protection against oxidative insults but not β‐amyloid peptides. Next, we examined whether β‐amyloid causes oxidative stress, by comparing levels of lipid peroxidation after exposure to either iron or β‐amyloid. In a cell‐free system, iron but not β‐amyloid generated lipid peroxidation. In culture, both insults caused rapid increases in lipid peroxidation, with iron inducing higher levels at later time points. Pretreatment with the antioxidant probucol significantly reduced lipid peroxidation caused by both insults but only attenuated iron toxicity, suggesting that lipid peroxidation does not contribute directly to cell death induced by β‐amyloid. Finally, we observed that increasing basal levels of oxidative stress by pretreating cultures with subtoxic doses of iron significantly increased neuronal vulnerability to β‐amyloid. The ability of β‐amyloid to induce oxidative stress and the demonstration that oxidative stress potentiates β‐amyloid toxicity support the clinical use of antioxidants for AD. However, these data do not support the theory that the primary mechanism of β‐amyloid toxicity involves oxidative pathways, indicating a continued need to identify additional cellular responses to β‐amyloid that underlie its neurodegenerative actions.
Characterization of the Palmitoylation Domain of SNAP‐25
Authors: Lane Stacie R. , Liu Yuechueng
Published: July 2010

Abstract: SNAP‐25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP‐25 is unclear. In this report, we show that the palmitate of SNAP‐25 is rapidly turned over in PC12 cells, with a half‐life of ∼3 h, and the half‐life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild‐type protein, respectively. Additional mutations of either Cys85,88 or Cys90,92 nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP‐25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four Cys residues are involved in palmitoylation and that membrane association of SNAP‐25 may be regulated through dynamic palmitoylation.
Decreased Zinc Affinity of Amyotrophic Lateral Sclerosis‐Associated Superoxide Dismutase Mutants Leads to Enhanced Catalysis of Tyrosine Nitration by Peroxynitrite
Authors: Crow John P. , Sampson Jacinda B. , Zhuang Yingxin , Thompson John A. , Beckman Joseph S.
Published: July 2010

Abstract: Mutations to Cu/Zn superoxide dismutase (SOD) linked to familial amyotrophic lateral sclerosis (ALS) enhance an unknown toxic reaction that leads to the selective degeneration of motor neurons. However, the question of how >50 different missense mutations produce a common toxic phenotype remains perplexing. We found that the zinc affinity of four ALS‐associated SOD mutants was decreased up to 30‐fold compared to wild‐type SOD but that both mutants and wild‐type SOD retained copper with similar affinity. Neurofilament‐L (NF‐L), one of the most abundant proteins in motor neurons, bound multiple zinc atoms with sufficient affinity to potentially remove zinc from both wild‐type and mutant SOD while having a lower affinity for copper. The loss of zinc from wild‐type SOD approximately doubled its efficiency for catalyzing peroxynitrite‐mediated tyrosine nitration, suggesting that one gained function by SOD in ALS may be an indirect consequence of zinc loss. Nitration of protein‐bound tyrosines is a permanent modification that can adversely affect protein function. Thus, the toxicity of ALS‐associated SOD mutants may be related to enhanced catalysis of protein nitration subsequent to zinc loss. By acting as a high‐capacity zinc sink, NF‐L could foster the formation of zinc‐deficient SOD within motor neurons.
Superoxide Dismutase Catalyzes Nitration of Tyrosines by Peroxynitrite in the Rod and Head Domains of Neurofilament‐L
Authors: Crow John P. , Ye Yao Zu , Strong Michael , Kirk Marion , Barnes Stephen , Beckman Joseph S.
Published: July 2010

