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Neurochemistry International (v.50, #1)
APP mRNA splicing is upregulated in the brain of biglycan transgenic mice by Annamária Bjelik; Magdolna Pákáski; Erika Bereczki; Szilvia Gonda; Anna Juhász; �gnes Rimanóczy; Marianna Zana; Zoltán Janka; Miklós Sántha; János Kálmán (pp. 1-4).
Many of the risk factors for cerebrovascular disease and atherosclerosis also increase the risk of Alzheimer's disease, characterized by the cerebral deposition of β-amyloid plaques resulting from the abnormal processing of the transmembrane amyloid precursor protein (APP). The initiating event of cholesterol-induced atherosclerosis is the retention and accumulation of atherogenic apolipoprotein B (apoB) together with low-density lipoproteins in the vascular intima. Biglycan, a member of the small leucine-rich protein family, was suspected of contributing to this process. The individual and combined overexpressions of biglycan and apoB-100 were therefore examined on the cortical APP mRNA levels of transgenic mice by means of semiquantitative PCR. As compared with the control littermates, transgenic biglycan mice had significantly increased cortical APP695 (122%) and APP770 (157%) mRNA levels, while the double transgenic (apoB+/−×biglycan+/−) mice did not exhibit any changes. These results provide the first experimental evidence that the atherogenic risk factor biglycan alters APP splicing and may participate in the pathogenesis of both Alzheimer and vascular dementias.
Keywords: Alzheimer's disease; Amyloid precursor protein; apoB; Biglycan; Transgenic mice; Atherosclerosis; Vascular
Systemic lupus erythematosus and the brain: What mice are telling us by Jessy J. Alexander; Richard J. Quigg (pp. 5-11).
Neuropsychiatric symptoms occur in systemic lupus erythematosus (SLE), a complex, autoimmune disease of unknown origin. Although several pathogenic mechanisms have been suggested to play a significant role in the etiology of the disease, the exact underlying mechanisms still remain elusive. Several inbred strains of mice are used as models to study SLE, which exhibit a diversity of central nervous system (CNS) manifestations similar to that observed in patients. This review will attempt to give a brief overview of the CNS alterations observed in these models, including biochemical, structural and behavioral changes.
Keywords: Abbreviations; SLE; systemic lupus erythematosus; CNS; central nervous system; NP-SLE; neuropsychiatric disease; MRL; medical research laboratories; NZ; New Zealand; BBB; blood–brain barrier; IC; immune complex; Cho; choline; GABA; gamma amino butyric acid; NAA; N; -acetyl aspartateSystemic lupus erythematosus; Animal models; Brain; Pathophysiology
Convergence of genes implicated in Alzheimer's disease on the cerebral cholesterol shuttle: APP, cholesterol, lipoproteins, and atherosclerosis by C.J. Carter (pp. 12-38).
Polymorphic genes associated with Alzheimer's disease (seewww.polygenicpathways.co.uk) delineate a clearly defined pathway related to cerebral and peripheral cholesterol and lipoprotein homoeostasis. They include all of the key components of a glia/neurone cholesterol shuttle including cholesterol binding lipoproteins APOA1, APOA4, APOC1, APOC2, APOC3, APOD, APOE and LPA, cholesterol transporters ABCA1, ABCA2, lipoprotein receptors LDLR, LRP1, LRP8 and VLDLR, and the cholesterol metabolising enzymes CYP46A1 and CH25H, whose oxysterol products activate the liver X receptor NR1H2 and are metabolised to esters by SOAT1. LIPA metabolises cholesterol esters, which are transported by the cholesteryl ester transport protein CETP. The transcription factor SREBF1 controls the expression of most enzymes of cholesterol synthesis. APP is involved in this shuttle as it metabolises cholesterol to 7-betahydroxycholesterol, a substrate of SOAT1 and HSD11B1, binds to APOE and is tethered to LRP1 via APPB1, APBB2 and APBB3 at the cytoplasmic domain and via LRPAP1 at the extracellular domain. APP cleavage products are also able to prevent cholesterol binding to APOE. BACE cleaves both APP and LRP1. Gamma-secretase (PSEN1, PSEN2, NCSTN) cleaves LRP1 and LRP8 as well as APP and their degradation products control transcription factor TFCP2, which regulates thymidylate synthase (TS) and GSK3B expression. GSK3B is known to phosphorylate the microtubule protein tau (MAPT). Dysfunction of this cascade, carved out by genes implicated in Alzheimer's disease, may play a major role in its pathology.Many other genes associated with Alzheimer's disease affect cholesterol or lipoprotein function and/or have also been implicated in atherosclerosis, a feature of Alzheimer's disease, and this duality may well explain the close links between vascular and cerebral pathology in Alzheimer's disease. The definition of many of these genes as risk factors is highly contested. However, when polymorphic susceptibility genes belong to the same signaling pathway, the risk associated with multigenic disease is better related to the integrated effects of multiple polymorphisms of genes within the same pathway than to variants in any single gene [Wu, X., Gu, J., Grossman, H.B., Amos, C.I., Etzel, C., Huang, M., Zhang, Q., Millikan, R.E., Lerner, S., Dinney, C.P., Spitz, M.R., 2006. Bladder cancer predisposition: a multigenic approach to DNA-repair and cell-cycle-control genes. Am. J. Hum. Genet. 78, 464–479.]. Thus, the fact that Alzheimer's disease susceptibility genes converge on a clearly defined signaling network has important implications for genetic association studies.
Keywords: Alzheimer's disease; Atherosclerosis; Cholesterol metabolism; Lipoprotein; Cholesterol shuttle
Parkinson disease IgG and C5a-induced synergistic dopaminergic neurotoxicity: Role of microglia by Xi-Jin Wang; Zhi-Qiang Yan; Guo-Qiang Lu; Smith Stuart; Sheng-Di Chen (pp. 39-50).
Increasing evidence suggests the involvement of immune/inflammatory system in Parkinson's disease (PD). Many immune/inflammatory factors may synergistically participate in PD. In this study, we demonstrated that immunoglobulin G from the serum of 4/11 PD patients (PDIgG, 60μg/ml) and recombinant human C5a (0.1nM) synergistically induced selective dopaminergic neurodegeneration in rat mesencephalic neuron–glia cultures, while that PDIgG alone or C5a alone was minimally toxic or nontoxic. IgG from 17 disease controls and from 7 normal controls did not significantly induce dopaminergic neurotoxicity in the cultures even in the presence of C5a. Using mesencephalic neuron-enriched cultures, we found that the synergistic dopaminergic neurotoxicity was mediated by glia. The results from microglia-supplemented neuronal cultures, astroglia-supplemented neuronal cultures and neuron–astroglia cocultures indicated that microglia, not astroglia, played a pivotal role in the neurotoxicity. Through immunocytochemistry analysis and assay of proinflammatory factors, we observed that each of the four PDIgGs (60μg/ml) and C5a (0.1nM) synergistically induced microglia activation and production of superoxide and nitric oxide (NO) in neuron–glia cultures. Further investigations indicated that superoxide and NO were both responsible for the synergistic neurotoxicity. Finally, using F(ab′)2 fragments of PDIgG, we demonstrated that microglial Fc receptors may play an important role in the neurotoxicity. Our work provides new evidence for the involvement of the immune/inflammatory system in PD and helpful clues for studying the combined effect of antibody and complement on microglia.
