bluuebunny/crossref_metadata_2025 huggingface dataset:
Ethanol Induces Apoptosis in Cerebellar Granule Neurons by Inhibiting Insulin‐Like Growth Factor 1 Signaling
Authors: Zhang Frank X. , Rubin Raphael , Rooney Thomas A.
Published: July 2010

Abstract: The ability of ethanol to interfere with insulin‐like growth factor 1 (IGF‐1)‐mediated cell survival was examined in primary cultured cerebellar granule neurons. Cells underwent apoptosis when switched from medium containing 25 mM K+ to one containing 5 mM K+. IGF‐1 protected granule neurons from apoptosis in medium containing 5 mM K+. Ethanol inhibited IGF‐1‐mediated neuronal survival but did not inhibit IGF‐1 receptor binding or the neurotrophic action of elevated K+, and failed to potentiate cell death in the presence of 5 mM K+. Inhibition of neuronal survival by ethanol was not reversed by increasing the concentration of IGF‐1. Significant inhibition by ethanol (15–20%) was observed at 1 mM and was half‐maximal at 45 mM. The inhibition of IGF‐1 protection by ethanol corresponded to a marked reduction in the phosphorylation of insulin receptor substrate 1, the binding of phosphatidylinositol 3‐kinase (PI 3‐kinase), and a block of IGF‐1‐stimulated PI 3‐kinase activity. The neurotrophic response of IGF‐1 was also inhibited by the PI 3‐kinase inhibitor LY294002, the protein kinase C inhibitor chelerythrine chloride, and the protein kinase A inhibitor KT5720, but unaffected by the mitogen‐activated protein kinase kinase inhibitor PD 98059. These data demonstrate that ethanol promotes cell death in cerebellar granule neurons by inhibiting the antiapoptotic action of IGF‐1.
Cyclopentyladenosine‐Induced Homologous Down‐Regulation of A1 Adenosine Receptors (A1AR) in Intact Neurons Is Accompanied by Receptor Sequestration but Not a Reduction in A1AR mRNA Expression or G Protein α‐Subunit Content
Authors: Hettinger Barbara D. , Leid Mark , Murray Thomas F.
Published: July 2010

Abstract: We showed previously that exposure of cerebellar granule cells to the A1 adenosine receptor (A1AR)‐selective agonist, cyclopentyladenosine, decreases A1AR density and G protein coupling corresponding to blunted agonist‐induced adenylyl cyclase (EC 4.6.1.1) inhibition. We have now determined that A1AR‐mediated adenylyl cyclase inhibition was desensitized in a homologous manner. Carbachol‐ and baclofen‐induced inhibition of adenylyl cyclase was unaffected by 48‐h exposure to 10 µM cyclopentyladenosine. Expression of G protein α‐subunits was not affected dramatically by agonist exposure. The fraction of sequestered A1AR was increased significantly at 4, 24, and 48 h of cyclopentyladenosine exposure (35, 57, and 81% increase over control, respectively). The time course of agonist‐induced A1AR sequestration was slower than that reported for other G protein‐coupled receptors. Incubation with the adenosine receptor antagonist, 8‐p‐sulfophenyltheophylline or adenosine deaminase did not alter sequestration significantly. Neither steady‐state A1AR mRNA levels nor transcript stability was affected by 48‐h agonist exposure. We determined that A1AR half‐life in cerebellar granule cells is 20.9 h, which is considerably longer than that reported for several other G protein‐coupled receptors. The slow time course of A1AR sequestration and the stability of the corresponding mRNA may be a reflection of the tonic inhibitory tone exerted by adenosine in brain.
Distinct Differences Between Morphine‐ and [d‐Ala2,N‐MePhe4,Gly‐ol5]‐Enkephalin‐ μ‐Opioid Receptor Complexes Demonstrated by Cyclic AMP‐Dependent Protein Kinase Phosphorylation
Authors: Chakrabarti Sumita , Law Ping‐Yee , Loh Horace H.
Published: July 2010

Abstract: The present study demonstrates a conditional, agonist‐dependent phosphorylation of the μ‐opioid receptor (MOR‐1) by cyclic AMP‐dependent protein kinase (PKA) in membrane preparations of MOR‐1‐transfected neuroblastoma Neuro2A cells. Opioid agonist‐dependent phosphorylation occurs in a time‐ and concentration‐dependent manner (EC50∼40 nM) and can be abolished by the receptor antagonist naloxone. Stoichiometric analysis indicates incorporation of a maximum of 6 mol of phosphate/mol of receptor in the presence of 1 µM morphine and 6 nM PKA. Although morphine and related alkaloids as well as some peptide agonists (PLO17 and β‐endorphin) stimulated phosphorylation of MOR‐1 by PKA, the potent μ‐opioid‐selective peptide [d‐Ala2,N‐MePhe4,Gly‐ol5]‐enkephalin (DAMGO) or other enkephalin analogues such as [d‐Ala2]‐Met5‐enkephalinamide (DALA), [d‐Ala2,d‐Leu5]‐enkephalin (DADLE), and Met5‐enkephalin had no effect. The lack of the effect of DAMGO on MOR‐1 phosphorylation state was evident also after chronic pretreatment. These results suggest the existence of different agonist‐dependent conformations of MOR‐1. Furthermore, phosphorylation may be a useful parameter with which to identify different agonist‐receptor conformations.
Pertussis Toxin Modification of PC12 Cells Inhibits a Protein Phosphatase 2A‐Like Phosphatase
Authors: Chen Fusheng , Vu Ngoc‐Diep , Wagner Paul D.
Published: July 2010

