bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidant Injury in PC12 Cells—A Possible Model of Calcium “Dysregulation” in Aging: I. Selectivity of Protection Against Oxidative Stress
Authors: Joseph J. A. , Strain J. G. , Jimenez N. D. , Fisher D.
Published: July 2010

Abstract: Previous research has suggested that the initial effects of cellular free radical neurotoxic insult involve large increases in intracellular Ca2+. However, the exact role of oxidative stress on the various parameters involved in these increases has not been specified. The present experiments were performed to examine these parameters in PC12 cells exposed to 5, 25, or 300 µM H2O2 for 30 min in growth medium alone or containing either nifedipine (L‐type Ca2+ antagonist), conotoxin (N‐type antagonist), Trolox (vitamin E analogue), or α‐phenyl‐n‐tert‐butylnitrone (nitrone trapping agent; PBN). The concentrations of H2O2 were chosen by examining the degree of cell killing induced by exposure to graded concentrations of H2O2. The 5 and 25 µM concentrations of H2O2 produced no significant cell killing at either 30 min or 24 h after treatment, whereas the 300 µM concentration produced a moderate degree of cell killing that did not increase between the two times. Fluorescent imaging was used to visualize intracellular Ca2+ changes in fura‐2‐loaded cells. Baseline (pre‐30 mM KCI) Ca2+ levels were increased significantly by H2O2 treatment (e.g., 300 µM, 200%), but the rise in the level of free intracellular Ca2+ after KCI stimulation (i.e., peak) was decreased (e.g., 300 µM, 50%) and the cell's ability to sequester or extrude the excess Ca2+ (i.e., Ca2+ recovery time) after depolarization was decreased significantly. All compounds prevented baseline Ca2+ increases and, with the exception of conotoxin, antagonized the peak decreases in Ca2+. It is interesting that after 300 µM H2O2 exposure, only Trolox was partially effective in preventing these deficits in recovery. Conotoxin increased the decrement recovery in the absence of H2O2. However, in cells exposed to 5 or 25 µM H2O2, conotoxin as well as the other agents were effective in preventing the deficits in recovery.
Activation of Phosphodiesterase IV During Desensitization of the A2A Adenosine Receptor‐Mediated Cyclic AMP Response in Rat Pheochromocytoma (PC12) Cells
Authors: Chang Yu‐Hsin , Conti Marco , Lee Yi‐Chao , Lai Hsing‐Lin , Ching Yung‐Hao , Chern Yijuang
Published: July 2010

Abstract: Prolonged activation of an A2A adenosine receptor significantly inhibits the cellular response to subsequent stimulation (A2A desensitization). We have reported previously that activation of phosphodiesterase (PDE) contributes to A2A desensitization in PC12 cells. In the present study, we show that a type IV PDE (PDE4)‐selective inhibitor (Ro 20‐1724) effectively blocks the increase in PDE activity in desensitized cells. Thus, PDE4 appears to be the PDE specifically activated during A2A desensitization in PC12 cells. Prolonged treatment of PC12 cells with an A2A‐selective agonist (CGS21680) leads to increased PDE4 activity in a dose‐dependent manner, which can be blocked by an A2A‐selective antagonist [8‐(3‐chlorostyryl)caffeine]. Using two PDE4 antibodies, we were able to demonstrate that the levels of two PDE4‐immunoreactive bands (72 and 79 kDa) were increased significantly during A2A desensitization. Prolonged treatment with forskolin to elevate intracellular cyclic AMP contents also resulted in increased PDE4 activity. In addition, activation of PDE4 activity during A2A desensitization could be blocked by a protein kinase A (PKA)‐selective inhibitor (H89) and was not observed in a PKA‐deficient PC12 cell line (A123). Taken together, activation of PDE4 via a cyclic AMP/PKA‐dependent pathway plays a critical role in dampening the signal of the A2A receptor.
A Comparative Study of 2′,3′‐Cyclic‐Nucleotide 3′‐Phosphodiesterase in Vertebrates: cDNA Cloning and Amino Acid Sequences for Chicken and Bullfrog Enzymes
Authors: Kasama‐Yoshida Hiromi , Tohyama Yoko , Kurihara Tadashi , Sakuma Mitsuhiro , Kojima Hisako , Tamai Yoichi
Published: July 2010

Abstract: In mammalian brain, two 2′,3′‐cyclic‐nucleotide 3′‐phosphodiesterase (EC 3.1.4.37) isoforms, CNP1 and CNP2, are translated, respectively, from the two mRNAs, which have been transcribed and processed by alternative use of the two transcription start points and by differential splicing. In the present study, the cDNAs encoding chicken CNP2 and bullfrog CNP1, respectively, were isolated, and the amino acid sequences of chicken CNP2 and bullfrog CNP1 were deduced. Western blot analysis showed that chicken brain contains a major CNP2‐type protein together with a minor unidentified isoform, and bullfrog brain contains only a CNP1‐type protein. All available amino acid sequences of vertebrate 2′,3′‐cyclic‐nucleotide 3′‐phosphodiesterases were aligned and compared. Three conserved motif sequences were noted: (a) an ATP‐binding site near the amino terminus, (b) an isoprenylation site at the carboxyl terminus, and (c) a probable catalytic site resembling the active site of β‐ketoacyl synthase (EC 2.3.1.41). The second and the third motifs are conserved also in goldfish RICH (regeneration‐induced 2′,3′‐cyclic‐nucleotide 3′‐phosphodiesterase homologue), which has been shown recently to have 2′,3′‐cyclic‐nucleotide 3′‐phosphodiesterase activity. The third motif (probably catalytic site) was assigned for the first time in the present report.
Cellular Acetylcholine Content and Neuronal Differentiation
Authors: Bignami F. , Bevilacqua P. , Biagioni S. , De Jaco A. , Casamenti F. , Felsani A. , Augusti‐Tocco G.
Published: July 2010

