bluuebunny/crossref_metadata_2025 huggingface dataset:
Transgenic Murine Dopaminergic Neurons Expressing Human Cu/Zn Superoxide Dismutase Exhibit Increased Density in Culture, but No Resistance to Methylphenylpyridinium‐Induced Degeneration
Authors: Sanchez‐Ramos Juan R. , Song Shije , Facca Alicia , Basit Abdul , Epstein Charles J.
Published: July 2010

Abstract: Primary dopaminergic neuronal cultures with increased superoxide dismutase (SOD) activity were established for studying the role of superoxide anion (O2−) in 1‐methyl‐4‐phenylpyridinium (MPP+)‐induced degeneration of dopamine (DA) neurons. Mean SOD activity in cultures prepared from transgenic (human) Cu/Zn SOD (hSOD1) mice was 2.46–2.60 times greater than in cultures prepared from nontransgenic control mice. After 1 and 2 weeks in culture, the mean density of DA neurons [number of tyrosine hydroxylase‐immunoreactive (TH‐ir) cells per visual field] was significantly higher in cultures prepared from transgenic mice compared with those prepared from nontransgenic control mice (4.55–5.63 TH‐ir neurons per field in hSOD1 cultures vs. 2.66–2.8 TH‐ir neurons per field in control cultures). However, uptake of [3H]DA relative to uptake of [3H]GABA was only slightly greater in hSOD1 cultures than in normal cultures (14.1 nmol of DA/100 nmol of GABA vs. 12.1 nmol of DA/100 nmol of GABA). Resistance to MPP+ toxicity was not significantly different from that in normal cultures when based on density of surviving TH‐ir cell bodies (EC50 = 0.54 µM in hSOD1 and EC50 = 0.37 µM in normal cultures). A more sensitive measure of DA neuron integrity and function ([3H]DA uptake) also failed to demonstrate increased resistance of hSOD1 cultures to the toxin (EC50 = 73.7 nM in hSOD1 and EC50 = 86.2 nM in controls). These results do not support the hypothesis that neurotoxicity of the active metabolite of MPTP, MPP+, is mediated by generation of O2− in the cytoplasm. Nevertheless, mesencephalic cultures with increased hSOD1 activity appear to survive better than normal control cultures in the oxidatively stressful environment of cell culture incubators, and such mesencephalic cells may be useful for cell grafting studies in animal models of Parkinson's disease.
Neurotrophin Protection Against Toxicity Induced by Low Potassium and Nitroprusside in Cultured Cerebellar Granule Neurons
Authors: Yu Onnfoh , Chuang De‐Maw
Published: July 2010

Abstract: Long‐term survival of cultured rat cerebellar granule neurons requires depolarizing concentrations of potassium (high potassium; 25 mM KCl). A high‐potassium culturing condition has been reported to increase the intracellular calcium concentration ([Ca2+]i) and the expression of brain‐derived neurotrophic factor (BDNF), which in turn induces the expression of neurotrophin‐3 (NT‐3) in these neurons. We therefore examined the neurotrophic effect of these two neurotrophins in low‐potassium (5 mM) cultures and their neuroprotective capabilities against sodium nitroprusside‐induced neurotoxicity in both low‐ and high‐potassium cultures. Neuronal survival and neurotrophic effects were monitored by [3H]ouabain binding and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assays. In low‐potassium cultures, the neurotrophic effect of BDNF approached that found in high‐potassium cultures but was much more robust than that of NT‐3. In contrast, undifferentiated neurons cultured in high‐potassium medium were much less responsive to BDNF and not responsive at all to NT‐3. Induction of nitroprusside neurotoxicity occurred more readily in low‐ than in high‐potassium cultures. BDNF, NT‐3, and a high potassium concentration, alone or in combination, were unable to protect neurons treated with nitroprusside at 50 or 100 µM. However, the neurotoxicity of a lower dose of nitroprusside (10 µM) was reversed by the combined actions of these two neurotrophins in low‐potassium cultures and by BDNF alone in high‐potassium cultures. Because nitroprusside neurotoxicity is less robust in high‐potassium cultures, high‐potassium‐induced BDNF expression and subsequent NT‐3 expression may participate in its neuroprotection and neurotrophism in these cultures. Also, we found that toxic doses of nitroprusside antagonized KCl‐ and NMDA‐induced rises in [Ca2+]i, suggesting that this effect is related to nitroprusside‐induced neurotoxicity.
K‐252b Potentiation of Neurotrophin‐3 Is trkA Specific in Cells Lacking p75NTR
Authors: Maroney Anna C. , Sanders Christa , Neff Nicola T. , Dionne Craig A.
Published: July 2010