Abstract: We have found that modification of rat PC12 cells with pertussis toxin resulted in an ∼50% inhibition of a protein phosphatase 2A‐like phosphatase. Protein phosphatase 2A (PP2A) is a major cellular serine/threonine‐specific protein phosphatase. Treatment of extracts from pertussis toxin‐modified PC12 cells with either immobilized alkaline phosphatase or Ca2+ reversed this inhibition. Reactivation of the PP2A‐like phosphatase in Ca2+ appears to result from the dephosphorylation of a protein by the Ca2+/calmodulin‐dependent protein phosphatase calcineurin. The PP2A‐like phosphatase in extracts from pertussis toxin‐modified PC12 cells eluted from a Mono Q column at a higher ionic strength than did the PP2A‐like phosphatase in extracts from control cells. After incubation in Ca2+, the PP2A‐like phosphatase in extracts from pertussis toxin‐modified cells eluted from a Mono Q column at the same ionic strength as did the PP2A‐like phosphatase in extracts from control cells. These results indicate that the effect of pertussis toxin on this PP2A‐like activity results from the phosphorylation of either one of the subunits of the PP2A‐like phosphatase or a protein that when phosphorylated binds to and inhibits this phosphatase. Pertussis toxin modification did not result in the phosphorylation of the catalytic subunit of PP2A. Because phosphorylation regulates the activities of many enzymes and cell surface receptors, a pertussis toxin‐induced decrease in PP2A activity could alter signaling pathways and other cellular processes in which G proteins are not directly involved.
Lobeline Displaces [3H]Dihydrotetrabenazine Binding and Releases [3H]Dopamine from Rat Striatal Synaptic Vesicles: Comparison with d‐Amphetamine
Authors: Teng LiHong , Crooks Peter A. , Dwoskin Linda P.
Published: July 2010

Abstract: Lobeline, an alkaloid from Indian tobacco (Lobelia inflata), is classified as a nicotinic agonist and is currently used as a smoking cessation agent. However, our previous in vitro studies demonstrate that lobeline does not act as a nicotinic agonist but alters presynaptic dopamine (DA) storage by potently inhibiting DA uptake into synaptic vesicles. Recently, d‐amphetamine has been reported to act at the level of the synaptic vesicle to alter presynaptic function. The present in vitro studies further elucidate the mechanism of lobeline's action and compare its effects with those of d‐amphetamine. [3H]Dihydrotetrabenazine ([3H]DTBZ), used routinely to probe a high‐affinity binding site on the vesicular monoamine transporter (VMAT2), bound to vesicle membranes from rat striatum with a KD of 1.67 nM and Bmax of 8.68 pmol/mg of protein. Lobeline inhibited [3H]DTBZ binding with an IC50 of 0.90 µM, consistent with its previously reported IC50 of 0.88 µM for inhibition of [3H]DA uptake into vesicles. These results suggest that lobeline specifically interacts with DTBZ sites on VMAT2 to inhibit DA uptake into synaptic vesicles. Interestingly, d‐amphetamine inhibited [3H]DTBZ binding to vesicle membranes with an IC50 of 39.4 µM, a concentration 20 times greater than reported for inhibition of VMAT2 function, suggesting that d‐amphetamine interacts with a different site than lobeline on VMAT2 to inhibit monoamine uptake. Kinetic analysis of [3H]DA release from [3H]DA‐preloaded synaptic vesicles in the absence of drug revealed a t1/2 of 2.12 min. Lobeline and d‐amphetamine evoked [3H]DA release with EC50 values of 25.3 and 2.22 µM, respectively. At a concentration 10 times the EC50, lobeline and d‐amphetamine significantly decreased the t1/2 of [3H]DA release to 1.58 and 1.48 min, respectively. Thus, in contrast to d‐amphetamine, which is equipotent in inhibiting DA uptake and promoting release from the synaptic vesicles, lobeline more potently (28‐fold) inhibits DA uptake (via an interaction with the DTBZ site on VMAT2) than it evokes DA release to redistribute presynaptic DA storage.
Regulation of Extracellular Dopamine by the Norepinephrine Transporter
Authors: Yamamoto Bryan K. , Novotney Susan
Published: July 2010

Abstract: There is growing evidence of an interaction between dopamine and norepinephrine. To test the hypothesis that norepinephrine terminals are involved in the uptake and removal of dopamine from the extracellular space, the norepinephrine uptake blocker desmethylimipramine (DMI) was infused locally while the extracellular concentrations of dopamine were simultaneously monitored. DMI increased the extracellular concentrations of dopamine in the medial prefrontal cortex and nucleus accumbens shell but had no effect in the striatum. The combined systemic administration of haloperidol and the local infusion of DMI produced an augmented increase in extracellular dopamine in the cortex compared with the increase produced by either drug alone. This synergistic increase in dopamine overflow is likely due to the combination of impulse‐mediated dopamine release produced by haloperidol and blockade of the norepinephrine transporter. No such synergistic effects were observed in the nucleus accumbens and striatum. Local perfusion of the α2‐antagonist idazoxan also increased the extracellular concentrations of dopamine in the cortex. Although the stimulation of extracellular dopamine by idazoxan and DMI could be due to the increased extracellular concentrations of norepinephrine produced by these drugs, an increase in dopamine also was observed in lesioned rats that were depleted of norepinephrine and challenged with haloperidol. This contrasted with the lack of an effect of haloperidol on cortical dopamine in unlesioned controls. These results suggest that norepinephrine terminals regulate extracellular dopamine concentrations in the medial prefrontal cortex and to a lesser extent in the nucleus accumbens shell through the uptake of dopamine by the norepinephrine transporter.
Oxidative Damage to Proteins, Lipids, and DNA in Cortical Brain Regions from Patients with Dementia with Lewy Bodies
Authors: Lyras Leonidas , Perry Robert H. , Perry Elaine K. , Ince Paul G. , Jenner Andrew , Jenner Peter , Halliwell Barry
Published: July 2010

