bluuebunny/crossref_metadata_2025 huggingface dataset:
Inter‐relationship of cytokine production and NOS2 expression in microglia
Authors: Dello Russo C. , Gavrilyuk V. , Feinstein D. L.
Published: July 2010

Under normal conditions, glial cells provide neurotrophic support, but can contribute to damage during neurodegenerative disorders such as multiple sclerosis and Alzheimer's disease. Once activated, glia produce and release inflammatory mediators and potentially neurotoxic substances (including cytokines, NO, and prostanoids) whose interactions could lead to sustained inflammation. We investigated the relationship between cytokine production and NO release using enriched cultures of rat microglia. Preliminary data suggest that low concentrations of endotoxin LPS (1–10 ng/mL) activated microglia by a complex mechanism involving NFκB activation, cAMP increase and PKA activation, and IL‐1β production and release. We characterized this system using pharmacological activators and inhibitors of NFκB and PKA, and IL‐1rα to reduce IL‐1β effects. Norepinephrine (NE) dose‐dependently inhibited LPS‐induced NOS2 expression and NO generation, via activation of β‐2 adrenergic receptors (β2‐ARs) and elevation of cAMP. Similarly, NE dose‐dependently blocked LPS‐dependent IL‐1β production. The addition of PKA inhibitors did not reverse the suppressive effects of NE on NO production, but did reverse its effects on IL‐1β. Addition of IL‐1rα also reduced NO production, and exogenous IL‐1β reversed the inhibitory effects of NE. These data suggest that effects of NE on LPS‐dependent NO release is, at least in part, mediated by blocking of IL‐1β secretion. At the same time, results with inhibitors suggest that PKA activation is necessary for LPS effects. Together, these results point to the existence of autocrine and paracrine regulatory mechanisms of microglia activation. The relationship between cytokines and NO could be an important mechanism of sustained and disruptive microglia activation.
Induction of nitric oxide synthase in cytokine‐stimulated human astrocytes by interleukin‐12 P40 monomer
Authors: Pahan K. , Jana M. , Dasgupta S. , Liu X.
Published: July 2010

Excessive production of IL‐12 participates in the neuroinflammatory disease process of Multiple Sclerosis (MS). IL‐12 is composed of two different subunits – p40 and p35. However, it has been found that p40 but not that p35 is expressed in excessive amount in the CNS of MS patients. However, the biological significance of this overexpression of p40 in the CNS of patients with MS is not known. The present study underlines the importance of human IL‐12 (p70, the heterodimeric form) and p40 monomer in inducing the expression of iNOS in IFN‐g‐stimulated human astrocytes. IL‐12, p40 or IFN‐γ alone was poor inducer of NO production in human U373MG astrocytoma cells. However, the combination of either IL‐12 and IFN‐γ or p40 and IFN‐γ but not that of the IL‐12 and p40 markedly induced the production of NO. This induction of NO production was accompanied by an induction of iNOS protein and mRNA. Similar to U373MG astrocytoma cells, IL‐12 or p40 also induced the production of NO in human primary astrocytes. Although IFN‐γ alone was a weak inducer of NF‐κB activation, it efficiently induced the activation of CCAAT/enhancer‐binding protein (C/EBP)b and g‐activation site (GAS). On the other hand, p40 alone induced the activation of NF‐κB and C/EBPb but not that of GAS suggesting that p40 alone cannot induce the expression of iNOS probably due to its inability to induce the activation of GAS. This study delineates a novel role of IL‐12 p70 or IL‐12 p40 monomer in inducing the expression of iNOS in human astrocytes which may participate in the pathogenesis of neuroinflammatory diseases.

Acknowledgements:  This study was supported by NIH grants (NS39940 and AG19487).
Lovastatin induced cytokine‐mediated inducible nitric oxide synthase (INOS) expression in brain transformed cell lines
Authors: Rattan R. , Giri S. , Singh I.
Published: July 2010

Nitric oxide (NO) produced by inducible nitric‐oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of neurodegenerative diseases. The present study underlines the involvement of RhoA protein in the expression of iNOS via NF‐κB in transformed brain cell lines [rat C6 glioma, human astrocytoma (T98G and A172) and immortal rat astrocytes]. Treatment of C6 glioma cells with lovastatin resulted in the induction of iNOS mRNA and increased NO production in a dose and time dependent manner. Addition of mevalonate and geranylgeranylpyrophosphate reversed the lovastatin‐mediated effect. A selective inhibitor of geranylgeranyltransferase inhibitor (GGTI 298) further enhanced the lovastatin as well as cytokine‐mediated iNOS expression suggesting the involvement of geranylgeranylated proteins in the down regulation of iNOS expression. Inactivators of Rho A family (bacterial toxin B), Rho A protein (C3 toxin from C. botulinum) and Rho A associated kinases (Y‐27632) increased the iNOS expression. Lovastatin induced NO production mediated by increasing IkB? degradation and NF‐κB translocation via inactivation of Rho A protein. Co‐transfection studies using dominant negative form of RhoA along with iNOS luciferase and NF‐κB luciferase reporter constructs further supported these observations. Taken together, these studies document a role for Rho A in lovastatin mediated induction of iNOS expression in brain glial cell lines.