Abstract: K‐252b potentiates the neurotrophic effects of neurotrophin‐3 (NT‐3) in primary cultures of rat central cholinergic and peripheral sensory neurons and in a rat pheochromocytoma PC12 cell line. The ligand and receptor specificity, and role of the low‐affinity neurotrophin receptor (p75NTR) in the potentiation response induced by K‐252b, are unknown. To address the issues of ligand and receptor specificity of K‐252b potentiation, we have examined neurotrophin‐induced DNA synthesis ([3H]thymidine incorporation) in NIH3T3 cells expressing trkA, trkB, or trkC. Neither NT‐3 nor K‐252b alone could stimulate mitogenic activity in the trkA‐overexpressing clone. However, coaddition of K‐252b (EC50 of ∼2 nM) with 10–100 ng/ml NT‐3 led to incorporation of [3H]thymidine in trkA expressing cells to a level induced by optimal concentrations of nerve growth factor (NGF). The K‐252b‐ and NT‐3‐induced [3H]thymidine incorporation correlated with an increase in the tyrosine autophosphorylation of the trkA receptor as well as tyrosine phosphorylation of trk‐associated phospholipase C‐γ1 and SH2‐containing proteins. K‐252b did not potentiate submaximal doses of NGF, or maximal doses of brain‐derived neurotrophic factor (BDNF) or neurotrophin‐4/5 (NT‐4/5) in trkA‐expressing cells. Furthermore, K‐252b did not potentiate DNA synthesis by submaximal doses of BDNF, NT‐4/5, or NT‐3 in trkB‐ or trkC‐expressing NIH3T3 cells, suggesting that the potentiation profile for K‐252b was specific for NT‐3 in trkA‐expressing cells. We found no expression of p75NTR in the trk‐expressing NIH3T3 cells. This is the first demonstration that K‐252b potentiates a trkA‐mediated biological nonneuronal response by NT‐3 that occurs independent of p75NTR and appears to be both ligand and receptor specific.
Real‐Time Measurement of Electrically Evoked Extracellular Dopamine in the Striatum of Freely Moving Rats
Authors: Garris Paul A. , Christensen John R. C. , Rebec George V. , Wightman R. Mark
Published: July 2010

Abstract: The real‐time measurement of electrically evoked dopamine was established in brain extracellular fluid of freely moving rats. Dopamine was monitored by fast‐scan cyclic voltammetry at carbon fiber microelectrodes lowered into the striatum by means of a detachable micromanipulator. A stimulating electrode, previously implanted in the substantia nigra, was used to evoke striatal dopamine efflux. Evoked extracellular dopamine was both current and frequency dependent. When low current intensities (±125 µA) and frequencies (10–20 Hz) were applied, detectable levels of dopamine were elicited without a perceptible behavioral response. Reproducible concentrations of extracellular dopamine could be evoked in the same rat for at least 2 months. These concentrations, moreover, were significantly higher in freely moving rats compared with rats anesthetized with Equithesin. Analysis of measured curves for dopamine uptake and release rates revealed that anesthesia inhibits release but does not affect uptake. It is concluded that (a) fast‐scan cyclic voltammetry at carbon fiber microelectrodes is a viable technique for the measurement of electrically evoked dopamine in brain extracellular fluid of freely moving rats, (b) it is possible to determine in situ rate constants for dopamine release and uptake from these temporally and spatially resolved measurements of levels of dopamine, and (c) transient changes in extracellular dopamine levels elicited by electrical stimulation are affected by anesthesia.
Coloboma Hyperactive Mutant Mice Exhibit Regional and Transmitter‐Specific Deficits in Neurotransmission
Authors: Raber Jacob , Mehta Prashant P. , Kreifeldt Max , Parsons Loren H. , Weiss Friebert , Bloom Floyd E. , Wilson Michael C.
Published: July 2010

Abstract: The mouse mutant coloboma (Cm/+), which exhibits profound spontaneous hyperactivity and bears a deletion mutation on chromosome 2, including the gene encoding synaptosomal protein SNAP‐25, has been proposed to model aspects of attention‐deficit hyperactivity disorder. Increasing evidence suggests a crucial role for SNAP‐25 in the release of both classical neurotransmitters and neuropeptides. In the present study, we compared the release of specific neurotransmitters in vitro from synaptosomes and slices of selected brain regions from Cm/+ mice with that of +/+ mice. The release of dopamine (DA) and serotonin (5‐HT) from striatum, and of arginine vasopressin and corticotropin‐releasing factor from hypothalamus and amygdala is calcium‐dependent. Glutamate release from and content in cortical synaptosomes of Cm/+ mice are greatly reduced, which might contribute to the learning deficits in these mutants. In dorsal striatum of Cm/+ mutants, but not ventral striatum, KCI‐induced release of DA is completely blocked and that of 5‐HT is significantly attenuated, suggesting that striatal DA and 5‐HT deficiencies may be involved in hyperactivity. Further, although acetylcholine failed to induce hypothalamic corticotropin‐releasing factor release from Cm/+ slices, restraint stress increased plasma corticosterone levels in Cm/+ mice to a significantly higher level than in +/+ mice, suggesting an important role for arginine vasopressin in hypothalamic‐pituitary‐adrenal axis activation. These results suggest that reduced SNAP‐25 expression may contribute to a region‐specific and neurotransmitter‐specific deficiency in neurotransmitter release.
Phorbol Esters Increase Dopamine Transporter Phosphorylation and Decrease Transport Vmax
Authors: Huff Robin A. , Vaughan Roxanne A. , Kuhar Michael J. , Uhl George R.
Published: July 2010

Abstract: Sodium‐ and chloride‐coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport Vmax in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC‐PK1 cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12‐myristate 13‐acetate (PMA) reduces transport Vmax by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter Vmax suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC‐activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.
Kainate‐Evoked Release of Adenosine from the Hippocampus of the Anaesthetised Rat: Possible Involvement of Free Radicals
Authors: Carswell H. V. , Graham D. I. , Stone T. W.
Published: July 2010

