bluuebunny/crossref_metadata_2025_split huggingface dataset:
Microarray assays reveal differential expression of hippocampal and amygdalal genes during contextual fear conditioning
Authors: Perrone‐Bizzozero Nora , Yang Y. , Merhege M. , Bolognani F. 
Published: July 2010

The hippocampus plays an important role in contextual learning. However, the gene expression alterations that subserve this function remain to be fully elucidated. We have previously shown that the levels of GAP‐43 and its mRNA‐binding protein, the ELAV‐like protein HuD, are increased in the hippocampus during contextual fear conditioning (CFC). In this study, we sought to identify additional changes in gene expression in the hippocampus and amygdala of CFC rats. Animals were subjected to a single trial CFC paradigm involving a paired presentation of a tone and footshock in a novel environment. Both untrained (naïve) rats and animals exposed to the shock followed by an unpaired presentation of the context and tone served as nonlearning controls. Twenty‐four hours after training, the hippocampus and amygdala of each animal were removed, RNAs were isolated and subjected to microarray analysis. Besides GAP‐43 and HuD, we found 150 neural genes in the hippocampal formation that were increased by at least 1.5 fold relative to controls. These include neurotrophic factors (e.g. BDNF), synaptic proteins (e.g. SNAP‐25, syntaxin), neurotransmitter receptors and transporters (e.g. glutamate transporter), transcription factors (e.g. zif268), cytoskeletal proteins (e.g. Arc, MAP2), cell cycle proteins (cyclins D1, D3 and E), signalling molecules (e.g, PKA, ERK2) and others (ion channel Kv9.3). In addition, we found 25 genes that were down‐regulated in the same tissues. Finally, a number of neuronal‐specific mRNAs were found to show differential expression in the amygdala after CFC.Acknowledgements: Supported by NIH NS30255 and AA11336 to NPB.
The influence of melatonin on the hippocampal glutamergic system
Authors: Wieraszko A. , Hogan M. V. , El‐Sherif Y. , Tesoriero J. , Raja H. 
Published: July 2010

We have demonstrated previously that melatonin and its analog chloromelatonin depressed evoked potentials (EPSP and the population spike) recorded from hippocampal slices during stimulation of Schaffer collaterals (Hogan et al. J. Pineal Res. 2001, 30, 87–96). The effect was concentration‐ and receptor‐dependent and occurred within minutes following melatonin application. Since Schaffer collaterals use glutamate as a neurotransmitter, we focused our research on the glutamergic system as a possible target of melatonin action. Experiments were performed on hippocamapal synaptosomes and/or synaptosomal membranes prepared from CD−1 mice. We evaluated the influence of melatonin on the binding of 3H‐glutamate to the glutamergic receptors and synaptosomal uptake of 3H‐D‐Aspartate used as a marker of glutamergic neurotransmission. When synaptosomal membranes were incubated with 1 mm melatonin (30 min, 37°C), the binding of 3H‐glutamate increased by 12.5 ± 7% (n = 7). In uptake studies the synaptosomes were exposed to 1 mm melatonin for 15 min at 37°C, and then the accumulation of 3H‐D‐Aspartate was tested at 5, 30, 60, 120 and 300 s. The incubation with 3H‐D‐Aspartate was followed by filtration and radioactivity absorbed on the filters was determined. Melatonin increased the uptake of 3H‐D‐Aspartate at all time intervals tested by 37.6 ± 5% (n = 5). Experiments are in progress to further characterize the melatonin actions and to evaluate melatonin effects on the release of 3H‐D‐Aspartate from synaptosomes. Supported partially by NIH grant RO1 ES011022‐01A1.
Functional glutaredoxin activity in rat brain & liver mitochondria
Authors: Zeevalk G. D. , Ehrhart J. 
Published: July 2010

Glutathione (GSH) serves as a major anitoxidant in cells to protect from oxidative damage. One less studied role for GSH is in GSH‐protein‐mixed disulfide (PrSSG) formation where GSH is covalently linked to sulfhydryl groups on a protein. This reversible glutathionylation is catalyzed by glutaredoxin‐1 (Grx1). Grx1 was identified in brain cytosol (J. Neurochem. 1999, 72, 1170). A 2nd glutaredoxin (Grx2) was recently cloned & expressed in E. coli (JBC 2001 276, 26 269, 30 374). Grx2 contains a mitochondrial (mito) leader sequence. Recombinant Grx2 is also a thiol‐disulfide oxidoreductase. Functional endogenous Grx2 activity in mito has not been demonstrated. To study this, subfractionation of liver & brain was carried out to obtain mito & microsome fractions. Marker enzyme activities as well as EM monitored fraction purity. Intact mito were inactive, but intramitochondrial matrix from lysed mito were able to dethiolate mixed disulfides. The specific activity for dethiolation for liver & brain cytosol was similar & greater than that of liver or brain mito. Grx activity in brain mito was 2‐fold higher than in liver mito. The microsomal fraction also contained Grx activity, but was significantly less than that found in cytosolic or mito fractions. Like cytosolic Grx, the mito enzyme showed a preference for GSH containing mixed disulfides, had a pH optima of ∼ 8.0 & was inhibited by aurothioglucose. Consistent with findings from recombinant Grx2, antibody directed against Grx1 cross reacted with brain & liver cytosolic enzyme, but not the mito form. These findings show that brain & liver mito contain functional glutathione‐dethiolating activity. In mito, this activity may play an important role in protecting against free radical damage of mito proteins. Acknowledgements: Supported by NS36157 & M.J. Fox Foundation for Parkinson's Research.
Apotransferrin enhances differentiation of two oligodendroglial cell lines
Authors: Paez P. , Pasquini J. , Soto E. 
Published: July 2010

