bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibitory regulation of glutamate aspartate transporter (GLAST) expression in astrocytes by cadmium‐induced calcium influx
Authors: Liu Yu‐Peng , Yang Chung‐Shi , Tzeng Shun‐Fen
Published: March 2008

After injury to the CNS, the accumulation of extracellular glutamate induces neuronal excitotoxicity, leading to secondary tissue damage. Astrocytes can reduce excess extracellular glutamate primarily through the astrocytic glutamate transporter‐1 and the Na+‐dependent glutamate/aspartate transporter (GLAST). In this study, we used an in vitro model of cadmium‐induced cellular stress and found that glutamate uptake activity of astrocytes was suppressed because of cadmium‐induced inhibition of GLAST expression. The blockage of cadmium‐triggered Ca2+ influx by Ca2+ chelators elevated GLAST transcription and glutamate uptake activity in astrocytes, suggesting that the suppression of GLAST expression in cadmium‐treated astrocytes was Ca2+‐dependent. This was supported by the findings showing the reduction of GLAST mRNA in astrocytes after treatment with Ca2+‐ionophore A23187. Cadmium reduced human GLAST promoter activity; however, it increased the binding of Ca2+‐sensitive activator protein‐1 (AP‐1) and cAMP response element binding protein (CREB) to their specific elements derived from the human GLAST promoter. These results demonstrate that AP‐1 and CREB may be coupled with Ca2+‐dependent pathway triggered by cadmium to mediate the inhibition of GLAST transcription. Our results suggest that Ca2+ influx into astrocytes after CNS injury could cause the down‐regulation of GLAST expression, thus reducing the astrocytic glutamate uptake function, which in turn may exacerbate secondary damage after CNS injury.
Csk‐homologous kinase interacts with SHPS‐1 and enhances neurite outgrowth of PC12 cells
Authors: Mitsuhashi Hiroaki , Futai Eugene , Sasagawa Noboru , Hayashi Yukiko , Nishino Ichizo , Ishiura Shoichi
Published: March 2008

SHPS‐1 is an immunoglobulin superfamily protein with four immunoreceptor tyrosine‐based inhibitory motifs (ITIMs) in its cytoplasmic region. Various neurotrophic factors induce the tyrosine phosphorylation of SHPS‐1 and the association of SHPS‐1 with the protein tyrosine phosphatase SHP‐2. Using a yeast two‐hybrid screen, we identified a protein tyrosine kinase, Csk‐homologous kinase (CHK), as an SHPS‐1‐interacting protein. Immunoprecipitation and pull‐down assays using glutathione S‐transferase (GST) fusion proteins containing the Src homology 2 (SH2) domain of CHK revealed that CHK associates with tyrosine‐phosphorylated SHPS‐1 via its SH2 domain. HIS3 assay in a yeast two‐hybrid system using the tyrosine‐to‐phenylalanine mutants of SHPS‐1 indicated that the first and second ITIMs of SHPS‐1 are required to bind CHK. Over‐expression of wild‐type CHK, but not a kinase‐inactive CHK mutant, enhanced the phosphorylation of SHPS‐1 and its subsequent association with SHP‐2. CHK phosphorylated each of four tyrosines in the cytoplasmic region of SHPS‐1 in vitro. Co‐expression of SHPS‐1 and CHK enhanced neurite outgrowth in PC12 cells. Thus, CHK phosphorylates and associates with SHPS‐1 and is involved in neural differentiation via SHP‐2 activation.
Over‐expression of the catalytic core of mitochondrial DNA (mtDNA) polymerase in the nervous system of Drosophila melanogaster reduces median life span by inducing mtDNA depletion
Authors: Martínez‐Azorín Francisco , Calleja Manuel , Hernández‐Sierra Rosana , Farr Carol L. , Kaguni Laurie S. , Garesse Rafael
Published: March 2008

DNA polymerase γ (pol γ) is the sole DNA polymerase devoted to mitochondrial DNA (mtDNA) replication. We have characterized the molecular and physiological effects of over‐expression of the catalytic subunit of pol γ, pol γ‐α, in the nervous system of Drosophila melanogaster using the upstream activation sequence (UAS)/yeast transcriptional activator by binding to UAS (GAL4) system. Tissue‐specific over‐expression of pol γ‐α was confirmed by immunoblot analysis, whereas the very low levels of endogenous protein are undetectable in UAS or GAL4 control lines. The transgenic flies over‐expressing pol γ‐α in the nervous system showed a moderate increase in pupal lethality, and a significant decrease in the median life span of adult flies. Moreover, these flies displayed a decrease in the rate of synthesis of mtDNA, which is accompanied by a significant mtDNA depletion, and a corresponding decrease in the levels of mitochondrial transcription factor A (mtTFA). Biochemical analysis showed an oxidative phosphorylation (OXPHOS) defect in transgenic flies, which were more susceptible to oxidative stress. Although we did not detect apoptosis in the nervous system of adult transgenic flies, brains of larvae over‐expressing pol γ‐α showed evidence of increased cell death that correlates with the observed phenotypes. Our data establish an animal model that mimics some of the features of human mtDNA depletion syndromes.
Complex I specific increase in superoxide formation and respiration rate by PrP‐null mouse brain mitochondria
Authors: Paterson Andrew W. J. , Curtis John C. , MacLeod Nikki K.
Published: March 2008

