bluuebunny/crossref_metadata_2025 huggingface dataset:
Microtubules and Microfilaments Participate in the Inhibition of Synaptosomal Noradrenaline Release by Tetanus Toxin
Authors: Ashton Anthony C. , Dolly J. Oliver
Published: July 2010

Abstract: Tetanus toxin (TeTX) has been demonstrated to inhibit transmitter release by two mechanisms: Zn2+‐dependent proteolytic cleavage of synaptobrevin and activation of a neuronal transglutaminase. Herein, attenuation of TeTX‐induced blockade of noradrenaline release from synaptosomes was achieved by prior disassembly of microfilaments with cytochalasin D or breakdown of microtubules by colchicine or nocodazole. These drugs and monodansylcadaverine, a transglutaminase inhibitor, displayed some additivity in antagonizing the inhibitory effect of the toxin on synaptosomal transmitter release; as none of them reduced synaptobrevin cleavage, all appear to work independently of the toxin's proteolytic action. Prior stabilization of microtubules with taxol prevented the antagonism seen with colchicine, highlighting that this cytoskeletal component is the locus of the effect of colchicine. Replacement of Ca2+ with Ba2+ caused disappearance of the fraction of evoked secretion whose inhibition by TeTX is reliant on polymerized actin but did not alter the blockade by toxin that is dependent on microtubules. Two temporally distinguished phases of release were reduced by TeTX, and colchicine lessened its effects on both. Blockade of the fast phase (≤10 s) of secretion by TeTX was unaffected by cytochalasin D, but it clearly antagonized the toxin‐induced inhibition of the slow (10‐s to ≥5‐min) component; it is notable that such antagonism was accentuated during a second bout of evoked release. These findings are consistent with sustained release requiring dissociation of synaptic vesicles from the microfilaments, a step that seems to be perturbed by TeTX.
Dopamine Agonist‐Induced Inhibition of Neurotransmitter Release from the Awake Squirrel Monkey Putamen as Measured by Microdialysis
Authors: Davis M. Duff , Heffner Thomas G. , Cooke Leonard W.
Published: July 2010

Abstract: Male squirrel monkeys (Saimiri sciureus) were surgically prepared with cranial guide cannulae for acute microdialysis sampling of the putamen nucleus, a dopamine (DA)‐rich brain region. On the day of an experiment, an animal was placed in a Plexiglas restraining chair and a microdialysis probe was inserted through the guide into the putamen. Perfusates of artificial cerebrospinal fluid were collected every 20 min over several hours and analyzed via HPLC with electrochemical detection. DA D2/D3 agonist drugs were administered either orally (p.o.) or subcutaneously (s.c.), and changes in levels of DA in the dialysates were measured. All of the drugs tested, i.e., quinpirole (0.5 mg/kg p.o.), talipexole (0.75 mg/kg p.o. or s.c.), and PD 135222 (7 mg/kg p.o.), decreased spontaneous DA overflow by ∼40–50% during the first 2 h following dosing. In animals that routinely underwent the microdialysis procedure up to 23 times over a 2‐year period, there was neither an appreciable change in basal DA overflow nor a significant change in the magnitude of drug response. These data suggest that DA D2/D3 agonists attenuate DA neuronal overflow in the primate brain, similar to effects seen in rodents. Furthermore, these results also demonstrate the utility of repeated intracerebral microdialysis as a tool to monitor dynamic changes in neurochemical activity in monkeys over a prolonged period of time.
Transient Elevation of Interstitial N‐Acetylaspartate in Reversible Global Brain Ischemia
Authors: Sager Thomas Nikolaj , Fink‐Jensen Anders , Hansen Ander Jon
Published: July 2010

Abstract: We evaluated the changes of interstitial N‐acetylaspartate (NAA) concentration ([NAA]e) in rat striatum by microdialysis following transient global ischemia and depolarization. The dialysate NAA concentration ([NAA]d) values were corrected for the in vivo recovery to obtain [NAA]e, by the use of [3H]mannitol in the perfusion fluid. During global ischemia the relative loss (RL) of [3H]mannitol decreased to 40% of preischemic values, reflecting the decrease in extracellular volume fraction. During reperfusion RL of [3H]mannitol quickly normalized. The [NAA]d doubled during transient ischemia, which, after correction for in vivo recovery, corresponds to a fivefold increase in [NAA]e (p < 0.05). Reperfusion induced a >10‐fold increase of [NAA]e (p < 0.01) with subsequent normalization after 45 min. KCI at 100 mM caused a reversible 50% reduction in RL of [3H]mannitol and a three times increase in [NAA]e (p < 0.05) but no further increase when normal perfusate was reintroduced. The mechanisms of NAA release from neurons are unknown but may involve the activation of unknown channels/carriers—possibly in relation to a volume regulatory response. The present study shows that the distribution of NAA in brain is dynamically regulated in acute ischemia and suggests that changes of NAA levels could be caused by other means than neuronal loss.
Production and Secretion of Adrenomedullin by Cultured Choroid Plexus Carcinoma Cells
Authors: Takahashi Kazuhiro , Satoh Fumitoshi , Hara Eishi , Murakami Osamu , Kumabe Toshihiro , Tominaga Teiji , Kayama Takamasa , Yoshimoto Takashi , Shibahara Shigeki
Published: July 2010