Acknowledgements:  Supported by NIH grants NS‐22576, NS‐34741 and NS‐37766.
Dietary restriction enhances neurogenesis and up‐regulates neurotrophin expression in the hippocampus of adult mice
Authors: Lee J. , Seroogy K. B. , Mattson M. P.
Published: July 2010

The brain has a limited regenerative capacity, making it vulnerable to injury and disease. However, the adult central nervous system contains small populations of neural precursor cells (NPC) that can give rise to new neurons and glia, and may play important roles in learning and memory, and recovery from injury. We now report that dietary restriction (DR) enhances neurogenesis in the dentate gyrus of adult male C57BL/6 mice and, in particular, increases the survival of newly generated dentate granule neurons. Triple‐labelling confocal immunofluorescence analysis revealed that the majority of newly synthesized cells in the granule cell layer of the dentate gyrus exhibited a neuronal phenotype. Levels of expression of brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) are increased by DR. We conclude that the increase in neurogenesis induced by DR results from increased survival of newly formed neurons associated with increased production of BDNF and NT‐3. In addition to these findings, DR has been shown to increase the resistance of neurons against excitotoxic insult and enhance the behavioral outcome after injury. Therefore, further experiments in kainic acid (KA)‐induced seizure model were performed to determine whether the enhanced neurogenesis by DR can repair damaged neuronal circuits in the adult CNS. We were unable to document replacement of new neuron in the damaged areas (regions CA3 and CA1 of the hippocampus) during a 4‐week period following KA administration in either DR or control mice. Our work is the first to show that neurogenesis can be affected by diet, suggesting a contribution of enhanced neurogenesis to the beneficial effects of DR on hippocampal plasticity and resistance to neurodegenerative disorders.
Characterization of mouse marrow stromal cells
Authors: Liour S. S , Suetake K. , Wen J. , Warsi S. , Kraemer S. , Yu R. K
Published: July 2010

Neural transplantation is a promising therapy for neurodegenerative diseases, including Parkinson's, Huntington's, Alzeheimer's, as well as mucopolysaccharidoses. However, neural transplantation is an invasive procedure in the early stages of research and development. In contrast, bone marrow transplantation has been used in medical treatment of immune and hematological disorders and genetic diseases. A increasing number of research reports suggest that cells derived from bone marrow, particularly mesenchymal stem cells, cannot only migrate into brains of recipient rodents after IV administration, but also differentiate into neurons and glia, to facilitate the functional recovery of rats after stroke or brain trauma. The lack of exclusive cell markers for mesenchymal stem cells makes them difficult to study. We isolated mouse marrow stromal cells and studied the expression of markers, particularly glycosphingolipids on their cell surface. Bone marrow was aspirated from femurs of two‐month‐old mice, and the stromal cells were propagated in attached cultures. Immuncytochemical analysis suggested that most stromal cells were immunopositive for antibodies against IGFR, flk‐1, and CD44. Analysis of the glycosphingolipid composition by HPTLC revealed that GM3, GM2, GM1, and GD1a were the major gangliosides expressed in stromal cell in culture. Glucosylceramide, lactosylceramide, and paragloboside were the major neutral glycolipids expressed in these cells. Combinations of these cell surface markers may prove useful in the isolation and characterization of mesenchymal stem cells.

Acknowledgements:  Supported by grants from NIH NS11853 and the Children's Medical Research Foundation.
Involvement of P38 MAP kinase in P2X7 receptor‐dependent cell death
Authors: Diaz‐Pimentel J. A. , Vivas P. , Weisman G. , Gonzalez F. A.
Published: July 2010

The P2X7 nucleotide receptor is a non‐selective ligand‐gated ion channel expressed primarily in hematopoietic cells, astrocytes, microglia, parotid acinar cells, and fibroblasts. P2X7 activation opens a channel, which triggers rapid depolarization and calcium influx. Additionally, it induces the formation of non‐selective pores able to pass molecules up to 900 Da, a phenomenon not observed in any other P2X receptors. Activation of the P2X7 receptor can lead to apoptosis or necrosis in a variety of cells including lymphocytes and microglia, but the biochemical steps linking channel/pore formation to cell death are poorly understood. Here we described the channel/pore formation upon P2X7 receptor activation in human promyelocitic HL‐60 and human 1321N1 astrocytoma cells. In addition we found that exposure of these cells to BzATP (10 min) was sufficient to trigger activation of p38 MAP Kinase. Our results show that BzATP activate p38 MAPK pathway and that the profile of activation is characterized by a strong and transient activation of this kinase. Pretreatment of the cells with the specific inhibitor of p38 MAPK, SB203580, inhibit BzATP‐dependent LDH liberation associated with necrotic cell death. We conclude that p38 MAPK activation is involved in the pathway conducing to the P2X7‐dependent necrotic cell death, and corroborate other observations that necrosis is not a passive form of cell death.
Role of PKCα in linking MAP kinase to caspase‐3 in 5‐HT1A receptor mediated signalling
Authors: Banerjee P. , Adayev T. , Sondhi R. , Sobocki T.
Published: July 2010

