bluuebunny/crossref_metadata_2025 huggingface dataset:
HSP27 but not HSP70 has a potent protective effect against α‐synuclein‐induced cell death in mammalian neuronal cells
Authors: Zourlidou Alexandra , Payne Smith Martin D. , Latchman David S.
Published: July 2010

α‐Synuclein is a neuronally expressed protein which is mutated in familial Parkinson's disease. We have previously shown that disease‐associated mutants of α‐synuclein cause enhanced neuronal cell death in response to a variety of stimuli, whereas wild‐type α‐synuclein has a protective effect against some stimuli, whilst enhancing the death response to others. We demonstrate, for the first time, that over‐expression of the heat shock protein HSP27 has a potent protective anti‐apoptotic effect against the damaging effects of wild‐type and particularly of mutant α‐synuclein. In contrast, HSP70 has some protective effect against the damaging effect of the wild‐type protein, but has no effect against the mutant proteins, whilst HSP56 has no protective effect in this system. Our results indicate that disease‐associated mutants of α‐synuclein enhance its death‐inducing properties and lead to increased apoptosis, which can be mitigated by either the use of specific caspase inhibitors or HSP27 over‐expression. This potent protective effect of HSP27 against the mutant and wild‐type proteins may be of potential therapeutic importance.
DNA methylation as an epigenetic regulator of neural 5‐lipoxygenase expression: evidence in human NT2 and NT2‐N cells
Authors: Zhang Zhijing , Chen Chang‐qing , Manev Hari
Published: July 2010

Increased expression of 5‐lipoxygenase is associated with various neuropathologies and may be related to epigenetic gene regulation. DNA methylation in promoter regions is typically associated with gene silencing. We found that human NT2 cells, which differentiate into neuron‐like NT2‐N cells, express 5‐lipoxygenase and we investigated the relationship between 5‐lipoxygenase expression and the methylation state of the 5‐lipoxygenase core promoter. We used the demethylating agent 5‐aza‐2′‐deoxycytidine and the histone deacetylase inhibitor valproate to alter DNA methylation and to induce histone modifications. 5‐Lipoxygenase expression and DNA methylation were assayed with RT‐PCR and bisulfite genomic sequencing, respectively. Neuronal differentiation of proliferating NT2 precursors decreased 5‐lipoxygenase expression. 5‐Aza‐2′‐deoxycytidine increased 5‐lipoxygenase mRNA levels only in proliferating cells, whereas valproate increased 5‐lipoxygenase mRNA levels in a cell cycle‐independent manner. In both precursors and differentiated cells, CpG dinucleotides of the promoter were poorly methylated. In precursors, both 5‐aza‐2′‐deoxycytidine and valproate further reduced the number of methylated CpGs. Moreover, we found evidence for cytosine methylation in CpWpG (W = adenine or thymine) and other asymmetrical sequences; CpWpG methylation was reduced by valproate in NT2‐N but not in NT2 cells. This is the first report demonstrating that the dynamics of DNA methylation relates to neural 5‐lipoxygenase gene expression.
Proteomic analysis of brain proteins in the gracile axonal dystrophy (gad) mouse, a syndrome that emanates from dysfunctional ubiquitin carboxyl‐terminal hydrolase L‐1, reveals oxidation of key proteins
Authors: Castegna Alessandra , Thongboonkerd Visith , Klein Jon , Lynn Bert C. , Wang Yu‐Lai , Osaka Hitoshi , Wada Keiji , Butterfield D. Allan
Published: July 2010