Keywords: Abbreviations; PD; Parkinson's disease; IgG; immunoglobulin G; DC; disease controls; NC; normal healthy controls; PDIgG; immunoglobulin G from the serum of PD patients; DCIgG; immunoglobulin G from the serum of disease controls; NCIgG; immunoglobulin G from the serum of normal healthy controls; SNc; substantia nigra compacta; CSF; cerebrospinal fluid; DA; dopamine; TH; tyrosine hydroxylase; OX-42; CR3 complement receptor; GFAP; glial fibrillary acidic protein; anti-C5a; the monoclonal antibody against C5a; yr; year; IRB; Institutional Review Board; FBS; fetal bovine serum; HS; horse serum; IR; immunoreactive; CAT; catalase; SOD; superoxide dismutase; l; -NAME; NG-nitro-; l; -arginine methyl ester; NO; nitric oxide; MEM; minimum essential medium; KRB; Krebs–Ringer buffer; ROS; reactive oxygen species; RNS; reactive nitrative species; ICs; immune complexes; FcRs; Fc receptorsImmunoglobulin; Complement; Dopaminergic neurotoxicity; Microglia; Free radicals
Presynaptic mechanisms underlying the α-lipoic acid facilitation of glutamate exocytosis in rat cerebral cortex nerve terminals by Su-Jane Wang; Hui-Hsin Chen (pp. 51-60).
The antioxidant α-lipoic acid has been reported to prevent and reverse age-related impairments in learning and memory. However, it is unclear how α-lipoic acid improves cognitive function. In this study, the effect of α-lipoic acid on the release of endogenous glutamate from rat cerebrocortical nerve terminals (synaptosomes) was examined. We found that α-lipoic acid potently facilitated 4-aminopyridine (4AP)-evoked glutamate release, and this release facilitation results from an enhancement of vesicular exocytosis and not from an increase of non-vesicular release. Examination of the effect of α-lipoic acid on cytosolic [Ca2+] revealed that the facilitation of glutamate release was associated with an increase in voltage-dependent Ca2+ influx. Consistent with this, α-lipoic acid-mediated facilitation of glutamate release was completely prevented in synaptosomes pretreated with a wide spectrum blocker of the N- and P/Q-type Ca2+ channels, ω-conotoxin MVIIC. The facilitatory effect of α-lipoic acid on Ca2+ influx was not due to an increase of synaptosomal excitability because α-lipoic acid did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential. In addition, both ionomycin and hypertonic sucrose-induced glutamate release were enhanced by α-lipoic acid. Furthermore, disruption of cytoskeleton organization with cytochalasin D occluded the facilitatory effect of α-lipoic acid on 4AP or ionomycin-evoked glutamate release. These results suggest that the antioxidant α-lipoic acid enhances the Ca2+ entry through presynaptic N- and P/Q-type Ca2+ channels as well as the vesicular release machinery to cause an increase in evoked glutamate release from rat cerebrocortical synaptosomes. Also, activation of PKA and PKC may underlie, at least in part, the α-lipoic acid-mediated facilitation of glutamate release observed here as α-lipoic acid-enhanced 4AP and ionomycin-evoked glutamate release were significantly attenuated by PKA and PKC inhibitors. This finding may provide some information regarding the mechanism of action of α-lipoic acid in the central nervous system (CNS).
Keywords: α-Lipoic acid; Antioxidant; Cognitive function; Glutamate exocytosis; Cerebrocortical nerve terminals
Mapping convulsants’ binding to the GABA-A receptor chloride ionophore: A proposed model for channel binding sites by A.V. Kalueff (pp. 61-68).
Gamma-aminobutyric acid (GABA) type A receptors play a key role in brain inhibitory neurotransmission, and are ligand-activated chloride channels blocked by numerous convulsant ligands. Here we summarize data on binding of picrotoxin, tetrazoles, β-lactams, bicyclophosphates, butyrolactones and neurotoxic pesticides to GABA-A ionophore, and discuss functional and structural overlapping of their binding sites. The paper reviews data on convulsants’ binding sensitivity to different point mutations in ionophore-lining second trans-membrane domains of GABA-A subunits, and maps possible location of convulsants’ sites within the chloride ionophore. We also discuss data on inhibition of glycine, glutamate, serotonin (5-HT3) and N-acetylcholine receptors by GABA-A channel blockers, and examine the applicability of this model to other homologous ionotropic receptors. Positioning various convulsant-binding sites within ionophore of GABA-A receptors, this model enables a better understanding of complex architectonics of ionotropic receptors, and may be used for developing new channel-modulating drugs.
Keywords: GABA-A receptors; Ionophore; Channel chemoconvulsants; Binding sites; Point mutagenesis
Cocaine-induced oxidative stress precedes cell death in human neuronal progenitor cells by H. Fai Poon; Laila Abdullah; Myles A. Mullan; Michael J. Mullan; Fiona C. Crawford (pp. 69-73).
By 2003, an estimated 34 million Americans had used cocaine according to the National Survey on Drug Use & Health. About 5.9 million of those had used in the past 12 months. Chronic cocaine users often develop addiction, dependency and tolerance to the drug. The psychological and physical effects of cocaine are due to the disruption of the limbic system in the central nervous system (CNS). Increased oxidative stress reported in the frontal cortex and the striatum of rats exposed to cocaine suggests that oxidative damage plays a significant role in cocaine-induced disruption of the CNS. Although it is evident that cocaine induces oxidative stress in the CNS, little has been learned about whether such increased oxidative stress is also relevant to apoptosis in cocaine-exposed models. To gain insight into the role of cocaine-induced oxidative stress in apoptosis, we hypothesized that oxidative stress precedes cell death when cocaine is administrated. To test this hypothesis, we have monitored the oxidative stress and apoptotic effects of acute cocaine exposure in human neuronal progenitor cells (HNPC). We found that oxidative stress was significantly increased at 48h after a 30min cocaine exposure compared to control cells, and that this was followed by cell death at 72h. Using the same experimental paradigm we have previously shown that pro-inflammatory genes are up-regulated in cocaine-exposed HNPC at 24h. Therefore, we suggest that the increased oxidative stress (possibly mediated by inflammatory responses) precedes cell death in cocaine-exposed HNPC. This may have implications for the consequences of cocaine abuse in situations where antioxidant capacity is compromised, as in the aging brain.
Keywords: Drug Addiction; Oxidative stress; Protein oxidation; Inflammatory; Drug abuse
Mitogen activated protein kinase and protein kinase C activation mediate promotion of sAPPα secretion by deprenyl by Hong-Qi Yang; Mao-Wen Ba; Ru-Jing Ren; Yu-Hong Zhang; Jian-Fang Ma; Jing Pan; Guo-Qiang Lu; Sheng-Di Chen (pp. 74-82).
The beta amyloid cascade plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Therefore, drugs that regulate amyloid precursor protein (APP) processing toward the nonamyloidgenic pathway may have therapeutic potential. Many anti-dementia drugs can regulate APP processing in addition to their pharmacological properties. Deprenyl is a neuroprotective agent used to treat some neurodegenerative diseases, including AD. In the present study, the effects of deprenyl on APP processing were investigated. Using SK-N-SH and PC12 cells, it was demonstrated that deprenyl stimulated the release of the nonamyloidogenic α-secretase form of soluble APP (sAPPα) in a dose-dependent manner without affecting cellular APP expression. The increase of sAPPα secretion by deprenyl was blocked by the mitogen activated protein (MAP) kinase inhibitor U0126 and PD98059, and by the protein kinase C (PKC) inhibitor GF109203X and staurosporine, suggesting the involvement of these signal transduction pathways. Deprenyl induced phosphorylation of p42/44 MAP kinase, which was abolished by specific inhibitors of MAP kinase and PKC. Deprenyl also phosphorylated PKC and its major substrate, and myristoylated alanine-rich C kinase (MARCKS) at specific amino acid residues. The data also indicated that 10μM deprenyl successfully induced two PKC isoforms involved in the pathogenesis of AD, PKCα and PKCɛ, to translocate from the cytosolic to the membrane fraction. This phenomenon was substantiated by immunocytochemistry staining. These data suggest a novel pharmacological mechanism in which deprenyl regulates the processing of APP via activation of the MAP kinase and PKC pathways, and that this mechanism may underlie the clinical efficacy of the drug in some AD patients.