Abstract: Using microdialysis in the hippocampus of anaesthetised rats, the concentration of extracellular adenosine was estimated to be 0.8 µM. Kainic acid (0.1–25 mM) in the perfusate evoked a concentration‐dependent release of adenosine with an EC50 of 940 µM. Two 5‐min pulses of 1 mM kainic acid in the perfusate increased the dialysate levels with an S2/S1 ratio of 0.52 ± 0.03. Kainate‐evoked release of adenosine was reduced significantly by 10 µM tetrodotoxin and by a κ‐receptor agonist, U50,488H (100 µM). The S2/S1 ratio was reduced by 4.5 µM 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, a non‐NMDA receptor antagonist, but not by the NMDA receptor blockers (+)‐MK‐801 (dizocilpine; 100 µM) or (±)‐2‐amino‐5‐phosphonopentanoic acid (1 mM), indicating a non‐NMDA receptor‐mediated process. The S2/S1 ratio was also reduced significantly by 10 mM ascorbic acid, 10 mM glutathione (a scavenger of hydroperoxides), and 1 mM oxypurinol (a xanthine oxidase inhibitor), indicating the possible involvement of free radicals. Neither the adenosine A1 receptor antagonist 8‐cyclopentyl‐1,3‐dimethylxanthine (100 µM) nor the A1 adenosine receptor agonist R(−)‐N6‐(2‐phenylisopropyl)adenosine (100 µM) affected release. Adenosine release evoked by kainic acid is therefore mediated by activation of non‐NMDA receptors and may involve the propagation of action potentials and the production of free radicals.
Amyloid β Protein Potentiates Ca2+ Influx Through L‐Type Voltage‐Sensitive Ca2+ Channels: A Possible Involvement of Free Radicals
Authors: Ueda Keiichi , Shinohara Shunji , Yagami Tatsurou , Asakura Kenji , Kawasaki Kazuo
Published: July 2010

Abstract: Amyloid β protein (Aβ), the central constituent of senile plaques in Alzheimer's disease (AD) brain, is known to exert toxic effects on cultured neurons. The role of the voltage‐sensitive Ca2+ channel (VSCC) in β(25–35) neurotoxicity was examined using rat cultured cortical and hippocampal neurons. When L‐type VSCCs were blocked by application of nimodipine, β(25–35) neurotoxicity was attenuated, whereas application of ω‐conotoxin GVIA (ω‐CgTX‐GVIA) or ω‐agatoxin IVA (ω‐Aga‐IVA), the blocker for N‐ or P/Q‐type VSCCs, had no effects. Whole‐cell patch‐clamp studies indicated that the Ca2+ current density of β(25–35)‐treated neurons is about twofold higher than that of control neurons. Also, β(25–35) increased Ca2+ uptake, which was sensitive to nimodipine. The 2′,7′‐dichlorofluorescin diacetate assay showed the ability of β(25–35) to produce reactive oxygen species. Nimodipine had no effect on the level of free radicals. In contrast, vitamin E, a radical scavenger, reduced the level of free radicals, neurotoxicity, and Ca2+ uptake. These results suggest that β(25–35) generates free radicals, which in turn, increase Ca2+ influx via the L‐type VSCC, thereby inducing neurotoxicity.
Effect of Ca2+ on In Vitro Astrocyte Injury
Authors: Rzigalinski Beverly A. , Liang Shi , McKinney Jerry S. , Willoughby Karen A. , Ellis Earl F.
Published: July 2010

Abstract: Current literature suggests that a massive influx of Ca2+ into the cells of the CNS induces cell damage associated with traumatic brain injury (TBI). Using an in vitro model for stretch‐induced cell injury developed by our laboratory, we have investigated the role of extracellular Ca2+ in astrocyte injury. The degree of injury was assessed by measurement of propidium iodide uptake and release of lactate dehydrogenase. Based on results of in vivo models of TBI developed by others, our initial hypothesis was that decreasing extracellular Ca2+ would result in a reduction in astrocyte injury. Quite unexpectedly, our results indicate that decreasing extracellular Ca2+ to levels observed after in vivo TBI increased astrocyte injury. Elevating the extracellular Ca2+ content to twofold above physiological levels (2 mM) produced a reduction in cell injury. The reduction in injury afforded by Ca2+ could not be mimicked with Ba2+, Mn2+, Zn2+, or Mg2+, suggesting that a Ca2+‐specific mechanism is involved. Using 45Ca2+, we demonstrate that injury induces a rapid influx of extracellular Ca2+ into the astrocyte, achieving an elevation in total cell‐associated Ca2+ content two‐ to threefold above basal levels. Pharmacological elevation of intracellular Ca2+ levels with the Ca2+ ionophore A23187 or thapsigargin before injury dramatically reduced astrocyte injury. Our data suggest that, contrary to popular assumptions, an elevation of total cell‐associated Ca2+ reduces astrocyte injury produced by a traumatic insult.
Effect of Pentylenetetrazole‐Induced Kindling on Acetylcholine Release in the Hippocampus of Freely Moving Rats
Authors: Serra Mariangela , Dazzi Laura , Cagetti Elisabetta , Chessa M. Francesca , Pisu M. Giuseppina , Sanna Angela , Biggio Giovanni
Published: July 2010

Abstract: The role of γ‐aminobutyric acid (GABA) modulation of septohippocampal cholinergic neurons in kindling was investigated. Hippocampal acetylcholine release was evaluated with the microdialysis technique in freely moving rats either after acute administration of isoniazid (an inhibitor of GABA synthesis) or pentylenetetrazole (PTZ)(a blocker of the GABAA receptor‐associated Cl− channel) or after chronic administration of PTZ. Short‐term treatment with PTZ (5–50 mg/kg, i.p.) or isoniazid (150–250 mg/kg, s.c.) increased hippocampal acetylcholine release in a dose‐dependent manner. In contrast, the basal concentration of acetylcholine in the dialysate from the hippocampus of rats chronically treated with PTZ (kindled animals) was significantly reduced relative to that of vehicle‐treated rats (2.39 ± 0.21 vs. 4.2 ± 0.31 pmol per 20‐min sample; p < 0.01). Moreover, the release of acetylcholine was markedly more sensitive to the effect of a challenge injection of PTZ (10 or 20 mg/kg, i.p.) in kindled rats than in naive rats or rats chronically treated with vehicle. Abecarnil, a selective benzodiazepine receptor agonist with marked anticonvulsant activity, was administered together with chronic PTZ to evaluate whether persistent activation of GABAA receptors and suppression of seizures during kindling might affect the sensitivity of septohippocampal cholinergic neurons to a challenge dose of PTZ. Abecarnil (1 mg/kg, i.p.) administered 40 min before each PTZ injection neither antagonized the decrease in basal acetylcholine release (2.26 ± 0.19 pmol per 20‐min sample) nor prevented the development of kindling. In contrast, abecarnil prevented the chronic PTZ‐induced increase in the sensitivity of acetylcholine release to a challenge dose of PTZ. These results provide novel in vivo data concerning the role of hippocampal acetylcholine function in the development of kindling and potentially in the learning and memory deficits associated with this phenomenon.
Inhibition of β‐Amyloid Formation by Haloperidol: A Possible Mechanism for Reduced Frequency of Alzheimer's Disease Pathology in Schizophrenia
Authors: Higaki Jeffrey , Murphy Greer M. , Cordell Barbara
Published: July 2010