Abstract: N18TG2 neuroblastoma clone is defective for biosynthetic neurotransmitter enzymes; its inability to establish functional synapses is overcome in the neuroblastoma × glioma 108CC15, where acetylcholine synthesis is also activated. These observations suggest a possible relation between the ability to produce acetylcholine and the capability to advance in the differentiation program and achieve a fully differentiated state. Here, we report the characterization of several clones after transfection of N18TG2 cells with a construct containing a cDNA for rat choline acetyltransferase (ChAT). The ability of these clones to synthesize acetylcholine is demonstrated by HPLC determination on cellular extracts. In the transfected clones, northern blot analysis shows increased expression of mRNAs for a specific neuronal protein associated with synaptic vesicles, synapsin I. Fiber outgrowth of transfected clones is also evaluated to establish whether there is any relation between ChAT levels and morphological differentiation. This analysis shows that the transfected clone 1/2, not expressing ChAT activity, displays a very immature morphology, and its ability to extend fibers also remains rather poor in the presence of “differentiation” agents such as retinoic acid. In contrast, clones 2/4, 3/1, and 3/2, exhibiting high ChAT levels, display higher fiber outgrowth compared with clone 1/2 in both the absence and the presence of differentiating agents.
Inhibitors of Trypsin‐Like Serine Proteases Inhibit Processing of the Caspase Nedd‐2 and Protect PC12 Cells and Sympathetic Neurons from Death Evoked by Withdrawal of Trophic Support
Authors: Stefanis Leonidas , Troy Carol M. , Qi Haiqing , Greene Lloyd A.
Published: July 2010

Abstract: Rat pheochromocytoma (PC12) cells and sympathetic neurons undergo apoptotic cell death upon withdrawal of trophic support. We have shown previously that selective cysteine aspartase (caspase) inhibitors protect PC12 cells and sympathetic neurons from such death, and that the caspase Nedd‐2 is required for this type of death to occur. We now show that 4‐(2‐aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) and Nα‐p‐tosyl‐l‐lysine chloromethyl ketone (TLCK), agents that inhibit another class of proteases, the trypsin‐like serine proteases, also suppress cell death in this paradigm. The site of action of these agents is upstream of the caspases, because the CPP32‐like and Nedd‐2‐cleaving activities that are induced upon withdrawal of trophic support in PC12 cells are inhibited when AEBSF and TLCK are applied to the cells. Both agents inhibit thymidine incorporation in PC12 cells at concentrations similar to those that promote survival, raising the possibility that they may promote survival in neuronal cells through inhibition of aberrant activation of cell cycle components.
The Metabotropic Glutamate Receptor mGluR5 Induces Calcium Oscillations in Cultured Astrocytes via Protein Kinase C Phosphorylation
Authors: Nakahara Kiyoshi , Okada Masamichi , Nakanishi Shigetada
Published: July 2010

Abstract: The metabotropic glutamate receptor mGluR5, but not the closely related mGluR1, is expressed in cultured astrocytes, and this expression is up‐regulated by specific growth factors. We investigated the capability and underlying mechanisms of mGluR5 to induce oscillatory responses of intracellular calcium concentration ([Ca2+]i) in cultured rat astrocytes. Single‐cell [Ca2+]i recordings indicated that an mGluR‐selective agonist, (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylate (1S,3R‐ACPD), elicits [Ca2+]i oscillations in good agreement with the growth factor‐induced up‐regulation of mGluR5 in cultured astrocytes. A protein kinase C (PKC) inhibitor, bisindolylmaleimide I, converted a 1S,3R‐ACPD‐mediated oscillatory response into a nonoscillatory response. In addition, the PKC activator phorbol 12‐myristate 13‐acetate completely abolished the [Ca2+]i increase. These and other pharmacological properties of 1S,3R‐ACPD‐induced [Ca2+]i oscillations correlate well with those of the cloned mGluR5 characterized in heterologous expression systems. Furthermore, the potential involvement of protein phosphatases in [Ca2+]i oscillations is suggested. The present study demonstrates that mGluR5 is capable of inducing [Ca2+]i oscillations in cultured astrocytes and that phosphorylation/dephosphorylation of mGluR5 is critical in [Ca2+]i oscillations, analogous to the cloned mGluR5 expressed in heterologous cell lines.
Species Heterogeneity Between Gerbils and Rats: Quinolinate Production by Microglia and Astrocytes and Accumulations in Response to Ischemic Brain Injury and Systemic Immune Activation
Authors: Heyes Melvyn P. , Saito Kuniaki , Chen Cai Y. , Proescholdt Margit G. , Nowak Thaddeus S. , Li Ju , Beagles Karen E. , Proescholdt Martin A. , Zito Mark A. , Kawai Kensuke , Markey Sanford P.
Published: July 2010