Keywords: Deprenyl; Alzheimer's disease; Amyloid precursor protein; Mitogen activated protein kinase; Protein kinase C; Translocation
In vitro evidence for an antioxidant role of 3-hydroxykynurenine and 3-hydroxyanthranilic acid in the brain by Guilhian Leipnitz; Cristiana Schumacher; Karina B. Dalcin; Karina Scussiato; Alexandre Solano; Cláudia Funchal; Carlos S. Dutra-Filho; Angela T.S. Wyse; Clóvis M.D. Wannmacher; Alexandra Latini; Moacir Wajner (pp. 83-94).
We investigated the in vitro effect of 3-hydroxykynurenine (3HKyn), 3-hydroxyanthranilic acid (3HAA), kynurenine (Kyn) and anthranilic acid (AA) on various parameters of oxidative stress in rat cerebral cortex and in cultured C6 glioma cells. It was demonstrated that 3HKyn and 3HAA significantly reduced the thiobarbituric acid-reactive substances (TBA-RS) and chemiluminescence measurements in rat cerebral cortex, indicating that these metabolites prevent lipid peroxidation in the brain. In addition, GSH spontaneous oxidation was significantly prevented by 3HAA, but not by the other kynurenines in cerebral cortex. We also verified that 3HKyn and 3HAA significantly decreased the peroxyl radicals induced by the thermolysis of 2,2′-azo-bis-(2-amidinopropane)-derived peroxyl radicals, and to a higher degree than the classical peroxyl scavenger trolox. 2-Deoxy-d-ribose degradation was also significantly prevented by 3HKyn, implying that this metabolite was able to scavenge hydroxyl radicals. Furthermore, the total antioxidant reactivity of C6 glioma cells was significantly increased when these cells were exposed from 1 to 48h to 3HKyn, being the effect more prominent at shorter incubation times. TBA-RS values in C6 cells were significantly reduced by 3HKyn when exposed from 1 to 6h with this kynurenine. However, C6 cell morphology was not altered by 3HKyn. Finally, we tested whether 3HKyn could prevent the increased free radical production induced by glutaric acid (GA), the major metabolite accumulating in glutaric acidemia type I, by evaluating the isolated and combined effects of these compounds on TBA-RS levels and 2′,7′-dihydrodichlorofluorescein (DCFH) oxidation in rat brain. GA provoked a significant increase of TBA-RS values and of DCFH oxidation, effects that were attenuated and fully prevented, respectively, by 3HKyn. The results strongly indicate that 3HKyn and 3HAA behave as antioxidants in cerebral cortex and C6 glioma cells from rats.
Keywords: 3-Hydroxykynurenine; 3-Hydroxyanthranilic acid; Glutaric acid; Oxidative stress; Antioxidant properties
Hyperammonemia induces transport of taurine and creatine and suppresses claudin-12 gene expression in brain capillary endothelial cells in vitro by Mireille Bélanger; Tomoko Asashima; Sumio Ohtsuki; Hirofumi Yamaguchi; Shingo Ito; Tetsuya Terasaki (pp. 95-101).
Ammonia is a key neurotoxin involved in the neurological complications of acute liver failure. The present study was undertaken to study the effects of exposure to pathophysiologically relevant concentrations of ammonium chloride on cultured brain capillary endothelial cells in order to identify mechanisms by which ammonia may alter blood–brain barrier function. Conditionally immortalized mouse brain capillary endothelial cells (TM-BBB) were used as an in vitro model of the blood–brain barrier. Gene expression of a series of blood–brain barrier transporters and tight junction proteins was assessed by quantitative real time PCR analysis. Exposure to ammonia (5mM for 72h) resulted in significant increases in mRNA levels of taurine transporter (TAUT; 2.0-fold increase) as well as creatine transporter (CRT; 1.9-fold increase) whereas claudin-12 mRNA expression was significantly reduced to 67.7% of control levels. Furthermore, [3H]taurine and [14C]creatine uptake were concomitantly increased following exposure to ammonia, suggesting that up-regulation of both TAUT and CRT under hyperammonemic conditions results in an increased function of these two transporters in TM-BBB cells. TAUT and CRT are respectively involved in osmoregulation and energy buffering in the brain, two systems that are thought to be affected in acute liver failure. Furthermore, claudin-12 down-regulation suggests that hyperammonemia may also affect tight junction integrity. Our results provide evidence that ammonia can alter brain capillary endothelial cell gene expression and transporter function. These findings may be relevant to pathological situations involving hyperammonemia, such as liver disease.
Keywords: Abbreviations; ABCG2; ATP-binding cassette G2 transporter; ALF; acute liver failure; ASCT2; alanine serine cysteine transporter 2; ATA2; Na; +; dependent small neutral amino acid transporter 2; BBB; blood–brain barrier; CNS; central nervous system; CRT; creatine transporter; GLUT1; glucose transporter 1; LAT1; large neutral amino acid transporter 1; MCT1; monocarboxylate transporter 1; MRP4; multidrug resistance-associated protein 4; TAUT; taurine transporter 1Ammonia; Blood-brain barrier; Brain capillary endothelial cells; Claudin-12; Creatine transporter; Taurine transporter
Immunoreactivity enhancement with chelators for increasing the detection sensitivity of human PrPSc by Western blotting by T. Kuczius; B. Brinkmann; J. Grassi (pp. 102-108).
Prion diseases are neurodegenerative disorders affecting humans as Creutzfeldt–Jakob disease. The host-encoded prion protein (PrPC) will be converted into a structurally altered isoform (PrPSc). PrPSc differ in sizes and glycoform patterns and can be identified using molecular typing with Western blotting. The electrophoretic mobility of PrPSc changes on treatment with metal ions or chelators prior to digestion with proteases. The effects of chelators applied to PrPSc after protease digestion had not been examined in detail, we investigated these effects in this study. Application of EDTA, NTA and DTPA, and to a lesser extent EGTA, significantly enhanced PrPSc signals in immunoblots. PrPSc intensities increased two- to three-fold compared with untreated PrPSc. Since the immunoblot method is highly specific, sensitivity is the limiting factor. Enhancing sensitivity might be important in the determination of PrPSc at levels close to or just below the limits of detection. It is to be expected that application of chelators to digested protein samples will increase the sensitivity of PrPSc detection using the Western blot technique.
Keywords: Prion protein; Signal enhancement; Chelator; Protease
Regulation of G proteins by human 5-HT1a receptor TM3/i2 and TM5/i3 loop peptides by Harish V. Thiagaraj; Thomas C. Ortiz; Marvin C. Devereaux Jr.; Ben Seaver; Brian Hall; Keith K. Parker (pp. 109-118).
A bioactive synthetic 11 amino acid peptide probe (P11) was constructed according to the published sequence of the human 5HT1a receptor. The probe was used to enhance understanding of cytoplasmic loop 2/G protein coupling and activation. Additionally, two peptides (P8, P9) from the cytoplasmic loop 3 region were synthesized and studied. These probes were tested in a model system of human 5HT1a receptor stably expressed in Chinese Hamster Ovary cells. In agonist inhibition studies, P11 was active in all three receptor preparations tested: whole cells, membrane bound, and solubilized. In analyses of the membrane bound receptor system, P11 demonstrated uncompetitive inhibition characteristics. When forskolin-stimulated cAMP levels were measured, P11 was inactive in this negatively coupled system. Utilizing a [35S]γ-S-GTP incorporation assay, P11 was unable to stimulate G protein incorporation of GTP. While P8 and P9 were also broadly active as non-competitive agonist inhibitors, their characteristics differed in the signal transduction system. P8 and P9 did not significantly change forskolin-stimulated cAMP levels. However, P8 increased [35S]γ-S-GTP incorporation, while P9 decreased incorporation. Thus, P11, a synthetic peptide from the TM3/i2 region of the receptor, provides suggestive evidence that this receptor region is involved in G protein coupling but not activation. On the other hand, P8 and P9 activities suggest that the TM5/i3 region is involved in both coupling to and regulation of G protein activity. The current evidence from these cytoplasmic loop regions is discussed in the overall context of an emerging model for human 5HT1a receptor–G protein interactions.
