bluuebunny/crossref_metadata_2025 huggingface dataset:
Presenilin‐1 and intracellular calcium stores regulate neuronal glutamate uptake
Authors: Yang Yaxiong , Kinney Gregory A. , Spain William J. , Breitner John C. S. , Cook David G.
Published: July 2010

Glutamate uptake by high affinity glutamate transporters is essential for preventing excitotoxicity and maintaining normal synaptic function. We have discovered a novel role for presenilin‐1 (PS1) as a regulator of glutamate transport. PS1‐deficient neurons showed a decrease in glutamate uptake of approximately 50% compared to wild‐type neurons. Gamma‐secretase inhibitor treatment mimicked the effects of PS1 deficiency on glutamate uptake. PS1 loss‐of‐function, accomplished by PS1 deficiency or γ‐secretase inhibitor treatment, caused a corresponding decrease in cell surface expression of the neuronal glutamate transporter, EAAC1. PS1 deficiency is known to reduce intracellular calcium stores. To explore the possibility that PS1 influences glutamate uptake via regulation of intracellular calcium stores, we examined the effects of treating neurons with caffeine, thapsigargin, and SKF‐96365. These compounds depleted intracellular calcium stores by distinct means. Nonetheless, each treatment mimicked PS1 loss‐of‐function by impairing glutamate uptake and reducing EAAC1 expression at the cell surface. Blockade of voltage‐gated calcium channels, activation and inhibition of protein kinase C (PKC), and protein kinase A (PKA) all had no effect on glutamate uptake in neurons. Taken together, these findings indicate that PS1 and intracellular calcium stores may play a significant role in regulating glutamate uptake and therefore may be important in limiting glutamate toxicity in the brain.
Pseudophosphorylation of tau protein alters its ability for self‐aggregation
Authors: Haase C. , Stieler J. T. , Arendt T. , Holzer M.
Published: July 2010

Filamentous tau protein deposits are a pathological hallmark of a group of neurodegenerative disorders (tauopathies). Tau protein in these aggregates is highly phosphorylated at different phosphorylation sites. Although tau filaments can be formed by heparin‐induced aggregation of unphosphorylated recombinant tau, it is not known how tau phosphorylation modulates aggregation behaviour. Analysis of the effect of tau phosphorylation at defined single or multiple sites is hampered by the low specificity of protein kinases and the highly dynamic turnover of phosphorylation in vivo. To overcome this problem we employed site‐directed mutagenesis to convert serine and threonine to aspartic acid or glutamic acid, which introduce a negative charge and conformational change that mimic phosphorylation. We tested 14 different mutated tau proteins for their propensity for self‐aggregation and formation of tau filaments. Tau aggregation was monitored with thioflavin S fluorescence in the presence of different inducers such as heparin, Al3+, Fe2+ and Fe3+. We found that mutations in the N‐terminal portion up to amino acid 208 mainly suppress tau aggregation, whereas mutations in the C‐terminal region mainly lead to an enhanced aggregation. Mutations in the middle portion of tau showed a mixed picture of suppression and enhancement of aggregation. A single amino acid change Ser422Glu has aggregation‐favouring properties with all four inducers.
From hematopoiesis to neuropoiesis: evidence of overlapping genetic programs
Authors: Terskikh A. V. , Easterday M. C. , Li L. , Hood L. , Kornblum H. I. , Geschwind D. H. , Weissman I. L.
Published: July 2010

Hematopoietic stem cells (HSC) are clonogenic cells that can self‐renew and give rise to both lymphoid and hematopoietic systems throughout the life span. Using cDNA subtraction, we have identified a set of genes selectively expressed in mouse adult HSC as opposed to its direct microenvironment, whole bone marrow. Some of these genes were found to be expressed in the subventricular zone of fetal and adult brains, one of the regions of continious neurogenesis. Using cDNA microarray techniques we confirmed gene expression in mouse neurospheres, a population enriched for neural stem cells, and down‐regulation in differentiated cells. We propose that transcripts enriched in several types of stem cells define a functionally conserved group of genes likely to include the regulatory genes involved in self‐renewal of stem cells. To address the self‐renewal potential of stem cell associated genes we have used retroviral transfer into highly purified HSC and primitive neural progenitor cells.
A necessary poison: a mechanism by which retinoic acid can be both essential and harmful for the developing hindbrain
Authors: McCaffery P. , Zhang J. , Hayakawa Y. , Smith D. , Yamamoto M.
Published: July 2010

Retinoic acid (RA) is known to many as a Sigma reagent to be purchased to induce differentiation of a favorite progenitor cell line, whether it be the hemopoietic cell line HL60 or a neural stem cell. The developing embryo uses RA in the same way, to regulate cell differentiation and patterning. The amount of this potent transcriptional activator is determined through regulation of the enzymes that either synthesize RA from vitamin A or inactivate RA. This dependence on precisely regulated amounts of RA renders the embryo acutely sensitive to RA as a teratogen. Exposure of the human embryo can occur because RA is used to treat chronic cystic acne. The abnormalities in the CNS are many but the hindbrain is particularly sensitive. Our studies show that RA exposure of the mouse at a specific embryonic stage alters morphological structures that derive from the wall of the IV ventricle and form the cerebellar system of the hindbrain (i.e. the cerebellum, the inferior olivary nucleus and the pontine nucleus). The loss of the cerebellar vermis in the mouse requires chronic exposure of the embryo to RA whereas abnormalities in the pontine nuclei and inferior olive results from exposure at a critical period of embryonic day 9.5 and 10.5. The abnormalities in the pontine nuclei are due to a failure in their usual neurophilic migration. The compact stream of cells, which leads from the anterior rhombic lip to the ventral pons, is instead scattered widely over the anterior medulla. Given that the RA exposure occurs after the resolution of rhombomere identity this suggests that teratogenic RA interferes with a regulatory event which overlays this original pattern.
Viral vectors carrying NR1 sequences injected into rat hippocampus interfered with learning and memory
Authors: Cheli V. , Adrover M. , Guyot‐Revol V. , Blanco C. , Vidal R. , Vázquez P. , Alché L. , Epstein A. , Jerusalinsky D.
Published: July 2010

