bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of extracellular signal‐regulated kinase in the ventral tegmental area in the suppression of the morphine‐induced rewarding effect in mice with sciatic nerve ligation
Authors: Ozaki Satoru , Narita Minoru , Narita Michiko , Ozaki Masahiko , Khotib Junaidi , Suzuki Tsutomu
Published: July 2010

We recently reported that μ‐opioid receptor agonist morphine failed to induce its rewarding effects in rodents with sciatic nerve injury. In the present study, we investigated whether a state of neuropathic pain induced by sciatic nerve ligation could change the activities of the extracellular signal‐regulated kinase (ERK) and p38 in the mouse lower midbrain area including the ventral tegmental area (VTA), and these changes could directly affect the development of the morphine‐induced rewarding effect in mice. The sciatic nerve ligation caused a long‐lasting and profound thermal hyperalgesia. A dose‐dependent place preference induced by s.c. administration of morphine was observed in sham‐operated mice, but not in sciatic nerve‐ligated mice. We found here for the first time that nerve injury produces a sustained and significant reduction in protein levels of phosphorylated‐ERK and ‐p38 in cytosolic preparations of the mouse lower midbrain. The inhibition of ERK activity by i.c.v. pre‐treatment with either PD98059 or U0126 impaired the morphine‐induced place preference. In contrast, i.c.v. treatment with a specific inhibitor of p38, SB203580, did not interfere with the morphine‐induced rewarding effect. Immunohistochemical study showed a drastic reduction in phosphorylated‐ERK immunoreactivity within tyrosine hydroxylase‐positive cells of the VTA. These results suggest that a sustained reduction in the ERK‐dependent signalling pathway in dopamine cells of the VTA may be implicated in the suppression of the morphine‐induced rewarding effect under neuropathic pain.
Glutathione S‐transferase is a novel target for mood stabilizing drugs in primary cultured neurons
Authors: Wang Jun‐Feng , Shao Li , Sun Xiujun , Young L. Trevor
Published: July 2010

Oligonucleotide microarray technology was used to analyze gene expression profiles after chronic treatment with the mood stabilizing drug valproate at a therapeutically relevant concentration in primary cultured rat cerebral cortical cells. We discovered that valproate regulates expression of 28 genes, including three isoenzymes (M1, A3 and A4) of glutathione S‐transferase (GST), an important protective factor against oxidative stress. Because previous studies in our laboratory found that chronic valproate treatment protected cultured neurons against oxidative stress, further experiments on the regulation of GST were performed. Regulation of GST M1, GST A3 and GST A4 was verified using northern blotting hybridization. Chronic valproate treatment increased mRNA levels of M1 and A4, but decreased the A3 mRNA level dose‐dependently, indicating further complexities in the regulation of GST by valproate. The level of GST M1 protein and GST activity were also increased by chronic valproate treatment. In addition, chronic treatment with lithium, another commonly prescribed mood stabilizer, also increased levels of GST M1 mRNA and protein. The present findings suggest that regulation of GST M1, and possibly GST A4, may mediate the anti‐oxidative effects of valproate treatment, and regulation of GST may be involved in the mood stabilizing effect of valproate and lithium.
Aggregate formation and synaptic abnormality induced by DSCR1
Authors: Ma Hong , Xiong Hui , Liu Tong , Zhang Lingyan , Godzik Adam , Zhang Zhuohua
Published: July 2010

Aggregation of conformation‐abnormal peptides probably plays a key role in the pathogenesis of many neurodegenerative diseases. DSCR1 Down syndrome (DS) critical region 1, was identified from a chromosomal region (21q22.1‐q22.2) for the clinical manifestations of DS when an extra‐copy is present. We report that expression of DSCR1 in several cell types, including primary neurons, causes microtubule‐dependent aggresome‐like inclusion body formation. Disease‐associated huntingtin (Q148) and ataxin‐3 (Q84) co‐localize with DSCR1 aggregates. Neurons bearing DSCR1 aggregates show reduced synaptophysin staining in processes. DSCR1 residues 31–90 constitute an aggregation‐prone domain that is predicted to form a hydrophobic patch on the protein surface when residues 1–30 are removed. This study identifies a novel function of DSCR1 that may underlie DS neuropathology.
Neuroprotective properties of copper binding fragments of amyloid precursor protein and prion
Authors: Inestrosa N. C. , Chacon M. , Barria M. I. , Varela‐Nallar L. , Opazo C.
Published: July 2010