Keywords: Serotonin; 5HT1a receptor; G proteins; Synthetic peptides; Cyclic AMP; [35S]γ-S-GTP
Dopaminergic neuronal loss in transgenic mice expressing the Parkinson's disease-associated UCH-L1 I93M mutant by Rieko Setsuie; Yu-Lai Wang; Hideki Mochizuki; Hitoshi Osaka; Hideki Hayakawa; Nobutsune Ichihara; Hang Li; Akiko Furuta; Yae Sano; Ying-Jie Sun; Jungkee Kwon; Tomohiro Kabuta; Kenji Yoshimi; Shunsuke Aoki; Yoshikuni Mizuno; Mami Noda; Keiji Wada (pp. 119-129).
The I93M mutation in ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) was reported in one German family with autosomal dominant Parkinson's disease (PD). The causative role of the mutation has, however, been questioned. We generated transgenic (Tg) mice carrying human UCHL1 under control of the PDGF-B promoter; two independent lines were generated with the I93M mutation (a high- and low-expressing line) and one line with wild-type human UCH-L1. We found a significant reduction in the dopaminergic neurons in the substantia nigra and the dopamine content in the striatum in the high-expressing I93M Tg mice as compared with non-Tg mice at 20 weeks of age. Although these changes were absent in the low-expressing I93M Tg mice, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment profoundly reduced dopaminergic neurons in this line as compared with wild-type Tg or non-Tg mice. Abnormal neuropathologies were also observed, such as silver staining-positive argyrophilic grains in the perikarya of degenerating dopaminergic neurons, in I93M Tg mice. The midbrains of I93M Tg mice contained increased amounts of insoluble UCH-L1 as compared with those of non-Tg mice, perhaps resulting in a toxic gain of function. Collectively, our data represent in vivo evidence that expression of UCHL1I93M leads to the degeneration of dopaminergic neurons.
Keywords: Ubiquitin carboxy-terminal hydrolase L1; Animal model; Parkinson's disease; Dopaminergic neuron
CCK-8 induces NGF and BDNF synthesis and modulates TrkA and TrkB expression in the rat hippocampus and septum: Effects on kindling development by Paola Tirassa; Nicola Costa (pp. 130-138).
In our previous studies, we demonstrated that intraperitoneal (i.p.) injections with the neurotransmitter/neuromodulatory peptide Cholecystokinin-8 (CCK-8) stimulate the synthesis of the neurotrophin nerve growth factor (NGF) resulting in the structural and functional recovery of neuronal damage. This neurotrophin-mediated neuroprotective action of CCK-8 has opened a new perspective for a better understanding of the CCK neurobiological and pharmacological properties.To explore the possible beneficial effects of the CCK-induced increase of neurotrophin availability in brain, we compared the effects of i.p. CCK-8 in healthy rats and in a chemical kindling model using a subconvulsive dose of pentylenetetrazol (PTZ). Behavioural changes were monitored during treatment and classified according to a six-point scale.After 3 weeks of treatment (12 trials), the PTZ group of rats manifested generalized clonic–tonic seizures (Class 5 behaviour). For this reason, this time point was chosen to compare the effects of CCK-8 treatment on the expression of NGF, the brain derived neurotrophin factor (BDNF) and their receptors in the septum and hippocampus.We found that repeated i.p. injections with CCK-8 in adult rats result in: (1) an increase of NGF and BDNF protein and mRNA levels in the septum and hippocampus; (2) a down-regulation of TrkA and p75NTR and an up-regulation of TrkB; (3) reduced susceptibility to develop chemical kindling; (4) recovery of the PTZ-induced changes in the expression of neurotrophin receptors in the septal and hippocampal tissues.This data clearly indicates that CCK-induced variation of neurotrophin synthesis in brain is able to influence the susceptibility to develop seizures in adult rats most probably by counteracting the progressive neuronal dysfunction and/or damage.
Keywords: CCK-8; Neurotrophins; Trk receptors; Kindling; Hippocampus
The effect of bovine serum albumin on the membrane potential and reactive oxygen species generation in succinate-supported isolated brain mitochondria by Laszlo Tretter; Dora Mayer-Takacs; Vera Adam-Vizi (pp. 139-147).
Characteristics of the succinate-supported H2O2 formation were compared in mitochondria prepared from guinea-pig brain either by Percoll gradient centrifugation or using digitonin. The high rate of H2O2 generation measured in mitochondria prepared with digitonin (600.6±26.8pmol/min/mg protein) was inhibited by rotenone, consistently with a reverse flow of electrons via complex I. The rate of H2O2 formation was significantly smaller in Percoll-purified mitochondria (252.6±17.3pmol/min/mg protein) and this was stimulated by rotenone. Since bovine serum albumin (BSA) is usually present in the isolation medium used in the digitonin method, systematic study was performed addressing the effect of BSA on H2O2 formation. Mitochondria prepared by the digitonin method (BSA present in the isolation medium) were highly polarized (185±3.2mV) and addition of BSA (0.025%) to the assay medium increased H2O2 generation by only 50%. In Percoll-purified mitochondria Δ Ψm was more depolarized (171±2mV) and BSA caused hyperpolarization by 10.7±1.9mV. H2O2 formation, which was largely independent of Δ Ψm, was stimulated by 400%, became highly dependent on Δ Ψm and could be inhibited by rotenone in the presence of BSA. This shows that in Percoll-purified mitochondria ROS formation via reverse electron flow is preferred only when BSA is present in the assay medium. It is demonstrated that (i) the presence or absence of BSA could determine the mechanism by which ROS is generated in succinate-supported mitochondria and (ii) depolarization by about 10mV eliminates reverse electron flow and the remaining ROS formation, which is smaller but still significant, is no longer dependent on Δ Ψm.
Keywords: Abbreviations; BSA; bovine serum albumin; FCCP; carbonyl cyanide-; p; -trifluoromethoxyphenylhydrazone; TMRM; tetramethylrhodamine methyl ester; TPP; +; tetraphenylphosphonium ion; RCR; respiratory control ratio; ROS; reactive oxygen species; UCP; uncoupling proteinMitochondria, Reactive oxygen species, ROS, BSA, Bovine serum albumin, Mitochondrial, Membrane potential, Reverse electron transfer
Emergence of a spermine-sensitive, non-inactivating conductance in mature hippocampal CA1 pyramidal neurons upon reduction of extracellular Ca2+: Dependence on intracellular Mg2+ and ATP by Christos Chinopoulos; John A. Connor; C. William Shuttleworth (pp. 148-158).