An imbalance in free radical production and removal is considered by many to be an important factor in the etiology of many degenerative diseases. Since mitochondria are a major source of free radicals, we have examined mitochondrial free radical production in relation to oxidative phosphorylation in PrP‐null mice. Quantitative electron paramagnetic resonance spectroscopy revealed up to a 70% increase in superoxide production from Complex I of submitochondrial particles prepared from PrP‐null mice. This was accompanied by elevated respiratory capacity through Complex I without any discernible alteration in respiratory efficiency. These differences are associated with changes in superoxide dismutase levels and defects in mitochondrial morphology, confirming previously reported results. Our results demonstrate a clear difference in free radical production and oxygen consumption by mitochondrial Complex I between PrP‐null mice and wild‐type controls, pointing to Complex I as a potential target for pathological change, suggesting similarities between prion‐related and other neurodegenerative diseases.
Myosin IIA is required for neurite outgrowth inhibition produced by repulsive guidance molecule
Authors: Kubo Takekazu , Endo Mitsuharu , Hata Katsuhiko , Taniguchi Junko , Kitajo Keiko , Tomura Sayaka , Yamaguchi Atsushi , Mueller Bernhard K. , Yamashita Toshihide
Published: March 2008

Although myelin‐associated neurite outgrowth inhibitors express their effects through RhoA/Rho‐kinase, the downstream targets of Rho‐kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho‐kinase, by using blebbistatin – a specific myosin II inhibitor – and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho‐kinase‐dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up‐regulated, and this effect depends on Rho‐kinase activity. Further, RGMa‐induced F‐actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho‐kinase‐dependent activation of MIIA via MLC phosphorylation induces F‐actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa.
Accelerated release of exosome‐associated GM1 ganglioside (GM1) by endocytic pathway abnormality: another putative pathway for GM1‐induced amyloid fibril formation
Authors: Yuyama Kohei , Yamamoto Naoki , Yanagisawa Katsuhiko
Published: March 2008

Exosomes are extracellularly released small vesicles that are derived from multivesicular bodies formed via the endocytic pathway. We treated pheochromocytoma PC12 cells with chloroquine, an acidotropic agent, which potently perturbs membrane trafficking from endosomes to lysosomes. Chloroquine treatment increased the level of GM1 ganglioside in cell media only when the cells were exposed to KCl for depolarization, which is known to enhance exosome release from neurons. In the sucrose‐density‐gradient fractionation of cell media, GM1 ganglioside was exclusively recovered with Alix, a specific marker of exosomes, in the fractions with the density corrresponding to that of exosomes. Notably, amyloid‐β assembly was markedly accelerated when incubated with the exosome fraction prepared from the culture media of PC12 cells treated with chloroquine and KCl. Furthermore, amyloid‐β assembly was significantly suppressed by the co‐incubation with an antibody specific to GM1‐bound amyloid‐β, an endogenous seed for amyloid formation of Alzheimer’s disease. Together with our previous finding that chloroquine treatment induces the accumulation of GM1 ganglioside in early endosomes, results of this study suggest that endocytic pathway abnormality accelerates the release of exosome‐associated GM1 ganglioside following its accumulation in early endosomes. Furthermore, this study also suggests that extracellular amyloid fibril formation is induced by not only GM1 gangliosides accumulated on the surface of the cells but also those released in association with exosomes.
Neurokinin‐3 peptide instead of neurokinin‐1 synergistically exacerbates kainic acid‐inducing degeneration of neurons in the substantia nigra of mice
Authors: Chen Liang‐Wei , Wang Yan‐Qin , Bian Gan‐Lan , Wei Li‐Chun , Yung Kin‐Lam
Published: March 2008

Neurokinin peptides neurokinin‐1 (NK1), neurokinin‐3 (NK3), and related receptors are abundantly distributed in the substantia nigra (SN) and evidenced by their possible roles in the Parkinson’s disease. Differential intervention roles of NK3 on kainic acid (KA)‐induced neuronal injury in the SN of mice were thus in vitro and in vivo studied by Fluoro‐Jade C (FJC) staining, immunohistochemistry to tyrosine hydroxylase (TH) or phospho‐NMDA receptor, and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. It revealed that (i) in contrast to protective effect of NK1 agonist septide that reduced FJC‐positive degenerative neurons and lesion volume insulted by KA, NK3 agonist senktide significantly increased FJC‐positive ones and lesion volume, and this effect was sufficiently reversed by NK3 antagonist SB218795; (ii) similarly, senktide reduced TH‐positive neurons and this effect was antagonized by SB218795, but septide increased TH‐positive ones; (iii) mechanistic observation showed differential influences of NK1 and NK3 agonists on phosphorylated‐NMDA receptor subunit 1 (phospho‐NMDAR1) and glial fibrillary acidic protein‐expressing astrocytes, i.e. senktide enhanced of NMDA receptor phosphorylation and astrocyte activity, while septide reduced NMDA receptor phosphorylation and astrocytic response; (iv) cell culture further confirmed the exacerbating effect of NK3 agonist on KA‐induced lesion of nigral cells or dopaminergic neurons, in which administration of senktide alone did not show significant cell toxicity. This study presents new evidence that neurokinin NK3 instead of NK1 synergistically exacerbate excitotoxic neuronal degeneration in the SN in a dose‐dependent manner and possibly through modulation of NMDA receptor phosphorylation and astrocyte activity, suggesting their potential significance in novel pharmaceutical therapy against Parkinson’s disease.
Enhanced behavioral sensitivity to the competitive GABA agonist, gaboxadol, in transgenic mice over‐expressing hippocampal extrasynaptic α6β GABAAreceptors
Authors: Saarelainen Kati S. , Ranna Martin , Rabe Holger , Sinkkonen Saku T. , Möykkynen Tommi , Uusi‐Oukari Mikko , Linden Anni‐Maija , Lüddens Hartmut , Korpi Esa R.
Published: March 2008