Abstract: Dementia with Lewy bodies (DLB) forms the second most common pathological subgroup of dementia after Alzheimer's disease. The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases in cortical brain areas from patients with DLB with levels in matched control tissues. Overall, there was a trend for protein carbonyl levels to be increased in all areas, but a significant difference was found only in the parietal and temporal lobes. No differences were observed in the levels of lipid peroxidation. Measurement of products of damage to DNA bases showed increased levels of thymine glycol, 8‐hydroxyguanine, 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine, 5‐hydroxycytosine, 5‐hydroxyuracil, 5‐hydroxymethyluracil, and xanthine. Xanthine levels were increased in the DLB group in the parietal, occipital, and temporal lobes, indicating that peroxynitrite or other deaminating species may be involved. The finding of increased protein carbonyls and increased DNA base products in cortical regions from DLB patients indicates that oxidative stress may play a role in DLB.
Mutant Presenilin 2 Transgenic Mouse: Effect on an Age‐Dependent Increase of Amyloid β‐Protein 42 in the Brain
Authors: Oyama Fumitaka , Sawamura Naoya , Kobayashi Kimio , Morishima‐Kawashima Maho , Kuramochi Takashi , Ito Mamoru , Tomita Taisuke , Maruyama Kei , Saido Takaomi C. , Iwatsubo Takeshi , Capell Anja , Walter Jochen , Grünberg Jürgen , Ueyama Yoshito , Haass Christian , Ihara Yasuo
Published: July 2010

Abstract: The N141I missense mutation in presenilin (PS) 2 is tightly linked with a form of autosomal dominant familial Alzheimer's disease (AD) in the Volga German families. We have generated transgenic mouse lines overexpressing human wild‐type or mutant PS2 under transcriptional control of the chicken β‐actin promoter. In the brains of transgenic mice, the levels of human PS2 mRNA were found to be five‐ to 15‐fold higher than that of endogenous mouse PS2 mRNA. The amyloid β‐protein (Aβ) 42 levels in the brains of mutant PS2 transgenic mice were higher than those in wild‐type PS2 transgenic mice at the age of 2, 5, or 8 months. In addition, the Aβ42 levels appeared to increase steadily in the mutant PS2 transgenic mouse brains from 2 to 8 months of age, whereas there was only a small increase in wild‐type transgenic mice between the ages of 5 and 8 months. There was no definite difference in the levels of N‐terminal and C‐terminal fragments between wild‐type and mutant PS2 transgenic mice at the age of 2, 5, or 8 months. These data show a definite effect of the PS2 mutation on an age‐dependent increase of Aβ42 content in the brain.
Expression Pattern of Synucleins (Non‐Aβ Component of Alzheimer's Disease Amyloid Precursor Protein/α‐Synuclein) During Murine Brain Development
Authors: Hsu Leigh J. , Mallory Margaret , Xia Yu , Veinbergs Isaac , Hashimoto Makoto , Yoshimoto Makoto , Thal Leon J. , Saitoh Tsunao , Masliah Eliezer
Published: July 2010

Abstract: The non‐Aβ component of Alzheimer's disease amyloid precursor protein (NACP) is predominantly a neuron‐specific presynaptic protein that may play a central role in neurodegeneration because NACP fragments are found in Alzheimer's disease amyloid and a mutation in the NACP gene is associated with familial Parkinson's disease. In addition, NACP may play an important role during synaptogenesis and CNS development. To understand better the patterns of NACP expression during development, we analyzed the levels of this protein as well as the levels of another synaptic protein (synaptophysin) by ribonuclease protection assay, western blotting, and immunocytochemistry in fetal, juvenile, and adult mouse brain. From embryonic day 12 to 15, there was a slight increase, which was then followed by a more dramatic increase at later time points. Immunocytochemical staining for NACP increases throughout these stages as well. Although NACP appeared early in CNS development, synaptophysin levels started to rise at a later stage. These findings support the contention that NACP might be important for CNS development. Furthermore, the cytosolic component of NACP precedes the particulate component in development, indicating that a redistribution of the protein to the membrane fraction may be important for events later in neuronal development and in synaptogenesis.
Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain
Authors: Liang Jin‐Jun , Cockett Mark , Khawaja Xavier Z.
Published: July 2010

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti‐Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double‐fluorescence immunostaining with anti‐Gγ3 antibody and individual anti‐Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.
Manipulation of Disulfide Bonds Differentially Affects the Intracellular Transport, Sorting, and Processing of Neuroendocrine Secretory Proteins
Authors: Van Horssen A. Martin , Van Kuppeveld Frank J. M. , Martens Gerard J. M.
Published: July 2010

Abstract: To investigate if the prevention of disulfide bond formation affects the intracellular transport, sorting, and processing of a distinct set of neuroendocrine proteins in the regulated secretory pathway, we have treated Xenopus intermediate pituitaries with the thiol‐reducing agent dithiothreitol. Pulse‐chase incubations in combination with immunoprecipitation analysis were used to monitor the fates of the prohormone proopiomelanocortin (POMC), prohormone convertase PC2 and its helper protein 7B2, as well as secretogranin III. Manipulation of the disulfide bonds in POMC and proPC2 blocked their transport to the trans‐Golgi network and strongly inhibited their processing. Reduction of the single disulfide bond in 7B2 did not disturb its transport and cleavage, but caused its missorting to the constitutive secretory pathway. Moreover, the liaison between proPC2 and 7B2 was prevented. Dithiothreitol did not affect transport, sorting, and cleavage of secretogranin III, which lacks disulfide bonds. When the reducing agent was washed away, POMC processing, proPC2 maturation, and the association between proPC2 and 7B2 were reestablished. Collectively, our findings indicate that manipulation of disulfide bonds differentially affects the fates of neuroendocrine proteins during their transit through the secretory pathway.
Glial Cell Line‐Derived Neurotrophic Growth Factor Inhibits Apoptotic Death of Postnatal Substantia Nigra Dopamine Neurons in Primary Culture
Authors: Burke Robert E. , Antonelli Megan , Sulzer David
Published: July 2010