Activation of caspases is a key step in initiation and execution of neuronal apoptosis following injury. Our earlier studies showed that the 5‐HT1A agonist (8‐OH‐DPAT) stimulates a PI‐3K‐independent mode of inhibition of caspase‐3. This results in an inhibition of anoxia‐induced apoptosis in the hippocampal neuron‐derived HN2‐5 cells through the extracellular signal‐regulated kinases (ERK1/2). Moreover, 5‐HT1A agonist treatment also blocks caspase‐3 activation in an ERK‐dependent manner during hydrogen peroxide mediated oxidative stress. This protective ERK activation involves Ca/Calmodulin (CaM) dependent endocytosis, since Fluphenazine, W‐7 (Ca/CaM inhibitors) and Monodansylcadaverine (an endocytosis inhibitor) effectively block the 8‐OH‐DPAT‐triggered ERK1/2 phosphorylation. A focal adhesion kinase (e.g. Pyk2) participates in this signal transduction pathway, since 8‐OH‐DPAT–mediated ERK1/2 activation is blocked in the presence of Cytochalasin D (a focal adhesion kinase inhibitor). Both a general inhibitor of PKCs (GFX) and a selective inhibitor of the calcium‐sensitive PKCs (Gö6976) reversed agonist‐induced suppresion of caspase‐3 activity, although GFX has no effect on the 5‐HT1A‐R‐mediated activation of ERK1/2. PKCα, but not PKCβ, immunoprecipitated from 8‐OH‐DPAT‐treated cells showed ERK1/2‐dependent stimulation, indicating the involvement of the PKCα isozyme. Transient expression of kinase‐negative PKCα in HN2‐5 cells caused elimination of the agonist‐evoked caspase‐3 inhibition, confirming that PKCα links the 5‐HT1A‐R‐MAPK pathway to the inhibition of caspase‐3. A study of this 5‐HT1A‐R‐mediated signalling cascade in other neuronal cells will demonstrate the generality of this novel pathway.
Poster Session BP07: Neurodegenerative Diseases. The role of α‐synuclein in cell division, differentiation and survival
Authors: Jayman F. , Duchatellier R. , Anantharam V. , Kanthasamy A. , Banerjee P.
Published: July 2010

Presynaptic terminals contain an abundant 140‐amino acid phosphoprotein, dubbed α‐synuclein, which is accumulated in Lewy bodies typically observed in neurons in neurodegenerative diseases, such as Parkinson's disease. In this study, the role of α‐synuclein in regulating cycle, differentiation, and survival of neuronal cells was studied using a rat dopaminergic cell line ZN27D. To delineate specific effects of α‐synuclein the same cell line was engineered to express human α‐synuclein and a vector‐transfected cell line RK27 was used as a second control. All three cell lines showed significant proliferation even in serum‐free medium, and complete inhibition of cell division and differentiation could be achieved in the ZN27D cells only when both dibutyryl cAMP (dbcAMP) and retinoic acid were present. In contrast, the α‐synuclein expressing cells could be differentiated in the presence of only dbcAMP. Dose dependence of MPP+(1‐methyl‐4‐phenylpyridinium iodide)‐mediated caspase3 activation was studied in undifferentiated ZN27D cells. At 200 μm MPP+ a significant cleavage of the caspase3 substrate PARP was observed and it was reversed in the presence of α‐synuclein. MPP+ also inhibited aminophospholipid translocase (APTL), a P‐type ATPase that is responsible for inner plasma membrane localization of phophotidylserine in healthy cells. The role of α‐synuclein in regulating cell cycle, differentiation, APTL activity and cell death is being investigated further in the dopaminergic ZN27D cell line.
Displacement toward NFT of a key enzyme of the ubiquitin proteolytic pathway in Alzheimer's brain
Authors: Lopez Salon M. , Castaño E. , Soto E. , Pasquini J.
Published: July 2010

Recent findings have led to a better understanding of the roles of the ubiquitin (Ub) proteasome system in the progression of different human degenerative diseases. The neuropathological hallmarks of Alzheimer's disease (AD) are the neurofibrillary tangles (NFT) and the senile plaques (SP). NFT accumulate within neurons and are composed of filamentous aggregates (PHF) of the microtubule‐associated protein tau (Layfield 2001). The intraneuronal inclusions contain deposits of ubiquitylated proteins, indicating that perturbations of Ub‐dependent proteolysis may be occurring. We have demonstrated the existence of a defective ubiquitination in the cytosol of AD brains, due to a decrease in normal Ub activating enzyme (E1) activity in the cytosol as a consequence of its de‐localization to the particulate fraction (Lopez Salon 2000). Immunohistochemical experiments using an antibody against PHF, showed a great number of NFT in AD. Using an anti E1 antibody we demonstrated the presence of this enzyme in the NFT. In order to corroborate the above mentioned results, we have carried out biochemical studies to investigate the pattern of solubilization of the proteins present in NFT in different fractions isolated by differential centrifugation. In AD almost 60% of E1 is obtained in a particulate fraction, while in control samples E1 is mainly located at the supernatant. The de‐localization of E1 could be a late event occurring during the progression of AD. A secondary impairment of Ub‐dependent degradation due to a decrease in normal E1 activity in the brain cytosol may contribute to the abnormal accumulation of proteins in AD and deserves future research as a possible disease mechanism.
Synaptosomal glutamate transport studies in a transgenic rat model of amyotrophic lateral sclerosis
Authors: McIlvain H. B. , She Y. , Howland D. S. , Dunlop J.
Published: July 2010

Transgenic rats over‐expressing the G93A Co/Zn superoxide dismutase (SOD1) mutation have recently been generated (Howland et al. PNAS99, 1604–1609) and found to replicate an amyotrophic lateral sclerosis (ALS) – like motor neuron disease previously described in transgenic mice harboring the same mutation. Impairments in synaptosomal glutamate transport capacity have been documented both in the transgenic mouse model and with tissue obtained from human ALS patients. We have measured synaptosomal glutamate transport in the G93A SOD1 rat to further characterize these animals and to provide a comparison with the data described above. We observed a 36% decrease in the maximum velocity of glutamate uptake in synaptosomes prepared from spinal cord of G93A SOD1 rats compared with age matched control animals (Vmax values for glutamate transport were 152 and 98 pmol/min/mg, respectively, in control and transgenic animals) while the affinity for glutamate (Km 3–5 μm) was unchanged. In contrast, no differences in Vmax values were observed when synaptosomes were prepared from cortex, hippocampus, striatum, brainstem or cerebellum. Pharmacological sensitivity to the selective GLT‐1 transport inhibitor dihydrokainate (IC50 74 μm) demonstrates that glutamate uptake in spinal cord synaptosomes is predominantly mediated by this subtype. Incubation of spinal cord synaptosomes with riluzole (100 μm) resulted in a 20–30% increase in glutamate uptake in the control animals while in the G93A SOD1 rats this effect was less pronounced. Our results indicate both a reduction in spinal cord glutamate transport capacity in the SOD1 rats and an altered functional response to riluzole.
Characterization of α‐synuclein in immune system
Authors: Kim S. H. , Heo C. J. , Im P. S. , Suh Y.‐H.
Published: July 2010