Abstract: Superoxide dismutase (SOD) catalyzes the nitration of specific tyrosine residues in proteins by peroxynitrite (ONOO−), which may be the damaging gain‐of‐function resulting from mutations to SOD associated with familial amyotrophic lateral sclerosis (ALS). We found that disassembled neurofilament‐L (light subunit) was more susceptible to tyrosine nitration catalyzed by SOD in vitro. Neurofilament‐L was selectively nitrated compared with the majority of other proteins present in brain homogenates. Assembled neurofilament‐L was more resistant to nitration, suggesting that the susceptible tyrosine residues were protected by intersubunit contacts in assembled neurofilaments. Electrospray mass spectrometry of trypsin‐digested neurofilament‐L showed that tyrosine 17 in the head region and tyrosines 138, 177, and 265 in α‐helical coil regions of the rod domain of neurofilament‐L were particularly susceptible to SOD‐catalyzed nitration. Nitrated neurofilament‐L inhibited the assembly of unmodified neurofilament subunits, suggesting that the affected tyrosines are located in regions important for intersubunit contacts. Neurofilaments are major structural proteins expressed in motor neurons and known to be important for their survival in vivo. We suggest that SOD‐catalyzed nitration of neurofilament‐L may have a significant role in the pathogenesis of ALS.
Low Thiamine Diphosphate Levels in Brains of Patients with Frontal Lobe Degeneration of the Non‐Alzheimer's Type
Authors: Bettendorff L. , Mastrogiacomo F. , Wins P. , Kish S. J. , Grisar T. , Ball M. J.
Published: July 2010

Abstract: We compared the thiamine and thiamine phosphate contents in the frontal, temporal, parietal, and occipital cortex of six patients with frontal lobe degeneration of the non‐Alzheimer's type (FNAD) or frontotemporal dementia with five age‐, postmortem delay‐, and agonal status‐matched control subjects. Our results reveal a 40–50% decrease in thiamine diphosphate (TDP) in the cortex of FNAD patients, whereas thiamine monophosphate was increased 49–119%. TDP synthesizing and hydrolyzing enzymes were unaffected. The activity of citrate synthase, a mitochondrial marker enzyme, was decreased in the frontal cortex of patients with FNAD, but no correlation with TDP content was found. These results suggest that decreased contents of TDP, which is essentially mitochondrial, is a specific feature of FNAD. As TDP is an essential cofactor for oxidative metabolism and neurotransmitter synthesis, and because low thiamine status (compared with other species) is a constant feature in humans, a nearly 50% decrease in cortical TDP content may contribute significantly to the clinical symptoms observed in FNAD. This study also provides a basis for a trial of thiamine, to improve the cognitive status of the patients.
Amidation of β‐Amyloid Peptide Strongly Reduced the Amyloidogenic Activity Without Alteration of the Neurotoxicity
Authors: Forloni Gianluigi , Lucca Elisa , Angeretti Nadia , Della Torre Paola , Salmona Mario
Published: July 2010

Abstract: β‐Amyloid accumulates in cerebral deposits in Alzheimer's disease, so to test the correlation between the neurotoxic and fibrillogenic capacity of β‐amyloid, we synthesized a peptide homologous to fragment 25–35 of β‐amyloid (β25–35) and amidated at the C‐terminus (β25–35‐NH2). As the amidation strongly reduced the amyloidogenic capacity of β25–35, we compared its neurotoxic activity in the amidated (β25–35‐NH2) and nonamidated forms. The viability of primary cultures from fetal rat hippocampus was reduced in a dose‐related manner (10–100 µM) similarly by β25–35 and β25–35‐NH2, whereas a scrambled peptide, amidated or nonamidated, did not alter the neuronal viability. The neurotoxic activity of β25–35‐NH2 is mediated by apoptosis as demonstrated by morphological and biochemical investigations. Electron microscopy examination of culture media with β25–35 or β25–35‐NH2 incubated with neuronal cells for 7 days confirmed the high level of fibrillogenic activity of β25–35 and the almost total absence of fibrils in the solution with β25–35‐NH2. Furthermore, staining with thioflavine S was used to identify amyloid fibrils, and only the cultures exposed to β25–35 exhibited intense staining associated with neuronal membranes. These data indicate that the neurotoxic activity of the β‐amyloid fragment is independent of the aggregated state of the peptide.
Role of Aromatic l‐Amino Acid Decarboxylase for Dopamine Replacement by Genetically Modified Fibroblasts in a Rat Model of Parkinson's Disease
Authors: Wachtel Stephen R. , Bencsics Craig , Kang Un Jung
Published: July 2010

