bluuebunny/crossref_metadata_2025 huggingface dataset:
Immediate Early Gene Expression in PC12 Cells Exposed to Lead: Requirement for Protein Kinase C
Authors: Kim Kyung‐Ah , Chakraborti Tamal , Goldstein Gary W. , Bressler Joseph P.
Published: July 2010

We previously demonstrated induction of c‐fos mRNA in PC12 cells exposed to lead that was dependent on new transcription. In the current work, we examined two signal transduction mechanisms that are activated by lead and have been shown to mediate induction of c‐fos mRNA. One mechanism involves protein kinase C, and the other requires calmodulin‐dependent protein kinase II. Significant increases in the levels of c‐fos, c‐jun, and egr‐1 but not NGFIB mRNA were observed in PC12 cells exposed to lead or phorbol 12‐myristate 13‐acetate. In contrast, PC12 cells depolarized with 56 mM K+ displayed an increase in c‐fos, egr‐
1, and NGFIB but not c‐jun mRNA. Similar to other activators of protein kinase C, lead increased AP‐1 and Egr‐1 DNA binding activity. Additionally, lead increased luciferase activity in cerebellar granule cells transfected with an AP‐1 luciferase reporter construct. Lead did not increase c‐fos mRNA in PC12 cells that were depleted of protein kinase C by a 24‐h treatment with phorbol 12,13‐dibutyrate or incubated with the protein kinase C inhibitor H‐7. In contrast, an inhibitor of calmodulin‐dependent protein kinase, KN‐62, and an inhibitor of calmodulin, W‐7, did not block the induction of c‐fos mRNA by lead. An increase in serum‐response element DNA‐binding activity was observed in nuclear extracts from PC12 cells exposed to lead. It is interesting that lead activated protein kinase C isoforms δ and ε , but not isoforms αand β. In conclusion, lead appears to induce the expression of immediate early genes by a mechanism that requires protein kinase C.
C3‐Fullero‐tris‐Methanodicarboxylic Acid Protects Cerebellar Granule Cells from Apoptosis
Authors: Bisaglia M. , Natalini B. , Pellicciari R. , Straface E. , Malorni W. , Monti C. , Franceschi D. , Schettini G.
Published: July 2010

Buckminsterfullerenols were recently investigated for their protective properties in different models of acute and chronic neurodegeneration. We tested C3‐fullero‐tris‐methanodicarboxylic acid in our in vitro model of apoptotic neuronal death, which consists of shifting the culture K+ concentration from 25 to 5 mM for rat cerebellar granule cells. The impairment of mitochondrial respiratory function as well as chromatin derangement and fragmentation of DNA in apoptotic oligonucleosomes that occur in these conditions were protected by this compound in a concentration‐dependent way. To assess whether antioxidant activity could account for the rescue of cerebellar granule cells from apoptosis, we tested the fullerene derivative under FeSO4‐induced oxidative stress and found significant protection. Thus, we visualized membrane and cytoplasmic peroxides and reactive oxygen species and found a significant reduction of the species after 24 h in 5 mM K+ with the fullerene derivative. Such evidence suggests that this compound exerts a protective role in cerebellar granule cell apoptosis, likely reducing the oxidative stress.
Ascorbate Inhibits Edema in Brain Slices
Authors: Brahma B. , Forman R.E. , Stewart E.E. , Nicholson C. , Rice M.E.
Published: July 2010

Ascorbate is an essential antioxidant in the CNS, localized predominantly in neuronal cytosol. Slices of mammalian brain rapidly lose ascorbate, however, when incubated in ascorbate‐free media; brain slices also take up water and swell. Here we investigated water gain in coronal slices of rat forebrain incubated with and without ascorbate for 1‐3 h at 34°C. Slices progressively gained water in ascorbate‐free media, with a significant 12% water increase after 3 h at 34°C, compared with the water content of slices after a 1‐h recovery period at 24°C, immediately following slice preparation. Inclusion of 400 μM ascorbate in the medium led to an increase in tissue ascorbate content and prevented water gain at 34°C. By contrast, water gain was not inhibited by isoascorbate or thiourea, which are antioxidants that are not accumulated in brain cells. The oxidant H2O2 enhanced water gain, whereas a cocktail of NMDA and non‐NMDA receptor blockers inhibited edema formation to the same extent as ascorbate. These data demonstrate that brain edema, linked to glutamate‐receptor activation, can result from intracellular oxidative stress and that this can be prevented by ascorbate.
Characterization of Carrier‐Mediated Efflux in Human Embryonic Kidney 293 Cells Stably Expressing the Rat Serotonin Transporter: A Superfusion Study
Authors: Sitte H.H. , Scholze P. , Schloss P. , Pifl C. , Singer E.A.
Published: July 2010