Abstract: Quinolinic acid is an excitotoxic kynurenine pathway metabolite, the concentration of which increases in human brain during immune activation. The present study compared quinolinate responses to systemic and brain immune activation in gerbils and rats. Global cerebral ischemia in gerbils, but not rats, increased hippocampus indoleamine‐2,3‐dioxygenase activity and quinolinate levels 4 days postinjury. In a rat focal ischemia model, small increases in quinolinate concentrations occurred in infarcted regions on days 1, 3, and 7, although concentrations remained below serum values. In gerbils, systemic immune activation by an intraperitoneal injection of endotoxin (1 mg/kg of body weight) increased quinolinate levels in brain, blood, lung, liver, and spleen, with proportional increases in lung indoleamine‐2,3‐dioxygenase activity at 24 h postinjection. In rats, however, no significant quinolinate content changes occurred, whereas lung indoleamine‐2,3‐dioxygenase activity increased slightly. Gerbil, but not rat, brain microglia and peritoneal monocytes produced large quantities of [13C6]‐quinolinate from l‐[13C6]tryptophan. Gerbil astrocytes produced relatively small quantities of quinolinate, whereas rat astrocytes produced no detectable amounts. These results demonstrate that the limited capacity of rats to replicate elevations in brain and blood quinolinic acid levels in response to immune activation is attributable to blunted increases in local indoleamine‐2,3‐dioxygenase activity and a low capacity of microglia, astrocytes, and macrophages to convert l‐tryptophan to quinolinate.
Irreversible Inhibition of Mitochondrial Complex I by 7‐(2‐Aminoethyl)‐3,4‐Dihydro‐5‐Hydroxy‐2H‐1,4‐Benzothiazine‐3‐Carboxylic Acid (DHBT‐1): A Putative Nigral Endotoxin of Relevance to Parkinson's Disease
Authors: Li Hong , Dryhurst Glenn
Published: July 2010

Abstract: Based on a number of lines of evidence, we have proposed recently that a very early step in the pathogenesis of idiopathic Parkinson's disease might be elevated translocation of l‐cysteine into neuromelanin‐pigmented dopaminergic cell bodies in the substantia nigra. In vitro studies suggest that such an influx of l‐cysteine would divert the neuromelanin pathway by scavenging dopamine‐o‐quinone, the proximate autoxidation product of dopamine, to give 5‐S‐cysteinyldopamine, which is oxidized further to 7‐(2‐aminoethyl)‐3,4‐dihydro‐5‐hydroxy‐2H‐1,4‐benzothiazine‐3‐carboxylic acid (DHBT‐1) and other cysteinyldopamines and dihydrobenzothiazines. In this study, it is demonstrated that DHBT‐1 inhibits ADP‐stimulated oxidation of malate and pyruvate (state 3 or complex I respiration) when incubated with intact rat brain mitochondria with an IC50 of ∼0.80 mM. Incubation of DHBT‐1 with freeze‐thawed rat brain mitochondria in both the presence and absence of KCN and/or NADH causes an irreversible, time‐dependent decrease of NADH‐coenzyme Q1 reductase activity. Significantly lower concentrations of DHBT‐1 are necessary to cause this effect when mitochondrial membranes are incubated in the absence of KCN and NADH. The irreversible inhibition of mitochondrial complex I caused by DHBT‐1 under the latter conditions could be blocked only partially by glutathione, ascorbic acid, superoxide dismutase, or catalase. Together, these results suggest that DHBT‐1 can cross the outer mitochondrial membrane and irreversibly inhibit complex I by a mechanism that is not primarily related to oxygen radical‐mediated damage. Formation of DHBT‐1 requires only dopamine, l‐cysteine, and an oxidizing environment, conditions that may well exist in the cytoplasm of neuromelanin‐pigmented dopaminergic neurons in the parkinsonian substantia nigra. The results of this study raise the possibility that DHBT‐1 might be an endotoxin formed specifically in pigmented dopaminergic neurons that can contribute to irreversible damage to mitochondrial complex I and substantia nigra cell death in Parkinson's disease.
Tau Released from Paired Helical Filaments with Formic Acid or Guanidine Is Susceptible to Calpain‐Mediated Proteolysis
Authors: Yang Liang‐Sheng , Gordon‐Krajcer Wanda , Ksiezak‐Reding Hanna
Published: July 2010

Abstract: Paired helical filaments (PHFs), a characteristic neuropathologic finding in Alzheimer's disease brain, are abnormal fibrillary forms of hyperphosphorylated tau (PHF‐tau), which have been shown to be highly resistant to calpain digestion. Either excessive phosphorylation or fibrillary arrangement of tau proteins in PHFs may play a role in proteolytic resistance by limiting access to calpain recognition/digestion sites. To determine the contribution of the fibrillary conformation, isolated PHFs were subjected to treatment with either formic acid or guanidine. Both procedures effectively abolished the fibrillary structure of PHF but preserved PHF‐tau immunoreactivity using a panel of antibodies that recognize nonphosphorylated and phosphorylated epitopes. These treatments also significantly increased the sensitivity of PHF‐tau polypeptides to calpain proteolysis as shown by significant decreases in the half‐life (t1/2) from the infinite with native PHF to 44 min and 4.4 min in formic acid‐ or guanidine‐treated samples, respectively. In contrast, the sensitivity of normal fetal tau (3.4 min) was either decreased (5.9 min) or unaffected (3.6 min) by similar treatment. Our results indicate that after guanidine treatment, the sensitivity of PHF to calpain resembles that of fetal tau. These results strongly suggest that the fibrillary structure of PHF‐tau, rather than hyperphosphorylation, is the major factor responsible for the resistance of abnormal filaments to calpain‐mediated proteolysis.
Mitochondrial Free Radical Signal in Ceramide‐Dependent Apoptosis: A Putative Mechanism for Neuronal Death in Parkinson's Disease
Authors: France‐Lanord Valentine , Brugg Bernard , Michel Patrick P. , Agid Yves , Ruberg Merle
Published: July 2010