The behavioral and functional significance of the extrasynaptic inhibitory GABAAreceptors in the brain is still poorly known. We used a transgenic mouse line expressing the GABAAreceptor α6 subunit gene in the forebrain under the Thy‐1.2 promoter (Thy1α6) mice ectopically expressing α6 subunits especially in the hippocampus to study how extrasynaptically enriched αβ(γ2)‐type receptors alter animal behavior and receptor responses. In these mice extrasynaptic α6β receptors make up about 10% of the hippocampal GABAAreceptors resulting in imbalance between synaptic and extrasynaptic inhibition. The synthetic GABA‐site competitive agonist gaboxadol (4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol; 3 mg/kg) induced remarkable anxiolytic‐like response in the light : dark exploration and elevated plus‐maze tests in Thy1α6 mice, while being almost inactive in wild‐type mice. The transgenic mice also lost quicker and for longer time their righting reflex after 25 mg/kg gaboxadol than wild‐type mice. In hippocampal sections of Thy1α6 mice, the α6β receptors could be visualized autoradiographically by interactions between gaboxadol and GABA via [35S]TBPS binding to the GABAAreceptor ionophore. Gaboxadol inhibition of the binding could be partially prevented by GABA. Electrophysiology of recombinant GABAAreceptors revealed that GABA was a partial agonist at α6β3 and α6β3δ receptors, but a full agonist at α6β3γ2 receptors when compared with gaboxadol. The results suggest strong behavioral effects via selective pharmacological activation of enriched extrasynaptic αβ GABAAreceptors, and the mouse model represents an example of the functional consequences of altered balance between extrasynaptic and synaptic inhibition.
Novel Brn3a cis‐acting sequences mediate transcription of human trkA in neurons
Authors: Valderrama Ximena , Misra Vikram
Published: March 2008

trkA, the receptor tropomyosin‐related kinase for nerve growth factor, is critical not only for the correct spatial and temporal development of sensory neurons during embryogenesis but also for the survival of sensory neurons, the differentiation and apoptosis of neuronal tumors and suppression of latent herpes simplex virus genomes. While the regulation of the expression of trkA is a complex process, the transcription factor Brn3a is known to play an important role as an enhancer of trkA transcription during development in the mouse. Despite considerable information on the regulation of trkA during embryogenesis, the mechanisms by which the expression of trkA is regulated in differentiated neurons, or the factors that influence its expression in tumor cells, have not been identified. We initiated studies to determine whether Brn3a/trkA promoter interactions may be important in a model of differentiated neurons and in medulloblastoma cells. We constructed a plasmid that contains 1043 base pairs of genomic sequences that extend to 30 nucleotides upstream of trkA coding region. In contrast to previous data, a short 190 bp region that lies proximal to the trkA initiation codon was sufficient for Brn3a responsiveness in Vero cells. This region was also sufficient for Brn3a trans‐activation in nerve growth factor‐differentiated PC12 cells. At least two portions of the 190 bp fragment bind to Brn3a with an affinity high enough to be detected in electromobility shift assays. In addition, Brn3a increased levels of endogenous trkA transcripts in PC12 cells and initiated trkA expression in medulloblastoma cells, which normally do not express trkA.
Gene expression profile of quinacrine‐cured prion‐infected mouse neuronal cells
Authors: Fasano Carlo , Campana Vincenza , Griffiths Beatrice , Kelly Gavin , Schiavo Giampietro , Zurzolo Chiara
Published: March 2008

Prion diseases are transmissible fatal neurodegenerative diseases of humans and animals, characterised by the presence of an abnormal isoform (scrapie prion protein; PrPSc) of the endogenous cellular prion protein (PrPC). The pathological mechanisms at the basis of prion diseases remain elusive, although the accumulation of PrPSc has been linked to neurodegeneration. Different genomic approaches have been applied to carry out large‐scale expression analysis in prion‐infected brains and cell lines, in order to define factors potentially involved in pathogenesis. However, the general lack of overlap between the genes found in these studies prompted us to carry an analysis of gene expression using an alternative approach. Specifically, in order to avoid the complexities of shifting gene expression in a heterogeneous cell population, we used a single clone of GT1 cells that was de novo infected with mouse prion‐infected brain homogenate and then treated with quinacrine to clear PrPSc. By comparing the gene expression profiles of about 15 000 genes in quinacrine‐cured and not cured prion‐infected GT1 cells, we investigated the influence of the presence or the absence of PrPSc. By real‐time PCR, we confirmed that the gene encoding for laminin was down‐regulated as a consequence of the elimination of PrPSc by the quinacrine treatment. Thus, we speculate that this protein could be a specific candidate for further analysis of its role in prion infection and pathogenesis.
Seizure resistance in fat‐1 transgenic mice endogenously synthesizing high levels of omega‐3 polyunsaturated fatty acids
Authors: Taha Ameer Y. , Huot Pedro S. P. , Reza‐López Sandra , Prayitno Nita R. , Kang Jing X. , Burnham W. McIntyre , Ma David W. L.
Published: March 2008