Human embryonic kidney 293 cells stably transfected with the rat plasmalemmal serotonin transporter (rSERT) were incubated with 5‐[3H]hydroxytryptamine ([3H]5‐HT) and superfused. Substrates of the rSERT, such as

p‐chloroamphetamine (PCA) or methylenedioxymethamphetamine, concentration‐dependently increased basal efflux of [3H]5‐HT. 5‐HT reuptake blockers (e.g., imipramine, citalopram) also caused an enhancement of [3H]5‐HT efflux, reaching about half the maximal effect of the rSERT substrates. In uptake experiments, both groups of substances concentration‐dependently inhibited 5‐HT uptake. EC50 values obtained in superfusion experiments significantly correlated with IC50 values from uptake studies (r2 = 0.92). Addition of the Na+,K+‐ATPase inhibitor ouabain (100 μM) to or the omission of K+ from the superfusion buffer accelerated basal efflux. The effect of PCA (10 μM) was markedly enhanced by both measures, whereas the effect of uptake inhibitors remained unchanged. When [3H]MPP+, a substrate with low affinity for the rSERT, was used instead of [3H]5‐HT for labeling the cells, uptake inhibitors failed to augment efflux. By contrast, PCA accelerated [3H]MPP+ efflux, and its effect was strongly enhanced in the presence of ouabain. The results suggest that the [3H]5‐HT efflux caused by substrates of rSERT is carrier‐mediated, whereas efflux induced by uptake inhibitors is a consequence of interrupted high‐affinity reuptake that is ongoing even under superfusion conditions.
Angiotensin II Modulates the Activity of Na+,K+‐ATPase in Cultured Rat Astrocytes via the AT1 Receptor and Protein Kinase C‐δ Activation
Authors: Muscella Antonella , Aloisi Francesca , Marsigliante Santo , Levi Giulio
Published: July 2010

In astrocytes the activity of the Na+,K+‐ATPase pump maintains an inwardly directed electrochemical sodium gradient used by the Na+‐dependent transporters and regulates the extracellular K+ concentration essential for neuronal excitability. We show here that incubation of cultured rat astrocytes with angiotensin II (Ang II) modulates Na+,K+‐ATPase activity, in a dose‐ and time‐dependent manner. Na+,K+‐ATPase activation was mediated by binding of Ang II to AT1 receptors as it was completely blocked by DuP 753, a specific AT1 receptor subtype antagonist. Stimulation of Na+,K+‐ATPase activity by Ang II was dependent on protein kinase C (PKC) activation because PKC antagonists abolished the inducing effect of Ang II and the PKC activator phorbol 12‐myristate 13‐acetate enhanced transporter activity. Ang II stimulated translocation of PKC‐δ but not that of other PKC isoforms from the cytosol to the plasma membrane. These results indicate that the activity of Na+,K+‐ATPase in astrocytes is increased by physiological concentrations of Ang II and that the AT1 receptor subtype mediates the Na+,K+‐ATPase response to Ang II via PKC‐δ activation.
Involvement of multiple molecular events in methamphetamine‐induced neurodegeneration: evidence from cDNA array analysis
Authors: Cadet J. L.
Published: July 2010

Methamphetamine (METH) is a neurotoxic drug of abuse that can cause terminal degeneration. Our laboratory recently showed that METH can also cause widespread apoptosis in the rodent brain. Current concepts of the molecular neurotoxicity of this illicit substance have earlier suggested the participation of reactive oxygen species, inflammatory processes and immediate early genes. Recent cDNA studies in our laboratory have hinted to the possibility that METH‐induced neurodegeneration might also include the participation of cell death might also include the participation of cell death genes such as BAX and BCL‐2. Activation of multiple caspases appears to also occur during METH‐induced neurodegeneration. Furthermore, DNA repair pathways seem to also be involved in attempts to protect against METH‐induced DNA damage. These results will be discussed in terms of the possible involvement of multiple transduction mechanisms in the appearance of METH neurotoxicity.
Workshop 2: Approaches for Studying the Biological Roles of Sphingolipids. Probing apoptosis during neuronal development with novel ceramide analogs
Authors: Bieberich E. , MacKinnon S. , Silva J. , Yu R. K.
Published: July 2010

Lipid analysis of gestational day E14.5 mouse brain revealed elevation of ceramide to a tissue concentration that induced apoptosis when added to the medium of neuroprogenitor cells grown in cell culture. Elevation of ceramide was coincident with the first appearance of β‐series complex gangliosides (BCGs). Expression of BCGs by stable transfection of murine neuroblastoma (F‐11) cells with sialyltransferase‐II (ST2) resulted in a 70% reduction of apoptosis that was induced with the novel ceramide analog (NCA) N‐oleoyl serinol (S18). This was most likely due to an 80% reduced expression of prostate apoptotis response‐4 (PAR‐4). PAR‐4 expression and apoptosis were restored by preincubation of ST2‐transfected cells with N‐butyl deoxinojirimycin (NB‐DNJ) or PD98059, two inhibitors of ganglioside biosynthesis or p42/44 MAPK‐kinase, respectively. In sections of day E14.5 mouse brain, the intermediate zone showed intensive staining for complex gangliosides, but only low staining for apoptosis (TUNEL) and PAR‐4. Apoptosis and PAR‐4 expression, however, were elevated in the ventricular zone, which only weakly stained for complex gangliosides. Complex gangliosides may thus activate a cell survival mechanism that selectively protects developing neurons against ceramide‐induced apoptosis by up‐regulation of MAPK and reduction of PAR‐4 expression. NCAs may be useful to analyse the molecular mechanisms that underlie or counteract ceramide‐induced apoptosis during neuronal development.