Abstract: Activation of the apoptogenic sphingomyelin‐dependent signaling pathway in neuronally differentiated PC12 cells with cell‐permeant C2‐ceramide resulted in a transient and short‐lived emission of reactive oxygen species that was maximal 6 h after the beginning of treatment, followed immediately by nuclear translocation of the transcription factor nuclear factor κB. The production of reactive oxygen species was necessary for cell death to occur. The origin of the reactive oxygen species was identified as complex I of the mitochondrial electron transport chain. The mitochondria were not dysfunctional, however. They maintained normal membrane potentials and ATP synthesis until the cells began to die and the cell nuclei to condense and to fragment, ∼12 h after the beginning of treatment. We conclude that a mitochondrial free radical signal plays a role in the sphingomyelin‐dependent transduction pathway. Convergent data from postmortem brain suggest that this signaling pathway may be activated in the dopaminergic neurons that die in patients with Parkinson's disease and would provide a mechanism for oxidative stress implicating the mitochondria, both of which have long been hypothesized to play a role in the pathogenesis of this disease.
Identification of Two Corticotropin‐Releasing Factor Receptors from Xenopus laevis with High Ligand Selectivity: Unusual Pharmacology of the Type 1 Receptor
Authors: Dautzenberg Frank M. , Dietrich Konstanze , Palchaudhuri Monika R. , Spiess Joachim
Published: July 2010

Abstract: Two cDNA clones encoding distinct members of the corticotropin‐releasing factor (CRF) receptor family have been isolated from Xenopus laevis with PCR‐based approaches. The first full‐length cDNA amplified from Xenopus brain encoded a 415‐amino acid protein with ∼80% identity to mammalian CRF receptor type 1 (CRF‐R1). The second full‐length cDNA isolated from Xenopus brain and heart encoded a 413‐amino acid protein with ∼81% identity to the α‐variant of mammalian CRF receptor, type 2 (CRF‐R2). No evidence could be obtained that the β‐variant of CRF‐R2 existed in Xenopus laevis. Binding studies using human embryonic kidney 293 (HEK 293) cells stably transfected with xenopus CRF‐R2 showed that the CRF analogues urotensin I, urocortin, and sauvagine were bound with higher affinities than human/rat CRF, xenopus CRF, and ovine CRF. In contrast to human CRF‐R1, xenopus CRF‐R1 (xCRF‐R1) was very selective for different CRF ligands. Urotensin I, urocortin, human/rat CRF, and xenopus CRF were bound with significantly (10–22‐fold) higher affinities than ovine CRF (KD = 31.7 nM) and sauvagine (KD = 51.4 nM). In agreement with these binding data, EC50 values of 39.7 and 1.1 nM were found for sauvagine and for human/rat CRF or xenopus CRF, respectively, when the cyclic AMP production in HEK 293 cells stably transfected with xCRF‐R1 was determined.
Transbilayer Distribution of Cholesterol Is Modified in Brain Synaptic Plasma Membranes of Knockout Mice Deficient in the Low‐Density Lipoprotein Receptor, Apolipoprotein E, or Both Proteins
Authors: Igbavboa Urule , Avdulov Nicolai A. , Chochina Svetlana V. , Wood W. Gibson
Published: July 2010

Abstract: Both apolipoprotein E (apoE) and the low‐density lipoprotein (LDL) receptor are present in brain; however, little is known regarding the function of these proteins in brain, in particular with respect to brain cholesterol. The role of apoE and the LDL receptor in modulating the transbilayer or asymmetric distribution of cholesterol in the exofacial and cytofacial leaflets of synaptic plasma membranes (SPMs) was examined in mutant mice deficient in apoE, the LDL receptor, or both proteins by using the fluorescent sterol dehydroergosterol and fluorescent quenching procedures. Fluidity of the exofacial and cytofacial leaflets was also measured. Cholesterol asymmetry of SPMs was altered in the mutant mice, with the largest effect observed in the LDL receptor‐deficient mice. There was an approximately twofold increase in the percent distribution of cholesterol in the exofacial leaflet of the LDL receptor‐deficient mice (32%) compared with C57BL/6J mice (15%). Mice deficient in apoE or both proteins also showed a significantly higher percent distribution of cholesterol (23 and 26%, respectively) in the exofacial leaflet compared with the C57BL/6J mice. Although the percent distribution of cholesterol was highest in the exofacial leaflet of the LDL receptor‐deficient mice, fluidity of the exofacial leaflet of that group was significantly lower. However, the cholesterol‐to‐phospholipid ratio of SPMs of the LDL receptor‐deficient mice was significantly lower, and this difference was largely the result of a significant increase in the total amount of SPM phospholipid. This study demonstrates for the first time that SPM lipid structure is altered in mice deficient in apoE or the LDL receptor. Although the mechanism that maintains the asymmetric distribution of cholesterol in plasma membranes is not well understood, data of the present experiments indicate that both apoE and the LDL receptor are involved in maintaining the transbilayer distribution of cholesterol.
Characterization of In Vitro Glycation Sites of Tau
Authors: Nacharaju Parimala , Ko Li‐wen , Yen Shu‐Hui C.
Published: July 2010

Abstract: Tau is a microtubule‐associated protein that loses microtubule binding activity and aggregates into paired helical filaments (PHFs) in Alzheimer's disease. Nonenzymic glycation is one of the posttranslational modifications detected in PHF‐tau, but not in normal tau. PHF‐tau has reduced ability to bind to microtubules. To determine whether glycation of tau occurs in its microtubule binding domains, we have characterized in vitro glycation sites of the longest isoform of tau, which has four microtubule binding domains (Tau‐4). The identified glycation sites are Lys‐87, 132, 150, 163, 174, 225, 234, 259, 280, 281, 347, 353, and 369. We have also studied glycation of another isoform of tau, which has only three microtubule binding domains (Tau‐3). This isoform is modified by glucose 15–20% more slowly than Tau‐4. However, the glycation sites appear to be the same in both isoforms, except for Lys‐280 and 281; these are located in the second microtubule binding domain, which is missing in Tau‐3. Lys‐150, 163, and 174 are located within or proximal to the sequence of tau that is involved in the microtubule nucleation activity, and Lys‐259, 280, 281, 347, 353, and 369 are located in the microtubule binding domains. Glycation at these sites can affect the functional properties of tau, and advanced glycation at these sites might lead to the formation of insoluble aggregates similar to the ones seen in Alzheimer's disease.
Amino‐Terminal Analysis of Tryptophan Hydroxylase: Protein Kinase Phosphorylation Occurs at Serine‐58
Authors: Kumer Sean C. , Mockus Susan M. , Rucker Paul J. , Vrana Kent E.
Published: July 2010