Abstract: Adrenomedullin is a potent vasodilator peptide that was originally isolated from pheochromocytoma. The production and secretion of adrenomedullin by cultured choroid plexus carcinoma cells were studied by radioimmunoassay and northern blot hybridization. Choroid plexus carcinoma is a rare malignant tumor derived from the epithelium of the choroid plexus. Immunoreactive adrenomedullin was detected in the conditioned medium of choroid plexus carcinoma cells (40.8 ± 7.5 fmol/105 cells/24 h; mean ± SEM, n = 5). Reverse‐phase HPLC of the conditioned medium showed one major peak of the immunoreactive peptide eluting in the position of synthetic human adrenomedullin and two smaller peaks eluting earlier. Addition of interleukin‐1β (10 ng/ml) alone or in combination with three cytokines, interferon‐γ (100 U/ml), tumor necrosis factor‐α (20 ng/ml), and interleukin‐1β (10 ng/ml), caused significant increases in the immunoreactive adrenomedullin concentrations in the medium (∼175 and 293% of the control level, respectively). Northern blot analysis showed the expression of 1.6‐kb adrenomedullin mRNA in the total RNA sample prepared from cultured choroid plexus carcinoma cells. Treatment with either interleukin‐1β or the combination of three cytokines caused significant increases in levels of adrenomedullin mRNA in parallel with those in immunoreactive adrenomedullin concentrations in the conditioned medium. These findings raise a possibility that adrenomedullin is secreted from the choroid plexus and has physiological roles in the CNS via the CSF. In addition, adrenomedullin secreted from choroid plexus carcinoma may be related to the pathophysiology of the tumor.
Evidence for Circadian Variations of Thyroid Hormone Concentrations and Type II 5′‐Iodothyronine Deiodinase Activity in the Rat Central Nervous System
Authors: Campos‐Barros Angel , Musa Alberto , Flechner Anke , Hessenius Carsten , Gaio Ulla , Meinhold Harald , Baumgartner Andreas
Published: July 2010

Abstract: The 24‐h patterns of tissue thyroid hormone concentrations and type II 5′‐ and type III 5‐iodothyronine deiodinase (5′D‐II and 5D‐III, respectively) activities were determined at 4‐h intervals in different brain regions of male euthyroid rats entrained to a regular 12‐h light/12‐h dark cycle (lights on at 6:00 a.m.). Activity of 5′D‐II, which catalyzes the intracellular conversion of thyroxine (T4) to 3,3′,5‐triiodo‐l‐thyronine (T3) in the CNS, and the tissue concentrations of both T4 and T3 exhibited significant daily variations in all brain regions examined. Periodic regression analysis revealed significant circadian rhythms with amplitudes ranging from 9 to 23% (for T3) and from 15 to 40% (for T4 and 5′D‐II) of the daily mean value. 5′D‐II activity showed a marked nocturnal increase (1.3–2.1‐fold vs. daytime basal value), with a maximum at the end of the dark period and a minimum between noon and 4:00 p.m. 5D‐III did not exhibit circadian patterns of variation in any of the brain tissues investigated. Our results disclose circadian rhythms of 5′D‐II activity and thyroid hormone concentrations in discrete brain regions of rats entrained to a regular 12:12‐h light‐dark cycle and reveal that, in the rat CNS, T3 biosynthesis is activated during the dark phase of the photoperiod. For all parameters under investigation, the patterns of variation observed were in part regionally specific, indicating that different regulatory mechanisms may be involved in generating the observed rhythms.
Preparation and Binding Properties of Radioiodinated Analogues of Dermorphin and Deltorphin with High Specificity for the μ‐ and δ‐Opioid Receptors
Authors: Gaudriault Georges , Zsürger Nicole , Vincent Jean‐Pierre
Published: July 2010

Abstract: The synthesis, purification, chemical characterization, and binding properties of two 125I‐labeled analogues of dermorphin and deltorphin‐I are described. Native deltorphin‐I and [Lys7]dermorphin sequences were elongated by an aminopentyl chain on their C‐terminal amide function and alkylated with the 125I‐labeled monoiodinated derivative of Bolton‐Hunter reagent (BH*). The resulting radiolabeled peptides, ε‐BH* [Lys7]dermorphin 5‐aminopentylamide and ω‐BH* deltorphin‐I 5‐aminopentylamide, have kept most of the original properties of the parent peptides. They bind with high selectivity and specificity to the μ‐ (dermorphin analogue) or δ‐ (deltorphin‐I analogue) opioid receptors from rat brain or from cells transfected with cDNAs encoding the μ and δ receptors. The autoradiographic distribution of specific binding sites for the 125I‐labeled dermorphin and deltorphin‐I analogues in rat brain is in complete agreement with previously reported localizations of μ‐ and δ‐opioid receptors. The two radiolabeled peptides are the best ligands of μ‐ and δ‐opioid receptors currently available in terms of sensitivity, specificity, and selectivity.
Posttranslational Maturation of the Prohormone Convertase SPC3 In Vitro
Authors: Coates Leigh C. , Birch Nigel P.
Published: July 2010

Abstract: The subtilisin‐like prohormone convertase SPC3 is likely to play a role in the biosynthesis of a variety of biologically active peptides. SPC3 undergoes a series of posttranslational processing events during its biosynthesis. Multiple forms have been identified that show varying degrees of truncation at the carboxyl terminus. In this study we show that the 86‐kDa form of recombinant SPC3 with an intact carboxyl terminus can undergo rapid carboxyl‐terminus truncation to produce a 64‐kDa form. We have defined the optimal conditions for carboxyl‐terminus truncation in vitro. The carboxyl‐terminus truncation reaction was less calcium sensitive, active over a broader pH range, and showed differences in inhibitor sensitivity compared with the enzymatic activities of full‐length and truncated forms of SPC3 toward a fluorescent peptide substrate. Increases in enzymatic activity of 86‐kDa SPC3 were also measured over a time frame consistent with conversion to the 64‐kDa form. However, similar specific activities for both forms of the enzyme suggest such activity increases may not be due to carboxyl‐terminus truncation. The different enzymatic properties of the major molecular forms of SPC3 highlight the importance of understanding the molecular events regulating carboxyl‐terminal processing of this endoprotease.
Localization and Characterization of Nitric Oxide Synthase in the Rat Suprachiasmatic Nucleus: Evidence for a Nitrergic Plexus in the Biological Clock
Authors: Chen Dong , Hurst William J. , Ding Jian M. , Faiman Lia E. , Mayer Bernd , Gillette Martha U.
Published: July 2010