n‐3 polyunsaturated fatty acids (PUFA), derived from marine oils, have been shown to protect against various neurological diseases. However, very little is known about their potential anticonvulsant properties. The objective of the present study was to determine whether enrichment of brain lipids with n‐3 PUFA inhibits seizures induced by pentylenetetrazol. We demonstrate that increased brain levels of n‐3 PUFA in transgenic fat‐1 male mice, which are capable of de novo synthesis of n‐3 PUFA from n‐6 PUFA, increases latency to seizure onset by 45%, relative to wildtype controls (p = 0.08). Compared with wildtype littermates, transgenic fat‐1 mice have significantly (p < 0.05) higher levels of docosahexaenoic acid and total n‐3 PUFA in brain total lipid extracts and phospholipids. Levels of brain docosahexaenoic acid were positively correlated to seizure latency (p < 0.05). These findings demonstrate that n‐3 PUFA have anticonvulsant properties and suggest the possibility of a novel, non‐drug dietary approach for the treatment of epilepsy.
Age‐related decrease in stimulated glutamate release and vesicular glutamate transporters in APP/PS1 transgenic and wild‐type mice
Authors: Minkeviciene R. , Ihalainen J. , Malm T. , Matilainen O. , Keksa‐Goldsteine V. , Goldsteins G. , Iivonen H. , Leguit N. , Glennon J. , Koistinaho J. , Banerjee P. , Tanila H.
Published: March 2008

We assessed baseline and KCl‐stimulated glutamate release by using microdialysis in freely moving young adult (7 months) and middle‐aged (17 months) transgenic mice carrying mutated human amyloid precursor protein and presenilin genes (APdE9 mice) and their wild‐type littermates. In addition, we assessed the age‐related development of amyloid pathology and spatial memory impaired in the water maze and changes in glutamate transporters. APdE9 mice showed gradual spatial memory impairment between 6 and 15 months of age. The stimulated glutamate release declined very robustly in 17‐month‐old APdE9 mice as compared to 7‐month‐old APdE9 mice. This age‐dependent decrease in stimulated glutamate release was also evident in wild‐type mice, although it was not as robust as in APdE9 mice. When compared to individual baselines, all aged wild‐type mice showed 25% or greater increase in glutamate release upon KCl stimulation, but none of the aged APdE9 mice. There was an age‐dependent decline in VGLUT1 levels, but not in the levels of VGLUT2, GLT‐1 or synaptophysin. Astrocyte activation as measured by glial acidic fibrillary protein was increased in middle‐aged APdE9 mice. Blunted pre‐synaptic glutamate response may contribute to memory deficit in middle‐aged APdE9 mice.
Simvastatin prevents the inflammatory process and the dopaminergic degeneration induced by the intranigral injection of lipopolysaccharide
Authors: Hernández‐Romero María del Carmen , Argüelles Sandro , Villarán Ruth F. , De Pablos Rocío M. , Delgado‐Cortés María José , Santiago Marti , Herrera Antonio J. , Cano Josefina , Machado Alberto
Published: March 2008

Anti‐inflammatory strategies have attracted much interest for their potential to prevent further deterioration of Parkinson’s disease. Recent experimental and clinical evidence indicate that statins – extensively used in medical practice as effective lipid‐lowering agents – have also anti‐inflammatory effects. In this study, we investigated the influence of simvastatin on the degenerative process of the dopaminergic neurons of the rat following intranigral injection of lipopolysaccharide (LPS), a potent inductor of inflammation that we have previously used as an animal model of Parkinson’s disease. We evaluated TH positive neurons, astroglial, and microglial populations and found that simvastatin prevented the inflammatory processes, as the induction of interleukin‐1β, tumor necrosis factor‐α, and iNOS and the consequent dopaminergic degeneration induced by LPS. Moreover, simvastatin produced the activation of the neurotrophic factor BDNF, along with the prevention of the oxidative damage to proteins. Moreover, it also prevents the main changes produced by LPS on different mitogen‐activated protein kinases, featured as increases of P‐c‐Jun N‐terminal protein kinase, P‐extracellular signal‐regulated kinase, p‐38, and P‐glycogen synthase kinase and the decrease of the promotion of cell survival signals such as cAMP response element‐binding protein and Akt. Our results suggest that statins could delay the progression of dopaminergic degeneration in disorders involving inflammatory processes.
BDNF impairment in the hippocampus is related to enhanced despair behavior in CB1 knockout mice
Authors: Aso Ester , Ozaita Andrés , Valdizán Elsa M. , Ledent Catherine , Pazos Ángel , Maldonado Rafael , Valverde Olga
Published: March 2008

Stress can cause damage and atrophy of neurons in the hippocampus by deregulating the expression of neurotrophic factors that promote neuronal plasticity. The endocannabinoid system represents a physiological substrate involved in neuroprotection at both cellular and emotional levels. The lack of CB1 receptor alters neuronal plasticity and originates an anxiety‐like phenotype in mice. In the present study, CB1 knockout mice exhibited an augmented response to stress revealed by the increased despair behavior and corticosterone levels showed in the tail suspension test and decreased brain derived neurotrophic factor (BDNF) levels in the hippocampus. Interestingly, local administration of BDNF in the hippocampus reversed the increased despair behavior of CB1 knockout mice, confirming the crucial role played by BDNF on the emotional impairment of these mutants. The neurotrophic deficiency seems to be specific for BDNF as no differences were found in the levels of nerve growth factor and NT‐3, two additional neurotrophic factors. Moreover, BDNF impairment is not related to the activity of its specific tyrosine kinase receptor or the activity of the transcription factor cAMP responsive element binding. These results suggest that the lack of CB1 receptor originates an enhanced response to stress and deficiency in neuronal plasticity by decreasing BDNF levels in the hippocampus that lead to impairment in the responses to emotional disturbances.
Photoreceptor‐specific expression, light‐dependent localization, and transcriptional targets of the zinc‐finger protein Yin Yang 1 in the chicken retina
Authors: Bernard Marianne , Voisin Pierre
Published: March 2008