In the CNS, transferrin (Tf) is produced by oligodendroglial cells (OLGc) and is essential for their survival. A single intracranial injection of apoTf in neonatal rats has been shown to accelerate differentiation of OLG (Marta et al. 2000). Our interest was to study the effect of apoTf at different stages of OLG maturation in two immortalized cell lines, obtained from primary cultures: N19 and N20.1 (Bongarzone et al. 1996). These cell lines grow constantly at 34°C (permissive temperature) and differentiate into mature OLGc at 39°C (nonpermissive temperature). At 34°C, N19 exhibit the characteristics of OLGc precursors (O2A), while N20.1 express markers of mature OLGc (O1, CNPase). In vitro addition of apoTf to the cultures at 39°C during 3 days, showed an increased expression of O1, O4, MBP in both cell lines, a decrease in transferrin receptors and of endogenous Tf. These effects were more evident in the N19 line. ApoTf induces a more mature phenotype of OLGc development in both cells lines. Previous studies have shown that the action of aTf is mediated by its receptor, by the OLGc cytoskeleton and by the activation of protein kinases. OLGc differentation is stimulated by cAMP (Raible and Mc Morris 1990) followed by activation of PKA and elevated levels of CREB (Sato‐Bigbee et al. 1999). In N19 and N20.1 cell lines after seven days in culture in the presence of apoTf, actin expression was mainly located in the OLGc processes, and in the growth cones, while there were no changes in tubulin expression. In the N19, there was a 10‐fold increase in cAMP levels, compared to controls while in the N20.1 there were no changes. These results agree with our previous studies in in vivo experiments and in OLGc primary cultures.
Poster Sessions BP03: Myelin, Demyelination and Diseases of Myelin. Myelin and myelination in PLP‐null mice
Authors: Toews A. , Jurevics H. , Hostettler J. , Sammond D. , Morell P. 
Published: July 2010

The myelin proteolipid protein (PLP) is the major structural protein of CNS myelin, accounting for approximately half of total myelin protein. We studied synthesis and accumulation of myelin components for two months postnatally in PLP‐null mice and age‐matched controls. Accumulation of myelin, as assayed by levels of whole brain cerebroside and myelin basic protein, was normal in the knockout mice. The rate of cerebroside synthesis in the knockout mice was also normal. Myelin was isolated at several ages during development, using a standard subcellular fractionation protocol. The yield of ‘purified myelin’ isolated from a large particle (crude mitochondrial) fraction was reduced in PLP‐null mice, but increased amounts of ‘myelin’ were obtained in the small particle (crude microsomal) fraction. This ‘myelin’ in the crude microsomal fraction was identified as such by flotation on 0.85 m sucrose and the myelin‐characteristic 2 : 1 molar ratio of cholesterol to cerebroside. This suggests myelin from PLP‐null mice is physically more fragile than normal myelin, and that during tissue dispersion, much more PLP‐null myelin is fragmented into small vesicles than is the case for normal myelin. Three hours after intracranial injection of tritiated acetate into PLP‐null mice, cerebroside in myelin isolated from the large particle fraction was at a similar specific radioactivity to that isolated from the small particle (crude microsomal) fraction, suggesting that the most recently deposited PLP‐null myelin is not preferentially unstable. The increased fragility evident during tissue dispersion is indicative of an underlying structural abnormality in PLP‐null myelin. Whether this inherent structural instability affects myelin metabolism is under investigation.Acknowledgements:  Supported by USPHS & NMSS grants.
Metabolism and gene expression during cuprizone‐induced demyelination/remyelination
Authors: Morell P. , Jurevics H. , Largent C. , Hostettler J. , Sammond D. , Matsushima G. , Toews A. 
Published: July 2010