Acknowledgements:  Supported by grants MH61934‐04 (to E.B.) and NS11853 (to R.K.Y.).
The role of GM1 and other gangliosides in neuronal function
Authors: Ledeen R. , Wu G. , Lu Z‐H.
Published: July 2010

Study of neuronal ganglioside function at the cellular level has involved observation of morphological/biochemical changes induced by application of exogenous ganglioside(s) or manipulation of endogenous gangliosides. The first approach was used by several laboratories to study the neuritogenic properties of gangliosides, until it was recognized that this effect was nonspecific and required relatively high (e.g. nonphysiological) concentrations. More recently it was shown that the nature of the induced neurites in neuroblastoma cells differs, those created by exogenous GM1 resembling dendrites in contrast to those accompanying elevation of endogenous GM1 which were axonic (J. Neurocytol. 26: 1–14). Alteration of endogenous gangliosides is accomplished in a number of ways: (a) use of metabolic inhibitors; (b) use of neuraminidase to increase of cell surface GM1; (c) creation of mutant cells. An example of the latter was selection of a cholera toxin resistant clone of NG108‐15 cells that lacks GM1 and is defective in regulation of intracellular Ca2+ (J. Neurochem. 76: 690–702). In focusing on endogenous gangliosides, a key question is which particular rotein or membrane system is associated with and modulated by a specific ganglioside. The opioid receptor is one example, which is converted from inhibitory to excitatory mode through GM1 association (Mol. Brain Res. 44: 341–346). More recently GM1 in the nuclear envelope was found to tightly associate with and strongly potentiate a Na–Ca exchanger in the same membrane. Since this association survives SDS‐PAGE, a study has been undertaken to determine the nature of this very tight linkage. There are other examples of tight association between ganglioside and protein that require similar study.

Acknowledgements:  Supported by NIH grant NS33912.
Workshop 6: Microglial Activators and Inactivators. Mechanisms of induction and repression of CD40 in microglia/macrophages
Authors: Benveniste E. , Nguyen V. T. , Wesemann D. , O'Keefe G. M.
Published: July 2010

Microglia and macrophages are important antigen‐presenting cells (APCs) in the central nervous system (CNS). By virtue of their ability to express class II MHC antigens and costimulatory molecules such as CD40 and B7, microglia/macrophages promote Th1 cell activation and subsequent immune/inflammatory responses within the CNS. We have previously demonstrated that IFN‐γ is the most potent inducer of CD40 expression on microglia. Our more recent studies have focused on the molecular basis of IFN‐γ induced CD40 expression, and mechanisms by which this gene can be inhibited. The suppressive effects of IL‐4 on CD40 expression will be discussed, as will the involvement of SH2 containing proteins called SOCS (for Suppressors Of Cytokine Signalling). Expression of CD40 by activated microglia/macrophages may contribute to the complex neuroimmunologic cascades that result in inflammation, demyelination and neuronal dysfunction. As such, understanding the mechanisms of inhibition of this molecule will be beneficial in diseases such as multiple sclerosis, HIV‐1 associated dementia and Alzheimer's disease.
Neuropeptide regulation of microglial function
Authors: Jonakait G. M. , Kim W.‐K. , Delgado M. , Su Y. , Ganea D.
Published: July 2010

Activated microglia are major producers of cytokines and chemokines in the injured or diseased brain. Neuropeptides vasoactive intestinal peptide (VIP) and the related pituitary adenylate cyclase‐activating polypeptide (PACAP) have potent anti‐inflammatory actions on peripheral immune cells. Microglia, too, have receptors for these peptides. When ligated, they inhibit the production of the pro‐inflammatory cytokine TNF‐α at both the protein and mRNA levels. VIP/PACAP also down‐regulate the expression of microglial‐ derived chemokines MIP‐2, KC, MIP‐1, MCP‐1 and RANTES. Inhibition of chemokine gene expression correlates with an inhibitory effect on NFkB binding, is mediated through the VPAC1 receptor, and involves a cAMP‐dependent pathway. VIP/PACAP also decrease expression of costimulatory molecules CD40 and B7‐2 on stimulated microglia. The inclusion of an anti‐IL‐10 antibody completely abrogates PACAP inhibition of LPS‐induced CD40 expression, suggesting that PACAP inhibition is at least partly mediated by IL‐10. Indeed, PACAP enhances LPS‐induced IL‐10 mRNA and protein levels suggesting that neuropeptides, through an increase in IL‐10 protein, down‐regulate costimulatory molecule expression on microglia. Another neuropeptide, galanin, also down‐regulates TNF‐α, but only at the protein level. Analyses of poly(A) acquisition and mRNA degradation suggest that galanin inhibits TNF‐α protein by preventing the addition of the poly(A) tail and/or by destabilization of the mRNA. These data taken together suggest that neuropeptides potently influence microglial activity.

Acknowledgements:  Supported by PHS AI 041786, Charles and Johanna Busch Foundation, and Johnson & Johnson.
Behavioural and gene transcription alterations induced by spontaneous cannabinoid withdrawal in mice
Authors: Oliva José M. , Ortiz Sergio , Palomo Tomás , Manzanares Jorge
Published: July 2010

This study examined behavioural signs that occur during tolerance development to cannabinoid treatment and hormonal and gene expression alterations induced by spontaneous cannabinoid withdrawal in mice. Tolerance to CP‐55,940 treatment developed for hypothermia, ambulatory and exploratory locomotor activity. Cessation of cannabinoid treatment resulted in a behavioural withdrawal syndrome characterized by a pronounced increase in ambulatory activity and rearings. Corticosterone plasma concentrations dramatically increased 24 and 72 h after cessation of cannabinoid treatment. Similarly, an increase (40%) in cannabinoid [35S]GTPγS binding autoradiography was detected on days 1 and 3 of abstinence. Spontaneous cannabinoid withdrawal produced time‐related significant alterations in gene transcription: (i) decreased (20%) tyrosine hydroxylase (TH) mRNA levels in the ventral tegmental area and increased (50%) in substantia nigra; (ii) increased proenkephalin (PENK) gene expression more than 100% in caudate‐putamen, nucleus accumbens, olfactory tubercle and piriform cortex; (iii) increased (20–40%) pro‐opiomelanocortin (POMC) gene expression in the arcuate nucleus of the hypothalamus. These results suggest that spontaneous cannabinoid withdrawal occur after cessation of CP‐55,940 treatment. This ‘syndrome’ includes behavioural, hormonal and gene transcription alterations that seems to be part of the regulation of neuronal plasticity induced by spontaneous cannabinoid withdrawal.
Neuronal activity‐dependent nucleocytoplasmic shuttling of HDAC4 and HDAC5
Authors: Chawla Sangeeta , Vanhoutte Peter , Arnold Fiona J. L. , Huang Christopher L.‐H. , Bading Hilmar
Published: July 2010