Abstract: Tryptophan hydroxylase (TPH) catalyzes the rate‐limiting and committed step in serotonin biosynthesis. Within this enzyme, two distinct domains have been hypothesized to exist, an amino‐terminal regulatory domain and a carboxyl‐terminal catalytic domain. In the present experiments, the functional boundary between the putative domains was defined using deletion muta‐genesis. A full‐length cDNA clone for rabbit TPH was engineered for expression in bacteria. Five amino‐terminal deletions were constructed using PCR, i.e., NΔ50, NΔ60, NΔ90, NΔ106, and NΔ116 (referring to the number of amino acids deleted from the amino terminus). Enzymatic activity was determined for each mutant after expression in bacteria. Whereas deletion of 116 amino acids (NΔ116) abolished enzyme activity, all of the other amino‐terminal deletions exhibited increased specific activity relative to the recombinant wild‐type TPH. The ability of the cyclic AMP‐dependent protein kinase (PKA) to phosphorylate members of the deletion series was also examined. Deletion of the first 60 amino‐terminal residues abolished the ability of the enzyme to serve as a substrate for PKA, yet the native and NΔ50 enzymes were phosphorylated. Moreover, a serine‐58 point mutant (S58A) was not phosphorylated by PKA. In conclusion, the first 106 amino acids comprise a regulatory domain that is phosphorylated by PKA at serine‐58. In addition, the boundary between regulatory and catalytic domains is analogous to the domain structure observed for the related enzyme tyrosine hydroxylase.
SNARE Proteins‐Why So Many, Why So Few?
Authors: Linial Michal
Published: July 2010

Abstract: Both trafficking and secretion critically depend on accurate and specific membrane recognition and fusion. A key step in these processes is the assembly of a complex consisting of a small number of proteins, i.e., the exocytic core complex. In nerve terminals, this set consists of VAMP and synaptotagmin, which reside at membranes of synaptic vesicles, and syntaxin and SNAP‐25 at the plasma membrane. In this survey, different secretory systems that depend on the exocytic core proteins are considered. The possibility that specificity in membrane recognition and fusion is achieved by the numerous variants of proteins of the exocytic core is discussed. Variability of the core complex proteins is determined by the complexity of gene families, isoform‐specific localization, and posttranslational modifications. Basic biochemical properties depend on specific isoforms, and the possible protein‐protein interactions are determined, in turn, by the compatibility of different isoforms. A correlation between specific variants and distinct biochemical or cellular properties is shown. The outcome of this survey is that heterogeneity in secretion may be dictated by the large number of possible combinations of variants of only a few proteins.
The Role of Glutathione in Dopaminergic Neuronal Survival
Authors: Nakamura Ken , Wang Wenyi , Kang Un Jung
Published: July 2010

Abstract: An increased production of reactive oxygen species is thought to be critical to the pathogenesis of Parkinson's disease. At autopsy, patients with either presymptomatic or symptomatic Parkinson's disease have a decreased level of glutathione in the substantia nigra pars compacta. This change represents the earliest index of oxidative stress in Parkinson's disease discovered to this point. This study compares the sensitivity of dopaminergic and nondopaminergic neurons in dissociated mesencephalic cultures to the depletion of glutathione. We have found that dopaminergic neurons are more resistant to the toxicity of glutathione depletion than nondopaminergic neurons. The possibility that dopaminergic neurons have a higher baseline glutathione level than nondopaminergic neurons is suggested by measurements of levels of cellular glutathione in a parallel system of immortalized embryonic dopaminergic and nondopaminergic cell lines. We also examined the role of glutathione in 1‐methyl‐4‐phenylpyridinium toxicity. Decreasing the glutathione level of dopaminergic neurons potentiates their susceptibility to 1‐methyl‐4‐phenylpyridinium toxicity, although 1‐methyl‐4‐phenylpyridinium does not deplete glutathione from primary mesencephalic cultures. Our data suggest that although a decreased glutathione content is not likely to be the sole cause of dopaminergic neuronal loss in Parkinson's disease, decreased glutathione content may act in conjunction with other factors such as 1‐methyl‐4‐phenylpyridinium to cause the selective death of dopaminergic neurons.
In Vitro Induction of Apoptosis or Differentiation by Dopamine in an Immortalized Olfactory Neuronal Cell Line
Authors: Coronas V. , Féron F. , Hen R. , Sicard G. , Jourdan F. , Moyse E.
Published: July 2010