Ubiquitin carboxyl‐terminal hydrolase L‐1 (UCH L‐1) is a crucial enzyme for proteasomal protein degradation that generates free monomeric ubiquitin. Our previous proteomic study identified UCH L‐1 as one specific target of protein oxidation in Alzheimer's disease (AD) brain, establishing a link between the effect of oxidative stress on protein and the proteasomal dysfunction in AD. However, it is unclear how protein oxidation affects function, owing to the different responses of proteins to oxidation. Analysis of systems in which the oxidized protein displays lowered or null activity might be an excellent model for investigating the effect of the protein of interest in cellular metabolism and evaluating how the cell responds to the stress caused by oxidation of a specific protein. The gracile axonal dystrophy (gad) mouse is an autosomal recessive spontaneous mutant with a deletion on chromosome 5 within the gene encoding UCH L‐1. The mouse displays axonal degeneration of the gracile tract. The aim of this proteomic study on gad mouse brain, with dysfunctional UCH L‐1, was to determine differences in brain protein oxidation levels between control and gad samples. The results showed increased protein oxidation in thioredoxin peroxidase (peroxiredoxin), phosphoglycerate mutase, Rab GDP dissociation inhibitor α/ATP synthase and neurofilament‐L in the gad mouse brain. These findings are discussed with reference to the effect of specific protein oxidation on potential mechanisms of neurodegeneration that pertain to the gad mouse.
Detection of basal and potassium‐evoked acetylcholine release from embryonic DRG explants
Authors: Bernardini Nadia , Tomassy Giulio Srubek , Tata Ada Maria , Augusti‐Tocco Gabriella , Biagioni Stefano
Published: July 2010

Spontaneous and potassium‐induced acetylcholine release from embryonic (E12 and E18) chick dorsal root ganglia explants at 3 and 7 days in culture was investigated using a chemiluminescent procedure. A basal release ranging from 2.4 to 13.8 pm/ganglion/5 min was detected. Potassium application always induced a significant increase over the basal release. The acetylcholine levels measured in E12 explants were 6.3 and 38.4 pm/ganglion/5 min at 3 and 7 days in culture, respectively, while in E18 explant cultures they were 10.7 and 15.5 pm/ganglion/5 min. In experiments performed in the absence of extracellular Ca2+ ions, acetylcholine release, both basal and potassium‐induced, was abolished and it was reduced by cholinergic antagonists. A morphometric analysis of explant fibre length suggested that acetylcholine release was directly correlated to neurite extension. Moreover, treatment of E12 dorsal root ganglion‐dissociated cell cultures with carbachol as cholinergic receptor agonist was shown to induce a higher neurite outgrowth compared with untreated cultures. The concomitant treatment with carbachol and the antagonists at muscarinic receptors atropine and at nicotinic receptors mecamylamine counteracted the increase in fibre outgrowth. Although the present data have not established whether acetylcholine is released by neurones or glial cells, these observations provide the first evidence of a regulated release of acetylcholine in dorsal root ganglia.
Glial soluble factors regulate the activity and expression of the neuronal glutamate transporter EAAC1: implication of cholesterol
Authors: Canolle B. , Masmejean F. , Melon C. , Nieoullon A. , Pisano P. , Lortet S.
Published: July 2010

A co‐ordinated regulation between neurons and astrocytes is essential for the control of extracellular glutamate concentration. Here, we have investigated the influence of astrocytes and glia‐derived cholesterol on the regulation of glutamate transport in primary neuronal cultures from rat embryonic cortices. Glutamate uptake rate and expression of the neuronal glutamate transporter EAAC1 were low when neurons were grown without astrocytes and neurons were unable to clear extracellular glutamate. Treatment of the neuronal cultures with glial conditioned medium (GCM) increased glutamate uptake Vmax, EAAC1 expression and restored the capacity of neurons to eliminate extracellular glutamate. Thus, astrocytes up‐regulate the activity and expression of EAAC1 in neurons. We further showed that cholesterol, present in GCM, increased glutamate uptake activity when added directly to neurons and had no effect on glutamate transporter expression. Furthermore, part of the GCM‐induced effect on glutamate transport activity was lost when cholesterol was removed from GCM (low cholesterol‐GCM) and was restored when cholesterol was added to low cholesterol‐GCM. This demonstrates that glia‐derived cholesterol regulates glutamate transport activity. With these experiments, we provide new evidences for neuronal glutamate transport regulation by astrocytes and identified cholesterol as one of the factors implicated in this regulation.
The three sorCS genes are differentially expressed and regulated by synaptic activity
Authors: Hermey Guido , Plath Niels , Hübner Christian A. , Kuhl Dietmar , Schaller H. Chica , Hermans‐Borgmeyer Irm
Published: July 2010