The zinc‐finger transcription factor Yin Yang 1 (YY1) is a multifunctional protein that plays a critical role in embryonic development. Although it has been shown to play a role in eye development, its expression in the retina was not previously described. Here, we investigated YY1 expression in chicken tissues and we identified the neural retina as one of the tissues with highest YY1 protein levels. Immunohistochemical detection of YY1 in the retina revealed a clear‐cut photoreceptor specificity and day/night differences in the cytoplasmic localization of the protein. YY1 was also present at high concentration in the nuclei of some photoreceptors. Gel‐shift assays indicated YY1 bound to regulatory regions of several genes specifically expressed in photoreceptors. One of these genes, hydroxyindole‐O‐methyltransferase (EC 2.1.1.4), encodes the last enzyme of the melatonin synthesis pathway. Although over‐expression of chicken YY1 was not sufficient to activate the chicken hydroxyindole‐O‐methyltransferase promoter in HEK293 cells, the YY1‐binding site contained in this promoter was clearly required for full transcriptional activity in chicken embryonic retinal cells. These results suggest a role of YY1 in regulating the melatoninergic function of retinal photoreceptors.
Agonist‐ and competitive antagonist‐induced movement of loop 5 on the α subunit of the neuronal α4β4 nicotinic acetylcholine receptor
Authors: Mourot Alexandre , Bamberg Ernst , Rettinger Jürgen
Published: March 2008

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand‐gated ion channels that rapidly convert a chemical signal into an electrical signal. Although the structure of the nAChR is quite well described, the coupling between agonist binding and channel gating is still under debate. In this study, we probed local conformational transitions on the neuronal α4β4 nAChR by specifically tethering a conformation‐sensitive fluorescent dye on αG98C located on loop 5 (L5), and simultaneously monitoring fluorescence intensity and current after expression inXenopusoocytes. The potency of acetylcholine (ACh) was significantly higher in the cysteine mutant and further increased upon tetramethylrhodamine‐6‐maleimide labeling, suggesting a role of L5 in binding or gating. Structural reorganizations of L5 were shown to occur upon activation, as revealed by the fluorescence intensity increase during ACh exposure. Fluorescence changes were also detected at ACh concentrations lower than needed for current activation, suggesting a movement of L5 for a closed, resting or desensitized state. The competitive antagonist dihydro‐β‐erythroidine also induced a movement of L5 although at concentrations significantly higher than needed for current inhibition. Consequently L5, located inside the lumen of the pentamer, plays a role in both activation and inhibition of the nAChR.
Increased social interaction in mice deficient of the striatal medium spiny neuron‐specific phosphodiesterase 10A2
Authors: Sano Hiromi , Nagai Yumiko , Miyakawa Tsuyoshi , Shigemoto Ryuichi , Yokoi Mineto
Published: March 2008

Cyclic nucleotide phosphodiesterase 10A (PDE10A) is a member of phosphodiesterase families that degrade cAMP and/or cGMP in distinct intracellular sites. PDE10A has a dual activity on hydrolysis of both cAMP and cGMP, and is prominently expressed in the striatum and the testis. Previous studies suggested that PDE10A is involved in regulation of locomotor activity and potentially related to psychosis, but concrete physiological roles of PDE10A remains elusive yet. In this study, we genetically inactivated PDE10A2, a prominent isoform of PDE10A in the brain, in mice, and demonstrate that PDE10A2 deficiency results in increased social interaction without any major influence on different other behaviors, along with increased levels of striatal cAMP. We also demonstrate that PDE10A2 is selectively distributed in medium spiny neurons, but not interneurons, of the striatal complex. Thus, our results establish a physiological role for PDE10A2 in regulating cAMP pathway and social interaction, and suggest that cAMP signaling cascade in striatal medium spiny neurons might be involved in regulating social interaction behavior in mice.
Dopaminergic differentiation of human neural stem cells mediated by co‐cultured rat striatal brain slices
Authors: Anwar Mohammad Raffaqat , Andreasen Christian Maaløv , Lippert Solvej Kølvraa , Zimmer Jens , Martinez‐Serrano Alberto , Meyer Morten
Published: March 2008

Properly committed neural stem cells constitute a promising source of cells for transplantation in Parkinson’s disease, but a protocol for controlled dopaminergic differentiation is not yet available. To establish a setting for identification of secreted neural compounds promoting dopaminergic differentiation, we co‐cultured cells from a human neural forebrain‐derived stem cell line (hNS1) with rat striatal brain slices. In brief, coronal slices of neonatal rat striatum were cultured on semiporous membrane inserts placed in six‐well trays overlying monolayers of hNS1 cells. After 12 days of co‐culture, large numbers of tyrosine hydroxylase (TH)‐immunoreactive, catecholaminergic cells could be found underneath individual striatal slices. Cell counting revealed that up to 25.3% (average 16.1%) of the total number of cells in these areas were TH‐positive, contrasting a few TH‐positive cells (<1%) in non‐induced areas. The presence of dopamine in the conditioned culture medium was confirmed by HPLC analysis. Interestingly, not all striatal slice cultures induced TH‐expression in underlying hNS1 cells. Common to TH‐inductive cultures was, however, the presence of degenerating, necrotic areas, suggesting that factors released during striatal degeneration were responsible for the dopaminergic induction of the hNS1 cells. Ongoing experiments aim to identify such factors by comparing protein profiles of media conditioned by degenerating (necrotic) versus healthy striatal slice cultures.
Validation of a robust and sensitive method for detecting hydroxyl radical formation together with evoked neurotransmitter release in brain microdialysis
Authors: Freinbichler Wolfhardt , Colivicchi Maria A. , Fattori Manuela , Ballini Chiara , Tipton Keith F. , Linert Wolfgang , Della Corte Laura
Published: March 2008