Abstract: Glial cell line‐derived neurotrophic factor (GDNF) was identified on the basis of its ability to enhance the development of embryonic mesencephalic dopamine neurons. It remains unknown whether GDNF is a physiologically relevant trophic factor for these neurons. We have shown that natural cell death among dopamine neurons of the substantia nigra occurs largely postnatally. To investigate whether GDNF may have the ability to support these neurons during their period of natural cell death, we have used a postnatal primary culture model. We find that GDNF is able to support the viability of postnatal nigral dopamine neurons by inhibiting apoptotic death. This ability of GDNF shows both regional specificity for the nigra and cellular specificity for the dopamine phenotype. Among eight other neurotrophic factors previously reported to support embryonic dopamine neurons, GDNF was unique in this ability. Thus, GDNF meets this criterion for a physiologically relevant trophic factor for dopamine neurons of the substantia nigra.
Adenosine, Inosine, and Guanosine Protect Glial Cells During Glucose Deprivation and Mitochondrial Inhibition: Correlation Between Protection and ATP Preservation
Authors: Jurkowitz Marianne S. , Litsky Monica L. , Browning Megan J. , Hohl Charlene M.
Published: July 2010

Abstract: The purpose of this study was to determine the mechanism by which adenosine, inosine, and guanosine delay cell death in glial cells (ROC‐1) that are subjected to glucose deprivation and mitochondrial respiratory chain inhibition with amobarbital (GDMI). ROC‐1 cells are hybrid cells formed by fusion of a rat oligodendrocyte and a rat C6 glioma cell. Under GDMI, ATP was depleted rapidly from ROC‐1 cells, followed on a much larger time scale by a loss of cell viability. Restoration of ATP synthesis during this interlude between ATP depletion and cell death prevented further loss of viability. Moreover, the addition of adenosine, inosine, or guanosine immediately before the amobarbital retarded the decline in ATP and preserved cell viability. The protective effects on ATP and viability were dependent on nucleoside concentration between 50 and 1,500 µM. Furthermore, protection required nucleoside transport into the cell and the continued presence of nucleoside during GDMI. A significant positive correlation between ATP content at 16 min and cell viability at 350 min after the onset of GDMI was established (r = 0.98). Modest increases in cellular lactate levels were observed during GDMI (1.2 nmol/mg/min lactate produced); however, incubation with 1,500 µM inosine or guanosine increased lactate accumulation sixfold. The protective effects of inosine and guanosine on cell viability and ATP were >90% blocked after treatment with 50 µM BCX‐34, a nucleoside phosphorylase inhibitor. Accordingly, lactate levels also were lower in BCX‐34‐treated cells incubated with inosine or guanosine. We conclude that under GDMI, the ribose moiety of inosine and guanosine is converted to phosphorylated glycolytic intermediates via the pentose phosphate pathway, and its subsequent catabolism in glycolysis provides the ATP necessary for maintaining plasmalemmal integrity.
Substrates of Energy Metabolism Attenuate Methamphetamine‐Induced Neurotoxicity in Striatum
Authors: Stephans Stacy E. , Whittingham Tim S. , Douglas Albertina J. , Lust W. David , Yamamoto Bryan K.
Published: July 2010

Abstract: High doses of methamphetamine (METH) produce a long‐term depletion in striatal tissue dopamine content. The mechanism mediating this toxicity has been associated with increased concentrations of dopamine and glutamate and altered energy metabolism. In vivo microdialysis was used to assess and alter the metabolic environment of the brain during high doses of METH. METH significantly increased extracellular concentrations of lactate in striatum and prefrontal cortex. This increase was significantly greater in striatum and coincided with the greater vulnerability of this brain region to the toxic effects of METH. To examine the effect of supplementing energy metabolism on METH‐induced dopamine content depletions, the striatum was perfused directly with decylubiquinone or nicotinamide to enhance the energetic capacity of the tissue during or after a neurotoxic dosing regimen of METH. When decylubiquinone or nicotinamide was perfused into striatum during the administration of METH, there was no significant effect on METH‐induced striatal dopamine efflux, glutamate efflux, or the long‐term dopamine depletions measured 7 days later. However, a delayed perfusion with decylubiquinone or nicotinamide for 6 h beginning immediately after the last METH injection attenuated the METH‐induced striatal dopamine depletions measured 1 week later. These results support the hypothesis that the compromised metabolic state produced by METH administration predisposes dopamine terminals to the neurotoxic effects of glutamate, dopamine, and/or free radicals.
Chronic Dizocilpine (MK‐801) Reversibly Delays GABAA Receptor Maturation in Cerebellar Granule Neurons In Vitro
Authors: Wang Xiao‐Hui , Zhu Wei Jian , Corsi Lorenzo , Ikonomovic Snezana , Paljug William R. , Vicini Stefano , Grayson Dennis R.
Published: July 2010