Abstract: Investigations of gene therapy for Parkinson's disease have focused primarily on strategies that replace tyrosine hydroxylase. In the present study, the role of aromatic l‐amino acid decarboxylase in gene therapy with tyrosine hydroxylase was examined by adding the gene for aromatic l‐amino acid decarboxylase to our paradigm using primary fibroblasts transduced with both tyrosine hydroxylase and GTP cyclohydrolase I. We compared catecholamine synthesis in vitro in cultures of cells with tyrosine hydroxylase and aromatic l‐amino acid decarboxylase together versus cocultures of cells containing these enzymes separately. l‐DOPA and dopamine levels were higher in the cocultures that separated the enzymes. To determine the role of aromatic l‐amino acid decarboxylase in vivo, cells containing tyrosine hydroxylase and GTP cyclohydrolase I were grafted alone or in combination with cells containing aromatic l‐amino acid decarboxylase into the 6‐hydroxydopamine‐denervated rat striatum. Grafts containing aromatic l‐amino acid decarboxylase produced less l‐DOPA and dopamine as monitored by microdialysis. These findings indicate that not only is there sufficient aromatic l‐amino acid decarboxylase near striatal grafts producing l‐DOPA, but also the close proximity of the enzyme to tyrosine hydroxylase is detrimental for optimal dopamine production. This is most likely due to feedback inhibition of tyrosine hydroxylase by dopamine.
A Conformation‐ and Phosphorylation‐Dependent Antibody Recognizing the Paired Helical Filaments of Alzheimer's Disease
Authors: Jicha Gregory A. , Lane Eric , Vincent Inez , Otvos Laszlo , Hoffmann Ralf , Davies Peter
Published: July 2010

Abstract: Hyperphosphorylated tau (PHF‐tau) is the major constituent of paired helical filaments (PHFs) from Alzheimer's disease (AD) brains. This conclusion has been based largely on the creation and characterization of monoclonal antibodies raised against PHFs, which can be classified in three categories: (a) those recognizing unmodified primary sequences of tau, (b) those recognizing phosphorylation‐dependent epitopes on tau, and (c) those recognizing conformation‐dependent epitopes on tau. Recent studies have suggested that the antibodies recognizing primary sequence and phosphorylation‐dependent epitopes on tau are unable to distinguish between normal adult biopsy tau and PHF‐tau. We now present evidence for a new fourth class of monoclonal antibodies recognizing conformation‐dependent phosphoepitopes on tau, typified by TG‐3, a monoclonal antibody raised to PHFs from AD brain homogenates. Studies using a series of deletional tau mutants, site‐directed tau mutants, and synthetic peptides enable the precise epitope mapping of TG‐3. Additional studies demonstrate that TG‐3 reacts with neonatal mouse tau and PHF‐tau but does not recognize adult mouse tau or tau derived from normal human autopsy or biopsy tissue. Further investigation reveals that TG‐3 recognizes a unique conformation of tau found almost exclusively in PHFs from AD brains.
Isoform‐Specific Up‐Regulation by Ouabain of Na+,K+‐ATPase in Cultured Rat Astrocytes
Authors: Hosoi Rie , Matsuda Toshio , Asano Shoichi , Nakamura Hiroaki , Hashimoto Hitoshi , Takuma Kazuhiro , Baba Akemichi
Published: July 2010