Abstract: Behavioral and electrophysiological evidence indicates that the biological clock in the hypothalamic suprachiasmatic nuclei (SCN) can be reset at night through release of glutamate from the retinohypothalamic tract and subsequent activation of nitric oxide synthase (NOS). However, previous studies using NADPH‐diaphorase staining or immunocytochemistry to localize NOS found either no or only a few positive cells in the SCN. By monitoring conversion of l‐[3H]arginine to l‐[3H]citrulline, this study demonstrates that extracts of SCN tissue exhibit NOS specific activity comparable to that of rat cerebellum. The enzymatic reaction requires the presence of NADPH and is Ca2+/calmodulin‐dependent. To distinguish the neuronal isoform (nNOS; type I) from the endothelial isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction was 6.7, a characteristic value for nNOS. No difference in nNOS levels was seen between SCN collected in day versus night, either by western blot or by enzyme activity measurement. Confocal microscopy revealed for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate that neuronal fibers within the rat SCN express abundant nNOS and that the level of the enzyme does not vary temporally. The distribution and quantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN.
Enhanced Expression of Amyloid Precursor Protein in Response to Dibutyryl Cyclic AMP Is Not Mediated by the Transcription Factor AP‐2
Authors: Bourbonnière Martin , Shekarabi Masoud , Nalbantoglu Josephine
Published: July 2010

Abstract: The gene for amyloid precursor protein (APP) is expressed almost ubiquitously, with high levels of mRNA being detected in brain. The basal expression level of the APP gene can be modulated by physiological stimuli, and in this report we demonstrate that the second messenger cyclic AMP can regulate APP mRNA through transcriptional mechanisms. Northern blot analysis showed a 1.8‐fold increase in steady‐state levels of APP mRNA when the neuroblastoma × glioma hybrid cell line NG108‐15 was treated with dibutyryl cyclic AMP. Although the upstream sequences of the APP gene do not contain a canonical cyclic AMP response element, transient transfection assays in NG108‐15 cells using different portions of the APP promoter showed an increase in reporter gene activity mediated by sequences located between −303 to −204 and −488 to −2991. Cotransfection assays carried out in HepG2 cells with AP‐2, a cyclic AMP‐regulated transcription factor, failed to activate the APP promoter through the AP‐2 consensus sequence (GCCNNNCGG) located at position −205. Electrophoretic mobility shift analysis revealed that the AP‐2 binding activity present in HeLa nuclear extracts fails to recognize the APP AP‐2 consensus sequence. We conclude that increases in cyclic AMP levels can lead to an up‐regulation of APP gene transcription through at least two different regions of the APP promoter. This increase does not involve the AP‐2 consensus sequence present in the APP promoter located at position −205, and, moreover, this putative site is not recognized by the transcription factor AP‐2.
Induction of a Distinct Morphology and Signal Transduction in TrkB/PC12 Cells by Nerve Growth Factor and Brain‐Derived Neurotrophic Factor
Authors: Iwasaki Yasuno , Ishikawa Mitsue , Okada Noriko , Koizumi Shinichi
Published: July 2010

Abstract: A clonal cell line stably expressing trkB (TrkB/PC12) was established from rat pheochromocytoma PC12 cells. Brain‐derived neurotrophic factor (BDNF), as well as nerve growth factor (NGF), stimulates neurite outgrowth in TrkB/PC12 cells. However, the morphology of BDNF‐differentiated cells was clearly different from NGF‐differentiated cells. BDNF treatment brought about longer and thicker neurites and induced a flattened soma and an increase in somatic size. This is not explained enough by the quantitative difference in the strength between TrkA and TrkB stimulation, because the level of BDNF‐stimulated tyrosine phosphorylation of TrkB was similar to that of TrkA stimulated with NGF in PC12/TrkB cells. There was no difference in major tyrosine phosphorylated proteins induced by NGF and BDNF. Signal proteins such as phosphatidylinositol 3‐kinase, phospholipase C‐γ1, Shc, and mitogen‐activated protein kinase seem to be involved in both TrkA‐ and TrkB‐mediated signaling pathways. However, a tyrosine‐phosphorylated 38‐kDa protein (pp38) was detected in anti‐pan‐Trk immunoprecipitation only after NGF stimulation. Immunoprecipitation using three distinct anti‐pan‐Trk antibodies suggests that pp38 is not a fragment of TrkA. These data indicate that TrkA has a unique signal transduction pathway that is not stimulated through TrkB in TrkB/PC12 cells and suggest distinct functions among neurotrophin receptors.
Cytokine‐ and Endotoxin‐Induced Nitric Oxide Synthase in Rat Astroglial Cultures: Differential Modulation by Angiotensin II
Authors: Kopnisky Kathy L. , Sumners Colin , Chandler L. Judson
Published: July 2010

Abstract: Recent studies have shown that the stimulatory effects of bacterial endotoxin [lipopolysaccharide (LPS)] on inducible nitric oxide (NO) synthase (iNOS) in astroglia are significantly reduced by the peptide angiotensin II (Ang II). In the present study we have compared the modulatory actions of Ang II on cytokine‐ and LPS‐stimulated iNOS in astroglia cultured from adult rat brain. Incubation of astroglia with LPS (100 ng/ml; 24 h) and/or combinations of interleukin‐1β (IL‐1β; 10 ng/ml, 24 h), interferon‐γ (IFN‐γ; 100 U/ml, 24 h), or tumor necrosis factor‐α (TNF‐α; 100 ng/ml, 24 h) resulted in significant increases of iNOS mRNA, iNOS protein, and NO production, with the latter indicated by increased nitrite accumulation. The effects of LPS, IL‐1β, and TNF‐α were significantly decreased by coincubation with Ang II (100 ng/ml, 24 h). In contrast, Ang II did not alter the stimulation of iNOS mRNA levels and NO production elicited by IFN‐γ. Therefore, Ang II differentially modulates the stimulatory actions of LPS and cytokines on iNOS, and subsequently NO production, in astroglia. These data suggest that Ang II may have an important modulatory role in intracerebral immune responses that involve production of NO by astroglia.
Role of Extracellular Signal‐Regulated Protein Kinases 1 and 2 in Oligodendroglial Process Extension
Authors: Stariha Rochelle L. , Kikuchi Seiji , Siow Yaw L. , Pelech Steven L. , Kim Myong , Kim Seung U.
Published: July 2010