Abstract: We investigated the effect of chronically blocking NMDA receptor stimulation to examine changes in GABAA receptor expression and pharmacology in cerebellar granule cells at different stages of maturation. We have previously shown that NMDA‐selective glutamate receptor stimulation alters GABAA receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunits. When NMDA receptor stimulation is blocked with MK‐801 during the first week in vitro, a decrease in the α1, γ2S, and γ2L receptor subunit mRNAs occurred. When present only during the second week, changes were limited to the α1 and γ2L mRNAs. Finally, if MK‐801 was present during the first week and removed during the second week, these changes reversed. Whole‐cell voltage‐clamp recordings showed that treatment with MK‐801 during either the first or second week increased the EC50 of the receptors for GABA and attenuated the potentiation mediated by flunitrazepam. Last, these properties were reversed if MK‐801 was removed after the first week in vitro. Our results suggest that MK‐801 reversibly inhibits GABAA receptor maturation by modulating receptor subunit expression and that the altered pharmacological responses appear to be dominated by changes in the levels of allosteric modulation mediated by the γ2 receptor subunit.
Phosphatidylinositol 3‐Kinase: Increased Activity and Protein Level in Amyotrophic Lateral Sclerosis
Authors: Wagey R. , Pelech S. L. , Duronio V. , Krieger C.
Published: July 2010

Abstract: Enzyme activities and protein levels of several protein and lipid kinases were measured in postmortem tissue from patients who died with amyotrophic lateral sclerosis (ALS) as well as from control subjects. Patients who died with ALS had increased activities and protein levels of phosphatidylinositol 3‐kinase (PI 3‐K) in particulate fractions of spinal cord tissue compared with control subjects. The PI 3‐K activity increased with PI 3‐K protein level, indicating no change in specific PI 3‐K activity in ALS. No differences in PI 3‐K activities were found in cytosolic fractions of spinal cord, or in motor and visual cortices, from ALS patients compared with those from controls. PI 3‐K activities and protein levels were unchanged in brain tissue from patients who died with Alzheimer's disease compared with those from controls. PI 3‐K is a lipid kinase that is important for cell survival and is activated in response to many growth factors. Increased PI 3‐K activities in particulate fractions of spinal cord from ALS patients may be related to the increase of PI 3‐K protein levels found in this tissue. The protein kinases Erk2, protein kinase B (PKB), and p70 ribosomal S6 kinase (S6K) showed no differences in activities in spinal cord tissue between ALS patients and controls. However, the amounts of PKB and S6K protein were significantly higher in ALS patients, whereas Erk2 protein amount was unchanged compared with controls. Protein kinase C activity was increased in spinal cord tissue from ALS patients, which is consistent with our previous report. The increased activity of PI 3‐K in spinal cord tissue from patients with ALS implicates the involvement or activation of PI 3‐K in ALS, as either a cause or a consequence of the neuron loss. The lack of up‐regulation in the activities of PKB and S6K in ALS tissue supports an impairment in signal transduction cascades mediated by PI 3‐K in this neurodegenerative disease.
p75 and TrkA Receptor Signaling Independently Regulate Amyloid Precursor Protein mRNA Expression, Isoform Composition, and Protein Secretion in PC12 Cells
Authors: Roßner S. , Ueberham U. , Schliebs R. , Perez‐Polo J. R. , Bigl V.
Published: July 2010

Abstract: The pheochromocytoma PC12 cell line was used as a model system to characterize the role of the p75 neurotrophin receptor (p75NTR) and tyrosine kinase (Trk) A nerve growth factor (NGF) receptors on amyloid precursor protein (APP) expression and processing. NGF increased in a dose‐dependent fashion neurite outgrowth, APP mRNA expression, and APP secretion with maximal effects at concentrations known to saturate TrkA receptor binding. Displacement of NGF binding to p75NTR by addition of an excess of brain‐derived neurotrophic factor abolished NGF's effects on neurite outgrowth and APP metabolism, whereas addition of brain‐derived neurotrophic factor alone did not induce neurite outgrowth or affect APP mRNA or protein processing. However, treatment of PC12 cells with C2‐ceramide, an analogue of ceramide, the endogenous product produced by the activity of p75NTR‐activated sphingomyelinase, mimicked the effects of NGF on cell morphology and stimulation of both APP mRNA levels and APP secretion. Specific stimulation of TrkA receptors by receptor cross‐linking, on the other hand, selectively stimulated neurite outgrowth and APP secretion but not APP mRNA levels, which were decreased. These findings demonstrate that in PC12 cells expressing p75NTR and TrkA receptors, binding of NGF to the p75NTR is required to mediate NGF effects on cell morphology and APP metabolism. Furthermore, our data are consistent with NGF having specific effects on p75NTR not shared with other neurotrophins. Lastly, we have shown that specific activation of TrkA receptors—in contrast to p75NTR‐associated signaling—stimulates neurite outgrowth and increases nonamyloidogenic secretory APP processing without increases in APP mRNA levels.
Depletion of Phospholipid Arachidonoyl‐Containing Molecular Species in a Human Schwann Cell Line Grown in Elevated Glucose and Their Restoration by an Aldose Reductase Inhibitor
Authors: Kuruvilla Rejji , Eichberg Joseph
Published: July 2010