NMDA receptors are relevant to learning and memory as has been shown both by pharmacological and genetic manipulations. Gene knockouts are useful for investigating in vivo functions, but genetic deletions unrestricted in time or region, may lead to developmental defects or death. The challenge is to control expression with temporal and spatial restrictions in the brain. Viral vectors derived from herpes type‐1 neurotropic virus are interesting candidates for it. To regulate gene expression of the NMDA receptor NR1 subunit, vectors carrying either sense NR1(+) or antisense NR1(–) sequences and that of the green fluorescent protein (GFP), were constructed. The protein or RNA expression were corroborated in cell culture by GFP autofluorescence, Western blots, immunofluorescence and RT‐PCR, and in rat brain, by Western blots and GFP autofluorescence. The vectors were injected into the dorsal hippocampus of adult male Wistar rats. After 6 days each rat was trained and evaluated for both habituation to an open field and inhibitory avoidance to a foot‐shock. The rats injected with GFP‐NR1(+) vectors showed habituation and learned the inhibitory avoidance, like sham operated rats; while animals injected with GFP‐NR1(–) vectors did not. The vectors were useful to modify endogenous gene expression at a defined period, in restricted regions, leading to investigate in vivo functions. NR1 subunit in the hippocampus is involved in mechanisms leading to habituation and to avoidance behaviour, since even a slight change in the availability of NR1 interfered with them.
M1 and M4 muscarinic receptors from hippocampus are positively involved in memory consolidation
Authors: Jerusalinsky D. , Kornisiuk E. , Romanelli Ferreira A. , Sánchez G. , Fürstenau L. , Blanco C. , Cerveñansky C. , Quillfeldt J.
Published: July 2010

The muscarinic antagonist scopolamine causes amnesia in rats, both systemically or administered into the hippocampus after training, supporting the involvement of muscarinic receptors (MAChR) in memory. The role of MAChR subtypes was poorly understood in vivo due to the lack of enough selective pharmacological tools. Central M1, M3 and M5 receptors are mainly coupled to the phosphoinositide pathway, while central M2 and M4 receptors couple to the inhibition of stimulated adenylate cyclase (AC).

Muscarinic toxins (MTs) from Dendroaspis snakes venom are selective for MAChR subtypes. MT1 and MT2, with affinity order M1M4 other subtypes, behaved as M1 agonists in various preparations. MT3 is highly selective for M4 receptors. These MTs fully antagonised the muscarinic inhibition of FSK‐stimulated hippocampal AC. As these MTs have negligible binding to M2 receptor, the M4 subtype must be responsible for most of the AC inhibition. Pirenzepine, an antagonist with similar affinity for M1 and M4, given into the hippocampus after an inhibitory avoidance task was amnesic. MT1 and MT2 facilitated memory consolidation, corroborating the positive involvement of M1 receptors. At a higher dose of MT2, the facilitation disappeared, suggesting that it was acting at another site, likely blocking M4 receptors. These results strongly support that M4 receptor of the hippocampus, responsible for muscarinic inhibition of AC, positively modulates memory consolidation.
Metabolic influence of gabapentin in brain
Authors: LaNoue K. F. , Kreiger J. , Xu Y. , Wood M. , Berkich D. A. , Hutson S. M.
Published: July 2010

De novo synthesis of glutamate in excised rat retinas results in incorporation of 14CO2 into neuronal glutamate that is partially (30%) inhibited by the neuroactive drug gabapentin (Lieth et al. 2001). Gabapentin inhibits the cytosolic form of branched chain aminotransferase (BCATc) and prevents formation of glutamate via transamination of α‐ketoglutarate by branched chain aminoacids. The present study was an evaluation of the influence of gabapentin on glutamate synthesis in the whole brain in vivo. Gabapentin or vehicle was given IP (100 mg/kg) 30 min prior to the experiment. The rats were continuously infused through the jugular vein with H14CO3 (1.0 mCi/h) and U13C‐glucose (1 mm) while blood samples were taken from the carotid artery. Blood H14CO3 specific activity and U13C glucose concentration remained constant throughout infusions and rats were sacrificed at 5, 10, 20, 40 and 60 min Heads were quick frozen in liquid nitrogen and frozen brains removed and extracted with perchloric acid. 14C‐metabolites were separated chromatographically and assayed as described previously. The 13C data are currently being analyzed to determine 13C‐glutamate turnover. The 14C‐glutamate specific activity increased with a half‐life of 6 ± 1 min in control rats and 9 ± min in gabapentin treated rats. At steady state, the H14CO3 in the control glutamate pool was 8.90 ± 0.3 and 7.92 ± 0.6 nmoles/mg protein in the gabapentin pool. The corresponding values in the glutamine pools were 9.48 ± 0.4 and 7.73 ± 0.6 nmoles/mg protein. The data suggest that gabapentin also inhibits de novo synthesis of glutamate in the whole brain but less so than in the intact excised retina. Nevertheless, gabapentin may induce larger changes in brain areas with high concentrations of glutamatergic neurons.
Voltammetric studies of basal and electrically evoked cathecolamine release from long‐term cultures of chromaffin cells
Authors: Pinzon Alberto , Riesgo Mirta I. , Noga Brian R.
Published: July 2010