We have isolated the murine sorCS3 gene, a new member of the family of receptors containing a Vps10p‐domain. Receptors of this family facilitate rapid endocytosis and are thought to be involved in intracellular sorting. SorCS3 and the highly homologous sorCS1 and sorCS2 genes were expressed in a combinatorial, mostly non‐overlapping pattern in both the developing and mature central nervous system. During development, distribution and abundancy of their transcripts was regulated. Moreover, their expression was differentially influenced by neuronal activity in the hippocampus of adult mice. Although kainic acid‐induced seizures had no effect on sorCS2 mRNA levels, they dramatically increased the expression of sorCS1 and sorCS3. The activity‐dependent induction of sorCS1 expression required de novo protein synthesis, whereas that of sorCS3 did not. Our results imply that the three sorCS genes have diverse, but partly overlapping functions in the developing and mature central nervous system.
Retrograde NGF signalling and survival in compartmentalized sympathetic neurons
Authors: Ginty D. D. , Ye H. , Kuruvilla R.
Published: July 2010

Previous work has shown that neurotrophins bind to and activate Trk receptors on distal axons, and that neurotrophin‐Trk complexes are internalized and retrogradely transported to cell bodies. Whether retrograde transport of neurotrophins and retrograde neurotrophin‐Trk signalling are necessary for survival remains unclear, and recently published findings are controversial. We are using compartmentalized cultures of sympathetic neurons to address the mechanism of retrograde NGF signalling and survival. We performed survival experiments using either the Trk kinase inhibitor K252a to inhibit TrkA activity in different cellular compartments, or a dominant‐negative form of dynamin, K44A dynamin, to block internalization of NGF‐TrkA complexes. We found that sympathetic neurons supported by NGF acting on distal axons undergo apoptosis when TrkA activity in either cell bodies or distal axons is inhibited by K252a, or when internalization is blocked by K44A dynamin. Results of experiments employing three‐compartment chambers indicate that TrkA signalling is required within cell bodies and distal axons, but not in proximal axons, for retrograde support of survival. Likewise, TrkA activity within distal axons, but not in proximal axons, is required for retrograde transport of [125I] NGF. Finally, peptide‐mediated delivery of affinity‐purified anti‐NGF into cell bodies results in apoptosis of neurons. Taken together, our results support a model in which NGF internalization and retrograde transport and retrograde TrkA signalling are necessary for survival of sympathetic neurons. This work is supported by the NIH and HHMI.
Colloquium 14: New Functions for Retinoic Acid in the Developing CNS. The role of retinoic acid in the patterning of the developing nervous system
Authors: Maden M. , Wilson L. , Gale E. A.
Published: July 2010

Retinoic acid (RA) is metabolised from its precursor, retinol (vitamin A). In mammalian embryos, retinol is provided by the mother via the placenta and in birds retinol comes from the yolk. We have studied the role of RA in CNS development in quail embryos by depriving adult quails of retinol in the diet which results in them laying eggs which have no retinol stores. The resulting embryos are therefore retinol and RA deficient. The CNS of these embryos is abnormal in three regards; patterning, neural crest production and neurite outgrowth. With regard to patterning, at an early stage of development prior to somitogenesis, hindbrain patterning genes are not induced which leads to the respecification of the posterior hindbrain territory. This region is not lost from the embryo but instead becomes transformed into an enlarged anterior hindbrain. Another aspect of patterning that is abnormal in these RA deficient embryos is the dorsoventral gene expression domains in the anterior spinal cord. These domains are required for the proper specification of motor neurons, sensory neurons and various classes of interneurons. Consequently these neuronal classes are mis‐specified in the RA deficient embryos. With regard to the neural crest, these cells often fail to migrate correctly and then die in the absence of RA. With regard to neurite outgrowth, very little outgrowth seems to take place in these deficient embryos which suggests that RA is involved in neurite outgrowth. Taking these experiments into the adult to examine the role of RA in neurite regeneration, we have had success in inducing neurite outgrowth in vitro from adult mouse spinal cord by manipulating the retinoic acid receptors which transduce the RA signal at the level of the nucleus.
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.
Transgenic and antisense manipulations of impairments in cholinergic balance
Authors: Soreq H. , Cohen O. , Meshorer E. , Ginzberg D. , Glick D. , Yirmiya R.
Published: July 2010