Abstract: Several reports have suggested that the frequency of Alzheimer's disease (AD) neuropathology is significantly reduced in elderly individuals with schizophrenia (SZ), and it has been proposed that medications used for treatment of SZ may be responsible. A central event in AD pathology is the formation of β‐amyloid (Aβ) peptide, which is derived by enzymatic processing of its precursor protein. Haloperidol, an antipsychotic medication commonly used in the treatment of SZ, can act as an inhibitor of select proteinases; hence, we examined the ability of this compound to inhibit Aβ formation by cultured cells. Haloperidol and, to a lesser extent, droperidol inhibited Aβ in a dose‐dependent manner. These results may explain the apparent reduction of AD neuropathological changes in elderly patients with SZ as well as provide a possible mechanism for this difference.
Portacaval Shunting and Hyperammonemia Stimulate the Uptake of l‐[3H]Arginine but Not of l‐[3H]Nitroarginine into Rat Brain Synaptosomes
Authors: Raghavendra Rao V. L. , Audet R. M. , Butterworth R. F.
Published: July 2010

Abstract: Elevated activities of nitric oxide synthase (NOS) have been reported previously in the brains of portacaval‐shunted (PCS) rats, a model of chronic hepatic encephalopathy (HE). As l‐arginine availability for nitric oxide synthesis depends on a specific uptake mechanism in neurons, we studied the kinetics of l‐[3H]‐arginine uptake into synaptosomes prepared from the brains of PCS rats. Results demonstrate that l‐arginine uptake is significantly increased in cerebellum (60%; p < 0.01), cerebral cortex (42%; p < 0.01), hippocampus (56%; p < 0.01), and striatum (51%; p < 0.01) of PCS rats compared with sham‐operated controls. Hyperammonemia in the absence of portacaval shunting also stimulated the transport of l‐[3H]arginine; kinetic analysis revealed that the elevated uptake was due to increased uptake capacity (Vmax) without any change in affinity (Km). Incubation of cerebellar synaptosomes with ammonium acetate for 10 min caused a dose‐dependent stimulation of l‐[3H]arginine uptake. Neither portacaval shunting nor hyperammonemia had any significant effect on the synaptosomal uptake of NG‐nitro‐l‐[3H]arginine. These studies demonstrate that increased NOS activity observed in experimental HE may result from increased availability of l‐arginine resulting from a direct stimulatory effect of ammonia on l‐arginine transport.
Characterization of CB1 Receptors on Rat Neuronal Cell Cultures: Binding and Functional Studies Using the Selective Receptor Antagonist SR 141716A
Authors: Jung M. , Calassi R. , Rinaldi‐Carmona M. , Chardenot P. , Le Fur G. , Soubrié P. , Oury‐Donat F.
Published: July 2010

Abstract: This study was undertaken to characterize further the central cannabinoid receptors in rat primary neuronal cell cultures from selected brain structures. By using [3H]SR 141716A, the specific CB1 receptor antagonist, we demonstrate in cortical neurons the presence of a high density of specific binding sites (Bmax = 139 ± 9 fmol/mg of protein) displaying a high affinity (KD = 0.76 ± 0.09 nM). The two cannabinoid receptor agonists, CP 55940 and WIN 55212‐2, inhibited in a concentration‐dependent manner cyclic AMP production induced by either 1 µM forskolin or isoproterenol with EC50 values in the nanomolar range (4.6 and 65 nM with forskolin and 1.0 and 5.1 nM with isoproterenol for CP 55940 and WIN 55212‐2, respectively). Moreover, in striatal neurons and cerebellar granule cells, CP 55940 was also able to reduce the cyclic AMP accumulation induced by 1 µM forskolin with a potency similar to that observed in cortical neurons (EC50 values of 3.5 and 1.9 nM in striatum and cerebellum, respectively). SR 141716A antagonized the CP 55940‐ and WIN 55212‐2‐induced inhibition of cyclic AMP accumulation, suggesting CB1 receptor‐specific mediation of these effects on all primary cultures tested. Furthermore, CP 55940 was unable to induce mitogen‐activated protein kinase activation in either cortical or striatal neurons. In conclusion, our results show nanomolar efficiencies for CP 55940 and WIN 55212‐2 on adenylyl cyclase activity and no effect on any other signal transduction pathway investigated in primary neuronal cultures.
Implication of Ca2+‐Dependent Protein Tyrosine Phosphorylation in Carbachol‐Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells
Authors: Ito Yuzuru , Nakashima Shigeru , Kanoh Hiroyuki , Nozawa Yoshinori
Published: July 2010