We are particularly interested in the neurobiology of copper as a start point to understand the role of this metal on neurodevelopmental as well as in neurodegenerative processes. Here, we summarize our evidence that support a role in copper homeostasis of the amyloid precursor protein (APP), a protein related to Alzheimer's disease, and the Prion protein (PrP), which is related to the Creutzfeldt–Jakob disease. First, APP and PrP possess copper reduction activity, measured by the bathocuproine assay, being cysteine and tryptophan key amino acids for oxidation (in APP and PrP, respectively). Second, PrP transcription is regulated by copper, both in primary neuronal cultures and in PC12 cell lines transfected with the PrP promoter. Finally, APP‐ and PrP‐copper binding domains rescue rats from copper induced neurotoxicity and spatial memory deficits induced by metal injection into the rat brain. Since APP and PrP proteins are localized at the plasma membrane, we proposed that their abilities to bind and reduce copper might play an important role as a primary neuronal response against copper neurotoxicity.

Acknowledgements: Supported by FONDECYT No. 2990087 to C.O. and CIMM‐ICA, FONDAP‐Biomedicine No. 13980001, MIFAB and a Presidential Chair in Science (1999–2001) from the Chilean Government to N.C.I.
Use of pharmacogenomics in clinical trials for multiple sclerosis
Authors: Martin R.
Published: July 2010

Multiple sclerosis (MS) is a T‐cell mediated autoimmune disease of the central nervous system. Its pathology is characterized by inflammation, demyelination, gliosis and axonal loss. Substantial heterogeneity exists at the levels of genetic susceptibility, clinical presentation and immunological factors as well. While we begin to realize the complexity of multiple sclerosis, it is currently not possible to assess the contributing factors appropriately since biomarkers other than magnetic resonance imaging are lacking. Standard treatment of MS includes interferon‐beta and glatiramer acetate for relapsing–remitting MS. Rapidly progressing and very active disease is treated by conventional immunosuppression with mitoxantrone, cyclophosphamide or other chemotherapeutics, and numerous experimental treatments are currently in testing. Given the complexity of the disease process and its variability between patients, it is no surprise that the response to each of the above treatments is overall modest. Recent developments of large scale gene expression arrays and proteomics approaches allow us to capture many markers and parts of entire biochemical pathways such as apoptosis or proinflammatory responses simultaneously in individual patients. We have begun to employ cDNA or oligonucleotide microarrays to assess the response to the above therapies first in vitro and later, after initiation of treatment, in vivo. These tools will be useful in defining new biomarkers for MS, to gain a better understanding of disease pathogenesis, but particularly also for developing new therapies for MS, identifying responders and nonresponders and rationale combination therapies.
Vitamins E and C prevent the impairment of retention of an inhibitory avoidance task caused by arginine administration
Authors: Reis E. A. , Zugno A. I. , Lamers M. L. , Franzon R. , Matté C. , Netto C. A. , Wyse A. T. S.
Published: July 2010

Hyperargininemia is an inherited metabolic disease of urea cycle caused by the deficiency of arginase I activity, resulting in tissue accumulation of arginine (Arg). Patients affected by this disease usually develop spasticity, epilepsy and mental retardation as principal symptoms. Previous studies from our laboratory have showed that acute administration of Arg impairs retention of the inhibitory avoidance task and that l‐NAME (NOS inhibitor) prevents this effect. In the present study, we investigated the effect of chronic treatment with antioxidants (vitamins E and C) on the retrieval of the inhibitory avoidance task in adults rats subjected to experimental model of acute hyperargininemia in order to investigate the participation of oxidative stress on this phenomenon. Sixty‐day‐old‐rats were treated for one week with i.p. injection of saline (0.9%) or vitamins E and C (vit E 40 mg/kg and vit C 100 mg/kg). Twelve hours after the last injection Arg (0.8 g/kg) or an equivalent volume of saline were administered 1 h before training, 1 h before testing or immediately after training sessions. Memory was significantly impaired in Arg‐treated group, whereas the rats chronically treated with vitamins E and C had this effect prevented. Present data strongly indicate that Arg administration impairs memory, an effect probably mediated by oxidative stress since treatment with vitamins E and C prevented amnesia. Assuming the possibility that this might occur in the human condition, reported results may be relevant to explain, at least in part, neurologic dysfunction associated with hyperargininemia.
Retinoic acid as a neurobehavioral teratogen
Authors: Adams J. , Lammer E. J.
Published: July 2010