Abstract: There are two α‐subunit isoforms (α1 and α2) and two β‐subunit isoforms (β1 and β2) of Na+,K+‐ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5–1.0 mM) increased the levels of α1 and β1 mRNAs, whereas it decreased those of α2 and β2 mRNAs in cultured rat astrocytes. The increases in α1 and β1 mRNAs were observed at 6–48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased α1 and β1, but not α2 and β2, proteins, and that the isoforms in control and ouabain‐treated cultures were of glial origin. Low extracellular K+ and monensin (20 µM) mimicked the effect of ouabain on α1 mRNA. The ouabain‐induced increase in α1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 µM), the intracellular Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid tetraacetoxymethyl ester (30 µM), and the calcineurin inhibitor FK506 (1 nM). These findings indicate that chronic inhibition of Na+,K+‐ATPase up‐regulates the α1 and β1, but not α2 and β2, isoforms in astrocytes, suggesting a functional coupling of α1β1 complex. They also suggest that intracellular Na+, Ca2+, and calcineurin may be involved in ouabain‐induced up‐regulation of the enzyme in astrocytes.
Increased Hippocampal 5‐Lipoxygenase mRNA Content in Melatonin‐Deficient, Pinealectomized Rats
Authors: Uz Tolga , Longone Patrizia , Manev Hari
Published: July 2010

Abstract: Melatonin is neuroprotective because of its antioxidative action, but it also can modify neuronal vulnerability by altering gene expression. 5‐Lipoxygenase (5‐LO) gene expression is suppressed by the binding of melatonin to its high‐affinity nuclear receptors. Recently, we reported that in rats the melatonin deficiency elicited by pinealectomy increases hippocampal susceptibility to excitotoxic injury. Here we have hypothesized that pinealectomy may increase hippocampal vulnerability by eliminating the tonic inhibitory action of melatonin on 5‐LO gene expression. Sham‐pinealectomized controls and pinealectomized rats were killed 15 days after surgery. Their hippocampi were dissected, and total RNA was extracted and processed for quantitative reverse transcription‐polymerase chain reaction assay of 5‐LO and cyclophilin mRNAs. Mutated primers were used as internal standards to assay attomole quantities of these two specific mRNAs per microgram of total RNA; the ratio 5‐LO/cycophilin was used to compare samples from control and pinealectomized rats. Pinealectomy increased hippocampal 5‐LO mRNA content by about threefold. These results support our hypothesis that melatonin deficiency may abate the tonic inhibition of 5‐LO mRNA expression and thereby up‐regulate 5‐LO gene expression, which in turn would increase the brain's synthesis rate of potentially harmful eicosanoids, leukotrienes.
Molecular Characterization and In Situ Localization of a Mouse Retinal Taurine Transporter
Authors: Vinnakota Shyamala , Qian Xiaojun , Egal Hussein , Sarthy Vijay , Sarkar Hemanta K.
Published: July 2010