Abstract: The mechanism for carbachol (CCh)‐induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid‐labeled pheochromocytoma PC12 cells with respect to the involvement of protein tyrosine phosphorylation and Ca2+. PLD activity was assessed by measuring the formation of [3H]phosphatidylbutanol in the presence of 0.3% butanol. Pretreatment of cells with the tyrosine kinase inhibitors herbimycin A, genistein, and tyrphostin inhibited PLD activation by CCh. Western blot analysis revealed several apparent tyrosine‐phosphorylated protein bands (111, 91, 84, 74, 65–70, 44, and 42 kDa) in PC12 cells treated with CCh. Phosphorylation of the 111‐, 91‐, 84‐, and 65–70‐kDa proteins peaked within 1 min, and their time‐dependent changes seemingly correlated with that of PLD activation. Others (74, 44MAPK, and 42MAPK kDa) were phosphorylated rather slowly, and maximal tyrosine phosphorylation was observed at 2 min. Herbimycin A inhibited PLD activity and tyrosine phosphorylation of four proteins (111, 91, 84, and 65–70 kDa) in a preincubation time‐ and concentration‐dependent fashion. In Ca2+‐free buffer, CCh‐induced [3H]phosphatidylbutanol formation and protein tyrosine phosphorylation were abolished. A Ca2+ ionophore, A23187, caused PLD activation and tyrosine phosphorylation of four proteins of 111, 91, 84, and 65–70 kDa only in the presence of extracellular Ca2+. Extracellular Ca2+ dependency for CCh‐induced PLD activation was well correlated with that for tyrosine phosphorylation of the four proteins listed above, especially the 111‐kDa protein. These results suggest that Ca2+‐dependent protein tyrosine phosphorylation is closely implicated in CCh‐induced PLD activation in PC12 cells.
The τ Protein in Human Cerebrospinal Fluid in Alzheimer's Disease Consists of Proteolytically Derived Fragments
Authors: Johnson Gail V. W. , Seubert Peter , Cox Teresa M. , Motter Ruth , Brown Jason P. , Galasko Douglas
Published: July 2010

Abstract: Previous studies have shown that the levels of the microtubule‐associated protein τ in the CSF of patients with Alzheimer's disease (AD) are elevated compared with age‐matched controls. In spite of these findings, the nature of τ in CSF has not been well documented. In the present study, τ was immunoprecipitated from CSF of patients with AD or acute stroke, as well as normal elderly controls, followed by immunoblot analysis. In all cases, CSF τ consisted primarily of a band migrating at 26–28 kDa. In AD and stroke patients, several smaller τ fragments were also detected. No intact τ was detected in any of the CSF samples examined. Further immunoprecipitation studies showed that the majority of the τ fragments contained the amino terminus of the molecule. Treatment of CSF τ with alkaline phosphatase did not alter the electrophoretic properties of the fragments. These studies clearly demonstrate that CSF τ is truncated rather than intact.
Interactions of Muscarinic Receptors with the Heterotrimeric G Proteins Gq and G12: Transduction of Proliferative Signals
Authors: Burstein E. S. , Bräuner‐Osborne H. , Spalding T. A. , Conklin B. R. , Brann M. R.
Published: July 2010

Abstract: The proliferative and transforming properties of m2 and m5 muscarinic acetylcholine receptors and a series of wild‐type, chimeric, and mutant G proteins were measured alone or in combination in NIH 3T3 cells to determine which G proteins mediate these signals and to what extent these signals can be influenced by changing the stoichiometry of receptors and G proteins. Responses were measured using the focus‐forming assay and a novel assay called R‐SAT (Receptor Selection and Amplification Technology) in which proliferative responses are monitored using a reporter gene. Individually, GTPase‐deficient mutants (⋆) of Gαq and Gα12, wild‐type Gαq, and m5 were active in R‐SAT. Gα12⋆ and m5 also induced focus formation. m2 was inactive in both assays. The ability of m5 to induce foci was significantly reduced by coexpression of Gαq⋆. Synergistic effects of receptor/G protein combinations were not observed in focus‐forming assays but were readily detected by R‐SAT. Coexpression of Gαq with m5 induced constitutive activity in R‐SAT and increased the potency of agonists at m5 by 90‐fold. Gαq also evoked agonist‐dependent responses from m2 but not constitutive activity. Agonist potency was increased 10‐fold at m2 and decreased 15‐fold at m5 when these receptors were coexpressed with Gαqi5, a chimeric G protein containing the five C‐terminal residues of Gαi2, compared with coexpression with Gαq. Both Gαq and Gαqi5 had biphasic effects on the proliferative responses to m5 and m2, respectively, inhibiting responses at high agonist concentrations. Coexpression of Gα12 or Gα12i5 had no effect on the concentration‐response relationships of m5, but both elicited weak responses from m2. We conclude that although Gα12 is a more potent oncogene, Gαq transduces m5‐driven cellular responses. The demonstrations that proliferative responses can be elicited from a nonmitogenic receptor by altering the type and concentration of available G proteins and that constitutive responses can be induced by G proteins imply that both the magnitude and type of receptor‐initiated signal can be regulated at the level of G proteins in vivo.
L1/HNK‐1 Carbohydrate‐ and β1 Integrin‐Dependent Neural Cell Adhesion to Laminin‐1
Authors: Hall Heike , Carbonetto Salvatore , Schachner Melitta
Published: July 2010