Inhibitors of acetylcholinesterase (AChE) catalysis are increasingly used as insecticides as well as therapeutic and prophylactic agents. However, the long‐term consequences of their use are not yet known. To investigate this topic, we used cultured neurons and transgenic mouse pedigrees that over‐express natural variants of human AChE. Following exposure to an anti‐AChE, alternative splicing of the pre‐mRNA from the single ACHE gene produces the relatively rare AChE‐R mRNA variant in mice and in cultured cerebellar neurons. This promotes a rapid (minutes) yet long‐lasting (weeks) translocation of AChE‐R mRNA into neurites that is associated with extreme neuronal hypersensitivity to both anti‐AChEs and atropine (Meshorer et al. 2002, Science, 295, 508‐512). In transgenic mice, over‐expression of AChE‐R causes stress‐induced irregular bursts of increased locomotor activity and failure of short‐term memory (Cohen et al. Mol. Psych., in press). Nanomolar concentrations of an antisense oligonucleotide selectively suppress AChE‐R mRNA levels in both hyperproducing cultured neurons and mouse brain. Moreover, this treatment reverses the behavioral and cognitive deficits of the mice (effect apparent for > 24 h, vs. < 45 min for tacrine). The efficacy of our antisense experiments raises the possibility of eventually using such agents to achieve AChE variant‐specific suppression, long‐lasting effect, and, presumably, fewer side‐effects than is offered by conventional anticholinesterase therapy.
Rho‐associated kinase (ROCK) inhibitor, Y27632, promotes neurite outgrowth in PC12 cells in the absence of NGF
Authors: Nath R. , DeGuia F. , Akbar J. , Borosky S. A. , Wang K. K. W.
Published: July 2010

Neurite extension and retraction are very important processes in the formation of neuronal networks. A strategy for fostering axonal regrowth/regeneration of injured adult neurons is attractive therapeutically for various diseases such as traumatic brain injury, stroke and Alzheimer's disease. The Rho family of small GTPases, including Rac and Cdc42 have been shown to be involved in promoting neurite outgrowth. On the other hand, activation of RhoA induces collapse of growth cone and retraction of neurites. Rho‐associated kinase (ROCK) an effector molecule of RhoA, is downstream of a number of axonal outgrowth and growth cone collapse inhibition mechanisms. In the present study, we sought to identify the role of ROCK in neurite outgrowth in PC12 cells. Y27632, a specific inhibitor of ROCK, induced a robust increase in neurite outgrowth in these cells within 24–48 h as visualized by phase contrast microscopy. Staining with FITC‐tubulin or phalloidin show extended neurites in PC12 cells treated with Y27632, comparable to that with 100 ng/mL of NGF. Assessment of other biochemical markers of neurite outgrowth such as GAP43, neurofilament and tyrosine hydroxylase phosphorylation further indicates that inhibition of ROCK in PC12 cells causes differentiation of these cells to a neuronal phenotype.
Poster sessions AP11: Neurotransmitters, Transporter and Enzymes. Structure and activity of recombinant human glutamate carboxypeptidase II
Authors: Barinka C. , Rinnova M. , Sacha P. , Mlcochova P. , Rojas C. , Majer P. , Slusher B. , Konvalinka J.
Published: July 2010

Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as N‐acetylated‐α‐linked‐acidic dipeptidase, NAALADase) cleaves N‐acetyl‐aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. We report the mapping of the entire coding region of GCPII and identification of the region sufficient and necessary for the production of active recombinant protein. Extracellular portion of human glutamate carboxypeptidase II (amino acids 44–750) was expressed in Drosophila Schneider's cells and purified to homogeneity. A novel assay for hydrolytic activity of GCPII, based on fluorimetric detection of released alpha‐amino groups was established, and used for enzymological characterization of GCPII. The potential of this assay for high‐throughput inhibitor testing was evaluated and pH dependence for the enzymatic activity have been analysed. Using a complete set of protected dipeptides, substrate specificity of recombinant GCPII was elucidated. Ac‐Glu‐Met, Ac‐Asp‐Met and surprisingly Ac‐Ala‐Met were identified as novel substrates for GCPII. The glycosylation has been found indispensable for the activity of the enzyme. A series of point mutants of the enzyme has been expressed and purified and the glycosylation sites critical for the proteolytic activity have been identified.
Distribution of alpha 1 and alpha 2 GABAA receptor subunits in developing chick optic tectum
Authors: De Plazas Sara Fiszer , Vacotto M. , Siccolone G. , Flores V. , Rodríguez Gil D. J.
Published: July 2010

In a previous work we described a differential ontogeny of type I and type II benzodiazepine (BZD) receptors. Molecular cloning has led to the characterization of different GABAA receptor subunits, with the alpha subunit reportedly containing the BZD binding site. The aim of this work was to study the developmental pattern of expression of α1 and α2 GABAA receptor subunit genes in chick optic tectum. In situ hybridization, using a 45‐base antisense oligonucleotide complementary to each one of these subunits, was assayed. Expression levels of representative mRNAs were quantified in each lamina by measurement of X‐ray film optical densities. Both subunits were expressed as early as embryonic day (ED) 10, exhibiting higher levels of mRNA in compartment i during all days analyzed. While α2 levels remained high, α1 levels increased during development. The superficial transient cell compartment 3 showed that α2 expression peaked on ED 12 and then decreased concomitantly with the segregation pattern of this lamina into four others, while α1 subunit levels exhibited relatively constant high levels in the superficial laminae. Nevertheless, α2 levels compared to α1 were greater for each one of the laminae studied throughout development. In conclusion, the developmental pattern of alpha subunits expression progresses through a characteristic temporospatial pattern related to the laminar organization in each layer and correlates with the temporal expression of BZDI and BZDII receptors.
α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid‐mediated neuroprotection requires TRKB receptor activation
Authors: Marini A. M. , Banaudha K. , McCall S. , Zhu D. , Lipsky R.
Published: July 2010

α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) plus aniracetam, an AMPA potentiator, prevents glutamate‐mediated neurotoxicity and apoptosis in cultured rat cerebellar granule cells. However, the mechanism of AMPA‐mediated neuroprotection is unknown. N‐methyl‐d‐aspartate (NMDA) protects neurons against glutamate‐mediated excitotoxicity via a brain‐derived neurotrophic factor (BDNF) autocrine loop. Thus, we tested whether AMPA mediates neuronal survival in a manner similar to NMDA. We show that AMPA (500 μm) plus aniracetam (1 μm) increases BDNF mRNA in a time‐dependent manner as measured by a 5′nuclease assay. AMPA elicited two peaks of BDNF mRNA, one at 15 min (2‐fold) and a second peak at 4 h (1.8‐fold). AMPA and aniracetam alone actually reduced baseline levels of BDNF mRNA. Preincubation with the tyrosine phosphorylation antagonist K252a (10 nm) or the mitogen‐activated protein kinase pathway inhibitor, U0126 (10 μm) inhibited AMPA receptor‐mediated neuroprotection suggesting that neuronal survival evoked by AMPA requires activation of TrkB receptors and the MAPK signal transduction pathway. In summary, AMPA increases BDNF mRNA with two peaks of expression. The early rapid increase in BDNF expression may be ‘preformed’ messenger RNA while the latter peak may be new synthesis. TrkB receptor activation and the MAPK pathway are also required. These results suggest that ionotropic glutamate receptors may use similar mechanisms to activate intrinsic neuronal survival pathways.
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.
Are endogenous neural stem cells of hematopoietic origin?
Authors: Fallon J. H. , Kinyamu R.
Published: July 2010