Large and protracted elevations of intracellular [Ca2+] and [Na+] play a crucial role in neuronal injury in ischemic conditions. In addition to excessive glutamate receptor activation, other ion channels may contribute to disruption of intracellular ionic homeostasis. During episodes of ischemia, extracellular [Ca2+] falls significantly. Here we report the emergence of an inward current in hippocampal CA1 pyramidal neurons in acute brain slices from adult mice upon reduction/removal of [Ca2+]e. The magnitude of the current was 100–300pA at −65mV holding potential, depending on intracellular constituents. The current was accompanied by intense neuronal discharge, observed in both whole-cell and cell-attached patch configurations. Sustained currents and increased neuronal firing rates were both reversed by restoration of physiological levels of [Ca2+]e, or by application of spermine (1mM). The amplitudes of the sustained currents were strongly reduced by raising intracellular [Mg2+], but not by extracellular [Mg2+] increases. Elevated intracellular ATP also reduced the current. This conductance is similar in several respects to the “calcium-sensing, non-selective cation current� (csNSC), previously described in cultured mouse hippocampal neurons of embryonic origin. The dependence on intracellular [ATP] and [Mg2+] shown here, suggests a possible role for this current in disruption of ionic homeostasis during metabolic stress that accompanies excessive neuronal stimulation.
Keywords: Transient receptor potential; Store-operated Ca; 2+; entry; Spreading depression; Stroke; TRPM7; Ca; 2+; paradoxAbbreviations; APV; 2-amino-5-phosphonopentanoic acid; CNQX; 6-cyano-7-nitroquinoxaline-2,3-dione; csNSC; calcium-sensing non-selective channel; mGluR; metabotropic glutamate receptor; NMDAR; NMDA receptor; TEA; tetraethylammonium chloride; TRP; transient receptor potential
KATP-dependent neurotransmitter release in the neuronal network of the rat caudate nucleus by Mirja Steinkamp; Tianlang Li; Henriette Fuellgraf; Andreas Moser (pp. 159-163).
KATP channels can couple the bioenergetic metabolism of the cell to membrane excitability. Here, we show γ-aminobutyric acid (GABA) mediated inhibition of dopamine outflow from slices of the rat caudate nucleus that is regulated by extracellular glucose via high- and low-affinity KATP channels. During glucose reduction, a biphasic dopamine effect could be observed with first a dopamine increase followed by a decline at low glucose concentrations. Both phases were inhibited by glibenclamide. Pinacidil decreased DA outflow without an effect of glucose reduction implying an overall activation of KATP channels. The first phase with dopamine increase was related to reduced GABAergic activity and could be blocked by bicuculline. Our results may be explained by different types of KATP channels with low affinity of ATP and glibenclamide on inhibitory GABAergic and high-affinity on excitatory DAergic neurons. This led us to suggest a biological principle through which neuronal networks are functioning.
Keywords: Dopamine; GABA; Caudate nucleus; ATP-sensitive potassium channels; High/low-affinity; Neuronal network
Methylene blue prevents methylmalonate-induced seizures and oxidative damage in rat striatum by Ana Flávia Furian; Michele Rechia Fighera; Mauro Schneider Oliveira; Ana Paula de Oliveira Ferreira; Natália Gindri Fiorenza; Jociane de Carvalho Myskiw; João Carlos Petry; Rafael Correa Coelho; Carlos Fernando Mello; Luiz Fernando Freire Royes (pp. 164-171).
Methylene blue (MB) is a thiazine dye with cationic and lipophilic properties that acts as an electron transfer mediator in the mitochondria. Due to this metabolic improving activity and free radicals scavenging effects, MB has been used in the treatment of methemoglobinemia and ifosfamide-induced encephalopathy. Considering that methylmalonic acidemia consists of a group of inherited metabolic disorders biochemically characterized by impaired mitochondrial oxidative metabolism and reactive species production, we decided to investigate whether MB, protects against the behavioral and neurochemical alterations elicited by the intrastriatal injection of methylmalonate (MMA). In the present study we showed that intrastriatal injection of MB (0.015–1.5nmol/0.5μl) protected against seizures (evidenced by electrographic recording), protein carbonylation and Na+,K+-ATPase inhibition ex vivo induced by MMA (4.5μmol/1.5μl). Furthermore, we investigated whether convulsions elicited by intrastriatal MMA administration are accompanied by striatal protein carbonyl content increase and changes in Na+,K+-ATPase activity in rat striatum. The effect of MB (0.015–1.5nmol/0.5μl) and MMA (4.5μmol/0.5μl) on striatal NO x (NO2 plus NO3) content was also evaluated. Statistical analysis revealed that the MMA-induced NO x content increase was attenuated by intrastriatal injection of MB and the duration of convulsive episodes correlated with Na+,K+-ATPase inhibition, but not with MMA-induced total protein carbonylation. In view of that MB decreases MMA-induced neurotoxicity assessed by behavioral and neurochemical parameters, the authors suggest that MB may be of value to attenuate neurological deficits of methylmalonic acidemic patients.
Keywords: Convulsion; Protein carbonylation; Methylmalonate; Na; +; ,K; +; -ATPase; Methylene blue
The serine protease Omi/HtrA2 is involved in XIAP cleavage and in neuronal cell death following focal cerebral ischemia/reperfusion by J. Althaus; M.D. Siegelin; F. Dehghani; L. Cilenti; A.S. Zervos; A. Rami (pp. 172-180).
Omi/HtrA2 is a pro-apoptotic mitochondrial serine protease involved in both forms of apoptosis, caspase-dependent as well as caspase-independent cell death. However, the impact of Omi/HtrA2 in the apoptotic cell machinery that takes place in vivo under pathological conditions such as cerebral ischemia remains unknown. The present study was monitored in order to examine whether Omi/HtrA2 plays a decisive role in apoptosis observed after focal cerebral ischemia in rats. Male adult rats were subjected to 90min of focal cerebral ischemia followed by reperfusion and treated with vehicle or ucf-101, a novel and specific Omi/HtrA2 inhibitor, prior reperfusion. Focal cerebral ischemia/reperfusion induced a mitochondrial up-regulation of Omi/HtrA2 and significantly increased cytosolic accumulation of Omi/HtrA2. Furthermore, ischemia led to activation of caspase-3 and degradation X-linked inhibitor of apoptosis protein (XIAP). Treatment of animals prior ischemia with ucf-101, the specific inhibitor of Omi/HtrA2, was able to (1) reduce the number of TUNEL-positive cells, to (2) attenuate the XIAP-breakdown and to (3) reduce the infarct size. This study shows for the first time that focal cerebral ischemia in rats results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it participates in neuronal cell death. Blocking the proteolytic activity of Omi/HtrA2 with specific inhibitors, such as the ucf-101, could be a novel way to afford neuroprotection and minimize cellular damage in cerebral ischemia/reperfusion.
Keywords: Serine protease Omi/HtrA2; Ischemia; Apoptosis; ucf-101; Neuroprotection
Gene transfer into Purkinje cells using herpesviral amplicon vectors in cerebellar cultures by Alfredo Gimenez-Cassina; Filip Lim; Javier Diaz-Nido (pp. 181-188).
Purkinje cells play a crucial role in sensory motor coordination since they are the only output projection neurons in the cerebellar cortex and are affected in most spinocerebellar ataxias. They stand out in the central nervous system due to their large size and their profusely branched dendritic arbor. However, molecular and cellular studies on Purkinje cells are often hampered by the difficulty of maintaining these cells in culture. Here we report an easy, robust and reproducible method to obtain Purkinje-enriched mixed cerebellar cell cultures from day 16 mouse embryos using papain digestion and a semi-defined culture medium, being the composition of the culture approximately 20% Purkinje cells, 70% non-Purkinje neurons and 10% glial cells. We demonstrate that efficient gene transfer into Purkinje cells (as well as into other cerebellar populations) is possible using herpes simplex virus-1 (HSV-1)-derived vectors. Indeed, up to 50% of the Purkinje cells can be transduced and gene expression may persist for at least 14 days. As a result, this procedure permits functional gene expression studies to be carried out on cultured Purkinje neurons. To demonstrate this, we show that the expression of a dominant-negative form of glycogen synthase kinase-3 protects Purkinje neurons against cell death triggered by a chemical inhibitor of phosphatidylinositol-3 kinase. In summary, we have established reproducible and reliable cerebellar cell cultures enriched for Purkinje cells which enables gene transfer studies to be carried out using herpesviral vectors.