Abstract: We have shown recently that mouse small cerebellar neurons adhere to a short amino acid sequence of the G2 domain of the laminin α1 chain via the cell surface‐expressed HNK‐1 carbohydrate. Therefore, we were interested in identifying glycoproteins carrying the HNK‐1 carbohydrate at the cell surface of these neurons. Adhesion of small cerebellar neurons to laminin is partially dependent on Ca2+, Mn2+, and Mg2+, indicating the involvement of integrins, which were identified as β1, α3, and α6. They could be shown to bind to laminin by a β1‐dependent adhesion mechanism. None of these subunits was found to carry the HNK‐1 carbohydrate. HNK‐1‐immunoreactive glycoproteins were immunoprecipitated and shown to consist of predominantly one molecular species, which was identified as the neural cell recognition molecule L1. L1 was demonstrated to bind in a concentration‐dependent and saturating manner to laminin. The binding could be partially inhibited by Fab fragments of monoclonal antibodies against the HNK‐1 carbohydrate and against the Ig‐like domains of L1. Furthermore, antibodies to the Ig‐like domains of L1 and β1 integrin inhibited partially cell adhesion to laminin. Determination of the association of L1, β1 integrin, and the HNK‐1 carbohydrate on the cell surface of live cerebellar neurons by antibody‐induced patching and copatching revealed HNK‐1 to be linked to L1, but less so to β1 integrin. However, only negligible association was found between L1 and β1 integrin. Furthermore, it could be shown that adhesion to laminin is mediated by L1/HNK‐1‐ and β1 integrin‐dependent mechanisms that act at least partially independent of each other.
Expression of Endothelin‐Converting Enzyme in Both Neuroblastoma and Glial Cell Lines and Its Localization in Rat Hippocampus
Authors: Barnes Kay , Walkden Brendan J. , Wilkinson Tamsin C. , Turner Anthony J.
Published: July 2010

Abstract: Endothelin‐converting enzyme is a phosphoramidon‐sensitive metalloprotease that cleaves big endothelin to the potent vasoconstrictor peptide, endothelin. The converting enzyme is expressed in endothelial cells in a variety of tissues and in some secretory cells. In the present study, phosphoramidon‐sensitive endothelin‐converting enzyme activity has been demonstrated by radioimmunoassay in the neuroblastoma cell line, SH‐SY5Y, and in Bu17 and C6 glioma lines. The identity of the activity was confirmed by immunoblotting, revealing a polypeptide of ∼120 kDa in each of these lines, in D384 glioma cells, and in primary astrocytes. Immunofluorescence revealed the cell‐surface location of endothelin‐converting enzyme in the neuronal and glial cell lines and in primary astrocytes. Pretreatment of SH‐SY5Y and Bu17 cells with phosphoramidon resulted in an apparent concentration of the enzyme protein in an intracellular compartment. Immunoperoxidase‐staining of rat brain sections located this metalloprotease to the pyramidal cells of the hippocampus. Endothelin‐converting enzyme‐1 was revealed by in situ hybridisation in the neuronal and glial cell lines.
Expression of the Nicotinic Receptor α7 Gene in Tendon and Periosteum During Early Development
Authors: Romano Suzanne J. , Corriveau Roderick A. , Schwarz Richard I. , Berg Darwin K.
Published: July 2010

Abstract: One of the most abundant nicotinic acetylcholine receptors expressed in the central and peripheral nervous systems is a species that contains the α7 gene product, binds α‐bungarotoxin with high affinity, and has a high relative permeability to calcium. The α7 gene is also expressed at low levels in embryonic muscle tissue. We show here that the α7 gene is expressed in tendon fibroblasts and periosteal cells during development. In situ hybridizations identify α7 transcripts in tissue sections containing embryonic tendon and periosteum. RNase protection experiments demonstrate α7 mRNA in primary tendon cells grown in culture. Immunofluorescence with subunit‐specific monoclonal antibodies reveals α7 protein in embryonic tendon. Immunoprecipitation assays with the antibodies indicate that the α7‐containing species in tendon is capable of binding α‐bungarotoxin and that a similar species can be identified at low levels on the surface of fibroblasts in culture. The results show that the α7 gene product is expressed in a range of tissues, including cells thought to be nonexcitable. The distribution of α7 expression early in development and the ability of α7‐containing receptors to elevate intracellular calcium suggest that the gene may influence a variety of calcium‐dependent events during embryogenesis.
Monoamine Metabolism in the Locus Coeruleus Measured Concurrently with Behavior During Opiate Withdrawal: An In Vivo Microdialysis Study in Freely Moving Rats
Authors: Javelle Nathalie , Renaud Bernard , Lambás‐Señas Laura
Published: July 2010

Abstract: Using microdialysis, changes in monoamine metabolism were monitored in the locus coeruleus of freely moving rats during opiate withdrawal concomitantly with behavioral symptoms. Rats were infused with morphine (2 mg/kg/h, s.c.) or saline for 5 days and challenged with naltrexone (100 mg/kg, s.c.) on day 6. Following naltrexone challenge, the classic behavioral symptoms of morphine withdrawal were observed in rats treated with morphine but not in saline‐infused rats. In morphine‐dependent rats, naltrexone induced a marked increase (280%) in dialysate concentrations of 3,4‐dihydroxyphenylacetic acid, an index of the functional activity of the noradrenergic neurons in the locus coeruleus. The local concentrations of the serotonin metabolite 5‐hydroxyindoleacetic acid were also increased (70%) during morphine withdrawal. Taken together, these results (a) confirm in unanesthetized rats the hypothesis of an activation by opiate withdrawal of noradrenergic neurons in the locus coeruleus and (b) suggest an increase in serotonergic transmission in the same nucleus during morphine withdrawal.
Proteasome Contributes to the α‐Secretase Pathway of Amyloid Precursor Protein in Human Cells
Authors: Marambaud Philippe , Chevallier Nathalie , Barelli Helene , Wilk Sherwin , Checler Frederic
Published: July 2010