Abstract: The relationship between extracellular signal‐regulated protein kinase (ERK) activation and process extension in cultured bovine oligodendrocytes (OLGs) was investigated. Process extension was induced through the exposure of cultured OLGs to phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C (PKC), for various intervals. During the isolation of these OLGs from bovine brain, the original processes were lost. Therefore, any reinitiation of process extension via PMA stimulation was easily discernible through morphological monitoring. It was found that exposure of OLGs to PMA for 10 min was enough to induce OLG process extension 24–72 h later. Furthermore, this extension was still evident at least 1 week after the initial PMA stimulation, indicating that OLGs do not need continuous PKC activation to sustain process extension. Control and PMA‐stimulated OLGs were also subjected to immunocytochemistry using an anti‐ERK antibody selective for the mitogen‐activated protein kinases p42 Erk2 (ERK2) and p44 Erk1 (ERK1) isoforms. ERK immunoreactivity in the nucleus was evident after PMA stimulation of OLGs but not in control OLGs. In parallel experiments, the control and PMA‐stimulated OLGs were purified by Mono Q fractionation and subjected to ERK phosphotransferase assays using [γ‐32P]ATP and either myelin basic protein (MBP) or a synthetic peptide substrate based on the Thr97 phosphorylation site in MBP. These assays indicated that in PMA‐treated OLGs, ERK activation was at least 12‐fold higher than in control OLGs. Anti‐ERK and anti‐phosphotyrosine western blots of the assay fractions verified an enhanced phosphorylation of ERK1 and ERK2 in PMA‐treated fractions relative to control fractions. When OLGs were pretreated for 15 min with the ERK kinase (MEK) inhibitor PD 098059 before PMA stimulation, they exhibited a 67% decrease in ERK activation as compared with cells treated with PMA alone. Furthermore, these MEK inhibitor‐pretreated cells were still viable but showed no process extensions up to 1 week later. Therefore, we propose that a threshold level of ERK activity is required for the initiation of OLG process extension.
Transcriptional Regulation of Amyloid Precursor Protein During Dibutyryl Cyclic AMP‐Induced Differentiation of NG108‐15 Cells
Authors: Shekarabi Masoud , Bourbonnière Martin , Dagenais André , Nalbantoglu Josephine
Published: July 2010

Abstract: Early expression of amyloid precursor protein (APP) during development of the nervous system suggests that this protein may play an important role first in axogenesis and later in synaptogenesis. To study regulation of APP mRNA expression in neuronal cells, NG108‐15 neuroblastoma × glioma cells were induced to differentiate in the presence of dibutyryl cyclic AMP. Steady‐state levels of APP mRNA and APP isoforms increased gradually, concomitantly with the appearance of differentiated phenotype. Northern blot analysis showed a three‐fold increase in APP expression at day 6 of dibutyryl cyclic AMP treatment. Nuclear run‐on assays and transient transfections performed using APP promoter/reporter constructs confirmed a twofold increase in the rate of APP gene transcription. The stability of the mRNA was unchanged, with differentiated and nondifferentiated cells having the same half‐life of about 21 h. These results strongly suggest that APP mRNA induction in the differentiated NG108‐15 cells is due to an increase in the rate of transcription of the gene.
Muscarinic Acetylcholine Receptor‐Mediated Induction of zif268 mRNA in PC12D Cells Requires Protein Kinase C and the Influx of Extracellular Calcium
Authors: Ebihara Tatsuhiko , Saffen David
Published: July 2010

Abstract: The immediate‐early gene zif268 (egr‐1, NGFI‐A, krox‐24) encodes a transcription factor that has been proposed to play a role in differentiation and neuronal plasticity. zif268 mRNA is undetectable in unstimulated PC12D cells, a subline of PC12 characterized by accelerated differentiation in the presence of nerve growth factor, but is rapidly and robustly induced following exposure to muscarinic acetylcholine receptor (mAChR) agonists. Although PC12D cells express mRNAs for both m1 and m4 mAChR subtypes, induction of zif268 mRNA by mAChR agonists is apparently mediated exclusively by the m1 subtype because the induction is completely blocked by Dendroaspis angusticeps m1 toxin. Pretreatment of the cells with the specific protein kinase C (PKC) inhibitor GF109203X partially inhibits mAChR‐mediated induction of zif268 mRNA. The remaining induction is blocked by chelation of extracellular calcium with EGTA. EGTA prevents the influx of calcium without blocking mAChR‐mediated release of calcium from internal stores. These data indicate that both PKC and the influx of extracellular calcium play a role in the induction of zif268 mRNA following activation of the m1 mAChR. Transient increases in intracellular calcium levels resulting from the release of calcium from internal stores alone are not sufficient to induce zif268 mRNA. Rather, induction requires a prolonged elevation of intracellular calcium levels, which is sustained by the influx of extracellular calcium.
Cyclic AMP‐Mediated Enhancement of High‐Affinity Choline Transport and Acetylcholine Synthesis in Brain
Authors: Vogelsberg V. , Neff N. H. , Hadjiconstantinou M.
Published: July 2010