Human α‐synuclein is a 140 amino acid protein with little or no secondary structure. α‐Synuclein is expressed at high levels in the brain and enriched in neural synaptic terminals but its physiological function remains largely unknown. More recently, α‐synuclein has been shown to be one of the principal components of Lewy bodies, neuronal inclusions that are found in diverse human neurodegenerative disorder including the Lewy body variant of Alzeheimer's disease, diffuse Lewy body disease, Parkinson's disease, multiple system atrophy. Therefore, α‐synuclein and its abnormal protein aggregation have been thought to play a role in the pathogenesis of these neurodegenerative diseases known as α‐synucleinopathies. In order to understand their etiology and pathogenesis, it is crucial to identify the normal function of ?α‐synuclein. It was previously reported that the splicing variant of ?α‐synuclein is expressed in heart, skeletal muscle, and pancreas. In our study, we identified the expression in spleen and also confirm the expression of α‐synuclein in isolated human lymphocytes. The physiological role in lymphocytes showed relationship with glucocorticoid‐induced apoptosis. This result suggests that the accumulated ?α‐synuclein might be involved in regulation of cell viability by the interaction with signal transduction proteins such as p38 MAP or JNK.
Spinal cord neuron death in methyl‐4‐phenyl‐1,2,3,6 tetrahydropyridine induced Parkinsonism
Authors: Chera Bhishamjit S. , Schaecher K. , Ray S. , Rocchini A. , Imam S. , Ali S. , Banik N.
Published: July 2010

Parkinsonism is a movement disorder characterized by rigidity, tremor, and bradykinesia, originating from abnormalities in and death of monoaminergic neurons (dopamine, serotonin, and norepinephrine) in the substantia niagra, retrorubral area, and locus ceoruleus. Calpain has been implicated in the pathophysiology of neurodegenerative diseases (Alzheimers and Parkinsons Disease (PD)). Since the spinal cord (SC) and brain are integrally connected and calpain is involved in cell death, we hypothesized that SC neurons are also affected in PD. To test this hypothesis, we examined SC from 1‐methyl‐4‐phenyl‐1,2,3,6 tetrahydropyridine (MPTP) induced PD in mice. To identify cells expressing calpain, double immunofluorescent staining with calpain and cell marker antibodies (NeuN, OX42, and GFAP for neuron, microglia, and astrocytes, respectively) was used to observe changes in the SC. Double TUNEL staining was used to identify apoptotic cells. There was an increase in calpain expression in neurons, microglia, and astrocytes in brain and SC of MPTP‐treated animals at 1 and 7 days compared to controls. TUNEL positive neurons in the SC showed morphological apoptotic signs. The NeuN stain was less concentrated around the nuclear membrane at 1 day post exposure, while at 1 week calpain content in NeuN+ cells was greatly increased in substantia niagra and SC. Also, MPTP‐exposure up‐regulated calpain activity and expression in the SC. These results demonstrated neuron death in the SC of MPTP‐treated mice, and has provided evidence for a possible calpain‐mediated neuron death in SC of MPTP‐induced PD in mice.

Acknowledgements:  Supported in part by NIH and MUSC Summer Student Research Program.
Does tissue transglutaminase play a role in Huntington's disease?
Authors: Johnson G. V. W. , Tucholski J. , Lesort M. , Bailey C. D. C. , Chun W.
Published: July 2010

Tissue transglutaminase (tTG) catalyzes the incorporation of polyamines into substrates, or the formation of isopeptide bonds. tTG also binds and hydrolyzes GTP/ATP. Huntington's disease (HD) is caused by a pathological expansion of the polyglutamine domain in the protein huntingtin (htt). Because a polypeptide bound Gln residue is the primary determining factor for a tTG substrate, it has been hypothesized that due to the increase in Gln content, mutant htt may modified by tTG and this event may contribute to the pathogenesis of HD, possibly by facilitating the formation of htt aggregates. tTG is increased in HD, suggesting involvement in the pathogenic process. However, tTG is not required for aggregate formation. Further, tTG is excluded from htt aggregates and increasing or decreasing tTG has no effect on the frequency or localization of the aggregates. Considering these and other data, tTG is unlikely to play a major role in the formation of htt inclusions in HD brain. tTG may play a role in modulating neuronal cell death in response to specific stressors. If a stress increases the transamidating activity of tTG (e.g. increases in Ca++ levels), then tTG facilitates the cell death process. In contrast, if a stress does not result in an increase in the transamidating activity of tTG, then tTG protects against cell death. The protective effects of tTG are independent of its transamidating and hence likely dependent on its GTP/ATP binding and hydrolytic activity. Therefore the increase in tTG levels in HD brain could either be helpful or harmful depending on the cellular mechanisms that contribute to neuronal death.