Abstract: A new neuronal cell line was generated by transfection of rat olfactory epithelium with immortalizing recombinant oncogene E1A of adenovirus‐2. The resulting 13.S.1.24 line of transformed cells expressed an antigenic phenotype of olfactory neuronal progenitors. Addition of dopamine to 13.S.1.24 cultures induced reduction of cell number within 2 days. Two hallmarks of apoptosis were detected in dopamine‐treated cultures: internucleosomal DNA fragmentation and nuclear condensation. Dopamine did not alter the cell proliferation rate, as assessed by [3H]thymidine incorporation. Dopamine also stimulated differentiation of surviving 13.S.1.24 cells into bipolar olfactory marker protein‐immunoreactive neurons. Time‐dependency assessments over 1 week of treatment indicated that apoptosis and differentiation induced by dopamine were concomitant. Both apoptosis and differentiation triggered by dopamine were dose‐dependent, half‐maximal effects being obtained with ∼10 µM dopamine. Mediation of both effects by dopaminergic D2 receptors was supported by several observations: active dopamine doses in micromolar ranges, quinpirole agonism and eticlopride antagonism, D2‐characteristic rank order of potency among the three agonists tested, and specific binding of a selective D2‐like radioligand to 13.S.1.24 cells. The present data altogether indicated that dopamine commits immortalized olfactory neuronal cells in vitro either to apoptosis or to olfactory‐like differentiation via D2 dopaminergic receptors.
Disrupted [Ca2+]i Homeostasis Contributes to the Toxicity of Nitric Oxide in Cultured Hippocampal Neurons
Authors: Brorson James R. , Zhang He
Published: July 2010

Abstract: Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca2+ concentration ([Ca2+]i) homeostasis in cultured hippocampal neurons during 20‐min exposures. In this study, we examine the relationship between late alterations of [Ca2+]i homeostasis and the delayed toxicity produced by NO. The NO‐releasing agent S‐nitrosocysteine (SNOC; 300 µM) reduced survival by about one half 1 day after 20‐min exposures, as did other NO‐releasing agents. SNOC also was found to produce prolonged elevations of [Ca2+]i, persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca2+]i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca2+ during the 20‐min SNOC treatment failed to prevent the late [Ca2+]i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca2+ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca2+‐activated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca2+]i homeostatic mechanisms, and that the resulting persistent [Ca2+]i elevations contribute to the delayed neurotoxicity of NO.
Expression and Regulation of Types I and II Inositol 1,4,5‐Trisphosphate Receptors in Rat Cerebellar Granule Cell Preparations
Authors: Oberdorf Jon , Vallano Mary Lou , Wojcikiewicz Richard J. H.
Published: July 2010

Abstract: Previous studies have shown that as rat cerebellar granule cell cultures differentiate in the presence of 25 mM KCl, they “up‐regulate” their ability to form inositol phosphates and release Ca2+ from internal stores in response to the activation of phosphoinositidase C‐linked muscarinic and metabotropic receptors. Here we show that they simultaneously up‐regulate their ability to respond to inositol 1,4,5‐trisphosphate (InsP3) by increasing InsP3 receptor (InsP3R) expression. In contrast, if granule cells are maintained at the more physiological KCl concentration of 5 mM, most cells undergo apoptosis, although a significant number survive. The surviving cells, however, express few InsP3Rs, suggesting that an influx of Ca2+ through voltage‐dependent channels is required for InsP3R up‐regulation. In addition, we have determined that these cultures express two genetically distinct InsP3R types, but that only one, the type I receptor, is expressed in granule cells. Type II receptors are also present but are found exclusively in astrocytes, which are a minor contaminant of granule cell cultures. This segregation of InsP3R types explains a previous observation, showing that the muscarinic agonist carbachol causes the reduction or “down‐regulation” of type I but not type II InsP3Rs.
In Vivo Electrochemical Measurement of the Long‐Lasting Release of Dopamine and Serotonin Induced by Intrastriatal Kainic Acid
Authors: Nakazato Taizo , Akiyama Akitane
Published: July 2010

Abstract: Intrastriatal injection of the glutamate agonist kainic acid (KA) in rats has been used to produce an animal model to investigate the mechanism of acetylcholine and GABA cell death associated with Huntington's disease. In the present study, the time course of low (10−5M) and high (5 × 10−3M) concentrations of KA on striatal dopamine and serotonin release was studied in freely moving rats by using in vivo voltammetry. The response to low concentrations of KA varied between animals, either increasing dopamine release during the injection or increasing dopamine and serotonin after the injection for an extended time, suggesting that 10−5 KA is near the threshold for KA toxicity in the striatum in rats. High concentrations of KA suppressed dopamine release during injection, with both dopamine and serotonin release increasing and remaining elevated for 1–4 and 7–21 days, respectively. KA‐induced changes were inhibited by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and bicuculline increased the release of dopamine but not serotonin. These findings suggest that KA‐induced changes in dopamine release resulted from a disinhibition of dopamine neurons due to KA‐mediated toxicity of striatal GABA neurons. An alternate possibility is that the change in dopamine and serotonin release may have arisen from a functional modification or degeneration of presynaptic terminals.
Identification and Characterization of a New Serotonergic Recognition Site with High Affinity for 5‐Carboxamidotryptamine in Mammalian Brain
Authors: Castro Ma. Elena , Romón Tamara , Castillo Ma. Josefa , Del Olmo Elena , Pazos Angel , Del Arco Carmen
Published: July 2010