The class II histone deacetylases, HDAC4 and HDAC5, directly bind to and repress myogenic transcription factors of the myocyte enhancer factor‐2 (MEF‐2) family thereby inhibiting skeletal myogenesis. During muscle differentiation, repression of gene transcription by MEF‐2/HDAC complexes is relieved due to calcium/calmodulin‐dependent (CaM) kinase‐induced translocation of HDAC4 and HDAC5 to the cytoplasm. MEF‐2 proteins and HDACs are also highly expressed in the nervous system and have been implicated in neuronal survival and differentiation. Here we investigated the possibility that the subcellular localization of HDACs, and thus their ability to repress target genes, is controlled by synaptic activity in neurones. We found that, in cultured hippocampal neurones, the localization of HDAC4 and HDAC5 is dynamic and signal‐regulated. Spontaneous electrical activity was sufficient for nuclear export of HDAC4 but not of HDAC5. HDAC5 translocation to the cytoplasm was induced following stimulation of calcium flux through synaptic NMDA receptors or L‐type calcium channels; glutamate bath application (stimulating synaptic and extrasynaptic NMDA receptors) antagonized nuclear export. Activity‐induced nucleocytoplasmic shuttling of both HDACs was partially blocked by the CaM kinase inhibitor KN‐62 with HDAC5 nuclear export being more sensitive to CaM kinase inhibition than that of HDAC4. Thus, the subcellular localization of HDACs in neurones is specified by neuronal activity; differences in the activation thresholds for HDAC4 and HDAC5 nuclear export provides a mechanism for input‐specific gene expression.
Aggregate formation and the impairment of long‐term synaptic facilitation by ectopic expression of mutant huntingtin in Aplysia neurons
Authors: Lee Jin‐A , Lim Chae‐Seok , Lee Seung‐Hee , Kim Hyoung , Nukina Nobuyuki , Kaang Bong‐Kiun
Published: July 2010

Huntington's disease (HD) is caused by an expansion of a polyglutamine (polyQ) tract within huntingtin (htt) protein. To examine the cytotoxic effects of polyQ‐expanded htt, we overexpressed an enhanced green fluorescent protein (EGFP)‐tagged N‐terminal fragment of htt with 150 glutamine residues (Nhtt150Q‐EGFP) in Aplysia neurons. A combined confocal and electron microscopic study showed that Aplysia neurons expressing Nhtt150Q‐EGFP displayed numerous abnormal aggregates (diameter 0.5–5 µm) of filamentous structures, which were formed rapidly (approximately 2 h) but which were sustained for at least 18 days in the cytoplasm. Furthermore, the overexpression of Nhtt150Q‐EGFP in sensory cells impaired 5‐hydroxytryptamine (5‐HT)‐induced long‐term synaptic facilitation in sensori‐motor synapses without affecting basal synaptic strength or short‐term facilitation. This study demonstrates the stability of polyQ‐based aggregates and their specific effects on long‐term synaptic plasticity.
Interaction between flavonoids and the blood–brain barrier:in vitrostudies
Authors: Youdim Kuresh A. , Dobbie Michael S. , Kuhnle Gunter , Proteggente Anna R. , Abbott N. Joan , Rice‐Evans Catherine
Published: July 2010

There is considerable current interest in the neuroprotective effects of flavonoids. This study focuses on the potential for dietary flavonoids, and their known physiologically relevant metabolites, to enter the brain endothelium and cross the blood–brain barrier (BBB) using well‐establishedin vitromodels (brain endothelial cell lines and ECV304 monolayers co‐cultured with C6 glioma cells). We report that the citrus flavonoids, hesperetin, naringenin and their relevantin vivometabolites, as well as the dietary anthocyanins andin vivoforms, cyanidin‐3‐rutinoside and pelargonidin‐3‐glucoside, are taken up by two brain endothelial cell lines from mouse (b.END5) and rat (RBE4). In both cell types, uptake of hesperetin and naringenin was greatest, increasing significantly with time and as a function of concentration. In support of these observations we report for the first time high apparent permeability (Papp) of the citrus flavonoids, hesperetin and naringenin, across thein vitroBBB model (apical to basolateral) relative to their more polar glucuronidated conjugates, as well as those of epicatechin and itsin vivometabolites, the dietary anthocyanins and to specific phenolic acids derived from colonic biotransformation of flavonoids. The results demonstrate that flavonoids and some metabolites are able to traverse the BBB, and that the potential for permeation is consistent with compound lipophilicity.
Peripherin‐mediated death of motor neurons rescued by overexpression of neurofilament NF‐H proteins
Authors: Beaulieu Jean‐Martin , Julien Jean‐Pierre
Published: July 2010