Animal research on retinoic acids (RA) has helped to establish a principle of neurobehavioral teratology: embryonic exposure to a CNS teratogen produces a continuum of outcomes, ranging from death, to malformation, to subtle functional alterations. Demonstrating this principle in human studies was problematic prior to the introduction in 1982 of 13‐cis RA (Accutane, Hoffman‐LaRoche) for the treatment of severe cystic acne. 13 cis RA is a potent human teratogen that causes a 40% rate of spontaneous abortion, and, among liveborn infants, a 35% rate of major malformation (Lammer et al, 1985; 2001). The characteristic pattern includes hindbrain, craniofacial, cardiac, and thymic abnormalities. Longitudinal follow‐up of this original cohort and the matched controls has focused on neuropsychological characteristics at 5 and 10 years of age, and the relationships between malformation and cognitive status. Of 35 children exposed to RA between postconception days 14 and 60 and tested at age 5, 46% scored in the below average range of general mental ability, as compared to 10% of the controls. Boys were more frequently affected than girls with respect to major malformations and reduced intelligence. Presence of a major malformation was associated with reduced intellectual performance, however, 6 of 16 children scoring in the below average range had no detectable malformations. Thus, as in the animal studies, features at birth, such as major malformations, do not fully characterize the adverse consequences of embryonic RA exposure. Likewise, the effects cannot be fully characterized by examining only reduced intelligence. As compared to control children, RA exposed children with average mental ability were three times more likely to have a specific, non‐verbal learning disability and boys were again over‐represented.
Selective neuronal vulnerability, protease‐activated receptors (PARS) and thrombin signalling
Authors: Festoff B. W. , Wu M. , Wong A. , Citron B. A. , Suo Z.
Published: July 2010

In order to investigate the roles of thrombin and its signalling via PARs in defining selective neuronal vulnerability we compared two different murine clonal neuronal lines: NSC34 motor and HT22 hippocampal neurons. RT‐PCR revealed mRNA expression all four PARS. Responses of both cell lines for thrombin effects on neurite outgrowth, cell survival and signalling changes through mitogen activated protein kinases (MAPKs) were evaluated. Ability of thrombin and PAR‐specific active peptides (agonists): PAR1 (SFLLRN) and PAR4 (GYPGQV), were compared for these parameters and for the presence of aggregated microtubule‐associated protein (MAP) tau neurofibrillary tangles (NFTs). Motor neuronal PAR1 or PAR4 activation induced rapid neurite retraction and neuronal apoptosis. This was accompanied by tau degradation. In contrast, hippocampal neuronal PAR1 or PAR4 activation resulted in mild effects on neurite outgrowth but rapid tau aggregation, which was dependent on phosphorylation and positive for NFT‐specific antibody. Delayed reduction in cell viability occurred 24–48 h after tau aggregation. Evaluation of signalling pathways revealed p38 MAPK in NSC‐34 and p44/42 MAPK activation in HT22 cells were closely related to observed differential neuronal morphology in response to thrombin. We conclude that different intracellular signalling pathways coupled to PARs in different neuronal sub‐populations mediate selective thrombin vulnerability.