Abstract: We analyzed the existence of an additional serotonin (5‐HT) receptor subtype, sensitive to 5‐carboxamidotryptamine, in the mammalian brain. Radioligand binding studies with [3H]5‐HT were carried out in rat, guinea pig, and human brain membranes, in the presence of unlabeled drugs to mask the binding to all known 5‐HT receptors, with the exception of 5‐HT1E sites. Under these conditions, unlabeled 5‐carboxamidotryptamine still showed a biphasic competition curve with a nanomolar affinity component. Saturation studies with 5‐[3H]carboxamidotryptamine were carried out in the presence of (±)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin, mesulergine, and ergotamine, to mask the binding to all receptors known to be labeled by 5‐carboxamidotryptamine. These studies showed the existence in cortex and hippocampus from guinea pig and human brain of a remaining binding site with high affinity (pKD = 7.8–8.1) and a unique pharmacological profile. 5‐HT and 5‐carboxamidotryptamine showed nanomolar affinity, whereas 5‐methoxytryptamine recognized this binding site with intermediate affinity. Other drugs exhibited low or very low potency in inhibiting this binding. The addition of 5′‐guanylylimidodiphosphate significantly reduced the number of binding sites labeled by 5‐[3H]carboxamidotryptamine, in the presence of the masking drugs described above, indicating the interaction with a GTP‐binding protein. Preliminary autoradiographic studies in human brain appear to indicate that this 5‐HT binding site is present in areas such as the globus pallidus, neocortex, and hippocampus, among others.
Molecular Dissection of Native Mammalian Forebrain NMDA Receptors Containing the NR1 C2 Exon: Direct Demonstration of NMDA Receptors Comprising NR1, NR2A, and NR2B Subunits Within the Same Complex
Authors: Chazot Paul L. , Stephenson F. Anne
Published: July 2010

Abstract: The subunit compositions of the NR1 C2 exon‐containing N‐methyl‐d‐aspartate (NMDA) receptors of adult mammalian forebrain were determined by using a combination of immunoaffinity chromatography and immunoprecipitation studies with NMDA receptor subunit‐specific antibodies. NMDA receptors were solubilised by sodium deoxycholate, pH 9, and purified by anti‐NR1 C2 antibody affinity chromatography. The purified receptor subpopulation showed immunoreactivity with anti‐NR1 C2, anti‐NR1 N1, anti‐NR1 C2′, anti‐NR2A, and anti‐NR2B NMDA receptor antibodies. The NR1 C2‐receptor subpopulation was subjected to immunoprecipitation using anti‐NR2B antibodies and the resultant immune pellets analysed by immunoblotting where anti‐NR1 C2, anti‐NR1 C2′, anti‐NR2A, and anti‐NR2B immunoreactivities were all found. Quantification of the immunoblots showed that 46% of the NR1 C2 immunoreactivity was associated with the NR2B subunit. Of this, 87% (i.e., 40% of total) were NR1 C2/NR2B receptors and 13% (6% of total) were NR1 C2/NR2A/NR2B, thus identifying the triple combination as a minor receptor subset. These results demonstrate directly, for the first time, the coexistence of the NR2A and NR2B subunits in native NMDA receptors. They show the coexistence of two splice forms of the NR1 subunit, i.e., NR1 C2 and NR1 C2′, in native receptors and, in addition, they imply an NMDA receptor subpopulation containing four types of NMDA receptor subunit, NR1 C2, NR1 C2′, NR2A, and NR2B, which, in accord with molecular size determinations, predicts that the NMDA receptor is at least tetrameric. These results are the first quantitative study of NMDA receptor subtypes and demonstrate molecular heterogeneity for both the NR1 and the NR2 subunits in native forebrain NMDA receptors.
Differential Regulation of Neuronal Nicotinic Receptor Binding Sites Following Chronic Nicotine Administration
Authors: Flores Christopher M. , Dávila‐García Martha I. , Ulrich Yvonne M. , Kellar Kenneth J.
Published: July 2010

Abstract: Chronic nicotine administration to rats produces an increase in neuronal nicotinic receptors in the CNS. Moreover, the up‐regulated sites labeled by [3H]cytisine in cerebral cortex appear to be composed exclusively of α4 and β2 subunits. It is unknown whether receptor subtypes that do not bind [3H]‐cytisine with high affinity are also affected. In the present studies, we tested the hypothesis that nicotine treatment differentially alters the density of neuronal nicotinic receptor subtypes in rat nervous tissues. Thus, we compared the binding of [3H]cytisine with that of [3H]epibatidine to nicotinic receptors in brain, spinal cord, and adrenal gland from rats that had been injected twice daily with nicotine or saline vehicle for 10 days. Chronic nicotine treatment led to an increase in nicotinic receptor binding sites in the cerebral cortex and in the dorsal lumbar spinal cord, but not in the thalamus. It is important that virtually all of the observed increases could be accounted for by a selective effect on the fraction of receptors exhibiting high affinity for both [3H]‐cytisine and [3H]epibatidine. In contrast, no change in [3H]‐epibatidine binding was seen in the adrenal gland, a tissue that does not exhibit high‐affinity [3H]cytisine binding. These data indicate that, under the conditions used here, nicotine up‐regulates the α4β2 nicotinic receptor subtype, which can be labeled by [3H]cytisine and [3H]epibatidine, but not non‐α4β2 subtypes, which can be labeled by [3H]epibatidine.
Differential Expression of Egr‐1‐Like DNA‐Binding Activities in the Naive Rat Brain and After Excitatory Stimulation
Authors: Beckmann Alison M. , Davidson Mark S. , Goodenough Sharon , Wilce Peter A.
Published: July 2010

Abstract: Egr‐1 and related proteins are inducible transcription factors within the brain recognizing the same consensus DNA sequence. Three Egr DNA‐binding activities were observed in regions of the naive rat brain. Egr‐1 was present in all brain regions examined. Bands composed, at least in part, of Egr‐2 and Egr‐3 were present in different relative amounts in the cerebral cortex, striatum, hippocampus, thalamus, and midbrain. All had similar affinity and specificity for the Egr consensus DNA recognition sequence. Administration of the convulsants NMDA, kainate, and pentylenetetrazole differentially induced Egr‐1 and Egr‐2/3 DNA‐binding activities in the cerebral cortex, hippocampus, and cerebellum. All convulsants induced Egr‐1 and Egr‐2 immunoreactivity in the cerebral cortex and hippocampus. These data indicate that the members of the Egr family are regulated at different levels and may interact at promoters containing the Egr consensus sequence to fine tune a program of gene expression resulting from excitatory stimuli.
S100β Induces Neuronal Cell Death Through Nitric Oxide Release from Astrocytes
Authors: Hu Jingru , Ferreira Adriana , Van Eldik Linda J.
Published: July 2010