Previous in vitro studies have revealed basal and evoked release from cultures of chromaffin cells (CC). However no basal and evoked release of catecholamines from long‐term permanent cultures of CC have been studied. The objectives of the present study were to compare basal and evoked release of catecholamines from these type of cultures and to achieve a long period of survival of permanent microcultures. CC were obtained from adult bovine adrenal glands and plated (50 k cells, n = 3; 100 k cells, n = 9) in microculture dishes with a platinum sputter‐coated base. Basal and electrical evoked release (50 Hz, 100–250 mA/15 s) from CC microcultures were recorded using fast cyclic voltammetry with carbon‐fiber microelectrodes at different intervals of time (20, 40, 60, 90 days). Cell counts at each time period were assessed using an inverted microscope. After at least 120 days, CC were fixed, marked for TH, PNMT, and the nuclei stained. CC count decreased slightly over the entire period of culture. The basal release of electroactive compounds from the long‐term microcultures was always present although a decrease was associated with the decrease in CC count. The electrical stimulation of cells did show an increased release (best between 200 and 250 mA stimuli) in the nanomolar range compared to that of the basal release. Confocal microscopy of labeled cultures revealed that most of the CC present were adrenergic and noradrenergic. This study shows that permanent populations of CC microcultures can be maintained long‐term and the catecholamine release enhanced with electrical stimulation.
Hormonal control of cerebral amyloidogenesis in Alzheimer's diseases
Authors: Xu H. , Greenfield J. P. , Cai D. , Leung L. W. , Kassik K. , Rodriguez‐Boulan E. , Greengard P.
Published: July 2010

Estrogen reduces the risk of Alzheimer disease (AD) in postmenopausal women, β‐amyloid (Aβ) burden in animal models of AD, and secretion of Aβ from neuronal cultures. The biological basis for these effects remains unknown. Aβ is proteolytically derived from the β‐amyloid precursor protein (βAPP) within the secretory pathway by distinct enzymatic activities known as β‐ and gamma‐secretase. Aggregated Aβ peptides are found predominantly within extraneuronal space and are believed to initiate toxic and inflammatory cascades leading to neuronal death. The major population of secreted Aβ peptides is generated within the trans‐Golgi‐network (TGN), also the major site of βAPP residence in neurons at steady state. Utilizing cell‐free systems derived from both neuroblastoma cells and primary neurons, we demonstrate that 17β‐estradiol (17β‐E2) stimulates formation of vesicles containing βAPP, from the TGN. Accelerated βAPP trafficking precludes maximal Aβ generation within the TGN. 17β‐E2 appears to modulate TGN phospholipid levels, particularly those of phosphatidylinositol, and recruit soluble trafficking factors, such as Rab11, to the TGN. Together, these results suggest that estrogen may exert its anti‐Aβ effects by regulating βAPP trafficking within the late secretory pathway. These results suggest a novel mechanism through which 17β‐E2 may act in estrogen‐responsive tissues and illustrate how altering the kinetics of a protein's transport can influence its metabolic fate.
Exchange of local and global neuronal information processing by recurrent fractal neural networks
Authors: Bieberich E.
Published: July 2010

The regulation of neural networks relies significantly on convergent feedback signalling loops that render a global output locally accessible. Ideally, the recurrent connectivity within these systems is self‐organized by a time‐dependent phase‐locking mechanism. This study analyzes recurrent fractal neural networks (RFNNs), which utilize a self‐similar or fractal branching structure of dendrites and downstream networks for phase‐locking of reciprocal feedback loops: output from outer branch nodes of the network tree enters inner branch nodes of the dendritic tree in single neurons. This structural organization enables RFNNs to amplify re‐entrant input by over‐the‐threshold signal summation from feedback loops with equivalent signal travelling times. The columnar organization of pyramidal neurons in the neocortical layers V and III is discussed as␣the structural substrate for this network architecture. RFNNs self‐organize spike trains and render the entire neural network output accessible to the dendritic tree of each neuron within this network. As the result of a contraction mapping operation, the local dendritic input pattern contains a downscaled version of the network output coding structure. Contraction mapping may even proceed to the molecular level and program neurotransmitter release or receptor activation by a downscaled, global coding pattern. RFNNs perform robust, fractal data compression, thereby providing a strategy for the exchange of global and local information processing in the human brain. This property may reconcile neuronal computation in separate neurons with a unified instance of experience in consciousness, which has been discussed as significant step toward the solution of the brain/mind dichotomy.
Perforin and granzyme mediated toxicity on cultured oligodendrocytes
Authors: Knapp P. E. , Adams M. H. , Holder J. M. , Hudig D.
Published: July 2010

Oligodendrocytes (OLs) are major targets of immune‐mediated demyelination in multiple sclerosis. Perforin, a pore‐forming protein released from cytolytic granules of cytotoxic T‐cells and NK cells, is implicated as an effector in inflammatory conditions leading to demyelination. Cytolytic granules contain other proteins which may also promote demyelination, including the serine dependent protease granzyme B. We used a repeated measures assay to test effects of enriched perforin, granzymes and total granule extracts on survival of individual OLs over 72 h. Granules were isolated from rat RNK‐16 cells. Granzymes and perforin were isolated by Cu + 2‐immobilized metal affinity chromatography. Lytic activity/mL at 4 h was calculated for all preparations. We assayed survival of both mature and immature OLs to determine developmental sensitivity. Cells were exposed to perforin, granzymes, perforin + granzymes, granule extract or control buffers for 1–3 h. Survival of mature OLs with large processes and membrane expansions was reduced by granule extracts but unaffected by perforin and granzymes. Immature OLs with a bipolar morphology or with multiple, thin processes were vulnerable to both perforin and granzymes alone and in combination, and to granule extracts. The majority of cell death in all cases occurred by 24 h. Results suggest that cytolytic granules contain factors that are toxic to OLs at all developmental stages and that immature OLs are differentially sensitive to perforin and/or granzyme mediated toxicity. Cytolytic granules may thus have a dual effect, to promote demyelination and to limit remyelination by targeting newly generated OLs.