Sodium terephthalate was shown to be a new robust and sensitive chemical trap for highly reactive oxygen species (hROS), which lacks the drawbacks of the salicylic acid method. Reaction of the almost non‐fluorescent terephthalate (TA2−) with hydroxyl radicals or ferryl‐oxo species resulted in the stoichiometric formation of the brilliant fluorophor, 2‐hydroxyterephthalate (OH‐TA). Neither hydrogen peroxide nor superoxide reacts in this system. This procedure was validated for determining hROS formation during microdialysis under in vivo conditions as well as by in vitro studies. The detection limit of OH‐TA in microdialysis samples was 0.5 fmol/μL. Derivatization of samples with o‐phthalaldehyde, for amino acid detection, had no effect on OH‐TA fluorescence, which could easily be resolved from the amino acid derivatives by HPLC, allowing determination in a single chromatogram. Use of terephthalate in microdialysis experiments showed the neurotoxin kainate to evoke hROS formation in a dose‐dependent manner. The presence of TA2− in the perfusion fluid did not affect basal or evoked release of aspartate, glutamate, taurine and GABA. Assessment of cell death ‘ex vivo’ showed TA2− to be non‐toxic at concentrations up to 1 mM. The in vitro results in the Fenton system (Fe2+ + H2O2) indicate a mechanism whereby TA2− forms a primary complex with Fe2+ followed by an intramolecular hydroxylation accompanied by intramolecular electron transfer.
Molecular characterization and localization of the RIC‐3 protein, an effector of nicotinic acetylcholine receptor expression
Authors: Castelán Francisco , Castillo Mar , Mulet José , Sala Salvador , Sala Francisco , Domínguez del Toro Eduardo , Criado Manuel
Published: March 2008

The RIC‐3 protein acts as a regulator of acetylcholine nicotinic receptor (nAChR) expression. In Xenopus laevis oocytes the human RIC‐3 (hRIC‐3) protein enhances expression of α7 receptors and abolishes expression of α4β2 receptors. In vitro translation of hRIC‐3 evidenced its membrane insertion but not the role as signal peptide of its first transmembrane domain (TMD). When the TMDs of hRIC‐3 were substituted, its effects on nAChR expression were attenuated. A certain linker length between the TMDs was also needed for α7 expression enhancement but not for α4β2 inhibition. A combination of increased α7 receptor steady state levels, facilitated transport and reduced receptor internalization appears to be responsible for the increase in α7 membrane expression induced by hRIC‐3. Antibodies against hRIC‐3 showed its expression in SH‐SY5Y and PC12 cells and its induction upon differentiation. Immunohistochemistry demonstrated the presence of RIC‐3 in rat brain localized, in general, in places where α7 nAChRs were found.
The intrastriatal injection of thrombin in rat induced a retrograde apoptotic degeneration of nigral dopaminergic neurons through synaptic elimination
Authors: Herrera Antonio J. , De Pablos Rocío M. , Carreño‐Müller Eloisa , Villarán Ruth F. , Venero José L. , Tomás‐Camardiel Mayka , Cano Josefina , Machado Alberto
Published: March 2008

We have performed intrastriatal injection of thrombin and searched for distant effects in the cell body region. In striatum, thrombin produced a slight loss of striatal neurons as demonstrated by neural nuclei immunostaining – a non‐specific neuronal marker – and the expression of glutamic acid decarboxylase 67 mRNA, a specific marker for striatal GABAergic interneurons, the most abundant phenotype in this brain area. Interestingly, striatal neuropil contained many boutons immunostained for synaptic vesicle protein 2 and synaptophysin which colocalize with tyrosine hydroxylase (TH), suggesting a degenerative process with pre‐synaptic accumulation of synaptic vesicles. When we studied the effects on substantia nigra, we found the disappearance of dopaminergic neurons, shown by loss of TH immunoreactivity, loss of expression of TH and dopamine transporter mRNAs, and disappearance of FluoroGold‐labelled nigral neurons. The degeneration of substantia nigra dopaminergic neurons was produced through up‐regulation of cFos mRNA, apoptosis and accumulation of α‐synuclein shown by colocalization experiments. Thrombin effects could be mediated by protease‐activated receptor 4 activation, as protease‐activated receptor 4‐activating peptide mimicked thrombin effects. Our results point out the possible relationship between synapse elimination and retrograde degeneration in the nigral dopaminergic system.
Silencing of the ADNP‐family member, ADNP2, results in changes in cellular viability under oxidative stress
Authors: Kushnir Michal , Dresner Efrat , Mandel Shmuel , Gozes Illana
Published: March 2008