Abstract: The glial‐derived neurotrophic protein S100β has been implicated in the development and maintenance of the nervous system. S100β has also been postulated to play a role in mechanisms of neuropathology because of its specific localization and selective overexpression in Alzheimer's disease. However, the exact relationship between S100β overexpression and neurodegeneration is unclear. Recent data have demonstrated that treatment of cultured rat astrocytes with high concentrations of S100β results in a potent activation of inducible nitric oxide synthase (iNOS) and a subsequent generation of nitric oxide (NO), which can lead to astrocytic cell death. To investigate whether S100β‐induced NO release from astrocytes might influence neurons, we studied S100β effects on neuroblastoma B104 cells or primary hippocampal neurons co‐cultured with astrocytes. We found that S100β treatment of astrocyte‐neuron co‐cultures resulted in neuronal cell death by both necrosis and apoptosis. Neuronal cell death induced by S100β required the presence of astrocytes and depended on activation of iNOS. Cell death correlated with the levels of NO and was blocked by a specific NOS inhibitor. Our data support the idea that overexpression of S100β may be an exacerbating factor in the neurodegeneration of Alzheimer's disease.
Effects of the Dopamine D3 Antagonist PD 58491 and Its Interaction with the Dopamine D3 Agonist PD 128907 on Brain Dopamine Synthesis in Rat
Authors: Whetzel S. Z. , Shih Y. H. , Georgic L. M. , Akunne H. C. , Pugsley T. A.
Published: July 2010

Abstract: The dopamine (DA) D3 receptor antagonist PD 58491 {3‐[4‐[1‐[4‐[2‐[4‐(3‐diethylaminopropoxy)phenyl]‐benzoimidazol‐1‐yl‐butyl]‐1H‐benzoimidazol‐2‐yl]‐phenoxy]propyl]diethylamine} bound with high affinity and selectivity to recombinant human DA D3 versus D2L and D4.2 receptors transfected into Chinese hamster ovary cells: Ki values of 19.5 nM versus 2,362 and >3,000 nM, respectively. In contrast, the putative DA D3 receptor antagonist (+)‐AJ76 displayed low affinity and selectivity for D3 versus D2L and D4.2 receptors (91 nM vs. 253 and 193 nM, respectively). In vitro, PD 58491 (1 nM−1µM) exhibited D3 receptor antagonist activity, reversing the quinpirole (10 nM)‐induced stimulation of [3H]thymidine uptake in D3 CHOpro‐5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the γ‐butyrolactone‐induced increase in DA synthesis (l‐3,4‐dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D2/D3 receptor agonist action at DA autoreceptors. PD 58491 (3–30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D3‐preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D3 autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.
Casein Kinase 1 Is Tightly Associated with Paired‐Helical Filaments Isolated from Alzheimer's Disease Brain
Authors: Kuret Jeff , Johnson Ginger S. , Cha Donald , Christenson Erik R. , DeMaggio Anthony J. , Hoekstra Merl F.
Published: July 2010

Abstract: The protein kinase activity tightly associated with paired helical filaments (PHFs) purified from the brain tissue of individuals with Alzheimer's disease has been characterized in vitro. The activity is shown to phosphorylate casein, an exogenous substrate, with a maximal velocity of ∼2 nmol/min/mg, suggesting it comprises a significant component of the total protein in the PHF preparation. On the basis of substrate selectivity, isoquinoline sulfonamide inhibitor selectivity, in‐gel renaturation assays, and western analysis, the activity consists of closely related members of the α branch of the casein kinase 1 family of protein kinases. Because of its tight association with PHFs and its phosphate‐directed substrate selectivity, casein kinase 1 is positioned to participate in the pathological hyperphosphorylation of tau protein that is observed in neurodegenerative diseases such as Alzheimer's disease.
Threshold Effects in Synaptosomal and Nonsynaptic Mitochondria from Hippocampal CA1 and Paramedian Neocortex Brain Regions
Authors: Davey Gavin P. , Canevari Laura , Clark John B.
Published: July 2010

Abstract: After a brief period of global ischemia, the hippocampal CA1 region is more susceptible to irreversible damage than the paramedian neocortex. To test whether primary differences in bioenergetic parameters may be present between these regions, respiration rates and respiratory control activities were measured. In synaptosomal and nonsynaptic mitochondria isolated from the hippocampal CA1 region, state 3 respiration rates and complex IV activities were significantly lower than those present in synaptosomal and nonsynaptic mitochondria from the paramedian neocortex. These results suggest that mitochondria from the CA1 hippocampal area differ in some properties of metabolism compared with the neocortex area, which may render them more susceptible to a toxic insult such as that of ischemia. In addition, when complex I and IV activities were titrated with specific inhibitors, thresholds in ATP synthesis and oxygen respiration became apparent. Complex I and IV activities were decreased by 60% in nonsynaptic mitochondria from the hippocampal CA1 region and paramedian neocortex before oxidative phosphorylation was severely compromised; however, in synaptosomes from these regions, complex I activities had a threshold of 25%, indicating heterogenous behaviour for brain mitochondria. Reduced complex I thresholds in mitochondria, in association with other constitutive defects in energy metabolism, may induce a decreased ATP supply in the synaptic region. The implications of these findings are discussed in relation to delayed neuronal death and processes of neurodegeneration.