Abstract: A major histopathological hallmark in Alzheimer's disease consists of the extracellular deposition of the amyloid β‐peptide (Aβ) that is proteolytically derived from the β‐amyloid precursor protein (βAPP). An alternative, nonamyloidogenic cleavage, elicited by a protease called α‐secretase, occurs inside the Aβ sequence and gives rise to APPα, a major secreted C‐terminal‐truncated form of βAPP. Here, we demonstrate that human embryonic kidney 293 (HK293) cells contain a chymotryptic‐like activity that can be ascribed to the proteasome and that selective inhibitors of this enzyme reduce the phorbol 12,13‐dibutyrate‐sensitive APPα secretion by these cells. Furthermore, we establish that a specific proteasome blocker, lactacystin, also induces increased secretion of Aβ peptide in stably transfected HK293 cells overexpressing wild‐type βAPP751. Altogether, this study represents the first identification of a proteolytic activity, namely, the proteasome, contributing likely through yet unknown intracellular relays, to the α‐secretase pathway in human cells.
Neuroprotection of Lubeluzole Is Mediated Through the Signal Transduction Pathways of Nitric Oxide
Authors: Maiese Kenneth , TenBroeke Michelle , Kue Ia
Published: July 2010

Abstract: Neuronal survival after ischemic injury is determined through the induction of several biological pathways. We examined whether lubeluzole, an agent efficacious in both clinical and experimental models of cerebral ischemia, modulated the signal transduction mechanisms of nitric oxide (NO), a downstream mediator of anoxic neurodegeneration. Both pretreatment [NO survival = 23 ± 3%, NO/lubeluzole (750 nM) survival = 63 ± 2%, p < 0.001] and coadministration [NO survival = 25 ± 3%, NO/lubeluzole (750 nM) survival = 59 ± 3%, p < 0.001] of lubeluzole with NO generators equally protected cultured hippocampal neurons in a dose‐dependent manner against the toxic effects of NO, suggesting that the agent protects by acutely modifying toxic cellular pathways rather than preconditioning the neuron before injury. The protection observed with lubeluzole was stereospecific but was not limited to pre‐ or coadministration. Lubeluzole also was found to significantly protect against the toxicity of NO for a period of 4–6 h after NO exposure [NO survival = 31 ± 2%, NO/lubeluzole (750 nM) survival at 6 h = 56 ± 3%, p < 0.001]. We conclude that the neuroprotective ability of lubeluzole is unique and involves the direct modulation of the NO pathway. In addition, the mechanisms of NO toxicity are dynamic and reversible processes that, if left unaltered, will lead to neuronal injury. Further investigation of the downstream signal transduction mechanisms below the level of NO generation may elucidate the specific cellular events responsible for neurodegeneration.
Characterization of the Binding of Amyloid‐β Peptide to Cell Culture‐Derived Native Apolipoprotein E2, E3, and E4 Isoforms and to Isoforms from Human Plasma
Authors: Yang Dun‐Sheng , Smith Jonathan D. , Zhou Zhongmin , Gandy Samuel E. , Martins Ralph N.
Published: July 2010

Abstract: The ε4 allele of apolipoprotein E (apoE, protein; APOE, gene) is a major risk factor for Alzheimer's disease (AD). Genetically, the frequency of the ε4 allele is enriched in early‐onset sporadic, late‐onset familial, and common late‐onset sporadic AD. ApoE is found in the extracellular amyloid‐β (Aβ) deposits that are characteristic features of AD. In this study, we examined the interaction between Aβ and apoE isoforms. The apoE isoforms used in this study were either produced by stably transfected Chinese hamster ovary cells (CHO) or were from human plasma. We report that when similar concentrations of the apoE isoforms were used, native nonpurified apoE3 from recombinant CHO‐derived sources bound Aβ, but apoE4 did not. In fact, in our system, binding of recombinant apoE4 to Aβ was never detectable, even after incubation for 4 days. Furthermore, using the same assay conditions, native apoE2, like apoE3, binds Aβ avidly. Furthermore, when human plasma apoE isoforms are tested in Aβ binding experiments, apoE3 bound Aβ more avidly than apoE4, and a major apoE/Aβ complex (the 40‐kDa form) was observed with plasma apoE3 but not apoE4. These data extend our understanding of apoE isoform‐dependent binding of Aβ by associating apoE2 with efficient apoE/Aβ complex formation and demonstrate that native apoE3 (whether recombinant or derived from human plasma) forms sodium dodecyl sulfate‐stable apoE/Aβ complexes more readily than native apoE4. The different Aβ‐binding properties of native apoE4 versus native apoE3 provide insight into the molecular mechanisms by which the APOE ε4 allele exerts its risk factor effects in AD.
Glucosylceramide Synthesis Is Required for Basic Fibroblast Growth Factor and Laminin to Stimulate Axonal Growth
Authors: Boldin Swetlana , Futerman Anthony H.
Published: July 2010

Abstract: To test the hypothesis that neuronal growth requires the synthesis and supply of new membrane components to the growing neurite, we have examined the relationship between the synthesis of sphingolipids and the ability of two growth factors, basic fibroblast growth factor (bFGF) and laminin, to stimulate axonal growth in cultured hippocampal neurons. Both bFGF and laminin stimulate axonal growth by approximately fourfold, but the stimulatory effects of both factors can be abolished completely by two inhibitors of sphingolipid synthesis, fumonisin B1 and d‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol. By using these inhibitors, together with two stereoisomers of short acyl chain derivatives of ceramide, only one of which is metabolized to glucosylceramide, we demonstrate that ongoing synthesis of glucosylceramide, the simplest glycosphingolipid, is a prerequisite for both bFGF and laminin to stimulate axon growth. These data imply that the ability of a growth factor to stimulate neuronal growth is dependent on the synthesis of an essential membrane lipid.
Evidence for Multiple, Local Functions of Ciliary Neurotrophic Factor (CNTF) in Retinal Development: Expression of CNTF and Its Receptor and In Vitro Effects on Target Cells
Authors: Kirsch Matthias , Lee Mun‐Yong , Meyer Verena , Wiese Annette , Hofmann Hans‐Dieter
Published: July 2010