Acknowledgements:  Supported by NIH grant AG12396.
Expression of the microtubule‐associated protein stop in oligodendrocytes
Authors: Galiano M. R. , Soto E. F. , Pasquini J. M. , Hallak M. E.
Published: July 2010

The STOP protein (stable tubule‐only polypeptide) is a calmodulin‐regulated protein which associates with microtubules and induces cold stabilization. There are different isoforms of this protein that arise from alternative splicing of STOP mRNA. Neurons express two major variants N‐STOP (125 kDa) and E‐STOP (84 kDa). NIH 3T3 fibroblasts contain a major F‐STOP isoform (42 kDa) and two minor STOP variants (48 and 89 kDa). Previously, we demonstrated the presence of N‐STOP in the cytoskeleton associated with myelin isolated from animals injected with apotransferrin. Since this protein was only described as a neuronal protein we decided to further investigate the expression of this protein in oligodendrocyte cultures. The analysis of the STOP protein expression in oligodendrocyte shows that STOP protein is expressed in the soma and processes of oligodendrocyte precursors, as well as in immature and mature oligodendroglial cells. In addition, we found that MBP shows a high degree of colocalization with STOP protein. By Western blot analysis, it was found that these cells express a major STOP variant (89 kDa). When the cultures were exposed to cold temperature we found that STOP protein associates with microtubules and induces microtubule cold stabilization. Under these experimental conditions, we found that MBP associates with microtubules too, and maintains its colocalization with STOP protein. At present, we are doing new assays directed to further characterize STOP (89 kDa) protein and to elucidate how this protein participates in the formation of myelin by oligodendrocytes.
Effects of apotransferrin upon different components of the cytoskeleton in primary oligodendroglial cell cultures
Authors: Ortiz E. , Pasquini L. , Galiano M. , Hallak M. , Soto E. , Pasquini J.
Published: July 2010

Apotransferrin (aTf) produces and acceleration of oligodendroglial cell (OLGc) differentiation (Marta 2000).This effect seems to be directly mediated by an action of aTf upon the OLGc cytoskeleton (CSK) (Escobar 2000).When tissue slice cultures, obtained from 3 to 4‐day‐old‐rats, were treated with 125I‐aTf, the presence of the radioactive aTf was bound to elements of the CSK isolated from these preparations. Using the same experimental design and after the isolation of microtubules (MT) and associated proteins, the radioactive aTf was associated with MT. In primary OLGc cultures,the expression of different components of the myelin CSK such as actin, tubulin and microtubule associated proteins as well as the ‘stable tubulin only peptide’ (STOP) were increased by treatment with aTf. This increase was followed by an increase in the expression of myelin basic protein and 2′3′cyclic nucleotide phosphohydrolase. In the same cultures and using immunocytochemical techniques,we analyzed the localization of endogenous (rat) and of exogenously added (human) aTf, as well as tubulin and actin, after treating the cultures with different drugs affecting the CSK (colchicine,cytochalasine and taxol). The patterns of association of these components observed in control cultures changed significantly in the cultures treated with drugs affecting the CSK. aTf seems to be acting in close association with the CSK of the OLGc, increasing the expression of tubulin and STOP in order to stabilize the MT. It produces an increase in the number of OLGc processes resulting as a consequence, in a more differentiated cell.
Poster Sessions CP01: Genomics and Proteomics. Global gene chip analysis of estrogen and tamoxifen‐regulated genes in the rat cerebral cortex
Authors: Dhandapani K. M. , Wade F. M. , De Sevilla L. , Mahesh V. B. , Brann D. W.
Published: July 2010

The ovarian steroid hormone, 17β‐estradiol (17β‐E2), and selective estrogen receptor modulators (SERMs), such as tamoxifen, have recently been implicated in regulating synaptic plasticity and in the protection of the brain against neurodegenerative diseases such as stroke and Alzheimer's disease. Although the mechanisms of these actions remain poorly understood, changes in gene expression are hypothesized to be involved in these processes. To address this issue, we identified genes that were regulated by physiological doses of 17β‐E2 and/or therapeutic doses of tamoxifen in the rat cerebral cortex of ovariectomized female rats. Gene chip analysis was performed using the Affymetrix U34A gene chip, which contains over 8800 genes and ESTs. Experiments were performed in duplicate to insure reproducibility of gene changes. Data analysis revealed 55 genes were similarly up‐regulated by both 17β‐E2 and tamoxifen whereas 61 genes were similarly down‐regulated, as compared to placebo treated animals. Interestingly, a greater number of genes were differentially regulated by 17β‐E2 and tamoxifen. Regulated genes spanned many different gene classes including signal transduction, transcriptional regulation, cell growth and maintenance, developmental processes, and apoptosis. As a whole, these gene changes may provide novel insights into the molecular mechanisms regulating the well‐documented ability of 17β‐E2 and SERMs to induce synaptic plasticity and neuroprotection in the cerebral cortex.
Poster Sessions CP02: Aging. Combination of NGF treatment and enrichment environment in old rats: biochemical and behavioral consequences
Authors: Fernández C. I. , Cruz R. , Collazo J. , López O. , Bauzá Y. , Castellanos M. R.
Published: July 2010

Brain glutathione system (Glut‐Syst) exhibits functional changes with age as well as during neurodegenerative diseases. After NGF treatment, cognitive functions and Glut‐Syst activity are favorably modified in aged rats. also, the environmental enrichment (E‐E) activates molecular mechanisms linked to cognition and sensorimotor coordination. We evaluate the functional repercussion of the combination of both factors. Old cognitively impaired rats received intracerebroventricular infusion of NGF (ICV, 22 mg/mL) or intraparenquimal (IP‐nbM, 5.5 mg/mL) during 14 days using ALZET osmotic minipumps. Simultaneously, these animals received a passive training in an E‐E during 4 weeks. A control group received training + saline infusion. Animals were assessed in the water maze task, avoidance passive test, open field test and transverse bridges test. At the end of the week 4, glutathione content and Choline Acetyltransferase (ChAT) activity were measured in brain areas of interest. E‐E or NGF treatments, particularly the IP route, improve the rat's overall behavioral performance but a synergic effect was observed when NGF and E‐E were applied simultaneously. A trend to hyperactivity was detected in the ICV group. Glutathione content and ChAT activity exhibited significant changes according to the group and brain area. It's well known that activity/levels of antioxidant enzymes and ChAT activity are related to age, brain region and neurotrophins activity. Results point out the possibilities of neurotrophic therapy if an adequate route of delivery is used as well as the benefit of combining a neurorehabilitation program on both, behavioural and protection from oxidative stress.
Age‐related alterations of extracellular signal‐regulated kinase 2 mRNA expression in the rat brain
Authors: Simonyi A. , Murch K. , Sun G. Y.
Published: July 2010