In previous studies, we showed that overexpression of peripherin, a neuronal intermediate filament (IF) protein, in mice deficient for neurofilament light (NF‐L) subunits induced a progressive adult‐onset degeneration of spinal motor neurons characterized by the presence of IF inclusion bodies reminiscent of axonal spheroids found in amyotrophic lateral sclerosis (ALS). In contrast, the overexpression of human neurofilament heavy (NF‐H) proteins provoked the formation of massive perikaryal IF protein accumulations with no loss of motor neurons. To further investigate the toxic properties of IF protein inclusions, we generated NF‐L null mice thatco‐express both peripherin and NF‐H transgenes. The axonal count in L5 ventral roots from 6 and 8‐month‐old transgenic mice showed that NF‐H overexpression rescued the peripherin‐mediated degeneration of motor neurons. Our analysis suggests that the protective effect of extra NF‐H proteins is related to the sequestration of peripherin into the perikaryon of motor neurons, thereby abolishing the development of axonal IF inclusions that might block transport. These findings illustrate the importance of IF protein stoichiometry in formation, localization and toxicity of neuronal inclusion bodies.
Regulation by cycloheximide and lowered temperature of cell‐surface α7‐nicotinic acetylcholine receptor expression on transfected SH‐EP1 cells
Authors: Schroeder Katherine M. , Wu Jie , Zhao Lingke , Lukas Ronald J.
Published: July 2010

Heterologous expression of functional, nicotinic acetylcholine receptors (nAChR) in mammalian cells has been difficult to achieve or optimize, even for nAChR containing only one kind of subunit. In this study, we determined effects of lowered temperature or of exposure to the protein synthesis inhibitor cycloheximide (CHX) on cell surface expression of homomeric α7‐nAChR in transfected SH‐EP1 human epithelial cells. We found that incubation of cells for 2 days at 25°C or in the presence of 0.5–2 μg/mL of CHX caused ∼four‐ or ∼eight‐fold increases, respectively, in surface binding sites for125I‐labeled α‐bungarotoxin (I‐Bgt). These increases were accompanied by increases in peak whole‐cell current responses to nicotinic agonists. Either treatment lowered protein synthesis and cell proliferation, but experiments using puromycin indicated that a reduction in protein synthesis or cell proliferation per se was not sufficient to increase surface binding. I‐Bgt binding to whole‐cell membrane pools increased in response to either treatment, suggesting that the increase in surface binding was due, at least in part, to an increase in intracellular receptor levels. The cyclophilin inhibitor cyclosporin A reduced surface expression in untreated as well as CHX‐ or 25°C‐treated cells. The results suggest practical means for increasing cell surface and functional expression of α7‐nAChR. Although these effects are not simply due to protein synthesis inhibition or reduced cell proliferation, they do involve an increase in intracellular receptor pool size.
Glutamate regulation of non‐quantal release of acetylcholine in the rat neuromuscular junction
Authors: Malomouzh Artem I. , Mukhtarov Marat R. , Nikolsky Eugen E. , Vyskočil František , Lieberman Edward M. , Urazaev Albert K.
Published: July 2010

Glutamate, previously demonstrated to participate in regulation of the resting membrane potential in skeletal muscles, also regulates non‐quantal acetylcholine (ACh) secretion from rat motor nerve endings. Non‐quantal ACh secretion was estimated by the amplitude of endplate hyperpolarization (H‐effect) following blockade of skeletal muscle post‐synaptic nicotinic receptors by (+)‐tubocurarine and cholinesterase by armin (diethoxy‐p‐nitrophenyl phosphate). Glutamate was shown to inhibit non‐quantal release but not spontaneous and evoked quantal secretion of ACh. Glutamate‐induced decrease of the H‐effect was enhanced by glycine. Glycine alone also lowered the H‐effect, probably due to potentiation of the effect of endogenous glutamate present in the synaptic cleft. Inhibition ofN‐methyl‐d‐aspartate (NMDA) receptors with (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzocyclohepten‐5,10‐imine (MK801),dl‐2‐amino‐5‐phosphopentanoic acid (AP5) and 7‐chlorokynurenic acid or the elimination of Ca2+from the bathing solution prevented the glutamate‐induced decrease of the H‐effect with or without glycine. Inhibition of muscle nitric oxide synthase byNG‐nitro‐l‐arginine methyl ester (l‐NAME), soluble guanylyl cyclase by 1H[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) and binding and inactivation of extracellular nitric oxide (NO) by haemoglobin removed the action of glutamate and glycine on the H‐effect. The results suggest that glutamate, acting on post‐synaptic NMDA receptors to induce sarcoplasmic synthesis and release of NO, selectively inhibits non‐quantal secretion of ACh from motor nerve terminals. Non‐quantal ACh is known to modulate the resting membrane potential of muscle membrane via control of activity of chloride transport and a decrease in secretion of non‐quantal transmitter following muscle denervation triggers the early post‐denervation depolarization of muscle fibres.
ASK1–p38 MAPK/JNK signaling cascade mediates anandamide‐induced PC12 cell death
Authors: Sarker Krishna Pada , Biswas Kamal Krishna , Yamakuchi Munekazu , Lee Ki‐Young , Hahiguchi Teruto , Kracht Michael , Kitajima Isao , Maruyama Ikuro
Published: July 2010

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide‐induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen‐activated protein kinase (MAPK), c‐Jun N‐terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn‐JNK, JNK1(A‐F) or SAPKβ(K‐R), blocked anandamide‐induced cell death, whereas a specific inhibitor of MEK‐1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide‐induced cell death. An important role for apoptosis signal‐regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn‐ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1ΔN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide‐induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochromecrelease from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide‐induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.
Contribution of two conserved glycine residues to fibrillogenesis of the 106–126 prion protein fragment. Evidence that a soluble variant of the 106–126 peptide is neurotoxic
Authors: Florio Tullio , Paludi Domenico , Villa Valentina , Principe Daniela Rossi , Corsaro Alessandro , Millo Enrico , Damonte Gianluca , D'Arrigo Cristina , Russo Claudio , Schettini Gennaro , Aceto Antonio
Published: July 2010