Abstract: Intracerebroventricular administration of N6, 2′‐O‐dibutyryladenosine 3′,5′‐cyclic monophosphate (db‐cyclic AMP) to mice increased high‐affinity choline transport (HAChT) into synaptosomal preparations from the hippocampus, striatum, and frontal cortex in a time‐dose‐, and brain region‐dependent manner. Similar observations were made when the cyclic AMP analogue 8‐bromo‐cyclic AMP, the adenylyl cyclase activator forskolin, and the phosphodiesterase inhibitor 3‐isobutyl‐1‐methylxanthine were administered. Inhibition of phosphatase 1 and 2A, with okadaic acid, increased basal choline transport and enhanced the response to db‐cyclic AMP. The early increase of HAChT activity induced by db‐cyclic AMP was blocked by H‐7 and H‐89, protein kinase A inhibitors, but not by cycloheximide, a protein synthesis inhibitor. Kinetic analysis of the early changes of HAChT revealed an increase in the apparent Vmax without a change of the Km for choline. Hemicholinium‐3 (HC‐3) binding was not altered when studied 1 h after db‐cyclic AMP administration. In contrast, HC‐3 binding and HAChT activity were both elevated when estimated 3 h after the treatment, and pretreatment with cycloheximide partially prevented the db‐cyclic AMP‐induced HAChT rise. As evidence that enhanced HAChT is associated with a direct action of cyclic AMP‐dependent pathways on the cholinergic nerve terminals, addition of 8‐bromocyclic AMP to isolated hippocampal synaptosomes induced an increase of HAChT that was prevented by H‐89. Choline acetyltransferase activity was not affected at any time during the studies. The synthesis of acetylcholine, however, was enhanced 1 h after db‐cyclic AMP addition. Our studies show that cyclic AMP‐mimetic compounds appear to modulate the choline carrier by a dual mode: an early increase of the maximal velocity without a change of the number of HC‐3 binding sites and a late rise of transport that is accompanied by an increase of HC‐3 binding. We postulate that HAChT and consequently acetylcholine synthesis in vivo is modulated, in part, by protein kinase A.
Rapid Degeneration of Cultured Human Brain Pericytes by Amyloid β Protein
Authors: Verbeek Marcel M. , De Waal Robert M. W. , Schipper Janine J. , Van Nostrand William E.
Published: July 2010

Abstract: Amyloid β protein (Aβ) deposition in the cerebral arterial and capillary walls is one of the major characteristics of brains from patients with Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis‐Dutch type (HCHWA‐D). Vascular Aβ deposition is accompanied by degeneration of smooth muscle cells and pericytes. In this study we found that Aβ1–40 carrying the “Dutch” mutation (HCHWA‐D Aβ1–40) as well as wild‐type Aβ1–42 induced degeneration of cultured human brain pericytes and human leptomeningeal smooth muscle cells, whereas wild‐type Aβ1–40 and HCHWA‐D Aβ1–42 were inactive. Cultured brain pericytes appeared to be much more vulnerable to Aβ‐induced degeneration than leptomeningeal smooth muscle cells, because in brain pericyte cultures cell viability already decreased after 2 days of exposure to HCHWA‐D Aβ1–40, whereas in leptomeningeal smooth muscle cell cultures cell death was prominent only after 4–5 days. Moreover, leptomeningeal smooth muscle cell cultures were better able to recover than brain pericyte cultures after short‐term treatment with HCHWA‐D Aβ1–40. Degeneration of either cell type was preceded by an increased production of cellular amyloid precursor protein. Both cell death and amyloid precursor protein production could be inhibited by the amyloid‐binding dye Congo red, suggesting that fibril assembly of Aβ is crucial for initiating its destructive effects. These data imply an important role for Aβ in inducing perivascular cell pathology as observed in the cerebral vasculature of patients with Alzheimer's disease or HCHWA‐D.
Differential Regulation of Fibroblast Growth Factor (FGF)‐2 and FGF Receptor 1 mRNAs and FGF‐2 Isoforms in Spinal Ganglia and Sciatic Nerve After Peripheral Nerve Lesion
Authors: Meisinger Christof , Grothe Claudia
Published: July 2010

Abstract: To study the functional role of endogenous basic fibroblast growth factor‐2 (FGF‐2) during degeneration and regeneration of the sensory system, we have determined the expression and regulation of FGF‐2 and FGF receptor (FGFR)‐1 mRNAs in spinal ganglia and sciatic nerve during experimental transection and crush injury of the sciatic nerve. In contrast to levels of the FGFR‐1 transcript, which is not altered, the level of FGF‐2 mRNA is dramatically up‐regulated in spinal ganglia after injury. In the proximal and distal nerve stumps both transcript levels are significantly elevated, albeit at different time points. The FGF‐2 isoforms are differently up‐regulated in spinal ganglia and sciatic nerve following peripheral nerve lesion. The differential response of FGF‐2 mRNA and protein and of FGFR‐1 mRNA in spinal ganglia and sciatic nerve after lesion is suggestive of different physiological functions: a local reaction at the lesion site where axonal regrowth occurs and a trophic reaction for the degenerating/regenerating sensory neurons.
Increased Density of Catalytic Sites and Expression of Brain Monoamine Oxidase A in Humans with Hepatic Encephalopathy
Authors: Mousseau D. D. , Baker G. B. , Butterworth R. F.
Published: July 2010

Abstract: Altered biogenic amine metabolism and function are believed to underlie certain of the neuropsychiatric symptoms, e.g., depression, mania, and anxiety, encountered in clinical hepatic encephalopathy (HE). We therefore investigated the activity of the degradative enzyme monoamine oxidase (MAO) and its binding parameters using [3H]Ro 41‐1049 (defining MAO‐A) and [3H]Ro 19‐6327 (Lazabemide; defining MAO‐B) in autopsied brain tissue from male cirrhotic patients with HE. The MAO‐B parameters in HE patient tissue were not significantly different from those determined for control tissue. In contrast, increases in MAO‐A activities in HE patient frontocortical (by ∼50%) and cerebellar (by ∼145%) tissues were observed, confirming our previous findings using comparable tissues. Increases in the abundance of the active MAO‐A protein were of the same order of magnitude, e.g., in frontal cortex by ∼85% and in cerebellum by ∼225%. Reverse transcriptase‐polymerase chain reaction indicated an increase in the level of gene expression (by ∼155%) and thus offers some of the first evidence of a transcriptional event potentially mediating MAO‐A function in human brain tissue. The levels of biogenic amine acid metabolites were increased as expected. As HE patients are most often treated for their hepatic symptoms rather than their neuropsychiatric manifestations, they represent an important “untreated” psychiatric population. The present findings are therefore not only important for our understanding of the pathophysiology of HE but also extremely relevant to our understanding of the pharmacotherapy of other neuropsychiatric disorders in which biogenic amine and MAO‐A dysfunction is indicated.
Inhibition of the High‐Affinity Brain Glutamate Transporter GLAST‐1 via Direct Phosphorylation
Authors: Conradt Marcus , Stoffel Wilhelm
Published: July 2010