Abstract: In experimental diabetic neuropathy, defective arachidonic acid metabolism characterized by a decrease in the proportion of glycerophospholipid arachidonoyl‐containing molecular species (ACMS) occurs and has been implicated in the pathogenesis of the disorder. In this study, we evaluated the suitability of a tumor‐derived human Schwann cell line (NF1T) as a model to investigate the mechanism underlying the loss of ACMS. NF1T cells grown in 30 versus 5.5 mM glucose undergo a marked reduction in ACMS in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, in a manner resembling that of diabetic nerve. The depletion of ACMS can be reversed on transferring the cells from 30 mM glucose to medium containing physiological levels of glucose. Cells maintained in 5.5 mM glucose plus 25 mM mannitol or sorbitol did not exhibit decreased ACMS levels, indicating that osmotic effects were not responsible for ACMS depletion. However, growth in 25 mM fructose elicited a reduction of ACMS similar to that produced by 30 mM glucose. Excessive glucose flux through the polyol pathway has been implicated in the neural and vascular abnormalities associated with diabetes. Therefore, we examined the effects of polyol pathway inhibitors, including two aldose reductase inhibitors, zopolrestat and sorbinil, and a sorbitol dehydrogenase inhibitor (SDI), CP166,572, on ACMS levels in NF1T cells cultured in elevated glucose concentrations. At 200 µM, zopolrestat fully and sorbinil partially corrected ACMS depletion. The SDI at concentrations up to 100 µM failed to affect diminished ACMS levels. Neither zopolrestat nor the SDI restored ACMS levels reduced in the presence of elevated fructose concentrations. These findings suggest that enhanced flux through the polyol pathway and, in particular, elevated aldose reductase activity may play a significant role in the reduction of ACMS levels in the cells brought about by elevated glucose levels.
Somatodendritic α2‐Adrenoceptors in the Locus Coeruleus Are Involved in the In Vivo Modulation of Cortical Noradrenaline Release by the Antidepressant Desipramine
Authors: Mateo Yolanda , Pineda Joseba , Meana J. Javier
Published: July 2010

Abstract: The effect of the antidepressant and selective noradrenaline reuptake blocker desipramine (DMI) on noradrenergic transmission was evaluated in vivo by dual‐probe microdialysis. DMI (1, 3, and 10 mg/kg, i.p.) dose‐dependently increased extracellular levels of noradrenaline (NA) in the locus coeruleus (LC) area. In the cingulate cortex (Cg), DMI (3 and 10 mg/kg, i.p.) also increased NA dialysate, but at the lowest dose (1 mg/kg, i.p.) it decreased NA levels. When the α2‐adrenoceptor antagonist RX821002 (1 µM) was perfused in the LC, DMI (1 mg/kg, i.p.) no longer decreased but rather increased NA dialysate in the Cg. In electrophysiological experiments, DMI (1 mg/kg, i.p.) inhibited the firing activity of LC neurons by a mechanism reversed by RX821002. Local DMI (0.01–100 µM) into the LC increased concentration‐dependently NA levels in the LC and simultaneously decreased NA levels in the Cg. This decrease was abolished by local RX821002 administration into the LC. The results demonstrate in vivo that DMI inhibits NA reuptake at somatodendritic and nerve terminal levels of noradrenergic cells. The increased NA dialysate in the LC inhibits noradrenergic activity, which in part counteracts the effects of DMI on the Cg. The modulation of cortical NA release by activity of DMI at the somatodendritic level is mediated through α2‐adrenoceptors located in the LC.
Constitutive Activity and Structural Instability of the Wild‐Type Human H2 Receptor
Authors: Alewijnse Astrid E. , Smit Martine J. , Hoffmann Marcel , Verzijl Dennis , Timmerman Henk , Leurs Rob
Published: July 2010

Abstract: Stable expression of the human H2 receptor in Chinese hamster ovary cells resulted in an increase in basal cyclic AMP (cAMP) production, which was inhibited by the inverse agonists cimetidine, famotidine, and ranitidine with potencies similar to those found for the rat H2 receptor. Burimamide, a neutral antagonist at the rat H2 receptor, behaved as a weak partial agonist at the human H2 receptor. Burimamide competitively antagonized both the histamine‐induced increase in cAMP and the cimetidine‐induced reduction of the basal cAMP level with apparent KB values that were similar to its H2 receptor affinity. Investigation of the modulation of receptor expression after long‐term drug treatment revealed that at low concentrations histamine induced a significant reduction in H2 receptor expression, whereas at high concentrations receptor expression was slightly increased. The partial agonist burimamide induced, like inverse agonists, an upregulation of the human H2 receptor after prolonged treatment. These findings suggest a structural instability of the constitutively active human H2 receptor in transfected Chinese hamster ovary cells. Occupation of the H2 receptor by any ligand reduces the instability, thus resulting in higher cellular expression levels.
Metabolism of Cysteine in Astroglial Cells: Synthesis of Hypotaurine and Taurine
Authors: Brand Annette , Leibfritz Dieter , Hamprecht Bernd , Dringen Ralf
Published: July 2010

Abstract: The synthesis of hypotaurine and taurine was investigated in astroglia‐rich primary cultures obtained from brains of neonatal Wistar rats using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Cell extracts of astroglial cultures analyzed by 1H NMR spectroscopy show prominent signals of hypotaurine. To identify cysteine as precursor for hypotaurine and taurine synthesis in astroglial cells, primary cultures were incubated with [3‐13C]cysteine for 24 or 72 h. Cell extracts and incubation media were then analyzed with 13C NMR spectroscopy. Labeled hypotaurine, taurine, glutathione, and lactate were identified in the cell extracts. Within 72 h, 35.0% of the total intracellular hypotaurine and 22.5% of taurine were newly synthesized from [3‐13C]cysteine. The presence of [1‐13C]hypotaurine and [1‐13C]taurine in the incubation medium proves the release of those products of cysteine metabolism into the medium. Minor amounts of the [3‐13C]cysteine were used for the synthesis of glutathione in astroglial cells or metabolized to [3‐13C]lactate, which was found in cell extracts and media. These results indicate that the formation of hypotaurine and taurine is a major pathway of cysteine metabolism in astroglial cells.
Dα3, a New Functional α Subunit of Nicotinic Acetylcholine Receptors from Drosophila
Authors: Schulz R. , Sawruk E. , Mülhardt C. , Bertrand S. , Baumann A. , Phannavong B. , Betz H. , Bertrand D. , Gundelfinger E. D. , Schmitt B.
Published: July 2010