The fibrillogenic peptide corresponding to the residues 106–126 of the prion protein sequence (PrP 106–126) is largely used to explore the neurotoxic mechanisms underlying the prion disease. However, whether the neuronal toxicity of PrP 106–126 is caused by a soluble or fibrillar form of this peptide is still unknown. The aim of this study was to correlate the structural state of this peptide with its neurotoxicity. Here we show that the two conserved Gly114 and Gly119 residues, in force of their intrinsic flexibility, prevent the peptide assuming a structured conformation, favouring its aggregation in amyloid fibrils. The substitution of both Gly114 and Gly119 with alanine residues (PrP 106–126 AA mutated peptide) reduces the flexibility of this prion fragment and results in a soluble, β‐structured peptide. Moreover, PrP 106–126 AA fragment was highly toxic when incubated with neuroblastoma cells, likely behaving as a neurotoxic protofibrillar intermediate of the wild‐type PrP 106–126. These data further confirm that the fibrillar aggregation is not necessary for the induction of the toxic effects of PrP 106–126.
High‐frequency stimulation of the subthalamic nucleus enhances striatal dopamine release and metabolism in rats
Authors: Meissner Wassilios , Harnack Daniel , Reese René , Paul Gesine , Reum Torsten , Ansorge Mark , Kusserow Heike , Winter Christine , Morgenstern Rudolf , Kupsch Andreas
Published: July 2010

High‐frequency stimulation of the subthalamic nucleus is believed to exert its main effects via the basal ganglia output structures. Previously, we have shown a concomitant increase in striatal dopamine (DA) metabolites in normal and 6‐hydroxydopamine‐lesioned rats. The present study was designed to determine whether this increase in striatal DA metabolites reflects enhanced intraneuronal DA turnover or, alternatively, is due to increased DA release with subsequent rapid and efficient reuptake and/or metabolism. Thus, high‐frequency stimulation of the subthalamic nucleus was performed in normal rats after inhibition of DA reuptake, metabolism or DA depletion. Extracellular levels of striatal DA and its metabolites were assessed using microdialysis. Our data suggest that subthalamic high‐frequency stimulation increases striatal DA release and activates independent striatal DA metabolism. Since such changes could be triggered by modification of either the activity or the gene expression of the rate‐limiting enzyme tyrosine hydroxylase, an activity assay and RT‐PCR of striatal and nigral samples were performed. Subthalamic stimulation increased striatal tyrosine hydroxylase activity without affecting gene expression. We, therefore, conclude that the application of subthalamic high‐frequency stimulation could partially compensate for the DA deficit by inducing increased striatal DA release and metabolism.
Tetrahydrobiopterin precursor sepiapterin provides protection against neurotoxicity of 1‐methyl‐4‐phenylpyridinium in nigral slice cultures
Authors: Madsen Jakob Torp , Jansen Pernille , Hesslinger Christian , Meyer Morten , Zimmer Jens , Gramsbergen Jan Bert
Published: July 2010

Complex‐I inhibition and oxidative processes have been implicated in the loss of nigral dopamine neurones in Parkinson's disease and the toxicity of MPTP and its metabolite MPP+. Tetrahydrobiopterin, an essential cofactor for tyrosine hydroxylase, may act as an antioxidant in dopaminergic neurones and protects against the toxic consequences of glutathione depletion. Here we studied the effects of manipulating tetrahydrobiopterin levels on MPP+toxicity in organotypic, rat ventral mesencephalic slice cultures. In cultures exposed to 30 µmMPP+for 2 days, followed by 8 days ‘recovery’ in control medium, we measured dopamine and its metabolites in the tissue and culture medium by HPLC, lactate dehydrogenase release to the culture medium, cellular uptake of propidium iodide and counted the tyrosine hydroxylase‐immunoreactive neurones. Inhibition of tetrahydrobiopterin synthesis by 2,4‐diamino‐6‐hydroxypyrimidine had no significant synergistic effect on MPP+toxicity. In contrast, the tetrahydrobiopterin precursorl‐sepiapterin attenuated the MPP+‐induced dopamine depletion and loss of tyrosine hydroxylase‐positive cells in a dose‐dependent manner with 40 µm l‐sepiapterin providing maximal protection. Accordingly, increasing intracellular tetrahydrobiopterin levels may protect against oxidative stress by complex‐I inhibition.
δ‐Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains
Authors: Bourova Lenka , Kostrnova Alexandra , Hejnova Lucie , Moravcova Zuzana , Moon Hyo‐Eun , Novotny Jiri , Milligan Graeme , Svoboda Petr
Published: July 2010