Abstract: Neurotransmission at excitatory glutamatergic synapses is terminated by the reuptake of the neurotransmitter by high‐affinity transporters, which keep the extracellular glutamate concentration below excitotoxic levels. The amino acid sequence of the recently isolated and cloned brain‐specific glutamate/aspartate transporter (GLAST‐1) of the rat reveals three consensus sequences of putative phosphorylation sites for protein kinase C (PKC). The PKC activator phorbol 12‐myristate 13‐acetate (PMA) decreased glutamate transport activity in Xenopus oocytes and human embryonic kidney cells (HEK293) expressing the cloned GLAST‐1 cDNA, within 20 min, to 25% of the initial transport activity. This down‐regulation was blocked by the PKC inhibitor staurosporine. GLAST‐1 transport activity remains unimpaired by phorbol 12‐monomyristate. Removal of all putative PKC sites of wild‐type GLAST‐1 by site‐directed mutagenesis did not abolish inhibition of glutamate transport. [32P]Phosphate‐labeled wild‐type and mutant transport proteins devoid of all predicted PKC sites were detected by immunoprecipitation after stimulation with PMA. Immunoprecipitation of [35S]methionine‐labeled transporter molecules indicates a similar stability of phosphorylated and nonphosphorylated GLAST‐1 protein. Immunofluorescence staining did not differentiate surface staining of HEK293 cells expressing GLAST‐1 with and without PMA treatment. These data suggest that the neurotransmitter transporter activity of GLAST‐1 is inhibited by phosphorylation at a non‐PKC consensus site.
Proteins Associated with α1‐Subunit‐Containing GABAA Receptors from Bovine Brain
Authors: Kannenberg Kai , Baur Roland , Sigel Erwin
Published: July 2010

Abstract: In contrast to some other ion channels, there are at present no proteins known that bind specifically to mature GABAA receptor channels. Such proteins may be important for the structural organization and cytoskeletal anchoring of GABAA receptors and could also be expected to have channel modulatory effects. To identify proteins that are associated with naturally occurring GABAA receptors we immunoprecipitated these receptors from detergent‐solubilized bovine brain membranes by an antibody directed against the α1‐subunit. Tubulin and actin were observed to coprecipitate specifically with the receptors. Nine additional proteins were detected, hinting at a complex protein network associated with α1‐subunit‐containing GABAA receptors. Results of a biochemical characterization of these GABAA receptor‐tubulin complex‐associated proteins (GTAPs) are presented here. Peptide mass fingerprinting analysis and microsequencing of tryptic peptides indicated that at least three GTAPs have not been described until the present.
V170 Area Plays an Essential Role in the Biological Activity of Human Ciliary Neurotrophic Factor
Authors: Inoue Makoto , Karita Hiromi , Kikuchi Kaoru , Nakayama Chikao , Noguchi Hiroshi
Published: July 2010

Abstract: The structure‐function relationships of human ciliary neurotrophic factor (CNTF) were analyzed by mutagenic means. Amino acid substitutions at helix D caused marked changes in the biological activity of CNTF, suggesting that the residues at helix D of CNTF participate in receptor recognition. In particular, both the cell survival‐promoting activity and receptor binding ability of V170 mutant CNTF proteins correlated well with the hydrophobicity of amino acids at position 170. The reduction of hydrophobicity at position 170 resulted in a loss of biological activity, indicating that the hydrophobicity of V170 is essential for the receptor binding and cell survival‐promoting activity. Substitutions of R171 or D175 evoked very little folding ability and negated the biological activity of CNTF. As R171 and D175 interact electrostatically with each other and with E75 and R72, respectively, these interactions would be indispensable for stabilizing the whole CNTF protein and for maintaining the structure of the receptor binding epitope.
Characterization of Calpain‐Mediated Proteolysis of GluR1 Subunits of α‐Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionate Receptors in Rat Brain
Authors: Bi Xiaoning , Chen Jing , Dang Sandeeb , Wenthold Robert J. , Tocco Georges , Baudry Michel
Published: July 2010

Abstract: Previous results have indicated that GluR1 subunits of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptors are targets of calpain. In the present study, we determined the effects of calpain treatment of synaptic membranes on GluR1 subunits using western blots with antibodies directed against the C‐terminal (C‐Ab) and the N‐terminal (N‐Ab) domains of the proteins, and compared them with the effects of calcium treatment of frozen‐thawed brain sections. Calpain treatment of synaptic membranes resulted in a large decrease in the GluR1 band (105 kDa) labeled with C‐Ab and in the formation of a doublet band labeled with N‐Ab due to the appearance of a new species of GluR1 (98 kDa). These effects were blocked almost completely by calpain inhibitors. Calpain‐induced changes in GluR1 immunological properties were not associated with modifications of [3H]AMPA or 6‐cyano‐7‐[3H]nitroquinoxaline‐2,3‐dione ([3H]CNQX) binding. Treatment of frozen‐thawed brain sections with concentrations of calcium as low as 0.2 mM resulted in a large decrease in the 105‐kDa GluR1 band and in the concurrent appearance of the 98‐kDa band. This treatment was associated with increased [3H]AMPA and [3H]CNQX binding. These results suggest that there exist several types/states of GluR1 subunits exhibiting different sensitivities to calpain. Our data also indicate the existence of additional calcium‐dependent processes regulating the characteristics of receptors in intact tissues.
Irreversible Inhibition of High‐Affinity [3H]Kainate Binding by a Novel Photoactivatable Analogue: (2′S,3′S,4′R)‐2′‐Carboxy‐4′‐(2‐Diazo‐1‐Oxo‐3,3,3‐Trifluoropropyl)‐3′‐Pyrrolidinyl Acetate
Authors: Willis Colin L. , Wacker Dean A. , Bartlett Richard D. , Bleakman David , Lodge David , Chamberlin A. Richard , Bridges Richard J.
Published: July 2010