Acknowledgements: Supported by Natl. MS Soc. (PEK) & NIH R01 CA38942 (DH).
Ca2+ blockers ameliorate disease in mouse model of multiple sclerosis (MS)
Authors: Brand‐Schieber E. , Werner P.
Published: July 2010

Influx of extracellular Ca2+ is implicated in CNS white matter injury. We studied the effects of clinically used Ca2+ blockers on mouse adoptive‐transfer EAE (AT‐EAE), a model of MS, an inflammatory white matter disease. Lymph node cells (LNC) from donor mice, sensitized to a CNS antigen, MBP, and clonally expanded in vitro with the same antigen, were injected I.V. to recipient SJL/J mice. Mouse AT‐EAE presents an initial acute ascending paralysis (onset 8–10 days, remission 18–20 days after LNC transfer) and a relapsing–remitting disease course thereafter. Bepridil (BEP), a Ca2+ blocker (3 mg/kg/day), reduced average clinical score of AT‐EAE by 1.5–2 points (out of 5; p < 0.05) compared with vehicle control (VEH), when treatment started on day 0, as well as, on day 3 post‐LNC transfer. Nitrendipine, a selective voltage‐gated Ca2+‐channel blocker (2.5 mg/kg/day), started on day 0, ameliorated AT‐EAE (p < 0.05) compared with VEH, in a manner similar to BEP. That two different calcium antagonists provided similar benefits points at a common target, likely voltage gated Ca2+ channels. As to mechanism, BEP reduced white matter pathology but not peripheral monocyte infiltration, assessed by histochemistry, suggesting that BEP may have suppressed secondary damage that followed the initial immune attack. However, we can not rule out an effect of BEP on immune cell competence since LNC, treated with BEP before transfer to recipient mice, only induced a much delayed and weaker EAE compared with vehicle treated cells. Moreover, besides attenuating the acute phase of AT‐EAE, long‐term BEP treatment seems to decrease the number of relapses. Our data implicates Ca2+ influx in AT‐EAE and suggest Ca2+ blockers as potential therapeutics in MS.
Hypochlorite‐induced white matter damage
Authors: Konat G. W. , Tirumalai P.
Published: July 2010

The extent and severity of inflammation‐mediated white matter destruction correlates with neutrophil infiltration. Although the mechanisms are not fully understood, the generation of vast quantities of hypochlorite (HC) by activated neutrophils may be the major damaging factor. This study was undertaken to determine the toxicity of HC to white matter using brain stem slices from 2 month‐old female rats. Because of its high volatility under the incubation conditions (half‐life of ∼ 7.5 min), HC was administered every 30 min as 1 mm boluses providing an average concentration of ∼ 0.27 mm. Exposure period (1–3 h) was followed by 1 h recovery in normal medium, and by 2 h labelling with 3H‐leucine. Subsequently, slices were homogenized, and myelin was isolated by sucrose gradient ultracentrifugation. HC exposure inhibited total homogenate protein synthesis in the concentration‐ and time‐dependent manner. Thus, following 1, 2 and 3 h of exposure, total homogenate protein synthesis was reduced to ∼ 65, 45 and 30% of control. HC exposure preferentially suppressed the assembly of major structural proteins into the myelin sheath. Thus, 1 h exposure reduced the assembly of proteolipid protein and basic protein to ∼ 30% of control value. A significant amount of 3H‐leucine was also incorporated into myelin lipids. Unexpectedly, HC exposure profoundly stimulated 3H‐labelling of myelin lipids. Thus, after 1 and 2 h of exposure the labelling of myelin lipids was increased by ∼ 40 and 90% over control, respectively. In contrast, the labelling of nonmyelin lipids decreased to ∼ 50% of control. These results demonstrate that HC drastically alters myelin metabolism, and thus, may underlie neutrophil‐mediated inflammatory demyelination in multiple sclerosis.

Acknowledgements:  Supported by NMSS research grant 3067A1.
Poster Sessions BP05: Neuroimmunology. Inhibition of adoptive transfer of EAE by nemo‐binding domain (NBD) peptides
Authors: Dasgupta S. , Zhou Y. , Jana M. , Pahan K.
Published: July 2010

Experimental allergic encephalomyelitis (EAE) is the animal model for Multiple Sclerosis (MS), the chronic autoimmune disease of the central nervous system (CNS). Activation of NF‐κB requires the activity of IkB kinase (IKK) complex containing (IKKa and IKKb) and the regulatory protein NEMO (NF‐κB essential modifier). Recently it has been shown that peptides corresponding to the NEMO‐binding domain (NBD) of IKKa or IKKb specifically inhibit the induction of NF‐κB activation without inhibiting the basal NF‐κB activity. The present study underlines the importance of cell‐permeable NBD peptides in inhibiting the disease process of adoptively‐transferred EAE in female SJL/J mice. Immunocytochemical analysis of spinal cords of EAE mice showed that there was marked induction of NF‐κB activation as evidenced by enhanced p65 (the RelA subunit of NF‐κB) expression compared to that of control mice. Double‐labelling analysis of p65 and cell‐specific markers showed that p65 was mainly expressed by astrocytes, microglia and infiltrating macrophages. Next we examined the effect of NBD peptides on the disease process of EAE. Interestingly, clinical symptoms of EAE were much lower in mice receiving wild type NBD peptides. In contrast, mutated NBD peptides had no effect on the clinical symptoms of EAE. Taken together, our results support the conclusion that activation of NF‐κB participates in the disease process of EAE and that inhibitors of NF‐κB activation may ameliorate the neuroinflammatory disease process in MS patients.