Aging is accompanied by the loss of memory and cognitive functions. Recent studies indicated that extracellular signal‐regulated kinases (ERKs) play an essential role in synaptic plasticity and memory. A selective activation of ERK2 has been demonstrated in the hippocampus during long‐term potentiation and in different in vivo learning paradigms. In this study, we investigated age differences in the expression of ERK2 by quantitative in situ hybridization using male Fisher 344 rats 3, 12 and 25 months of age. The results show an intense expression of ERK2 mRNA in many brain regions including the hippocampus, the cerebral cortex, the caudate‐putamen and the cerebellum. Moderate expression was found in the thalamus and the hypothalamus. No age‐dependent changes in ERK2 mRNA expression were detected in most cortical areas except for a slight decreasing trend in the pyriform cortex. ERK2 mRNA expression remained stable in the dentate gyrus and CA1 region of the hippocampus during aging. However, an early decline in ERK2 mRNA level was found in the CA3 region. Several behavioral studies have provided evidence that new protein synthesis in the CA3 region is necessary for memory consolidation during the first 1–2 days after learning. ERK2 is a regulator of neuronal gene expression and hippocampal ERK2 activation has been demonstrated in vivo in different learning paradigms. Thus, the early decrease in ERK2 mRNA in the CA3 region may underlie age‐related impairments in learning and memory.

Acknowledgements:  PO1 AG 18357 from NIH.
LIGA20 ganglioside prevents excitotoxicity through activation of TrkB receptor
Authors: Bachis Alessia , Rabin Stuart J. , Mocchetti Italo
Published: July 2010

Gangliosides protect cerebellar granule cells from excitotoxicity; however, their mechanism of action remains to be fully characterized. GM1 ganglioside has been shown to activate Trk, the tyrosine kinase receptor implicated in the neuroprotective properties of the neurotrophins. In these studies, we used primary cultures of cerebellar granule cells to determine whether gangliosides exert neuroprotective effect via the activation of Trk receptors. We first examined the relative potency of the neurotrophins, brain derived neurotrophic factor (BDNF), neurotrophin‐3 and nerve growth factor to prevent glutamate‐mediated apoptosis. BDNF was the only neurotrophin that elicited a complete neuronal protection against glutamate. GM1 and its semisynthetic compound LIGA20 also prevented glutamate toxicity, however, LIGA20 was more potent than GM1. Both LIGA20 and BDNF blocked glutamate‐mediated activation of caspase‐3 and consequently apoptosis; however, the anticaspase‐3 activity was seen only when these compounds were added to the cultures several hours before glutamate, suggesting that LIGA20 and BDNF share an identical molecular mechanism. To test this hypothesis, we compared the ability of LIGA20 and BDNF to activate TrkB. Both compounds elicited a similar time‐dependent increase in Trk tyrosine phosphorylation. Moreover, the neuroprotective effect of BDNF and LIGA20 was abolished in neurons exposed to the Trk tyrosine kinase inhibitor k252a, demonstrating a relationship between neuroprotection and activation of Trk receptors. Our data suggest that by activating the Trk neurotrophin receptors, gangliosides may be used as neuroprotective agents.
Poster Sessions CP04: Axonal Growth and Transport. Neurotrophin‐3 expressed in situ induces axonal regeneration in the injured spinal cord
Authors: Zhou L. , Zheng X. , McGowen L. R. , Baumgartner B. J. , Shine H. D.
Published: July 2010

Neurotrophins support neuronal survival and axonal regeneration after injury. To test whether local expression of Neurotrophin‐3 (NT‐3) would elicit axonal regeneration we lesioned the corticospinal tract (CST) at the level of the hindbrain and measured the number of axons that would grow from the unlesioned CST to the contralateral side where NT‐3 was over expressed at the lumbar level of the spinal cord. An adenoviral vector that carried the rat NT‐3 gene and the NGF signal peptide driven by the EF1α promoter (Adv.EF‐NT‐3) was used. This model enabled us to test the effects of NT‐3 on axonal regeneration without confounding injury processes. Biotinylated dextran amine (BDA) was injected into the rat cortex on unlesioned side to mark CST axons 10 days postlesion. Adenoviral vectors (1 × 109 pfu, Adv.EF‐NT‐3 or Adv.EF‐LacZ) were delivered to lumbar spinal cord by retrograde transport from the sciatic nerve 4 days later. Histological examination 3 weeks later revealed that more BDA‐labelled axons had grown from the unlesioned CST to the denervated side at the lumbar level. Morphometric measurements showed that a significantly larger number of BDA‐labelled CST axons (p < 0.001) were present in the animals that were treated with Adv.EF‐NT‐3 than those treated with Adv.EF‐LacZ. These data demonstrate that local expression of NT‐3 will support axonal regeneration in the injured spinal cord without adverse effects and suggest that gene delivery of neurotrophins may be an effective strategy for nervous system repair after injury.