Keywords: Purkinje; Gene transfer; Neuronal cell culture; Cerebellum; HSV-1; GSK-3; Neuronal cell death; Ataxia
A rat model of Parkinsonism shows depletion of dopamine in the retina by Oliver Biehlmaier; Mesbah Alam; Werner J. Schmidt (pp. 189-195).
The retinal dopamine (DA) deficiency is an important feature of the pathogenesis in Parkinson's disease (PD) visual dysfunction. Systemic inhibition of complex I (rotenone) in rats has been proposed as a model of PD. In this study, we investigated whether systemic inhibition of complex I can induce impairment of DA-ergic cells in the retina, similar to the destruction of retinal cells found in PD patients. Rotenone (2.5mg/kg i.p., daily) was administered over 60 days. Neurochemically, rotenone treated rats showed a depletion of DA in the striatum and substantia nigra (SN). In addition, the number of retinal DA-ergic amacrine cells was significantly reduced in the rotenone treated animals.This study is the first one giving highlight towards a deeper understanding of systemic complex I inhibition (rotenone as an environmental toxin) and the connection between both, DA-ergic degeneration in the nigrostriatal pathway, and in the DA-ergic amacrine cells of the retina.
Keywords: Rotenone; Animal models; Neurodegeneration; Amacrine cells; Parkinson disease
The protective effects of omega−3 fatty acids against MK-801-induced neurotoxicity in prefrontal cortex of rat by Birsen Ozyurt; Mustafa Sarsilmaz; Nusret Akpolat; Huseyin Ozyurt; Omer Akyol; Hasan Herken; Ilter Kus (pp. 196-202).
The aims of this study are to investigate the contribution effect of oxidative stress in MK-801-induced experimental psychosis model, and to show that prevention of oxidative stress may improve prognosis. Because oxidative damage has been suggested in the neuropathophysiology of schizophrenia, the possible protecting agents against lipid peroxidation are potential target for the studies in this field. For this purpose, Wistar Albino rats were divided into three groups: the first group was used as control, MK-801 was given to the rats in the second group and MK-801+ω−3 essential fatty acids (EFA) was given to the third group. MK-801 was given intraperitoneally at the dose of 0.5mg/(kgday) once a day for 5 days in experimental psychosis group. In the second group, 0.8g/(kgday), ω−3 FA (eicosapentaenoic acid, 18%, docosahexaenoic acid, 12%) was given to the rats while exposed MK-801. In control group, saline was given intraperitoneally at the same time. After 7 days, rats were killed by decapitation. Prefrontal brain area was removed for histological and biochemical analyses. As a result, malondialdehyde (MDA), as an indicator of lipid peroxidation, protein carbonyl (PC), as an indicator of protein oxidation, nitric oxide (NO) levels and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities as antioxidant enzymes, and xanthine oxidase (XO) and adenosine deaminase (AD) activities as an indicator of DNA oxidation was found to be increased significantly in prefrontal cortex (PFC) of MK-801 group ( P<0.0001) compared to control group. In ω−3 FA treated rats, prefrontal tissue MDA, PC and NO levels as well as SOD, GSH-Px, XO, and AD enzyme activities were significantly decreased when compared to MK-801 groups ( P<0.0001) whereas catalase (CAT) enzyme activity was not changed. Moreover, in the light of microscopic examination of MK-801 groups, a great number of apoptotic cells were observed. ω−3 FA supplementation decreased the apoptotic cell count in PFC.The results of this study revealed that oxidative stress and apoptotic changes in PFC may play an important role in the pathogenesis of MK-801-induced neuronal toxicity. This experimental study also provides some evidences for the protective effects of ω−3 FA on MK-801-induced changes in PFC of rats.
Keywords: Apoptosis; Malondialdehyde; MK-801; Omega; −; 3 fatty acids; Prefrontal cortex; Protein carbonyl; Oxidative stress
Neither energy collapse nor transcription underlie in vitro neurotoxicity of poly(ADP-ribose) polymerase hyper-activation by Silvia Fossati; Giulia Cipriani; Flavio Moroni; Alberto Chiarugi (pp. 203-210).
Poly(ADP-ribose)polymerase-1 (PARP-1) overactivation is a key event in neurodegeneration but the underlying molecular mechanisms wait to be unequivocally identified. Energy failure, transcriptional derangement and deadly nucleus-mitochondria cross-talk have been proposed as mechanisms responsible for PARP-1 neurotoxicity. In this study, we sought to determine how these mechanisms contributes to PARP-1-dependent neuronal death. We report that the PARP-1 activating agent methyl-nitrosoguanidine (MNNG) caused poly(ADP-ribosyl)ation-dependent death of pure mouse cortical neurons in culture. Upon PARP-1 hyperactivation, NAD and ATP storages only partially decreased, neurons rapidly acquired apoptotic morphology, apoptosis inducing factor and cytochrome c were released from mitochondria and caspase activation occurred. No evidence for p53 activation was found, lactate dehydrogenase release occurred only 18h later, and JNK kinase was constitutively activated and not affected by PARP-1 activation. The PARP-1 inhibitors 6-(5)H-phenanthridinone and N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)- N, N-dimethylacetamide (PJ-34) prevented nucleotide depletion and cell death, whereas the transcription inhibitor actinomycin D did not affect PARP-1-dependent neurotoxicity. Together, our findings provide the first evidence that neither energy collapse nor transcriptional changes are involved in PARP-1-dependent apoptotic neuronal death, and support the existence of a poly(ADP-ribose)-mediated death signaling targeting mitochondria.
Keywords: Neuronal apoptosis; PARP; AIF; Energy failure; Transcription
Norepinephrine acts as D1-dopaminergic agonist in the embryonic avian retina: Late expression of β1-adrenergic receptor shifts norepinephrine specificity in the adult tissue by Regina Celia Cussa Kubrusly; Ana Lúcia Marques Ventura; Ricardo Augusto de Melo Reis; Gracinda Conceição Fernandes Serra; Edna Nanami Yamasaki; PatrÃcia Franca Gardino; Maria Christina Fialho de Mello; Fernando Garcia de Mello (pp. 211-218).
Dopamine is the main catecholamine found in the chick retina whereas norepinephrine is only found in trace amounts. We compared the effectiveness of dopamine and norepinephrine in promoting cyclic AMP accumulation in retinas at embryonic day 13 (E13) and from post-hatched chicken (P15). Dopamine (EC50=10μM) and norepinephrine (EC50=30μM), but not the β1-adrenergic agonist isoproterenol, stimulated over seven-fold the production of cyclic AMP in E13 retina. The cyclic AMP accumulation induced by both catecholamines in embryonic tissue was entirely blocked by 2μM SCH23390, a D1 receptor antagonist, but not by alprenolol (β-adrenoceptor antagonist). In P15 retinas, 100μM isoproterenol stimulated five-fold the accumulation of cAMP. This effect was blocked by propanolol (10μM), but not by 2μM SCH23390. Embryonic and adult retina display β1 adrenergic receptor mRNA as detected by RT-PCR, but the β1 adrenergic receptor protein was detected only in post-hatched tissue. We conclude that norepinephrine cross-reacts with D1 dopaminergic receptor with affinity similar to that of dopamine in the embryonic retina. In the mature retina, however, D1 receptors become restricted to activation by dopamine. Moreover, as opposed to the embryonic tissue, norepinephrine seems to stimulate cAMP accumulation via β1-like adrenergic receptors in the mature tissue.