Abstract: There is increasing, although largely indirect, evidence that neurotrophic factors not only function as target‐derived survival factors for projection neurons, but also act locally to regulate developmental processes. We studied the expression of ciliary neurotrophic factor (CNTF) and the CNTF‐specific ligand‐binding α‐subunit of the CNTF receptor complex (CNTFRα) in the rat retina, a well‐defined CNS model system, and CNTF effects on cultured retinal neurons. Both CNTF and CNTFRα (mRNA and protein) are expressed during phases of retinal neurogenesis and differentiation. Retina‐specific Müller glia are immunocytochemically identified as the site of CNTF production and CNTFRα‐expressing, distinct neuronal cell types as potential CNTF targets. Biological effects on corresponding neurons in culture further support the conclusion that locally supplied CNTF plays a regulatory role in the development of various retinal cell types including ganglion cells and interneurons.
Identification of a Neuronal Cell Surface Receptor for a Growth Inhibitory Chondroitin Sulfate Proteoglycan (NG2)
Authors: Dou Chang‐Lin , Levine Joel M.
Published: July 2010

Abstract: The NG2 chondroitin sulfate proteoglycan inhibits neurite outgrowth from neonatal rat cerebellar granule neurons when presented to the neurons as a component of the substrate. To begin to understand the cellular mechanisms by which this inhibition occurs, we investigated the hypothesis that cerebellar granule neurons express cell surface receptors for NG2 and that these receptors are linked to cellular signaling pathways. Here, we show that the NG2 core protein binds specifically and with high affinity to cerebellar granule neurons. Using protein cross‐linking techniques and immunoprecipitation, a 280‐kDa membrane cell surface protein of granule neurons was identified as an NG2‐binding site. Treatment of the neurons with pertussis toxin reversed the growth inhibition, suggesting a role for pertussis toxin‐sensitive G proteins in the inhibitory response. Treatment of the neurons with pharmacological agents that increase either intracellular calcium or intracellular cyclic AMP levels partially reversed the growth inhibition induced by NG2. These results suggest that the growth‐inhibitory actions of NG2 proteoglycan are due to an interaction with a specific cell surface receptor that is linked, either directly or indirectly, to intracellular second messenger systems.
Gene Expression and Regulation of Aplysia californica Prohormone Convertases aPC1B and aPC2
Authors: Ouimet Tanja , Castellucci Vincent F.
Published: July 2010

Abstract: Peptide diversity can be regulated in many ways. One of the possible mechanisms of such a regulation may lie in the differential processing of a precursor in a tissue‐specific manner or in a modulated processing within a single cell. To address these questions the enzymes responsible for the release of active neuropeptides first need to be well characterized. In this study, we have established the cellular distributions of the recently cloned prohormone convertases aPC2 and aPC1B in Aplysia californica and found the distribution of these enzymes to be mainly neuronal and endocrine. We then investigated in two sets of neurons (bag cell neurons and sensory neurons) the possible regulation of these two prohormone convertases by a neurotransmitter known to modulate behavior in Aplysia. In these neurons, we found that only aPC2 is regulated by serotonin possibly via the cyclic AMP cascade. Finally, a study of the biosynthesis of the bag cell egg‐laying hormone precursor after treatment with serotonin suggests that such a treatment may increase its processing.
Regulation of μ‐Opioid Receptor in Neural Cells by Extracellular Sodium
Authors: Yabaluri Nirmala , Medzihradsky Fedor
Published: July 2010

Abstract: SH‐SY5Y neural cells expressing μ‐ and δ‐opioid receptors were maintained viable in isotonic, sodium‐free buffer in vitro. Intracellular sodium levels were manipulated by various methods, and ligand binding to intact cells was studied. In physiological buffer containing 118 mM sodium, [3H]Tyr‐d‐Ala‐Gly‐(Me)Phe‐Gly‐ol ([3H]‐DAMGO) and [3H]naltrexone bound to μ receptor with KD values of 3.1 and 0.32 nM and Bmax values of 94 and 264 fmol/mg of protein, respectively. Replacement of sodium by choline decreased the affinity of the antagonist and increased Bmax for [3H]DAMGO, without significantly affecting the other corresponding binding parameters. Depolarizing concentrations of KCl (34 mM) in physiological buffer decreased the intracellular sodium levels by 67%, but this did not decrease the [3H]DAMGO binding to the cells. Incubation of cells with monensin and ouabain increased the intracellular sodium levels dramatically (from 78 to 250 and 300 nmol/mg, respectively), with no changes in agonist binding parameters. Ethylisopropylamiloride inhibited [3H]DAMGO and [3H]naloxone binding to intact cells with EC50 values of 24 and 3,600 nM, respectively. Adenylyl cyclase activities measured in intact cells, at different concentrations of sodium, showed the physiological significance of this ion in signal transduction. Potency of DAMGO in inhibiting the forskolin‐stimulated adenylyl cyclase activity was significantly higher at lower concentrations of sodium. However, inhibition reached the maximal level only at 50 mM sodium, and typical sigmoidal dose‐response curves were obtained only in the presence of 118 mM sodium. Furthermore, even at low or high intracellular sodium levels, DAMGO inhibition of cyclic AMP levels was normal. These results support a role for extracellular sodium in regulating not only the ligand interactions with the receptor, but also the signal transduction through the μ receptor.