Exposure of mice to the copper chelator, Cuprizone, results in a CNS demyelination; remyelination occurs following removal of this metabolic insult. Using this model, we have demonstrated changes in content and metabolism of myelin lipids in whole brain. We now extend these biochemical studies to brain regions and identify corpus callosum as particularly severely affected; 65% of cerebroside is lost after 6 weeks of exposure. Animals can be rescued by removal of the toxicant and the extent of recovery of myelin, as well as recovery of ability to synthesize cerebroside and cholesterol, can be determined. The temporal pattern for concentration of myelin basic protein tracked that of cerebroside, supporting the assumption that cerebroside level is a good surrogate for myelin concentration. We also applied Affymetrix microarray technology to corpus callosum to track temporal changes in steady‐state levels of mRNA species related to demyelination and remyelination. Genes coding for myelin structural components were greatly down‐regulated during demyelination and up‐regulated during remyelination. Genes related to microglia/macrophages appeared in a time course (peaking at 6 weeks) correlating with that expected for phagocytosis of myelin. mRNAs coding for many cytokine genes had peak expression at 4 weeks, compatible with an intercellular signaling role. Of interest were other genes with temporal patterns correlating with one of the three patterns mentioned above, but with functions not obviously related to demyelination/remyelination. The ability to correlate gene expression with known pathophysiological and regenerative events should be of utility in elucidating further function of such genes.Acknowledgements:  Supported by USPHS & NMSS grants.
The role of inflammatory mediators in the pathophysiology of X‐ALD disease
Authors: Paintlia A. S. , Singh A. K. , Singh I. 
Published: July 2010

CER is the most frequent clinical phenotype of the defective X‐ALD (ALD; ABCD1) gene, which results in accumulation of VLCFAs (in plasma, brain, adrenal glands and testis), inflammatory demyelination and subsequent death in children. To understand the inflammatory mediators that play a role in the neuropathology of CER, we studied mRNA expressions of inflammatory mediators in CER brain by using super gene array. Total of nine tissue slices were taken from three different locations from CER brain starting with plaque, demyelinating edge (Plaque shadow) and normal looking area. Histopathological examinations showed intensive demyelination in plaque region and accumulation of infiltrates (Macrophages and T lymphocytes) in plaque and plaque shadow as compared to normal looking area. Biochemical studies indicated significant increase in the levels of VLCFAs (26 : 0) in plaque and plaque shadow regions in comparison to normal looking area. There was significant increase in the levels of inflammatory cytokines (IL‐1α, TNF‐α and IL‐6) in plaque shadow as compared with normal looking area and control brain tissue region. Other cytokines like IL‐2, IL‐3, GM‐CSF, IL‐11, IL‐12A were found to be increased significantly (p < 0.01) in plaque shadow. There was significant (p < 0.01) increase in the expression chemokines; MCP‐1, MCP‐3, MIP‐1β and MIP‐2 in the plaque shadow in comparison with normal looking area of CER brain. Chemokines; Fractalkine, Eotaxin, SDF‐2, MDC‐2, HCC‐4 were also observed to be higher in plaque shadow. Data was further evaluated using RT–PCR for MCP‐1 and CCR‐2, which showed elevated levels in plaque and plaque shadow. This study identified a group of inflammatory mediators that may play a role in pathophysiology of X‐ALD disease.Acknowledgements:  Supported by grants; NS‐22526, NS‐34741, NS‐37766 and NS‐40810.
Interleukin‐12 P40 induces the expression of TNF‐α in microglia and macrophages
Authors: Jana M. , Dasgupta S. , Liu X. , Pahan K. 
Published: July 2010

Recently, it has been found that overproduction of IL‐12 can be dangerous to the host as it is involved in the pathogenesis of a number of autoimmune inflammatory diseases such as multiple sclerosis. It is composed of two different subunits – p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the CNS of patients with Multiple Sclerosis (MS) suggests that IL‐12 p40 may have a role in the pathogenesis of the disease. The present study was undertaken to explore the role of p40 in the expression of TNF‐α in microglia. Interestingly, we have found that IL‐12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose‐dependently induced the production of TNF‐α in BV‐2 microglial cells. This induction of TNF‐α production was accompanied by an induction of TNF‐α mRNA. In addition to BV‐2 glial cells, p70, p402 and p40 also induced the production of TNF‐α in mouse primary microglia and peritoneal macrophages. Since the activation of both NF‐κB and C/EBPb is important for the expression of TNF‐α in microglial cells, we investigated the effect of p40 on the activation of NF‐κB as well as C/EBPb. Activation of NF‐κB as well as C/EBPb by p40 and inhibition of p40‐induced expression of TNF‐α by Dp65, a dominant‐negative mutant of p65, and DC/EBPb, a dominant‐negative mutant of C/EBPb, suggests that p40 induces the expression of TNF‐α through the activation of NF‐κB and C/EBPb. This study delineates a novel role of IL‐12 p40 in inducing the expression of TNF‐α in microglial cells which may participate in the pathogenesis of neuroinflammatory diseases.Acknowledgements:  This study was supported by NIH grants (NS39940 and AG19487).
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.
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.
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.