Acknowledgements:  This study was supported by NIH grants (NS39940 and AG19487.
Induction of nitric oxide synthase in microglial cells by MBP‐primed T cells
Authors: Dasgupta S. , Jana M. , Liu X. , Pahan K.
Published: July 2010

The present study was undertaken to investigate the role of MBP‐primed T cells in the expression of iNOS in microglial cells. MBP‐primed T cells alone markedly induced the production of NO and the expression of iNOS protein and mRNA in mouse BV‐2 microglial cells. This induction of NO production was primarily dependent on the contact between MBP‐primed T cells and microglia. Similarly, MBP‐primed T cells also induced the contact‐mediated production of NO in mouse primary microglia. To understand the mechanism of induction of iNOS, we investigated the role of NF‐κB, transcription factor responsible for the induction of iNOS. MBP‐primed T cells alone were able to induce the activation of NF‐κB. Consistently, Dp65, a dominant‐negative mutant of p65, and SN50, a specific inhibitor of p50 NF‐κB, but not SN50M, a nonfunctional peptide mutant, inhibited MBP‐primed T cell‐induced production of NO in BV‐2 microglial cells suggesting that MBP‐primed T cell‐microglia contact induces the expression of iNOS through the activation of NF‐κB. Since IFN‐β has been used to treat MS patients, we examined the effect of IFN‐β on T cell‐mediated production of NO and activation of NF‐κB. Surprisingly, IFN‐β alone induced the production of NO and the activation NF‐κB in microglial cells. However, the pretreatment of MBP‐primed T cells with IFN‐β markedly inhibited the ability of MBP‐primed T cells to induce the production of NO and the activation NF‐κB in microglial cells, mechanisms which can help account for its efficacy in MS.

Acknowledgements:  This study was supported by NIH grants (NS39940 and AG19487).
In vitro specification of CNS stem cells into either oligodendrocytes or neurons: their fate in vivo
Authors: Espinosa‐Jeffrey A. , Zhao P. M. , Becker‐Catania S. G. , Cole R. , De Vellis J.
Published: July 2010

Cell loss within the central nervous system due to disease, trauma, or aging results in severe disability. Recent research indicates that proliferating populations of neural precursors are a promising source of cells for neural grafting and CNS repair. In our study, neural stem cells (NSCs) isolated from embryonic day (ED) 16 Sprague–Dawley rats were cultured in a novel serum‐free stem cell medium that allowed their propagation for extended periods of time, retaining immunoreactivity for both nestin and PSA‐NCAM, two markers characteristic of the stem cell phenotype. Alternatively, oligospheres were obtained from NSCs neurospheres by using a combination of an oligodendrocyte (OL) specification medium and the NSC medium. When neurospheres or oligospheres were plated as single spheres on poly‐d‐lysine and fed with OL specification medium their progenies gave rise to only OL progenitors that matured into MBP+ cells. Preliminary data from transplantation studies showed that when either neurospheres or oligospheres were grafted into the md rat brain cells survived and migrated extensively. Some of the neurosphere derived cells differentiated into NeuN+ cells. In contrast, oligosphere grafted into the md brain gave rise to transferrin, but not to GFAP or NeuN expressing cells. These results indicate that oligosphere derived cells only gave rise to OL progenitors, but not to neurons or astrocytes.

Acknowledgements:  NICHD #06576 and AT Foundation.
Regulation of prion protein gene expression by heavy metals
Authors: Varela‐Nallar L. , Cuitino L. , Sagal J. P. , Inestrosa N. C.
Published: July 2010

Prion diseases are a family of neurodegenerative disorders characterized by neuronal cell death and gliosis. Despite its pathological relevance, the normal function of the prion protein (PrPc) is still unknown. Due to the ability of PrPc to bind and reduce copper it has been suggested that it could be involved in copper homeostasis. Since this metal regulates gene expression of copper‐related proteins, the objective of this work was to learn whether this metal regulates PrPc gene expression. With this purpose in mind, we used PC12 and C6 clones stably transfected with a reporter vector that contains the luciferase gene under control of the PrPc promoter. We observed that copper treatment increases PrPc promoter activity in all PC12 clones in a dose dependent manner, however, this effect was not observed for any of the C6 clones. We also evaluated the effect of other heavy metals on PrPc promoter activity and found that cadmium increases luciferase activity on PC12 clones but not on C6 clones, and zinc has no effect on any of the clones studied. These results show that there is an up‐regulation of PrPc gene expression by copper and cadmium and suggest that this regulation might be tissue specific. This up‐regulation may involve a metal‐responsive element (MRE) located on the PrPc promoter. Supported by CIMM‐ICA, FONDAP‐Biomedicine No. 13980001 and Millennium Institute (MIFAB) No. 2398969.
Myelin proteolipid protein (Plp) intron 1 DNA is required to temporally regulate Plp gene expression in the brain
Authors: Wight P. A. , Li S. , Moore C. L. , Dobretsova A.
Published: July 2010