Keywords: Norepinephrine; Dopamine; Cyclic AMP; Retina; β; 1; adrenergic receptor
Pentosan polysulfate protects brain endothelial cells against bacterial lipopolysaccharide-induced damages by Szilvia Veszelka; Mária Pásztói; Attila E. Farkas; István Krizbai; Ngo Thi Khue Dung; Masami Niwa; Csongor S. �brahám; Mária A. Deli (pp. 219-228).
Peripheral inflammation can aggravate local brain inflammation and neuronal death. The blood–brain barrier (BBB) is a key player in the event. On a relevant in vitro model of primary rat brain endothelial cells co-cultured with primary rat astroglia cells lipopolysaccharide (LPS)-induced changes in several BBB functions have been investigated. LPS-treatment resulted in a dose- and time-dependent decrease in the integrity of endothelial monolayers: transendothelial electrical resistance dropped, while flux of permeability markers fluorescein and albumin significantly increased. Immunostaining for junctional proteins ZO-1, claudin-5 and β-catenin was significantly weaker in LPS-treated endothelial cells than in control monolayers. LPS also reduced the intensity and changed the pattern of ZO-1 immunostaining in freshly isolated rat brain microvessels. The activity of P-glycoprotein, an important efflux pump at the BBB, was also inhibited by LPS. At the same time production of reactive oxygen species and nitric oxide was increased in brain endothelial cells treated with LPS. Pentosan polysulfate, a polyanionic polysaccharide could reduce the deleterious effects of LPS on BBB permeability, and P-glycoprotein activity. LPS-stimulated increase in the production of reactive oxygen species and nitric oxide was also decreased by pentosan treatment. The protective effect of pentosan for brain endothelium can be of therapeutical significance in bacterial infections affecting the BBB.
Keywords: Blood–brain barrier; Rat brain endothelial cell; Lipopolysaccharide; Pentosan polysulfate; Permeability; P-glycoprotein; Nitric oxide
β-Amyloid peptide toxicity in organotypic hippocampal slice culture involves Akt/PKB, GSK-3β, and PTEN by Melissa Nassif; Juliana Hoppe; Katiane Santin; Rudimar Frozza; Lauren L. Zamin; FabrÃcio Simão; Ana P. Horn; Christianne Salbego (pp. 229-235).
In the present study we investigated the toxicity induced by exposing organotypic slice culture to β-amyloid peptide 25–35 (25μM) for 1, 3, 6, 12, 24 and 48h. To elucidate a mechanism involved in its toxicity, we studied the PI3-K cell signaling pathway, particularly Akt/PKB, GSK-3β, and PTEN proteins. Cell death was quantified by propidium iodide uptake and proteins were analyzed by immunoblotting. Our results showed a significant cell death after 48h of β-amyloid 25–35 peptide exposition. The exposition of cultures to β-amyloid peptide resulted in an increase in the phosphorylation state of Akt and GSK-3β proteins after 6h, followed by a decrease of the phosphorylation state of these proteins after 12h of exposition. However, after 24h of peptide treatment, the phosphorylation of GSK-3β presented a new increase while the phosphorylation of Akt remained down. The immunocontent of the PTEN protein, an indirect Akt phosphatase, increased after 24 and 48h of β-amyloid exposition. These results suggest an involvement of Akt dephosphorylation/inactivation in the toxicity induced by the β-amyloid 25–35 peptide in organotypic slice hippocampal culture, probably induced by increasing PTEN immunocontent. Taken together, our results provide more information about the molecular mechanisms involved on β-amyloid peptide toxicity.
Keywords: Alzheimer's disease; β-Amyloid; Organotypic culture; Akt; GSK-3β; PTEN
Axonal transports of tripeptidyl peptidase II in rat sciatic nerves by Toshiyuki Chikuma; Maki Shimizu; Yukihiro Tsuchiya; Takeshi Kato; Hiroshi Hojo (pp. 236-242).
Axonal transport of tripeptidyl peptidase II, a putative cholecystokinin inactivating serine peptidase, was examined in the proximal, middle, and distal segments of rat sciatic nerves using a double ligation technique. Enzyme activity significantly increased not only in the proximal segment but also in the distal segment 12–72h after ligation, and the maximal enzyme activity was found in the proximal and distal segments at 72h. Western blot analysis of tripeptidyl peptidase II showed that its immunoreactivities in the proximal and distal segments were 3.1- and 1.7-fold higher than that in the middle segment. The immunohistochemical analysis of the segments also showed an increase in immunoreactive tripeptidyl peptidase II level in the proximal and distal segments in comparison with that in the middle segment, indicating that tripeptidyl peptidase II is transported by anterograde and retrograde axonal flow. The results suggest that tripeptidyl peptidase II may be involved in the metabolism of neuropeptides in nerve terminals or synaptic clefts.
Keywords: Tripeptidyl peptidase II; Double ligation technique; Immunohistochemistry; Rat sciatic nerve; Axonal flow
Bone marrow stromal cells mediate protection through stimulation of PI3-K/Akt and MAPK signaling in neurons by Nicola B. Isele; Hea-Sook Lee; Stefan Landshamer; Andreas Straube; Claudio S. Padovan; Nikolaus Plesnila; Carsten Culmsee / (pp. 243-250).
Application of adult bone marrow stromal cells (BMSC) improves functional outcome in animal models of cerebral ischemia, traumatic brain injury, and spinal cord injury. Accumulating evidence suggests that such functional recovery after BMSC treatment is mediated by enhanced trophic support of the injured neurons and improved neuronal plasticity rather than tissue replacement by bone marrow-derived stem cells. Therefore, the aim of the present study was to explore the potential of non-hematopoietic BMSC to stimulate signaling pathways in neurons that mediate trophic effects and neuroprotection. In primary embryonic rat neurons, BMSC conditioned medium (CM) attenuated staurosporine (STS) or amyloid-beta peptide-induced apoptosis in a concentration-dependent manner. The neuroprotective effect of CM required several hours of pretreatment and was abolished by heating over 90°C. Immunoblot analyses revealed that CM enhanced Erk1/2 and Akt phosphorylation in neurons, and the specific MEK1 inhibitor PD98059 or the phosphoinositide-3 kinase (PI3-K) inhibitor Ly294002 abolished the neuroprotective effect of CM. Further, double-conditioned medium (DCM) obtained from BMSC previously stimulated by medium from STS-challenged neurons showed a more potent anti-apoptotic effect compared to the single-conditioned medium. Overall, these findings demonstrate that BMSC trigger endogenous survival signaling pathways in neurons that mediate protection against apoptotic insults. Moreover, the interaction between stressed neurons and BMSC further amplifies the observed neuroprotective effect.
Keywords: Abbreviations; BDNF; brain-derived growth factor; BMSC; bone marrow stromal cells; CM; conditioned medium; DCM; double conditioned medium; DCM(STS); DCM from staurosporine-treated neurons; EBSS; Earle's balanced salt solution; Erk; extracellular signal-regulated kinase; HBSS; Hank's buffered saline solution; MAPK; mitogen-activated kinase; MEK; MAPK kinase; MSC; mesenchymal stem cells; NGF; nerve growth factor; NMDA; N; -methyl-; d; -aspartic acid; PI3-K; phosphoinositide-3-kinase; STS; staurosporine; Trk; tropomyosine-related kinase; VEGF; vascular endothelial growth factorApoptosis; Neuroprotection; Conditioned medium; Staurosporine; Amyloid-beta; Growth factors
The production of antibodies that distinguish rat choline acetyltransferase from its splice variant product of a peripheral type by Shin Kimura; Jean-Pierre Bellier; Akinori Matsuo; Ikuo Tooyama; Hiroshi Kimura (pp. 251-255).