Abstract: Nicotinic acetylcholine (ACh) receptors (nAChRs) are important excitatory neurotransmitter receptors in the insect CNS. We have isolated and characterized the gene and the cDNA of a new nAChR subunit from Drosophila. The predicted mature nAChR protein consists of 773 amino acid residues and has the structural features of an ACh‐binding α subunit. It was therefore named Dα3, for Drosophilaα‐subunit 3. The dα3 gene maps to the X chromosome at position 7E. The properties of the Dα3 protein were assessed by expression in Xenopus oocytes. Dα3 did not form functional receptors on its own or in combination with any Drosophilaβ‐type nAChR subunit. Nondesensitizing ACh‐evoked inward currents were observed when Dα3 was coexpressed with the chick β2 subunit. Half‐maximal responses were at ∼0.15 µM ACh with a Hill coefficient of ∼1.5. The snake venom component α‐bungarotoxin (100 nM) efficiently but reversibly blocked Dα3/β2 receptors, suggesting that Dα3 may be a component of one of the previously described two classes of toxin binding sites in the Drosophila CNS.
Inducible Galanin and GalR2 Receptor System in Motor Neuron Injury and Regeneration
Authors: Burazin Tanya C. D. , Gundlach Andrew L.
Published: July 2010

Abstract: Galanin has been ascribed several physiological roles that are thought to be mediated via multiple galanin receptors. Recently, two galanin receptors—galanin receptor‐1 (GalR1) and galanin receptor‐2 (GalR2)—have been cloned and characterized and shown to have differences in amino acid sequence, pharmacology, and second messenger signaling systems. Previous studies have demonstrated an up‐regulation of galanin expression in damaged neurons of several different types. Using in situ hybridization histochemistry this study investigated whether adult cranial motor neurons express mRNAs encoding GalR1 and/or GalR2 and explored possible time‐dependent changes in these transcripts following facial nerve injury. GalR2 mRNA levels were increased in the ipsilateral facial nucleus 3 (∼1.8‐fold) and 7 days (∼3.7‐fold) after unilateral facial nerve crush and had returned to levels equivalent to those in contralateral controls by 14–21 days. GalR1 mRNA was not detected in facial nuclei of naive, sham‐operated, or operated rats but was present in adjacent reticular nuclei. Galanin mRNA levels were also increased eight‐ to 10‐fold in the ipsilateral facial nucleus following nerve injury. These experiments confirm the putative importance of galanin signaling systems after nerve injury by demonstating a differential response of galanin receptor subtypes and suggest an important “autoreceptor” role for the GalR2 receptor in these processes.
Neurochemical and Neurogenetic Correlates of Parkinson's Disease
Authors: Mizuno Yoshikuni , Hattori Nobutaka , Matsumine Hiroto
Published: July 2010

Abstract: We discuss neurochemical and neurogenetic correlates of Parkinson's disease (PD) based on the recent progress in the study of its etiology and pathogenesis. Nigral degeneration with the presence of Lewy bodies in the remaining neurons is the pathologic hallmark of PD, and the resultant loss of striatal dopamine is responsible for most of the clinical manifestations. Although the primary cause is still unknown, mitochondrial respiratory failure and oxidative stress appear to be two major contributors to the nigral cell death. Many endogenous and exogenous compounds with structural similarity to MPTP have been postulated as potential neurotoxins inducing nigral cell death in PD, but there is little evidence of accumulation of such compounds in the nigra. Genetic influence has increasingly been recognized as an important risk factor for PD. In this respect, genetic linkage analysis and molecular cloning of the disease genes in familial parkinsonism are of utmost importance today. Recently, the disease gene for one of the autosomal dominant forms of familial PD was identified, and we cloned the gene for an autosomal recessive type of familial parkinsonism that had been mapped to the long arm of chromosome 6 by our group. Information obtained on familial parkinsonism will contribute to the studies on sporadic PD as well.
Dominant Expression of Rat Prostanoid DP Receptor mRNA in Leptomeninges, Inner Segments of Photoreceptor Cells, Iris Epithelium, and Ciliary Processes
Authors: Gerashchenko Dmitry , Beuckmann Carsten T. , Kanaoka Yoshihide , Eguchi Naomi , Gordon William C. , Urade Yoshihiro , Bazan Nicolas G. , Hayaishi Osamu
Published: July 2010

Abstract: Prostaglandin (PG) D2 is one of the major prostanoids in the mammalian brain and eye tissues. Its function is mediated by the prostanoid DP receptor, which is specific for PGD2 among the various prostanoids. In this study, we cloned the full‐length cDNA for the rat DP receptor and used it for detection of DP receptor mRNA in various rat tissues. Northern blotting and RT‐PCR analyses revealed that this DP receptor was expressed most intensely in the eye tissues, moderately in the leptomeninges and oviduct, and weakly in the epididymis. The tissue distribution profile of the mRNA for the rat DP receptor is overlapped with those of hematopoietic and lipocalin‐type PGD synthases. Among rat eye tissues, the expression was the highest in the iris. In situ hybridization and in situ RT‐PCR revealed DP receptor mRNA to be localized in the epithelium of the iris and ciliary body and in photoreceptor cells of the retina, suggesting the involvement of the receptor in the physiological regulation of intraocular pressure and the vision process. In the brain, DP receptor mRNA was dominantly expressed in the leptomeninges and was not detected in the brain parenchyma including the ventral rostral forebrain, the surface area of which is reportedly involved in sleep induction by PGD2.
Identification of a Homophilic Binding Site in Immunoglobulin‐Like Domain 2 of the Cell Adhesion Molecule L1
Authors: Zhao Xiaoning , Yip Paul M. , Siu Chi‐Hung
Published: July 2010