The myelin proteolipid protein (Plp) gene encodes the most abundant protein found in mature CNS myelin. Expression of the gene is regulated spatiotemporally, with maximal expression occurring in oligodendrocytes during the myelination period of CNS development. Plp gene expression is tightly controlled. Misregulation of the gene in humans can result in the dysmyelinating disorder Pelizaeus–Merzbacher disease, and in transgenic mice carrying a null mutation or extra copies of the gene can result in a variety of conditions, from late onset demyelination and axonopathy, to severe early onset dysmyelination. Previous transfection studies suggest that the first intron contains elements that are vital for directing Plp gene activity, spatiotemporally. Here we verify, in vivo, the importance of these regulatory elements by the use of a Plp‐lacZ transgene [PLP(–)Z], which does not contain any Plp intron 1 sequence. The developmental profile of PLP(–)Z expression in transgenic mice is flat throughout the active myelination period of brain development, whereas mice which harbor a related transgene [PLP(+)Z] that contains the first intron, demonstrate the expected surge in oligodendroglial expression during this period. Although lacZ expression was extremely low in the brains of PLP(–)Z mice (and could not be detected in oligodendrocytes by X‐gal staining), expression in the testes was far more abundant and restricted to Leydig cells. Similar levels of lacZ expression were detected in the testes of PLP(+)Z mice. Thus, intron 1 DNA contains regulatory sequences critical for Plp gene expression in oligodendrocytes, but may not be required for expression in other cell types.
Noradrenergic depletion potentiates β‐amyloid induced cortical inflammation: implications for Alzheimer's disease
Authors: Feinstein D. L. , Galea E. , Gavrilyuk V. , O'Banion M. K. , Heneka M. T.
Published: July 2010

Degeneration of locus ceruleus (LC) neurons and reduced levels of noradrenaline (NA) in LC projection areas is a well known feature of Alzheimer's disease (AD); however, the consequences of those losses are not clear. Since inflammatory mediators contribute to AD pathogenesis, and since NA can suppress inflammatory gene expression, we tested if LC loss influenced brain inflammatory gene expression elicited by amyloid β (Aβ). Adult rats were injected with the selective neurotoxin DSP4 to induce LC death, and subsequently injected in cortex with Aβ (aggregated 1–42 peptide). DSP4‐treatment potentiated the Aβ‐dependent induction of inflammatory nitric oxide synthase (iNOS), IL‐1β and IL6 expression compared to control animals. In contrast, the induction of cyclooxygenase‐2 expression was not modified by DSP4‐treatment. In control animals, injection of Aβ induced iNOS primarily in microglial cells, while in DSP4‐treated animals iNOS was localized to neurons, as is observed in AD brains. Injection of Aβ increased IL‐1β expression initially in microglia, and at later times in astrocytes, and expression levels were greater in DSP4 treated animals than controls. The potentiating effects of DSP4‐treatment on iNOS and IL‐1β expression were attenuated by coinjection with NA or the β‐adrenergic receptor agonist isoproterenol. These data demonstrate that LC loss and NA depletion augment inflammatory responses to Aβ, and suggest that LC loss in AD is permissive for increased inflammation and neuronal cell death.
Oxidative stress, bioenergetics and neurodegenerative diseases
Authors: Bliznakov E. G. , Bhagavan H. N.
Published: July 2010

Neurodegenerative diseases (NDD) are an assortment of clinical central nervous system (CNS) disorders, characterized by intraneuronal accumulation of fibrillary material, forming intracellular aggregates. These abnormal products play a prominent role in the dysfunction and neuronal destruction that is specific for NDD, such as Alzheimer's, Parkinson's, and prion‐related diseases, known as transmissible spongiform encephalopathies. Related to this group are some neurological syndromes that involve muscle and CNS; Huntington's chorea, Friedreich's ataxia, and amyotrophic lateral sclerosis. A number of mechanisms appear to initiate or contribute to the neurodegenerative process, including oxidative stress, resulting from free radical generation and mitochondrial dysfunction. Overall, the clinical data on antioxidant efficacy tend to be encouraging. Moreover, the brain is highly dependent on energy availability, and mitochondrial functional impairment results in tissue injury, with detrimental clinical manifestations. Coenzyme Q (coenzyme Q10 in humans) is an essential component of the electron tranport chain in the mitochondria and the intracellular energy generation. It is also a potent antioxidant and a membrane stabilizer. For this reason, the use of coenzyme Q10 (and its derivative idebenone) in the prevention and treatment of NDD is fundamentally justified. Anecdotal, as well as preliminary results from well‐designed long‐term coenzyme Q10 clinical trials reinforce the validity of this novel approach. Due to the aging population, NDD have become a major health and social problem, and conceiving treatment or even arresting progression has become a primary focus of medical research worldwide.
Poster Sessions AP02: Proteins, Amino Acids, Peptides and Metabolism. The effect of proline rich polypeptide on the interaction between activated factor X and anti‐thrombin III
Authors: Coggin M. H. , Srapionyan R. M. , Galoyan A. A. , Brecher A. S.
Published: July 2010

The effect of the hypothalamic neurosecretory Proline Rich Polypeptide (PRP) on the interaction of human Factor Xa (Xa) with human antithrombin III (ATIII) was investigated by SDS–PAGE and Western Blot analysis. Xa runs as two bands with masses of ∼51 and ∼47 kDa. A fraction of the ATIII (∼55 kDa) undergoes a conformational change upon interaction with Xa to run slower at ∼58 kDa. Xa and ATIII form a 1° duplex complex of ∼109 and ∼104 kDa, a 2° duplex complex of ∼99 and ∼94 kDa, and a 3° duplex complex of ∼66 and ∼62 kDa. Additionally, a Xa degradation product (∼35 kDa) is noted. Pre‐incubation of 1.5 μg of either Xa or ATIII with 8 μg PRP for 15′ at R.T., or essentially simultaneous addition of all three, increases the 2° complex upper band, 3° complex lower band, ATIII conformational change, and the Xa degradation product. It also decreases the 3° complex upper band and the native ATIII. Xa/PRP premixtures cause a decrease in unreacted Xa. ATIII/PRP premixtures produce an increase in the 1° complex lower band and a decrease in the unreacted Xa upper band. Xa/ATIII/PRP mixtures increase both bands of the 1° complex. Thus, 8 μg PRP promotes 1° complex formation as well as formation of the 2° and 3° degradation complexes. It also promotes modification of ATIII and degradation of Xa, possibly by autolysis and/or promotion of proteolysis of the 1° complex by free Xa. Data obtained indicate the role of PRP in the molecular mechanisms of blood clotting. (Supported, in part, from a grant by the Office of Sponsored Programs and Research, BGSU.)
Poster Sessions CP07: Ions, Channels, Pumps and Transporters. Functional relationship between Na/K‐pump and glutamate receptors in cerebellum granule cells
Authors: Boldyrev A. A. , Schoner W.
Published: July 2010