Acknowledgements:  Funded by NIH Grant NS35280 and by Mission Connect of the TIRR Foundation.
Poster Sessions CP05: Intracellular Transport and Vesicle Trafficking. MBP mRNA trafficking factor HNRNP A2 interacts with microtubules binding protein TOG
Authors: Barbarese E. , Kosturko L. , D'Sa C. , Carson J. H.
Published: July 2010

HnRNP A2 is an RNA trafficking protein that binds to a specific cis‐acting RNA trafficking element (A2RE) in myelin basic protein RNA and other transported RNAs. A2RE/hnRNPA2 determinants mediate several different steps in RNA trafficking including granule assembly, transport to the plus ends of microtubules and translational activation. A yeast two hybrid screen designed to identify proteins that interact with hnRNP A2 selected a clone corresponding to the carboxyl terminal portion of TOG (tumor overexpressed gene), a microtubule‐associated protein that regulates microtubule dynamics. Co‐immunostaining of oligodendrocytes with antibody to hnRNPA2 and TOG revealed extensive colocalization of TOG with hnRNP A2 granules in the dendrites. A small population of hnRNP A2 granules lacked TOG and some regions of TOG staining lacked hnRNP A2. In oligodendrocytes injected with fluorescent A2RE RNA and stained for TOG, granules containing fluorescent RNA colocalized with TOG. Co‐injection of anti‐TOG antibody with fluorescent A2RE RNA decreased colocalization with TOG and increased transport of the injected RNA. These observations suggest that molecular interaction between hnRNP A2 and TOG serves to anchor A2RE mRNAs/hnRNPA2 granules at plus ends of microtubules.

Acknowledgements:  Supported by NIH NS19943 (EB) and NS15190 (JHC), and NMSS RG2843 (EB).
Differences in the retrograde axonal transport of nerve growth factor and dopamine beta‐hydroxylase
Authors: Hendry I. A. , Weible II M. W. , Ozsarac N.
Published: July 2010

There are two potential populations of vesicles in the sympathetic nervous system that may contribute to the retrograde axonal transport of signals from the target tissue. These are derived from the population of recycled synaptic vesicles and endosomes derived from receptor mediated endocytosis of growth factors. We have analysed the retrograde axonal transport of two l125‐labelled marker proteins, nerve growth factor (NGF) and dopamine beta‐hydroxylase (d.b.h.), to determine whether these two populations are processed in the same way in the nerve terminal. Two models were used in the rat, either from the anterior eye chamber to the superior cervical ganglion and trigeminal ganglion or the ligated sciatic nerve after injection into the footpad. The results show that there are significant differences in the regulatory processes that control the targeting of these proteins for retrograde transport. Many inhibitors of the second messenger cascades inhibit the transport of both molecules, for example Latrunculin A and wortmannin. However, the retrograde axonal transport of NGF is inhibited by phenylarsine oxide but not by bis‐tyrphostin, FK506, dibutyryl‐cAMP, thapsigargin, cadmium and nickel whereas the transport of d.b.h. is the reverse, suggesting the mechanism of endocytosis and targeting the vesicles in the nerve terminal for retrograde axonal transport is different. We have isolated vesicles labelled with these iodinated proteins from the sciatic nerve and have shown that the density of the NGF containing vesicles is significantly lighter than those containing d.b.h. This suggests that there are at least two distinct pathways for retrograde axonal transport of vesicles.
Identification of a novel pathway for the in vivo regulation of fast anterograde axonal transport
Authors: Morfini Gerardo , Szebenyi G. , Brown H. , Wray R. , Reyna H. , Brady S. T.
Published: July 2010

Nerve cell function relies on fast axonal transport for the accurate delivery of organelles to specific compartments within the axon. Regulatory mechanisms for microtubule motor‐driven motility are poorly understood. Previous work has demonstrated an important role for Cyclin‐dependent protein kinase 5 (CDK5) in the regulation and maintenance of kinesin‐driven motility. However, the molecular mechanisms by which this kinase positively regulates axonal transport remained unknown. In this work, pharmacological, biochemical and in vivo experiments lead to the identification of a novel regulatory pathway for the regulation of kinesin‐driven motility. This pathway involves the actions of CDK5, protein phosphatase 1 (PP1) and Glycogen Synthase Kinase 3 (GSK‐3) enzymatic activities, which ultimately lead to detachment of kinesin from its transported cargo. We propose that, by regulating the activities and localization of individual components of this pathway, nerve cells can regulate organelle delivery within specific subcellular compartments.
HNK‐1 carbohydrate synthesis in retinoic acid‐induced P19 cells
Authors: Chou D. K. H. , Henion T. R. , Jungalwala F. B.
Published: July 2010

Sulfoglucuronyl carbohydrate (SGC) reactive with HNK‐1 antibody are expressed on glycoproteins and on glycolipids (SGGLs) in the nervous system. The interaction of SGC with SGC‐binding protein‐1 (SBP‐1) has been implicated in the regulation of neurite outgrowth and cell‐migration during development. To define the cell‐specific expression and function of SGC, SGC synthesis on glycoproteins and glycolipids was studied in embryonal carcinoma P19 cells after induction with retinoic acid (RA). With RA, P19 cells differentiate to neuron‐like cells with axonal and dendritic processes. The uninduced cells express SSEA‐1 glycoproteins, but are SGC negative. After RA, the induced neuronal population strongly expresses SGC‐glycoproteins. The appearance of SGC is accompanied by a 16‐fold up‐regulation in the activity of GlcNAc‐Tr (Lc3 synthase), which synthesizes the key intermediate LcOse3Cer, for lacto‐ and neolacto‐glycolipids. Despite up‐regulation of Lc3 synthase, the levels of SGGLs were not significantly up‐regulated after RA induction. Instead, the levels of other neolactoglycolipids, such as SSEA‐1 and disialosyl‐nLcOse4Cer, were up‐regulated. Treatment with RA also induced four fold higher synthesis of SBP‐1, which was specifically localized in neuron‐like cells along with SGC, similar to that in vivo. The results indicate that the induction of SGC in RA‐treated P19 neuronal cells is mostly due to SGC on glycoproteins and not SGGLs. The coordinated induction of SBP‐1 along with the SGC‐glycoproteins in the same cells support the hypothesis that their interaction is important in neurite outgrowth.
Differential regulation of brain protein phosphatase activities by nitric oxide and nitroxyl anion
Authors: Foley T. D. , Kupchak B. R. , Carlson D. M. , Cenera C. M.
Published: July 2010