Abstract: A photolabile trifluoromethyldiazoketone derivative of kainate (KA), (2′S,3′S,4′R)‐2′‐carboxy‐4′‐(2‐diazo‐1‐oxo‐3,3,3‐trifluoropropyl)‐3′‐pyrrolidinyl acetate (DZKA), was synthesized and evaluated as an irreversible inhibitor of the high‐affinity KA site on rat forebrain synaptic plasma membranes (SPMs). In the absence of UV irradiation, DZKA preferentially blocked [3H]KA binding with an IC50 of 0.63 µM, a concentration that produced little or no inhibition at AMPA or NMDA sites. At 100 µM, however, DZKA inhibited [3H]AMPA and l‐[3H]glutamate binding by ∼50%. When examined electrophysiologically in HEK293 cells expressing human KA (GluR6) or AMPA (GluR1) subtypes, DZKA acted preferentially at KA receptors as a weak agonist. DZKA also exhibited little or no excitotoxic activity in mixed rat cortical cultures. Irreversible inhibition was assessed by pretreating SPMs with DZKA (50 µM) in the presence of UV irradiation, removing unbound DZKA, and then assaying the reisolated SPMs for radioligand binding. This protocol produced a selective and irreversible loss of ∼50% of the [3H]KA sites. The binding was recoverable in SPMs pretreated with DZKA or UV alone. Coincubation with l‐glutamate prevented the loss in [3H]KA binding, suggesting that the inactivation occurred at or near the ligand binding site. These results are consistent with the action of DZKA as a photoaffinity ligand for the KA site and identify the analogue as a valuable probe for future investigations of receptor structure and function.
Insertion of Lysosomal Targeting Sequences to the Amyloid Precursor Protein Reduces Secretion of βA4
Authors: Schrader‐Fischer Gesine , Staufenbiel Matthias , Paganetti Paolo A.
Published: July 2010

Abstract: The processing of the amyloid precursor protein (APP) was investigated in cells stably expressing different APP hybrid proteins. The cytoplasmic domain of APP was either deleted or replaced by the corresponding domain of the membrane protein TGN38, lamp‐1, or LIMPII. The cytosolic domain of TGN38 in the APP molecule did not alter the secretion of βA4 when compared with the wild‐type APP; however, APP associated with the cell surface and the nonamyloidogenic processing of APP were reduced. With the APP molecules carrying the lysosomal targeting signals of lamp‐1 or LIMPII, a decrease in the secretion of βA4 was observed. Cell surface association and nonamyloidogenic processing were also impaired. This suggests increased degradation of APP and thus efficient targeting to the lysosomal system. Cells expressing the Swedish APP variant generated intracellular βA4 that accumulated after treatment with chloroquine. This effect was more dramatic with APP mutants carrying lysosomal targeting signals than with full‐length APP. Our data suggest the existence of an intracellular site of βA4 generation from where βA4 is degraded rather than secreted.
Apoptosis in Cerebellar Granule Neurones: Involvement of Interleukin‐1β Converting Enzyme‐Like Proteases
Authors: Taylor Joanne , Gatchalian Christine L. , Keen Gregory , Rubin Lee L.
Published: July 2010

Abstract: Proteases of the interleukin‐1β converting enzyme (ICE) family have been implicated as mediators of apoptosis in several cell types. Here we report the ability of peptide inhibitors of ICE‐like proteases to inhibit apoptosis of cultured cerebellar granule neurones caused by reduction of extracellular K+ levels and by the broad‐spectrum protein kinase inhibitor staurosporine. Unlike apoptosis induced by K+ deprivation, staurosporine‐induced neuronal death does not require new protein synthesis. The ICE‐like protease inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp (O‐methyl)fluoromethyl ketone (zVAD‐fmk) was found to be extremely effective at preventing staurosporine‐induced death of cerebellar granule neurones and yet was completely ineffective in preventing K+ deprivation‐induced death. Staurosporine induced cleavage of the 116‐kDa poly(ADP‐ribose) polymerase enzyme, a substrate of ICE‐like proteases, to the 85‐kDa product, and this cleavage was also blocked by zVAD. By comparison, K+ deprivation led to the disappearance of the 116‐kDa protein, with no detectable increase in level of the 85‐kDa cleavage product. Taken together, these results imply the existence of divergent ICE‐like protease pathways in a CNS model of neuronal apoptosis.
Nerve Tissue‐Specific Human Glutamate Dehydrogenase that Is Thermolabile and Highly Regulated by ADP
Authors: Shashidharan P. , Clarke Donald D. , Ahmed Naveed , Moschonas Nicholas , Plaitakis Andreas
Published: July 2010