Stationary level of reactive oxygen species (ROS) in cerebellum granule cells of 12‐day‐old‐rats was measured using three fluorescent dyes characteristic of different location within the neuronal cell: BODIPY 581/591 (for LOO.radicals), DCF‐DA (for H202) and DHR123 (OH‐radicals in mitochondria). When the neurons were activated by N‐methyl‐d‐aspartate (NMDA) a dose‐ and time‐dependent rise of the fluorescent signal was registered with each of the three dyes; the former dye provided the smallest and the latter the largest response. 3‐HPG, a ligand for metabotropic receptors decreases ROS fluorescence and suppressed the NMDA‐induced effect. NMDA and kainic acid presented simultaneously cumulatively increased ROS levels. Ouabain, specific inhibitors of Na/K‐pump induced a considerable increase in ROS fluorescence, which was decreased by 2.5–5 mm KCl, 50 mkM Vanadate or 10 mkM D‐AP5, an inhibitor of NMDA‐activated ionic channels. The K0.5 for activation of ROS generation by Ouabain was more than 250 mkM, which is much higher than that for inhibition of Na/K‐ATPase or its rubidium pumping activity. The data show that the Na/K‐pump protein regulates ROS production by NMDA‐receptors and that the E1(Na) conformation of the Na/K‐pump being less sensitive to ouabain may be responsible for the effects. The data illustrate functional interaction between ionotropic and metabotropic receptors and Na/K‐ATPase.

Acknowledgements:  Supported by DAAD, Grant 325‐sm, Germany.
Identification of a GM1/Sodium–Calcium exchanger complex in the nuclear envelope of non‐neuronal cells
Authors: Xie X. , Wu G. , Lu Z‐H. , Fontainhas A. , Ledeen R. W.
Published: July 2010

Our previous studies identified a Na–Ca exchanger (NCX) that is tightly associated with GM1 ganglioside and potentiated by it in the nuclear envelope (NE) of NG108‐15 cells and primary neurons. The purpose of the present study was to explore whether this is a general phenomena or limited to neurons. Non‐neuronal C6 (glioma), HeLa (Epithelial carcinoma) and NCTC (connective tissue) cell lines were used. Immunocytochemical staining with anti‐NCX antibody and cholera toxin B subunit revealed that NCX and GM1 coexist in the nuclei from all 3 cell lines; in relation to plasma membrane, only HeLa cells showed staining for both NCX and GM1. Purified NE and non‐nuclear membrane mixture (mainly plasma membrane) from the 3 cell lines were immunoprecipitated with a mouse monoclonal anti‐NCX antibody and the precipitated proteins separated on SDS–PAGE. Analysis by immunoblot, showed that NCX is tightly associated with GM1 in the NE of all 3 cell lines. In contrast, NCX and the more loosely associated GM1 from plasma membrane of HeLa cells were separated by SDS–PAGE. Isolated nuclei from C6 cells were used for 45Ca2+ uptake experiments, which provided functional evidence that this exchanger protein is strongly potentiated by GM1. In similar experiments with Jurkat cells (T lymphocyte), no NCX was found. These results suggest a possible new and widely distributed mechanism for regulation of nuclear calcium by NCX in association with GM1.

Acknowledgements:  supported by NIH grant NS 33912.
Prolactin‐dependent activation of ERK and TYR‐phosphorylation of cortactin in postmitotic neurons
Authors: Mangoura D. , Singh D. , Johnson S. , Sakellaridis N.
Published: July 2010

Prolactin (PRL), originally identified as a hormone involved in reproduction, is now investigated as a growth factor. We have previously demonstrated that PRL is a mitogen in PC12 cells and astrocytes, acting independently of ERK activation. To investigate the potential role of prolactin as a neurotrophic factor, we used cultured postmitotic cortical neurons derived from embryonic day 6 chick. Western blot analysis with antiphosphotyrosine antibodies established functional expression of PRL receptors by showing that several proteins were modified with time of PRL exposure, including the PRL receptor and JAK2 kinase. Using both kinase in‐gel assays and Western blots with antiactive ERK antibodies we found that PRL stimulated activation of ERK1/2 in a sustained fashion, typical of differentiation. While active ERK was detected in the nucleus, some was associated with the cytoskeleton. We then investigated in nuclei‐free fractions the identity of proteins that coimmunoprecipitated with ERK and were tyrosine‐phosphorylated in response to prolactin. The most prominent protein was identified as the F‐actin‐binding cortactin. Upon stimulation with PRL, an activated ERK coimmunoprecipitated cortactin as early as 2′ and as long as 15′ after exposure. Tyr‐phosphorylation of cortactin and translocation to the Triton X‐100 fraction was also established. In the long term PRL induced a small but significant increase of cortactin expression. In lieu of our recent data that the developmentally regulated cortactin may supress several parameters of the GABAergic program, these results suggest that prolactin may have a role in neuronal differentiation and that signal specificity may be a result of protein interactions between cytoskeleton and signaling molecules.