While nitric oxide (NO) is a physiological messenger molecule, increasing evidence suggests that its one‐electron reduction product, nitroxyl anion (NO–), may be particularly cytotoxic. We have examined the differential regulation of redox‐sensitive protein phosphatase activities in a rat brain soluble fraction by the nitric oxide donor dethylamine NONOate and the nitroxyl anion donor Angeli's salt using p‐nitrophenylphosphate (p‐NPP) as a nonspecific substrate. We report that micromolar NONOate but not Angeli's salt produced a 2–3‐fold increase in a calcineurin (CaN) phosphatase activity, defined as Ni2± dependent, calmidazolium‐sensitive, p‐NPPase activity. Activation of CaN by NONOate was (i) enhanced by carboxy‐PTIO, which promotes formation of the S‐nitrosylating agent N2O3 (ii) mimicked by S‐nitrosoglutathione (iii) partially reversed by the sulfhydryl reductant DTT, and (iv) accompanied by protein S‐nitrosylation. NONOate also produced a modest, largely DTT‐reversible, inhibition of the vanadate‐sensitive fraction of p‐NPPase that was attributed to protein tyrosine phosphatase (PTP) activity. We propose that regulation of protein phosphatase activities by reversible sulfhydryl modulation may contribute to the physiological actions of nitric oxide. In contrast to NONOate, Angeli's salt produced a marked and irreversible inhibition of PTP activity consistent with a possible higher order oxidation of an active‐site cysteine residue. Notably, Angeli's salt was at least a 10‐fold more potent inhibitor of PTP activity than was hydrogen peroxide. We propose that PTP inactivation by nitroxyl anion may promote excessive and neurotoxic protein tyrosine phosphorylation.
Hyposmotic‐induced glutamate release in different brain preparations: possible role of PKC
Authors: Franco R. , Ordaz B. , Pasantes‐Morales H.
Published: July 2010

Following hyposmotic swelling induced, cell volume regulation is achieved by the efflux of osmolytes and osmotically obligated water. The role of amino acids as organic osmolytes is widely reported in brain preparations. Results on mechanisms of the osmosensitive taurine release have been often extrapolated to other amino acids. In the present work we present results showing that amino acids such as glutamate, which serve the dual role of osmolytes and neurotransmitters deviate from the taurine release features in several brain preparations, including cultured astrocytes and neurons, and brain slices. Taurine efflux as previously reported is inhibited by chloride channel blockers and by tyrosine kinase and PI3K inhibitors, and was insensitive to manipulation of PKC activity. Glutamate efflux (followed as 3H‐d‐aspartate) was reduced by chloride channel blockers in cultured cell preparations, while it was unaffected in brain slices. PKC activation markedly enhanced hyposmotic‐induced glutamate release in all preparations, pointing to a role of this kinase in the osmosensitive glutamate release. These results suggest that in cell cultures glutamate may share a common efflux pathway with taurine, but in more integrated preparation, the hyposmotic challenge activates a different pathway modulated by PKC. This mechanism may be identical to the stimulation‐secretion vesicular release typical of neurotransmitter amino acids. However, no difference in the release mechanism was observed between neuroblasts and differentiated neurons. Moreover, the PKC‐sensitive pathway is also present in fibroblasts in which such vesicular release does not operate. However, this does not exclude the activation by hyposmolarity of exocytotic mechanisms dependent on PKC activity.
Regulation of JNK signalling pathway by GM1
Authors: Mo L. , Neff N. H. , Hadjiconstantinou M.
Published: July 2010

The ganglioside GM1 exerts pleitropic trophic actions in both the central and peripheral nervous systems in vivo and in vitro. Our previous research has shown that in brain tissue, GM1 is capable of inducing tyrosine phosphorylation of Trk receptors for the neurotrophins and initiating signaling via the MEK/ERK pathway. In order to better understand the intracellular signalling events associated with GM1, we investigated the effects of GM1 on the JNK pathway and its downstream transcriptional factor targets. Incubating slices from hippocampus of young or aged rats with GM1 increased the content of phosphorylated (p) JNK and augmented JNK kinase activity. The GM1‐induced phosphorylation/activation of JNK was accompanied by an increase in phosphorylated (p) c‐Jun and (p) Elk1, but not (p) ATF, transcription factors. Furthermore, addition of GM1 to hippocampal slices induced enhanced AP‐1 DNA binding activity. A similar response was observed when slices were treated with the neurotrophins NGF and BDNF. Both the GM1‐ and neurotrophin‐induced activation of the JNK pathway were blocked by the selective Trk kinase inhibitor AG879. Taken together, these data suggest that the molecular mechanisms of GM1 actions may include recruitment of the JNK pathway.

Acknowledgements:  Supported, in part, by grant AG10530.