Abstract: Various ocular tissues have a higher concentration of taurine than plasma. This taurine concentration gradient across the cell membrane is maintained by a high‐affinity taurine transporter. To understand the physiological role of the taurine transporter in the retina, we cloned a taurine transporter encoding cDNA from a mouse retinal library, determined its biochemical and pharmacological properties, and identified the specific cellular sites expressing the taurine transporter mRNA. The deduced protein sequence of the mouse retinal taurine transporter (mTAUT) revealed >93% sequence identity to the canine kidney, rat brain, mouse brain, and human placental taurine transporters. Our data suggest that the mTAUT and the mouse brain taurine transporter may be variants of one another. The mTAUT synthetic RNA induced Na+‐ and Cl−‐dependent [3H]taurine transport activity in Xenopus laevis oocytes that saturated with an average Km of 13.2 µM for taurine. Unlike the previous studies, we determined the rate of taurine uptake as the external concentration of Cl− was varied, a single saturation process with an average apparent equilibrium constant (KCl−) of 17.7 mM. In contrast, the rate of taurine uptake showed a sigmoidal dependence when the external concentration of Na+ was varied (apparent equilibrium constant, KNa+∼54.8 mM). Analyses of the Na+‐ and Cl−‐concentration dependence data suggest that at least two Na+ and one Cl− are required to transport one taurine molecule via the taurine transporter. Varying the pH of the transport buffer also affected the rate of taurine uptake; the rate showed a minimum between pH 6.0 and 6.5 and a maximum between pH 7.5 and 8.0. The taurine transport was inhibited by various inhibitors tested with the following order of potency: hypotaurine > β‐alanine > l‐diaminopropionic acid > guanidinoethane sulfonate > β‐guanidinopropionic acid > chloroquine > γ‐aminobutyric acid > 3‐amino‐1‐propanesulfonic acid (homotaurine). Furthermore, the mTAUT activity was not inhibited by the inactive phorbol ester 4α‐phorbol 12,13‐didecanoate but was inhibited significantly by the active phorbol ester phorbol 12‐myristate 13‐acetate, which was both concentration and time dependent. The cellular sites expressing the taurine transporter mRNA in the mouse eye, as determined by in situ hybridization technique, showed low levels of expression in many of the ocular tissues, specifically the retina and the retinal pigment epithelium. Unexpectedly, the highest expression levels of taurine transporter mRNA were found instead in the ciliary body of the mouse eye.
Cytotoxic Amyloid Peptides Inhibit Cellular 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide (MTT) Reduction by Enhancing MTT Formazan Exocytosis
Authors: Liu Yuanbin , Schubert David
Published: July 2010

Abstract: Amyloid β peptide (Aβ) neurotoxicity is believed to play a central role in the pathogenesis of Alzheimer's disease. An early indicator of Aβ toxicity is the inhibition of cellular 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction to MTT formazan, a widely used assay for measuring cell viability. In this report we show that Aβ and other cytotoxic amyloid peptides such as human amylin dramatically enhance MTT formazan exocytosis, resulting in the inhibition of cellular MTT reduction. Only the amyloid peptides that are known to be cytotoxic enhanced MTT formazan exocytosis. Basal MTT formazan exocytosis and amyloid peptide‐enhanced MTT formazan exocytosis are blocked by several drugs with diverse known effects. These and other data suggest that MTT formazan exocytosis is a multistep process and that cytotoxic amyloid peptides enhance MTT formazan exocytosis through an intracellular signal transduction pathway.
N‐Acetylaspartylglutamate Stimulates Metabotropic Glutamate Receptor 3 to Regulate Expression of the GABAAα6 Subunit in Cerebellar Granule Cells
Authors: Ghose Subroto , Wroblewska Barbara , Corsi Lorenzo , Grayson Dennis R. , De Blas Angel L. , Vicini Stefano , Neale Joseph H.
Published: July 2010

Abstract: We have shown that the vertebrate neuropeptide N‐acetylaspartylglutamate (NAAG) meets the criteria for a neurotransmitter, including function as a selective metabotropic glutamate receptor (mGluR) 3 agonist. Short‐term treatment of cerebellar granule cells with NAAG (30 µM) results in the transient increase in content of GABAAα6 subunit mRNA. Using quantitative PCR, this increase was determined to be up to 170% of control values. Similar effects are seen following treatment with trans‐1‐aminocyclopentane‐1,3‐dicarboxylate and glutamate and are blocked by the mGluR antagonists (2S,3S,4S)‐2‐methyl‐2‐(carboxycyclopropyl)glycine and (2S)‐α‐ethylglutamic acid. The effect is pertussis toxin‐sensitive. The increase in α6 subunit mRNA level can be simulated by activation of other receptors negatively linked to adenylate cyclase activity, such as adenosine A1, α2‐adrenergic, muscarinic, and GABAB receptors. Forskolin stimulation of cyclic AMP (cAMP) levels abolished the effect of NAAG. The change in α6 levels induced by 30 µM NAAG can be inhibited in a dose‐dependent manner by simultaneous application of increasing doses of the β‐adrenergic receptor agonist isoproterenol. The increase in α6 mRNA content is followed by a fourfold increase in α6 protein level 6 h posttreatment. Under voltage‐clamped conditions, NAAG‐treated granule cells demonstrate an increase in the furosemide‐induced inhibition of GABA‐gated currents in a concentration‐dependent manner, indicating an increase in functional α6‐containing GABAA receptors. These data support the hypothesis that NAAG, acting through mGluR3, regulates expression of the GABAAα6 subunit via a cAMP‐mediated pathway and that cAMP‐coupled receptors for other neurotransmitters may similarly influence GABAA receptor subunit composition.
Effects of Acute and Chronic Ethanol Exposure on Heteromeric N‐Methyl‐d‐Aspartate Receptors Expressed in HEK 293 Cells
Authors: Blevins Tana , Mirshahi Tooraj , Chandler L. Judson , Woodward John J.
Published: July 2010