Acknowledgements:  Support: HD‐09402 to DM.
Poster Sessions CP09: Kinases and Phosphatases. Protein kinase C inhibitors counteract ethanol effects on myelin basic protein expression in CG‐4 oligodendrocytes
Authors: Bichenkov E. , Ellingson J. S.
Published: July 2010

Abnormal myelin formation appears to be one defect contributing to the neuropathology associated with the fetal alcohol syndrome, the major cause of noncongenital mental retardation. Using the CG‐4 cell line we previously showed that 25–75 mm ethanol (EtOH) down‐regulates myelin basic protein (MBP) expression in differentiating oligodendrocytes (OLGs) without affecting the 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) expression or morphological development (Bichenkov and Ellingson 2001). Here we observed that a relatively low concentration of 12‐phorbol‐13‐myristate acetate (PMA) mimicked the EtOH‐caused inhibition of MBP expression without affecting CNP expression or morphology. The inhibition of MBP expression by 100 mm EtOH or 1 nm PMA was completely counteracted by three inhibitors of protein kinase C (PKC); bisinodoylmaleimide I, chelerythrine chloride, and calphostin C, indicating that EtOH down‐regulated MBP expression by activating PKC. We investigated whether the EtOH‐caused activation resulted in part from up‐regulation of the expression of PKC isozymes. Of 11 PKC isozymes examined, CG‐4 OLGs expressed nine; PKC α, β1, β2, δ, ε, λ, μ, nu and zeta; while PKC isozymes γ and theta were not detected. Only five PKC isozymes, α, β1, β2, μ, and nu, displayed developmental changes in expression. However, EtOH did not up‐regulate the early expression of any PKC isozyme during the first two days of differentiation, the developmental stage when it down‐regulates the MBP expression in CG‐4 cells. The results indicate that EtOH delays MBP expression by activating at least one phorbol ester‐sensitive PKC isozyme in differentiating oligodendrocytes without up‐regulating its expression.

Acknowledgements:  Support: NIAAA Grant AA072185.
Effects of ethanol on gangliosides in the plasma, liver, and brain of inbred mouse strains
Authors: Saito M. I. , Saito M. A. , Szakall I. , Vadasz C.
Published: July 2010

The mouse inbred strains C57BL/6ByJ and BALB/cJ show genetically different alcohol‐related behaviors. Using these strains, we examined ganglioside contents in the plasma, liver, and brain with or without acute ethanol treatment. The quantification of GM1 was performed with a TLC‐immunostaining procedure using choleragenoid, and the contents of other gangliosides were measured after staining with resorcinol reagents. It is known that there are polymorphisms in ganglioside compositions among inbred mouse strains. We found that the plasma GM1 level in BALB/cJ mice (1.6 ± 0.6 ng/μL) was 12 times higher than the level found in C57BL/6ByJ mice (0.13 ± 0.03 ng/μL) although the major ganglioside in both strains was GM2. The ganglioside profiles in the liver were similar to those of the plasma, and the GM1 level in BALB/cJ was 25 times higher than that of C57BL/6ByJ. The liver probably synthesizes the plasma gangliosides as suggested in other studies. The total brain ganglioside compositions were also different between BALB/cJ and C57BL/6ByJ. The levels of GD1b and GQ1b were higher in BALB/cJ although the GM1 contents were similar. These animals were injected with 20% ethanol intraperitoneally in a single dose of 3 g/kg and the ganglioside contents were measured after 4 h. The GM1 levels in the liver and plasma were lower in ethanol‐treated animals while the GM1 levels were higher in erythrocytes and brains. Since it has been shown that the administration of GM1 attenuates or modifies the effects of ethanol in several animal models, the difference in GM1 contents in the plasma between BALB/cJ and C57BL/6ByJ may contribute to some of the differences in ethanol‐related behaviors or toxicity between these strains.
Aromatic hydrocarbon 1,2‐diacetylbenzene cross‐links neurofilament and other proteins
Authors: Sabri M. I. , Hashemi S. , Kim M. S. , Spencer P. S.
Published: July 2010

Rats treated with 1,2‐diacetylbenzene (1,2‐DAB) develop giant proximal axonal swellings packed with 10‐nm neurofilaments in lumbar spinal motor neurons (Kim et al. 2001; Toxicol. Appl. Pharmacol. 177, 121–131), an early pathological feature in amyotrophic lateral sclerosis. This study examines the reactivity of 1,2‐DAB with a number of proteins. Incubation of purified bovine serum albumin or ribonuclease with DAB isomers (1–5 mm) revealed the formation of high molecular weight polymers with 1,2‐DAB, but not with the non‐neurotoxic analog 1,3‐DAB. Western blots of rat spinal cord revealed that 1,2‐DAB (1–10 mm), but not 1,3‐DAB, produces a concentration‐dependent decrease in neurofilament proteins with increased high‐molecular‐weight protein species. Native beta‐tubulin was least affected by 1,2‐DAB. Western blots of rat sciatic nerves exposed in situ to 1,2‐DAB (5–50 mm) showed a decrease of the NF‐M band with corresponding increased staining in the high‐molecular‐weight region. Protein polymerization was not detected with monoclonal antibodies to either NF‐L or beta‐tubulin. Preliminary results show that 1,2‐DAB, but not 1,3‐DAB, significantly reduces the levels of other axonal proteins (e.g. microtubule‐associated protein, tau and motor proteins kinesin and dynein). In summary, 1,2‐DAB reacts differentially with a number of axonal proteins; which reactions underlie the genesis of axonal pathology is unknown.

Acknowledgements:  Supported by NIEHS grants ES10338 and ES11384, and the State of Oregon's Worker's Benefit Fund.