Low‐density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a δ‐opioid (DOP) receptor‐Gi1α fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein‐linked Gi1α and endogenous pertussis‐toxin sensitive G proteins was measured asd‐Ala2,d‐Leu5‐enkephalin stimulated high‐affinity GTPase or guanosine‐5′‐[γ‐35S]triphosphate ([35S]GTPγS) binding. The maximumd‐Ala2‐d‐Leu5 enkephalin (DADLE)‐stimulated GTPase was two times higher in low‐density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPγS binding. Contrarily, the low‐density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low‐density domains exhibited four times higher agonist‐stimulated GTPase and [35S]GTPγS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain‐bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes – EC50 = 4.5 ± 0.1 × 10−8and 3.2 ± 1.4 × 10−8 mfor GTPase;Kd = 1.2 ± 0.1 and 1.3 ± 0.1 nmfor [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent‐insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE‐stimulated GTPase or GTPγS binding. Functional coupling between the DOP receptor and cognate G proteins was also blocked by high‐energy ultrasound and repeated freezing‐thawing. Our data indicate, for the first time, that membrane domains isolated using ‘detergent‐free’ procedures exhibit higher efficiency of coupling between a G protein‐coupled receptor and its corresponding G protein(s) than bulk plasma membranes. Detergent‐extraction diminishes these interactions, even when the receptor and G proteins are physically tethered together.
Fluorescent two‐dimensional difference gel electrophoresis and mass spectrometry identify age‐related protein expression differences for the primary visual cortex of kitten and adult cat
Authors: Van den Bergh Gert , Clerens Stefan , Cnops Lieselotte , Vandesande Frans , Arckens Lutgarde
Published: July 2010

The recent introduction of fluorescent two‐dimensional difference gel electrophoresis, combined with mass spectrometry, has greatly simplified the analysis and identification of differentially expressed proteins by eliminating intergel variability. In this report, we describe the successful application of this functional proteomics approach to compare protein expression levels in visual cortical area 17 of adult cats and 30‐day‐old kittens, in order to identify proteins expressed in an age‐related fashion. We identified 16 proteins that were more abundantly expressed in kitten striate cortex and 12 proteins with a pronounced expression in adult cat area 17. Among those isolated from kitten area 17 were proteins related to axon growth and growth cone guidance and to the formation of cytoskeletal filaments. Glial fibrillary acidic protein, as identified in adult cat area 17, has been implicated previously in the termination of the critical period for cortical plasticity in kittens. In situ hybridization experiments for two of the identified proteins, glial fibrillary acidic protein and collapsin response mediator protein 5, confirmed and extended their differential expression to the mRNA level. Our findings show that two‐dimensional difference gel electrophoresis combined with mass spectrometry is a powerful approach that permits the identification of small protein expression differences correlated to different physiological conditions.
State‐dependent phosphorylation of ε‐isozyme of protein kinase C in adult rat dorsal root ganglia after inflammation and nerve injury
Authors: Zhou Yu , Li Guo‐Dong , Zhao Zhi‐Qi
Published: July 2010

The ε‐isozyme of protein kinase C (PKCε) and the vanilloid receptor 1 (VR1) are both expressed in dorsal root ganglion (DRG) neurons and are reported to be predominantly and specifically involved in nociceptive function. Using phosphospecific antibody against the C‐terminal hydrophobic site Ser729 of PKCε as a marker of enzyme activation, the state‐dependent activation of PKCε, as well as the expression of VR1 in rat DRG neurons, was evaluated in different experimental pain models in vivo. Quantitative analysis showed that phosphorylation of PKCε in DRG neurons was significantly up‐regulated after carrageen‐ and Complete Freund's Adjuvant‐induced inflammation, while it was markedly down‐regulated after chronic constriction injury. A double‐labeling study showed that phosphorylation of PKCε was expressed predominantly in VR1 immunoreactivity positive small diameter DRG neurons mediating the nociceptive information from peripheral tissue to spinal cord. The VR1 protein expression showed no significant changes after either inflammation or chronic constriction injury. These data indicate that functional activation of PKCε has a close relationship with the production of inflammatory hyperalgesia and the sensitization of the nociceptors. Inflammatory mediator‐induced activation of PKCε and subsequent sensitization of VR1 to noxious stimuli by PKCε may be involved in nociceptor sensitization.
Zinc inhibition of cellular energy production: implications for mitochondria and neurodegeneration
Authors: Dineley Kirk E. , Votyakova Tatyana V. , Reynolds Ian J.
Published: July 2010

An increasing body of evidence suggests that high intracellular free zinc promotes neuronal death by inhibiting cellular energy production. A number of targets have been postulated, including complexes of the mitochondrial electron transport chain, components of the tricarboxylic acid cycle, and enzymes of glycolysis. Consequences of cellular zinc overload may include increased cellular reactive oxygen species (ROS) production, loss of mitochondrial membrane potential, and reduced cellular ATP levels. Additionally, zinc toxicity might involve zinc uptake by mitochondria and zinc induction of mitochondrial permeability transition. The present review discusses these processes with special emphasis on their potential involvement in brain injury.
Xenobiotic response element binding enriched in both nuclear and microsomal fractions of rat cerebellum
Authors: Kuramoto Nobuyuki , Baba Katsuhiro , Gion Keiko , Sugiyama Chie , Taniura Hideo , Yoneda Yukio
Published: July 2010

Xenobiotic response element (XRE) is a core nucleotide sequence at the upstream of inducible target genes for the transcription factor aryl hydrocarbon receptor (AhR) that is responsible for signal transduction of exogenous environmental pollutants in eukaryotic cells. Immunoblotting analysis revealed the constitutive expression of AhR‐related proteins in rat liver and brain, while specific binding of a radiolabelled probe containing XRE was detected in nuclear preparations of both liver and brain on gel retardation electrophoresis. Among discrete rat brain structures examined, cerebellum exhibited the highest XRE binding with less potent binding in hypothalamus, midbrain, medulla‐oblongata, hippocampus, cerebral cortex and striatum. In contrast to liver and hippocampus, cerebellum also contained unusually higher XRE binding in microsomal fractions than that in either nuclear or mitochondrial fractions. Limited proteolysis by V8 protease did not markedly affect XRE binding in cerebellar nuclear extracts, with concomitant diminution of that in hepatic and hippocampal nuclear extracts. In primary cultured cerebellar neurons, indigo was effective in significantly increasing XRE binding only when determined immediately after sustained exposure for 120 min in the presence of high potassium chloride. These results suggest the abundance of as‐yet unidentified proteins with high affinity for XRE and responsiveness to indigo in both nuclear and microsomal fractions of rat cerebellum.
Social isolation‐induced increase in the sensitivity of rats to the steroidogenic effect of ethanol
Authors: Serra Mariangela , Pisu M. Giuseppina , Floris Ivan , Cara Valeria , Purdy Robert H. , Biggio Giovanni
Published: July 2010