Abstract: Ion flux through native N‐methyl‐d‐aspartate (NMDA) receptors is inhibited by behaviorally relevant concentrations of ethanol (10–100 mM) in a variety of neuronal preparations. However, in animal tissues, it is often difficult to determine accurately which NMDA receptor subunits are responsible for the observed effect. In this study, human embryonic kidney 293 (HEK 293) cells normally devoid of NMDA receptors were transiently transfected with cDNA expression plasmids coding for specific rat NMDA receptor subunits. Brief application of an NMDA/glycine solution to cells markedly increased intracellular calcium in cells transfected with NR1/NR2A, NR1/NR2B, or NR1/NR2A/NR2B as measured by fura‐2 calcium imaging. This increase was both NMDA‐ and glycine‐dependent and was inhibited by competitive and noncompetitive NMDA antagonists, including 2‐amino‐5‐phosphopentanoic acid and MK‐801. The NR2B‐selective antagonist ifenprodil inhibited responses in cells transfected with NR1/NR2B or NR1/NR2A/NR2B, but not NR1/NR2A subunits. Increasing the transfection ratio of NR2B versus NR2A subunit in NR1/NR2A/NR2B‐transfected cells greatly increased their ifenprodil sensitivity. Acute exposure to ethanol (25–100 mM) inhibited the NMDA‐mediated increase in intracellular calcium in a dose‐dependent manner without affecting basal calcium concentrations. There were no statistically significant differences in ethanol's potency or maximal inhibition between any of the subunit combinations tested. HEK 293 cells transfected with NR1/NR2A/NR2B subunits showed an enhanced sensitivity to ifenprodil following a 24‐h exposure to concentrations of ethanol of 50 mM and greater. The enhanced ifenprodil sensitivity following ethanol exposure was not associated with changes in NR1, NR2A, or NR2B immunoreactivity. In contrast to results obtained in transfected HEK 293 cells, no effect of chronic ethanol was observed in oocytes expressing NR1/NR2A/NR2B subunits. These results demonstrate that recombinant NMDA receptors expressed in HEK 293 cells form functional receptors that, like native receptors, are sensitive to modulation by both acute and chronic ethanol treatment.
Evidence Against an Acute Inhibitory Role of nSec‐1 (Munc‐18) in Late Steps of Regulated Exocytosis in Chromaffin and PC12 Cells
Authors: Graham Margaret E. , Sudlow Allan W. , Burgoyne Robert D.
Published: July 2010