To produce antibodies that permit the immunohistochemical discrimination of choline acetyltransferase of the common type (cChAT) from its splice variant of a peripheral type (pChAT), we immunized rabbits with a cChAT specific recombinant protein encoded by ChAT exons 7 and 8 of the rat cChAT gene. Successful antibody production was proved by Western blotting on rat brain and on HEK293 cells expressing green fluorescent protein (GFP), cChAT-GFP and pChAT-GFP. By immunohistochemistry our antiserum clearly labeled known cholinergic structures in rat brain, but gave no positive staining in the trigeminal ganglion which contained many neurons positive with pChAT antiserum.
Keywords: Choline acetyltransferase; Immunohistochemistry; Recombinant fusion proteins; Antibody production; Western blotting; Green fluorescent protein
Cloning, molecular characterization and expression of ecto-nucleoside triphosphate diphosphohydrolase-1 from Torpedo electric organ by Mireia MartÃn-Satué; BenjamÃn Torrejón-Escribano; Antonio Felipe; Inmaculada Gómez de Aranda; Marc ElÃas; Jordi Marsal; Juan Blasi; Carles Solsona (pp. 256-263).
During synaptic transmission large amounts of ATP are released from pre- and post-synaptic sources of Torpedo electric organ. A chain reaction sequentially hydrolyses ATP to adenosine, which inhibits acetylcholine secretion. The first enzyme implicated in this extracellular ATP hydrolysis is an ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) that dephosphorylates both ATP and ADP to AMP. This enzyme has been biochemically characterized in the synaptosomal fraction of Torpedo electric organ, having almost equal affinity for ATP as for ADP, a fact that pointed to the type-1 NTPDase enzyme.In the present work we describe the cloning and molecular characterization of the cDNA for an NTPDase from Torpedo marmorata electric organ. The clone, obtained using the RACE-PCR technique, contains and open-reading frame of 1506bp and encodes a 502 amino acids protein that exhibits high homology with other NTPDases1 from vertebrates previously identified, including those of zebrafish and Xenopus, as well as human, rat and mouse. Topology analyses revealed the existence of two transmembrane regions, two short cytoplasmic tails and a long extracellular domain containing five apyrase-conserved regions. Gene expression studies revealed that this gene is expressed in all the Torpedo tissues analyzed. Finally, activity and cellular localization of the protein encoded by this newly cloned cDNA was assessed by heterologous expression experiments involving COS-7 and HeLa cells.
Keywords: E-NTPDase; ATP hydrolysis; Torpedo; electric organ; Cholinergic neurotransmission
Phosphorylation of methyl-CpG binding protein 2 (MeCP2) regulates the intracellular localization during neuronal cell differentiation by Kunio Miyake; Kaoru Nagai (pp. 264-270).
Methyl-CpG binding protein 2 (MeCP2) is a transcriptional repressor which recognizes methylated CpG dinucleotides. Mutations in the MeCP2 gene is known to cause human autistic disease Rett syndrome, but its molecular mechanisms remain to be elucidated. Since MeCP2 is a DNA-binding protein, it has been believed that MeCP2 functions only in the nucleus. We herein show that MeCP2 is localized in the cytosol as well as in the nucleus of neuronal cells. Through the use of immunofluorescence and Western blot analyses, MeCP2 was found to be localized both in the nucleus and cytosol of rat PC-12 and mouse Neuro2a cells before neuronal differentiation, and it was translocated into the nucleus during differentiation. In primary cultured neurons from mouse cortex, MeCP2 was expressed in whole cell bodies on the first day of culture while after 7 days of culture, MeCP2 was localized mainly in the nucleus. Furthermore, MeCP2 was re-localized in the nucleus and cytosol after 14 days of culture. To study the molecular mechanisms of translocation, we analyzed the post-translational modification of MeCP2. The cytosolic MeCP2 was Ser/Thr-phosphorylated, while the nuclear MeCP2 was not. Both the cytosolic and nuclear MeCP2 were SUMOylated, which has been reported to be a nuclear transport signal. Our data suggests that the nuclear translocation of neuronal MeCP2 was induced during differentiation and/or maturation, and that Ser/Thr-phosphorylation regulates its translocation.
Keywords: Abbreviations; DMEM; Dulbecco's modified Eagle medium; FBS; fetal bovine serum; HS; horse serum; IEF; isoelectric focusing; MeCP2; methyl-CpG binding protein 2; O-GlcNAc; O-linked; N; -acetylglucosamine; PBS; phosphate-buffered saline; PVDF; polyvinylidene fluoride; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SUMO; small ubiquitin-related modifierMeCP2; Intracellular localization; Neuronal differentiation; Phosphorylation; Post-translational modification; SUMOylation
A quantitative regional expression profile of EAAT2 known and novel splice variants reopens the question of aberrant EAAT2 splicing in disease by Tara L. Lauriat; Esther Richler; L. Alison McInnes (pp. 271-280).
The glutamate transporter 1 (GLT1) in rodents, or EAAT2 in humans, is alternatively spliced in a complex manner including the use of multiple 5′ and 3′ untranslated regions and several coding variants. We used quantitative RT-PCR to profile these splice variants in human and rat brain. We also used RT-PCR and Northern blotting to demonstrate that a novel isoform of GLT1b has an ∼11kb 3′ UTR extending through intron 9, exon 10 and approximately 5kb into the 3′ untranslated region of GLT1. However, our most important finding concerns an aberrant transcript lacking exon 9, which contains a motif permitting translocation from the endoplasmic reticulum. This variant had previously been associated with amyotrophic lateral sclerosis until several groups reported high levels in normal brain tissue. In contrast, our data shows that this aberrant transcript is present at 0.1–0.2% of the major EAAT2 isoforms.
Keywords: Quantitative RT-PCR; Alternative splicing; Gene expression; GLT1; EAAT2
Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1 by Thomas Christoph; Clemens Gillen; Joanna Mika; Arnold Grünweller; Martin K.-H. Schäfer; Klaus Schiene; Robert Frank; Ruth Jostock; Gregor Bahrenberg; Eberhard Weihe; Volker A. Erdmann; Jens Kurreck (pp. 281-290).
To examine the role of the vanilloid receptor TRPV1 in neuropathic pain, we assessed the effects of the receptor antagonist thioxo-BCTC and antisense oligonucleotides against the TRPV1 mRNA in a rat model of spinal nerve ligation. In order to identify accessible sites on the mRNA of TRPV1, the RNase H assay was used, leading to the successful identification of binding sites for antisense oligonucleotides. Cotransfection studies using Cos-7 cells were employed to identify the most effective antisense oligonucleotide efficiently inhibiting the expression of a fusion protein consisting of TRPV1 and the green fluorescent protein in a specific and concentration-dependent manner. In an in vivo rat model of spinal nerve ligation, intravenous application of the TRPV1 antagonist thioxo-BCTC reduced mechanical hypersensitivity yielding an ED50 value of 10.6mg/kg. Intrathecal administration of the antisense oligonucleotide against TRPV1, but not the mismatch oligonucleotide or a vehicle control, reduced mechanical hypersensitivity in rats with spinal nerve ligation in a similar manner. Immunohistochemical analysis revealed neuropathy- and antisense-associated regulation of TRPV1 protein expression in spinal cord and dorsal root ganglia. Our data demonstrate comparative analgesic effects of a TRPV1 anatagonist and a rationally designed TRPV1 antisense oligonucleotide in a spinal nerve ligation model of neuropathic pain and thus, lend support to the validation of TRPV1 as a promising target for the treatment of neuropathic pain.
Keywords: Antisense oligonucleotides; Capsaicin receptor; Thioxo-BCTC; Neuropathic pain; Vanilloid receptor