Abstract: The cell adhesion molecule L1 plays an important role in neural development, and mutations in human L1 have been implicated in X‐linked hydrocephalus and related neurological diseases. We have previously demonstrated that recombinant proteins containing the second immunoglobulin‐like domain (Ig2) of L1 contain both homophilic binding and neuritogenic activities. In this report, the involvement of L1 Ig2 in cell‐cell adhesion and neuritogenesis was further evaluated in cell transfection studies. Transfectants expressing intact L1 were capable of undergoing L1‐dependent self‐aggregation and promoting neurite outgrowth from neural retinal cells. However, both activities were abolished in transfectants expressing L1Δ2, a mutant L1 with Ig2 deleted. In competition experiments, the wild‐type Ig2 fusion protein inhibited L1‐dependent cell aggregation, whereas an Ig2 fusion protein containing the hydrocephalus mutation R184Q did not. Oligopeptides flanking Arg184 were therefore synthesized and assayed for their effects on L1‐mediated cell‐cell binding and neuritogenesis. The peptide L1‐A, spanning the residues His178 and Gly191, inhibited both L1‐ and Ig2 fusion protein‐mediated homophilic binding. When neural retinal cells were cultured on substrate‐coated Ig2 fusion protein, peptide L1‐A also abolished L1‐dependent neurite outgrowth. Substitutions of several charged residues and hydrophobic residues with alanine in peptide analogues led to the loss of inhibitory effects, suggesting that multiple amino acids might be involved in L1‐L1 binding. Taken together, these results identify an L1 homophilic binding site within the sequence HIKQDERVTMGQNG of Ig2 and demonstrate the requirement of L1 homophilic binding in the promotion of neurite outgrowth.
Regulation of Ganglioside Metabolism by Phosphorylation and Dephosphorylation
Authors: Bieberich Erhard , Freischütz Bettina , Liour Shyh‐Shyurng , Yu Robert K.
Published: July 2010

Abstract: Cell differentiation is frequently accompanied by alterations in the composition of gangliosides in the plasma membrane resulting from a regulation of the enzyme activities involved. The regulation of CMP‐NeuAc:GM1 α2‐3‐sialyltransferase (ST‐IV) and UDP‐GalNAc:GM3 N‐acetylgalactosaminyltransferase (Gal‐NAc‐T) by the degree of enzyme phosphorylation was analyzed by determination of the enzyme activity on incubation of NG108‐15 cells with various protein phosphatase inhibitors (okadaic acid and orthovanadate) or protein kinase activators (phorbol ester and forskolin). Incubation with okadaic acid, but not with orthovanadate, inhibited the ST‐IV activity to 45% of that of control cells with t1/2 = 60 min for the inactivation reaction. This indicates a rapid hyperphosphorylation of ST‐IV due to the inhibition of a serine/threonine‐specific phosphatase. A similar rate of inactivation was found on stimulation of protein kinase C with phorbol ester. In contrast to ST‐IV, the activity of GalNAc‐T was increased on stimulation of intracellular phosphorylation systems. The fastest activation of GalNAc‐T was achieved with forskolin, yielding up to 160% of the initial activity within 30 min of effector incubation. Up‐regulation of GalNAc‐T in conjunction with down‐regulation of ST‐IV by stimulation of phosphorylation is suggested to serve as a physiological mechanism to increase the concentration of GM1, which was found to be elevated in correlation with the cell density. This assumption was corroborated by metabolic labeling studies with radioactive ganglioside precursors indicating an enhancement of the relative amount of a‐series gangliosides subsequent to GM3 on phosphorylation stimulation. In particular, the biosynthesis of GM1 was specifically elevated within 2 h of incubation with forskolin. We conclude from the overall data that the ganglioside composition during the cell differentiation of NG108‐15 cells can be specifically regulated by both protein kinase A‐ and protein kinase C‐related phosphorylation systems.
Alkalinization Prolongs Recovery from Glutamate‐Induced Increases in Intracellular Ca2+ Concentration by Enhancing Ca2+ Efflux Through the Mitochondrial Na+/Ca2+ Exchanger in Cultured Rat Forebrain Neurons
Authors: Hoyt Kari R. , Reynolds Ian J.
Published: July 2010

Abstract: Increasing extracellular pH from 7.4 to 8.5 caused a dramatic increase in the time required to recover from a glutamate (3 µM, for 15 s)‐induced increase in intracellular Ca2+ concentration ([Ca2+]i) in indo‐1‐loaded cultured cortical neurons. Recovery time in pH 7.4 HEPES‐buffered saline solution (HBSS) was 126 ± 30 s, whereas recovery time was 216 ± 19 s when the pH was increased to 8.5. Removal of extracellular Ca2+ did not inhibit the prolongation of recovery caused by increasing pH. Extracellular alkalinization caused rapid intracellular alkalinization following glutamate exposure, suggesting that pH 8.5 HBSS may delay Ca2+ recovery by affecting intraneuronal Ca2+ buffering mechanisms, rather than an exclusively extracellular effect. The effect of pH 8.5 HBSS on Ca2+ recovery was similar to the effect of the mitochondrial uncoupler carbonyl cyanide p‐(trifluoromethoxyphenyl)hydrazone (FCCP; 750 nM). However, pH 8.5 HBSS did not have a quantitative effect on mitochondrial membrane potential comparable to that of FCCP in neurons loaded with a potential‐sensitive fluorescent indicator, 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolocarbocyanine iodide (JC‐1). We found that the effect of pH 8.5 HBSS on Ca2+ recovery was completely inhibited by the mitochondrial Na+/Ca2+ exchange inhibitor CGP‐37157 (25 µM). This suggests that increased mitochondrial Ca2+ efflux via the mitochondrial Na2+/Ca2+ exchanger is responsible for the prolongation of [Ca2+]i recovery caused by alkaline pH following glutamate exposure.