Abstract: nSec‐1 (munc‐18) is a mammalian homologue of proteins implicated in constitutive exocytosis in yeast and neurotransmission in Caenorhabditis elegans and Drosophila. Mutant phenotypes in these species suggest that nSec‐1 is likely to be required for neurotransmission. Various other data have been interpreted as suggesting that nSec‐1 could also be a negative regulator of Ca2+‐dependent exocytosis. We have tested this possibility by introducing exogenous nSec‐1 into permeabilised chromaffin or PC12 cells and examining its effects on Ca2+‐induced and α‐soluble N‐ethylmaleimide‐sensitive fusion protein attachment protein‐stimulated exocytosis. No effects of exogenous nSec‐1 were observed in these assays. In addition, the effect of nSec‐1 overexpression in transiently transfected PC12 cells on reporter growth hormone (GH) secretion was examined. Overexpression of nSec‐1 resulted in a marked increase in GH production, reflected in an increase in both cell‐associated and medium GH levels. The relative amounts retained in the cells were unaffected by nSec‐1 overexpression, indicating that GH storage was unaffected and that the major effect was on its synthesis. In contrast, nSec‐1 overexpression did not affect the proportion of GH that was released following stimulation in intact or permeabilised cells. These results suggest either that nSec‐1 is already expressed at sufficient levels and remains so following permeabilisation or that nSec‐1 may not be an acute inhibitory regulator of Ca2+‐dependent exocytosis in chromaffin or PC12 cells.
Tyrosine Hydroxylase Phosphorylation in Digitonin‐Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser19 and Ser40 Phosphorylation
Authors: Gonçalves Carlos‐Alberto , Hall Amanda , Sim Alistair T. R. , Bunn Stephen J. , Marley Philip D. , Cheah Tat B. , Dunkley Peter R.
Published: July 2010

Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ‐32P]ATP, in the presence or absence of 10 µM Ca2+, 1 µM cyclic AMP, 1 µM phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca2+ increased the phosphorylation of Ser19 and Ser40. Cyclic AMP, and phorbol dibutyrate in the presence of Ca2+, increased the phosphorylation of only Ser40. Ser31 and Ser8 were not phosphorylated. The Ca2+‐stimulated phosphorylation of Ser19 was incompletely reduced by inhibitors of calcium/calmodulin‐stimulated protein kinase II (46% with KN93 and 68% with CaM‐PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca2+‐stimulated phosphorylation of Ser40 was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31‐8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser19 and Ser40 were dephosphorylated by PP2A.
High Serotonin and 5‐Hydroxyindoleacetic Acid Levels in Limbic Brain Regions in a Rat Model of Depression; Normalization by Chronic Antidepressant Treatment
Authors: Zangen Abraham , Overstreet David H. , Yadid Gal
Published: July 2010

Abstract: Although alterations in serotonin levels and neurotransmission are associated with depressive disorders and effective antidepressant therapy, the exact cause of these disorders and the mode of action of anti‐depressant drugs are poorly understood. In a genetic rat model of depression [Flinders sensitive line (FSL) rats], deviations from normal serotonin (5‐HT) levels and metabolism in specific brain regions were determined. The levels of 5‐HT and its metabolite, 5‐hydroxyindoleacetic acid (5‐HIAA), in tissue punches of various brain regions were quantitated simultaneously with an HPLC apparatus coupled to an electrochemical detector. In the nucleus accumbens, prefrontal cortex, hippocampus, and hypothalamus of FSL rats, the levels of 5‐HT and 5‐HIAA were three‐ to eightfold higher than in control Sprague‐Dawley rats. Significant differences in the levels of 5‐HT and 5‐HIAA in the striatum and raphe nucleus of the “depressed” and normal rats were not observed. After chronic treatment with the antidepressant desipramine (5 mg/kg/day for 18 days), the immobility score in a swim test, as a measure of a behavioral deficit, and 5‐HT levels of the FSL rats became normalized, but these parameters in the control rats did not change. The [5‐HIAA]/[5‐HT] ratio was lower in the nucleus accumbens and hypothalamus of the FSL than in the control rats, and increased after desipramine treatment only in the nucleus accumbens of the FSL rats. These results indicate that the behavioral deficits expressed in the FSL model for depression correlate with increased 5‐HT levels in specific limbic sites and suggest the FSL rats as a novel model for clarification of the molecular mechanism of clinically used antidepressant drugs.