Abstract: Glutamate dehydrogenase (GDH), an enzyme that is central to the metabolism of glutamate, is present at high levels in the mammalian brain. Studies on human leukocytes and rat brain suggested the presence of two GDH activities differing in thermal stability and allosteric regulation, but molecular biological investigations led to the cloning of two human GDH‐specific genes encoding highly homologous polypeptides. The first gene, designated GLUD1, is expressed in all tissues (housekeeping GDH), whereas the second gene, designated GLUD2, is expressed specifically in neural and testicular tissues. In this study, we obtained both GDH isoenzymes in pure form by expressing a GLUD1 cDNA and a GLUD2 cDNA in Sf9 cells and studied their properties. The enzymes generated showed comparable catalytic properties when fully activated by 1 mM ADP. However, in the absence of ADP, the nerve tissue‐specific GDH showed only 5% of its maximal activity, compared with ∼40% showed by the housekeeping enzyme. Low physiological levels of ADP (0.05–0.25 mM) induced a concentration‐dependent enhancement of enzyme activity that was proportionally greater for the nerve tissue GDH (by 550–1,300%) than of the housekeeping enzyme (by 120–150%). Magnesium chloride (1–2 mM) inhibited the nonactivated housekeeping GDH (by 45–64%); this inhibition was reversed almost completely by ADP. In contrast, Mg2+ did not affect the nonstimulated nerve tissue‐specific GDH, although the cation prevented much of the allosteric activation of the enzyme at low ADP levels (0.05–0.25 mM). Heat‐inactivation experiments revealed that the half‐life of the housekeeping and nerve tissue‐specific GDH was 3.5 and 0.5 h, respectively. Hence, the nerve tissue‐specific GDH is relatively thermolabile and has evolved into a highly regulated enzyme. These allosteric properties may be of importance for regulating brain glutamate fluxes in vivo under changing energy demands.
Increased Phosphorylation of Myelin Basic Protein During Hippocampal Long‐Term Potentiation
Authors: Atkins Coleen M. , Chen Shu‐Jen , Klann Eric , Sweatt J. David
Published: July 2010

Abstract: Hippocampal long‐term potentiation (LTP) is a long‐lasting and rapidly induced increase in synaptic strength. Previous experiments have determined that persistent activation of protein kinase C (PKC) contributes to the early maintenance phase of LTP (E‐LTP). Using the back‐phosphorylation method, we observed an increase in the phosphorylation of a 21‐kDa PKC substrate, termed p21, 45 min after LTP was induced in the CA1 region of the hippocampus. p21 was found to have the same apparent molecular weight as the 18.5‐kDa isoform of myelin basic protein (MBP) and was recognized by an antibody to MBP in western blotting and immunoprecipitation. Furthermore, p21 from control and potentiated hippocampal slices and purified MBP have identical phosphopeptide maps when back‐phosphorylated and then digested with either endoproteinase Lys‐C or endoproteinase Asp‐N, suggesting that p21 and MBP are identical proteins. As there was no observed change in the amount of MBP in LTP, the increase in MBP phosphorylation during LTP cannot be explained by a change in the amount of protein. From these experiments, we conclude that the phosphorylation of the 18.5‐kDa isoform of MBP is increased during E‐LTP.
Effects of Single and Repeated Footshock on Dopamine Release and Metabolism in the Brains of Fischer Rats
Authors: Chrapusta Stanislaw J. , Wyatt Richard Jed , Masserano Joseph M.
Published: July 2010

Abstract: Changes in the tissue levels of 3‐methoxytyramine (3‐MT), 3,4‐dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and dopamine in the frontal cortex, hypothalamus, nucleus accumbens, and striatum were evaluated after 0.5‐4 h of footshock (2 mA, for 3 s every 30 ± 5 s) in Fischer rats. 3‐MT, DOPAC, and HVA levels in the four brain areas peaked at 0.5 h and in most cases returned to baseline values within 4 h. No changes were found in dopamine levels. Repeated footshock stress was evaluated by administering 10 footshock sessions (0.5 h, two per day for 5 days). At the end of the 10th footshock session, 3‐MT levels were higher than at the end of the first footshock session in three of the four brain regions, indicating sensitization of dopamine release. No differences were found between the first and 10th footshock sessions in DOPAC and HVA levels. Fourteen days after the 10th footshock session, the levels of 3‐MT, DOPAC, and HVA were the same as in control rats in all four brain regions. A 0.5‐h footshock challenge presented 14 days after the 10th footshock session attenuated DOPAC levels in the hypothalamus and nucleus accumbens. In contrast, DOPAC and HVA levels in the frontal cortex showed sensitization after footshock challenge, and a similar trend was apparent for 3‐MT levels. These results indicate that repeated footshock stress induces generalized sensitization of dopamine release and turnover in some areas of the brain of Fischer rats. This sensitization may persist in the cortical but not subcortical dopamine neurons after discontinuation of the treatment.
Effects of Methylphenidate on Extracellular Dopamine, Serotonin, and Norepinephrine: Comparison with Amphetamine
Authors: Kuczenski Ronald , Segal David S.
Published: July 2010

Abstract: Methylphenidate promotes a dose‐dependent behavioral profile that is very comparable to that of amphetamine. Amphetamine increases extracellular norepinephrine and serotonin, in addition to its effects on dopamine, and these latter effects may play a role in the behavioral effects of amphetamine‐like stimulants. To examine further the relative roles of dopamine, norepinephrine, and serotonin in the behavioral response to amphetamine‐like stimulants, we assessed extracellular dopamine and serotonin in caudate putamen and norepinephrine in hippocampus in response to various doses of methylphenidate (10, 20, and 30 mg/kg) that produce stereotyped behaviors, and compared the results with those of a dose of amphetamine (2.5 mg/kg) that produces a level of stereotypies comparable to the intermediate dose of methylphenidate. The methylphenidate‐induced changes in dopamine and its metabolites were consistent with changes induced by other uptake blockers, and the magnitude of the dopamine response for a behaviorally comparable dose was considerably less than that with amphetamine. Likewise, the dose‐dependent increase in norepinephrine in response to methylphenidate was also significantly less than that with amphetamine. However, in contrast to amphetamine, methylphenidate had no effect on extracellular serotonin. These results do not support the hypothesis that a stimulant‐induced increase in serotonin is necessary for the appearance of stereotyped behaviors.