Activity‐dependent neuroprotective protein (ADNP) 2 (KIAA0863; ZNF508) gene, a homeobox‐profile containing gene, was identified in a screen for homologous proteins to ADNP. The human ADNP2 contains 1131 amino acid residues with a molecular weight of 122.8 KDa. In silico analysis indicated that ortholgs to ADNP2 exist in different phyla, suggesting that ADNP2 might be evolutionary conserved. Here, we began to explore the molecular and functional characterization of ADNP2. Results showed that the mouse ADNP2 mRNA is ubiquitously expressed in distinct normal tissues with increased expression in the brain, particularly in the cerebral cortex. During development, a relatively high level of ADNP2 gene expression was found in the embryonic mouse brain and was sustained throughout embryogenesis and adulthood. An increase in the mRNA was detected in differentiated P19 neuronal/glial‐like cells as compared with the non‐differentiated cells. To gain insight into ADNP2 function, ADNP2‐deficient cell lines were established by the RNA silencing (small interfering RNA) technology. ADNP2 deficiency significantly changed the toxicity induced by hydrogen peroxide in P19 embryonic carcinoma cells, similar to what would be predicted for ADNP deficiency. These findings represent an initial characterization of ADNP2 and suggest that this gene product may have an important function in brain by playing a role in cellular survival pathways.
Excitotoxic damage, disrupted energy metabolism, and oxidative stress in the rat brain: antioxidant and neuroprotective effects ofl‐carnitine
Authors: Silva‐Adaya Daniela , Pérez‐De La Cruz Verónica , Herrera‐Mundo María Nieves , Mendoza‐Macedo Karina , Villeda‐Hernández Juana , Binienda Zbigniew , Ali Syed F. , Santamaría Abel
Published: March 2008

Excitotoxicity and disrupted energy metabolism are major events leading to nerve cell death in neurodegenerative disorders. These cooperative pathways share one common aspect: triggering of oxidative stress by free radical formation. In this work, we evaluated the effects of the antioxidant and energy precursor, levocarnitine (l‐CAR), on the oxidative damage and the behavioral, morphological, and neurochemical alterations produced in nerve tissue by the excitotoxin and free radical precursor, quinolinic acid (2,3‐pyrindin dicarboxylic acid; QUIN), and the mitochondrial toxin, 3‐nitropropionic acid (3‐NP). Oxidative damage was assessed by the estimation of reactive oxygen species formation, lipid peroxidation, and mitochondrial dysfunction in synaptosomal fractions. Behavioral, morphological, and neurochemical alterations were evaluated as markers of neurotoxicity in animals systemically administered withl‐CAR, chronically injected with 3‐NP and/or intrastriatally infused with QUIN. At micromolar concentrations,l‐CAR reduced the three markers of oxidative stress stimulated by both toxins alone or in combination.l‐CAR also prevented the rotation behavior evoked by QUIN and the hypokinetic pattern induced by 3‐NP in rats. Morphological alterations produced by both toxins (increased striatal glial fibrillary acidic protein‐immunoreactivity for QUIN and enhanced neuronal damage in different brain regions for 3‐NP) were reduced byl‐CAR. In addition,l‐CAR prevented the synergistic action of 3‐NP and QUIN to increase motor asymmetry and depleted striatal GABA levels. Our results suggest that the protective properties ofl‐CAR in the neurotoxic models tested are mostly mediated by its characteristics as an antioxidant agent.
AMP‐activated protein kinase phosphorylation in brain is dependent on method of killing and tissue preparation
Authors: Scharf Matthew T. , Mackiewicz Miroslaw , Naidoo Nirinjini , O’Callaghan James P. , Pack Allan I.
Published: March 2008

AMP‐activated protein kinase (AMPK) is activated when the catalytic α subunit is phosphorylated on Thr172 and therefore, phosphorylation of the α subunit is used as a measure of activation. However, measurement of α subunit of AMPK (α‐AMPK) phosphorylation in vivo can be technically challenging. To determine the most accurate method for measuring α‐AMPK phosphorylation in the mouse brain, we compared different methods of killing and tissue preparation. We found that freeze/thawing samples after homogenization on ice dramatically increased α‐AMPK phosphorylation in mice killed by cervical dislocation. Killing of mice by focused microwave irradiation, which rapidly heats the brain and causes enzymatic inactivation, prevented the freeze/thaw‐induced increase in α‐AMPK phosphorylation and similar levels of phosphorylation were observed compared with mice killed with cervical dislocation without freeze/thawing of samples. Sonication of samples in hot 1% sodium dodecyl sulfate blocked the freeze/thaw‐induced increase in α‐AMPK phosphorylation, but phosphorylation was higher in mice killed by cervical dislocation compared with mice killed by focused microwave irradiation. These results demonstrate that α‐AMPK phosphorylation is dependent on method of killing and tissue preparation and that α‐AMPK phosphorylation can increase in a manner that does not reflect biological alterations.
Substrates dissociate dopamine transporter oligomers
Authors: Chen Nianhang , Reith Maarten E. A.
Published: March 2008

Substrate‐induced endocytic trafficking of dopamine transporter (DAT) has been observed, but little is known about the regulation of DAT oligomerization by substrate. The present study investigates the effect on substrates on DAT oligomerization and explores a potential link with the presence of DAT at the cell surface in human embryonic kidney cells transiently or stably expressing N‐terminal tagged DAT constructs. Dopamine (100 μM) or amphetamine (2–10 μM) reduced Myc‐DAT coimmunoprecipitated along with Flag‐DAT (oligomeric DAT) in tandem with a reduction in surface DAT determined by biotinylation. Dopamine (10–1000 μM) and amphetamine (0.2–200 μM) reduced DAT oligomerization as assessed by cross‐linking with copper sulfate phenanthroline or Cu2+. Inhibition of endocytosis by 10 μM phenylarsine oxide or 450 mM sucrose counteracted the effect of 10 μM DA or 2 μM amphetamine in reducing DAT cross‐linking. In addition to overall similarities between the results with the two cross‐linking agents and between the results with the two different endocytosis inhibitors, some differences were noted as well, likely related to the efficiency of the cross‐linking process and the sulfhydryl‐reactive properties of phenylarsine oxide, respectively. The present results are the first to indicate regulation of oligomerization of an solute carrier family 6 transporter, the DAT, by substrates that act at DAT. In addition, the present study opens up the possibility of an important linkage between oligomerization of DAT and endocytic or other modulatory mechanisms impacting surface DAT.
Delayed neuronal preconditioning by NS1619 is independent of calcium activated potassium channels
Authors: Gáspár Tamás , Katakam Prasad , Snipes James A. , Kis Béla , Domoki Ferenc , Bari Ferenc , Busija David W.
Published: March 2008