Endogenous neural stem cells have been identified in diverse areas of the adult mammalian central nervous system including the subventricular zone, cerebral cortex and hippocampus. These cells have been demonstrated to participate actively in postnatal neurogenesis in restricted territories within the adult brain. They have further been characterized as having a committed neural fate in vivo, capable of generating neurons, astroglia and oligodendroglia. Endogenous CNS stem cells, when cultured in vitro, have been shown to have a much broader potential, capable of differentiating into diverse tissues such as blood, muscle, bone and kidney. Conversely, stem cells taken from other organs and grown in vitro have been demonstrated to differentiate into neurons, and hematopoietic stem cells injected intravenously have been shown to migrate into mature CNS, and differentiate into neurons. We have previously reported the mobilization of endogenous neural stem cells in vivo. Further work to determine if the stem cells so mobilized may include hematopoietic stem cells is reported here. Using immunohistochemical localization of antigens known to be present on primitive hematopoietic stem cells, or antigens present on neural stem cells, we report the presence of cells closely resembling hematopoietic stem cells in the mature CNS whose response to a mobilization paradigm is similar to that of endogenous neural stem cells. We further propose a lineage relationship between primitive hematopoietic stem cells and neural stem cells.
Regulation of SVZ‐derived gliogenesis by inflammatory‐demyelination
Authors: Baron‐Van Evercooren A. , Decker L. , Picard N. , Nait‐Oumesmar B. , Lachapelle F.
Published: July 2010

Identifying a source of cells with the capacity to generate oligodendrocytes in the adult CNS would help the development of strategies to promote myelin repair. During development of the neonate rodent forebrain, most oligodendrocytes derive from the subventricular zone (SVZ), a germinative area which also contributes to the genesis of astrocytes and neurons. While the SVZ persists in the adult brain, its size is largely reduced and its contribution to cell genesis is essentially restricted to the renewal of the granular and periglomerular neurons of the olfactory bulb. Lesion derived signals can have a profound impact on the behavior of the SVZ cells. While cortical trauma triggers their mobilization and differentiation in astrocytes in the lesioned cortex (Holmin et al. 1997), focaly‐induced demyelination promotes their migration in the demyelinated white matter and differentiation in astrocytes and oligodendrocytes (Nait‐Oumesmar et al. 1999). Using EAE, we will show that multifocal demyelination (i) promotes the proliferation of the SVZ precursors (ii) enhances their migration towards the olfactory bulb and triggers their mobilization to multiple sites of the diseased white matter, and (iii) induces their differentiation in neurons, astrocytes and oligodendrocytes in the olfactory bulb, and in oligodendrocytes and astrocytes in the demyelinated white matter. SVZ precursors could thus be a source of oligodendrocytes and contribute with oligodendrocyte progenitors to the replacement of lost oligodendrocytes in demyelinating diseases of the adult CNS.
Alterations in cerebral metabolism by the neurotoxin kainic acid studied by 13C MRS
Authors: Olstad E. , Qu H. , Sonnewald U.
Published: July 2010

Kainic acid is a potent agonist at the kainate subclass of ionotropic glutamate receptors, and functional kainate receptors have not only been demonstrated on neurons but also on glial cells in culture. Kainic acid injections are used to induce limbic seizures in rodents. When combined with injections of [1‐13C]glucose and [1,2‐13C]acetate followed by analyses of forebrain extracts using 13C magnetic resonance spectroscopy (MRS) and HPLC information about glial neuronal interaction can be obtained. Using kainic acid treatment and 24 h later injection of 13C label a significant increase in label derived from [1,2‐13C]acetate was observed in glutamine and glutamate. Label derived from [1‐13C]glucose was unchanged in most metabolites, however, a decrease was observed in [2‐13C]GABA. It should be noted that only astrocytes are able to utilize acetate as a substrate, whereas acetyl CoA derived from glucose is metabolized predominantly in the neuronal tricarboxylic acid cycle. These results indicate that turnover of metabolites was increased predominantly in astrocytes whereas glutamatergic neurons were not affected. However, GABAergic neurons showed decreased GABA labelling, possibly due to reduced GABA release 24 h after kainic acid injection. Taken together with results obtained 2 weeks after kainic acid injection, it can be suggested that increased astrocytic activity one day after epileptic seizures results, subsequently, in an increased amino acid turnover in neurons. Cell culture work was also performed, results will be presented at the meeting.
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.
Poster Sessions BP02: Oligodendrocytes and Schwann Cells. RAS activation regulates angiogenic and anti‐angiogenic factor expression in NF1‐derived Schwann cells
Authors: Thomas S. , Gollapudi L. , DeVries G. H.
Published: July 2010

Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro, the NF1‐derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1‐derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1‐derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1‐derived Schwann cells have increased RAS activity and a three‐fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1‐derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP‐related domain of neurofibromin into the NF1‐derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1‐derived Schwann cells the Schwann cells expressing the GAP‐related domain expressed about one‐half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1‐drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.
PPAR gamma activators induce differentiation of B12 oligodendrocyte‐like cells and rat spinal cord oligodendrocytes
Authors: Roth A. D. , Leisewitz A. V. , Inestrosa N. C. , Bronfman M.
Published: July 2010

The regulation of CNS lipid metabolism by nuclear receptors and their relation to cell differentiation remains undetermined. Since myelinating oligodendrocytes are the major lipid‐synthesizing cells in the CNS, we characterized the effect of PPAR activation in a CNS derived cell line that expresses oligodendrocyte markers and compared these effects with oligodendrocyte primary cultures (90–95% pure). The rat glioma derived B12 cell line express the three major PPAR isoforms (PPAR α, β and γ) and present a large number of peroxisomes, indicating an important lipid metabolism. Treatment with ciprofibrate, a general PPAR activator and clinical hypolipidemic, induces proliferation arrest, process extension and a moderate rise in the expression of acyl‐CoA oxidase, a specific marker of peroxisomal proliferation. Cell growth arrest by ciprofibrate is enhanced 100‐fold by low concentrations of retinoic acid (0.01 μm), suggesting the involvement of the PPAR‐retinoid acid receptor heterodimers. Since ciprofibrate possibly acts by modifying the concentration of endogenous PPAR ligands, we traced its effects to PPARγ by using isoform specific ligands: Troglitazone and 15‐deoxy‐prostaglandin J2, both of which induce growth arrest and process extension in B12 cells. These effects were corroborated on rat spinal cord derived oligodendrocytes primary cultures, where a significant rise in the number of mature oligodendrocytes is observed in response to PPARγ activators. These results show that PPARγ, a master gene in the differentiation of adipose tissue could be involved in the lipid metabolism of maturing oligodendrocytes.
Gas6 enhances human oligodendrocyte viability and maturation
Authors: Shafit‐Zagardo B. , Shankar S. Latha , O'Guin K. , McMorris F. A. , Stitt T. , Varnum B. , Basch R. S.
Published: July 2010

Oligodendrocyte development and survival occurs in response to signalling pathways activated by protein trophic factors. To identify novel factors that regulate human oligodendrocyte maturation/survival, we performed microarray analysis and identified genes expressed in moderate amounts in oligodendrocytes at 18–22 gestational weeks (gw). Among those characterized were the genes encoding the receptor tyrosine kinases, Axl and Mer. These receptors belong to the mammalian Axl/Mer/Rse receptor tyrosine kinase family and are developmentally regulated. The ligand for this family is the growth arrest‐specific 6 gene product (Gas6). Gas6 stimulates growth and prevents apoptosis in a variety of cell types. Oligodendrocytes isolated from 20 to 23 gw human fetal spinal cord were grown for 6 days on NIH 3T3 fibroblasts engineered to stably express Gas6 or on unmodified 3T3 cells. Oligodendrocytes were identified by immunostaining for proteins expressed by mature oligodendrocytes, CNPaseI and MBP. An increase in the numbers of CNPaseI+ and MBP+ oligodendrocytes, and the extent of process branching and extension, was observed when grown on irradiated 3T3‐cells secreting Gas6, but not on irradiated 3T3 cells. The effect of Gas6 was abolished in the presence of the Axl‐Fc receptor, but remained unchanged in the presence of TrkA‐Fc receptor. Our results suggest a role for the Gas6‐Axl signalling in regulating oligodendrocyte viability and maturation.
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).