Social isolation of rats for 30 days immediately after weaning results in marked decreases in the cerebrocortical and plasma concentrations of pregnenolone, progesterone, 3α‐hydroxy‐5α‐pregnan‐20‐one (3α,5α‐TH PROG), and 3α,5α‐tetrahydrodeoxycorticosterone (3α,5α‐TH DOC), as well as a moderate increase in the plasma concentration of corticosterone. This mildly stressful condition has now been shown to increase the sensitivity of rats to the effect of acute ethanol administration on the cerebrocortical and plasma concentrations of neuroactive steroids. The percentage increases in the brain and plasma concentrations of pregnenolone, progesterone, 3α,5α‐TH PROG, and 3α,5α‐TH DOC, apparent 20 min after a single intraperitoneal injection of ethanol (1 g/kg), were thus markedly greater in isolated rats than in group‐housed animals. A subcutaneous injection of isoniazid (300 mg/kg) also induced greater percentage increases in the concentrations of these steroids in isolated rats than in group‐housed animals. These results suggest that mild chronic stress, such as that induced by social isolation, enhances the steroidogenic effect of ethanol, a drug abused by humans under stress or affected by neuropsychiatric disorders. Social isolation also induced hyper‐responsiveness of the hypothalamic–pituitary–adrenal (HPA) axis, as was apparent after reduction of GABA‐mediated inhibitory tone by isoniazid administration.
Novel excitatoryConuspeptides define a new conotoxin superfamily
Authors: Jimenez Elsie C. , Shetty Reshma P. , Lirazan Marcelina , Rivier Jean , Walker Craig , Abogadie Fe C. , Yoshikami Doju , Cruz Lourdes J. , Olivera Baldomero M.
Published: July 2010

A new class ofConuspeptides, the I‐superfamily of conotoxins, has been characterized using biochemical, electrophysiological and molecular genetic methods. Peptides in this superfamily have a novel pattern of eight Cys residues. Five peptides that elicited excitatory symptomatology, r11a, r11b, r11c, r11d and r11e, were purified fromConus radiatusvenom; four were tested on amphibian peripheral axons and shown to elicit repetitive action potentials, consistent with being members of the ‘lightning‐strike cabal’ of toxins that effect instant immobilization of fish prey. A parallel analysis ofConuscDNA clones revealed a new class of conotoxin genes that was particularly enriched (with 18 identified paralogues) in aConus radiatusvenom duct library; severalC. radiatusclones encoded the excitatory peptides directly characterized from venom. The remarkable diversity of related I‐superfamily peptides within a singleConusspecies is unprecedented. When combined with the excitatory effects observed on peripheral circuitry, this unexpected diversity suggests a corresponding molecular complexity of the targeted signaling components in peripheral axons; the I‐conotoxin superfamily should provide a rich lode of pharmacological tools for dissecting and understanding these. Thus, the I‐superfamily conotoxins promise to provide a significant new technology platform for dissecting the molecular components of axons.
Changes in dialysate concentrations of glutamate and GABA in the brain: an index of volume transmission mediated actions?
Authors: Del Arco A. , Segovia G. , Fuxe K. , Mora F.
Published: July 2010

Brain microdialysis has become a frequently used method to study the extracellular concentrations of neurotransmitters in specific areas of the brain. For years, and this is still the case today, dialysate concentrations and hence extracellular concentrations of neurotransmitters have been interpreted as a direct index of the neuronal release of these specific neurotransmitter systems. Although this seems to be the case for neurotransmitters such as dopamine, serotonin and acetylcholine, the extracellular concentrations of glutamate and GABA do not provide a reliable index of their synaptic exocytotic release. However, many microdialysis studies show changes in extracellular concentrations of glutamate and GABA under specific pharmacological and behavioural stimuli that could be interpreted as a consequence of the activation of specific neurochemical circuits. Despite this, we still do not know the origin and physiological significance of these changes of glutamate and GABA in the extracellular space. Here we propose that the changes in dialysate concentrations of these two neurotransmitters found under specific treatments could be an expression of the activity of the neurone–astrocyte unit in specific circuits of the brain. It is further proposed that dialysate changes of glutamate and GABA could be used as an index of volume transmission mediated actions of these two neurotransmitters in the brain. This hypothesis is based firstly on the assumption that the activity of neurones is functionally linked to the activity of astrocytes, which can release glutamate and GABA to the extracellular space; secondly, on the existence of extrasynaptic glutamate and GABA receptors with functional properties different from those of GABA receptors located at the synapse; and thirdly, on the experimental evidence reporting specific electrophysiological and neurochemical effects of glutamate and GABA when their levels are increased in the extracellular space. According to this concept, glutamate and GABA, once released into the extracellular compartment, could diffuse and have long‐lasting effects modulating glutamatergic and/or GABAergic neurone‐astrocytic networks and their interactions with other neurotransmitter neurone networks in the same areas of the brain.