1,3‐Dihydro‐1‐[2‐hydroxy‐5‐(trifluoromethyl)phenyl]‐5‐(trifluoromethyl)‐2H‐benzimidazol‐2‐one (NS1619), a potent activator of the large conductance Ca2+ activated potassium (BKCa) channel, has been demonstrated to induce preconditioning (PC) in the heart. The aim of our study was to test the delayed PC effect of NS1619 in rat cortical neuronal cultures against oxygen‐glucose deprivation, H2O2, or glutamate excitotoxicity. We also investigated its actions on reactive oxygen species (ROS) generation, and on mitochondrial and plasma membrane potentials. Furthermore, we tested the activation of the phosphoinositide 3‐kinase (PI3K) signaling pathway, and the effect of NS1619 on caspase‐3/7. NS1619 dose‐dependently protected the cells against the toxic insults, and the protection was completely blocked by a superoxide dismutase mimetic and a PI3K antagonist, but not by BKCa channel inhibitors. Application of NS1619 increased ROS generation, depolarized isolated mitochondria, hyperpolarized the neuronal cell membrane, and activated the PI3K signaling cascade. However, only the effect on the cell membrane potential was antagonized by BKCa channel blockers. NS1619 inhibited the activation of capase‐3/7. In summary, NS1619 is a potent inducer of delayed neuronal PC. However, the neuroprotective effect seems to be independent of cell membrane and mitochondrial BKCa channels. Rather it is the consequence of ROS generation, activation of the PI3K pathway, and inhibition of caspase activation.
Purinergic receptor signaling regulates N‐cadherin expression in primary astrocyte cultures
Authors: Tran Minh D. , Wanner Ina B. , Neary Joseph T.
Published: March 2008

Extracellular ATP exerts both short‐term and long‐term effects in the CNS by stimulating cell‐surface purinergic receptors. Here we have examined the effect of purinergic receptor activation on N‐cadherin expression, a calcium‐dependent cell adhesion molecule involved in many processes, including glia‐glia and axon‐glia interactions. When primary cultures of rat cortical astrocytes were treated with ATP, N‐cadherin protein expression increased in a time‐ and concentration‐dependent manner. In addition, ATP treatment caused an increase in N‐cadherin immunoreactivity in both the cytoplasm and on the cell surface membrane. Interestingly, experiments with cycloheximide revealed that relocalization of N‐cadherin to the cell surface membrane were independent of protein synthesis. The ATP‐induced increase in N‐cadherin protein expression was blocked by reactive blue 2 and 8‐(p‐sulfophenyl)‐theophylline, suggesting involvement of both P2 and P1 purinergic receptors, respectively. In addition, N‐cadherin expression was partially blocked when signaling from purinergic receptors to extracellular signal regulated protein kinase or Akt was inhibited by 1,4‐diamino‐2,3‐dicyano‐1,4‐bis(2‐aminophenylthio)butadiene or wortmannin, respectively. By using an in vitro model of traumatic CNS injury, we found that N‐cadherin expression was increased when astrocytes were subjected to rapid and reversible mechanical strain. The findings presented here demonstrate a role for extracellular ATP, purinergic receptors and protein kinase signaling in regulating N‐cadherin expression and suggest a role for this mechanism in cell‐cell interactions.
Role of autophagy in G2019S‐LRRK2‐associated neurite shortening in differentiated SH‐SY5Y cells
Authors: Plowey Edward D. , Cherra Salvatore J. , Liu Yong‐Jian , Chu Charleen T.
Published: March 2008

Neuritic retraction represents a prominent feature of the degenerative phenotype associated with mutations in leucine rich repeat kinase 2 (LRRK2) that are implicated in autosomal dominant and some cases of sporadic Parkinson’s disease. Alterations in macroautophagy, the vacuolar catabolism of cytoplasmic constituents, have been described in Parkinson’s disease. In this study, we utilized retinoic‐acid differentiated SH‐SY5Y cells to determine whether autophagy contributes to mutant LRRK2‐associated neurite degeneration. Transfection of pre‐differentiated SH‐SY5Y cells with LRRK2 cDNA containing the common G2019S mutation resulted in significant decreases in neurite length, which were not observed in cells transfected with wild type LRRK2 or its kinase‐dead K1906M mutation. G2019S LRRK2 transfected cells also exhibited striking increases in autophagic vacuoles in both neuritic and somatic compartments, as demonstrated by fluorescence and western blot analysis of the autophagy marker green fluorescent protein‐tagged microtubule‐associated protein Light Chain 3 and by transmission electron microscopy. RNA interference knockdown of LC3 or Atg7, two essential components of the conserved autophagy machinery, reversed the effects of G2019S LRRK2 expression on neuronal process length, whereas rapamycin potentiated these effects. The mitogen activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) kinase (MEK) inhibitor 1,4‐diamino‐2,3‐dicyano‐1,4‐bis[2‐aminophenylthio]butadiene (U0126) reduced LRRK2‐induced neuritic autophagy and neurite shortening, implicating MAPK/ERK‐related signaling. These results indicate an active role for autophagy in neurite remodeling induced by pathogenic mutation of LRRK2.