Acknowledgements:  Supported by the DVA, Christopher Reeve Paralysis Foundation, Missouri Alzheimer's Res. Fund, ALS Association, Lied Endowment, KUMCRI and Midwest Biomedical Research Foundation.
Gly456 and Arg443 are essential for allosteric regulation of human GLUD1 glutamate dehydrogenase
Authors: Plaitakis A. J. , Zaganas I. V.
Published: July 2010

Human glutamate dehydrogenase (GDH) exists in housekeeping and brain‐specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. Although the two isoenzymes share all but 15 of their 505 amino acids, they differ markedly in their allosteric regulation characteristics. To identify the structural basis of these functional properties, we performed site‐directed mutagenesis of the GLUD1 at sites that differ from the GLUD2 gene. Mutated cDNAs were expressed in Sf21 cells using the baculovirus expression system. Results showed that replacement of Gly456 with Ala renders the GLUD1 isoenzyme markedly resistant to GTP inhibition (IC50 = 2.8 ± 0.15 μm) as compared to the wild type enzyme (IC50 = 0.19 ± 0.01 μm) and abolishes its cooperative behavior. In contrast, replacement of Arg443 with Ser caused the enzyme to be essentially inactive when assayed under baseline conditions (without allosteric activators). Also, the Arg443Ser mutant was markedly resistant to ADP activation (SC50 = 345.83 ± 16.35 μm) as compared to the wild type GDH (SC50 = 24.69 ± 3.84 μm). Three other mutants (Ser331Thr; Arg470His; Asn498Ser) showed kinetic and allosteric properties similar to those of the normal GLUD1 enzyme. Hence, change of Gly456 with Ala and Arg443 with Ser accounts for some of the functional characteristics that distinguish the nerve tissue specific (GLUD2) from the housekeeping (GLUD1) GDH. Since GLUD2 has evolved from the GLUD1 gene, these data identify some of the structural changes that permitted the GLUD2 GDH to adapt to the unique metabolic requirements of the nerve tissue.
Possible functions for adult hippocampal neurogenesis
Authors: Kempermann G.
Published: July 2010

Why does the adult brain go through the considerable effort to maintain the machinery necessary for neurogenesis? Why does neurogenesis persist in two brain regions and not in others? Whereas early reflections on this topic tended to regard adult hippocampal neurogenesis a mere atavism with little or no functional consequences, this evaluation has begun to change in the last few years. We now hypothesize that adult neurogenesis is necessary for hippocampal function. In the hippocampus, information is processed in order to consolidate memory, hence the emblematic description of the hippocampus as ‘the gateway to memory’. In our view, new neurons are added to the hippocampal dentate gyrus not to increase memory per se, but to optimize the network by strategic insertion of ‘new gatekeepers at the gateway to memory’. Optimization means the smallest (and fastest) possible network that is able to cope with the degree of novelty and complexity an individual experiences. Data on the experience‐ and activity‐dependent regulation of adult hippocampal neurogenesis support this view. Adult hippocampal neurogenesis is a long‐term adjustment of the hippocampus to functional needs, not means for acute changes in the sense of ‘putting in more memory, while the computer is running’. What lies ahead is to find out, which molecular rules govern this long‐term plasticity. To consider functional implications of adult hippocampal neurogenesis also begins to influence concepts of hippocampal disease, for example certain aspects of epilepsia and major depression.
NSAIDs protect dopaminergic neurons against 6‐OHDA and MPP+ toxicity
Authors: Werner P. , Carrasco E.
Published: July 2010

Endogenous and environmental neurotoxins are among the suspected causes of the loss of dopaminergic (DA) neurons in Parkinson's disease (PD). Non‐steroidal anti‐inflammatory drugs (NSAIDs) reduce inflammation by inhibiting cyclooxygenase (COX)‐dependent synthesis of prostaglandins (PG) from arachidonic acid. NSAIDs decrease the incidence of Alzheimer's disease, but little is known about their potential benefit for PD. Therefore, we examined whether NSAIDs could protect DA neurons from neurotoxic insults. NSAIDs can protect DA neurons against excitotoxicity (Casper et al. 2000), and against 6‐hydroxydopamine (6‐OHDA) toxicity (Carrasco et al. 2001). Here, we compared in primary mesencephalic/DA neuron cultures the effect of NSAIDs on the toxicity of 1‐methyl‐phenylpyridinium (MPP+) or 6‐OHDA. 6‐OHDA significantly (*p < 0.0001) increased PG production, whereas MPP+ did not (p < 0.05). We then compared the competitive/unspecific COX inhibitors ibuprofen and naproxen and the noncompetitive/unspecific inhibitor acetylsalicylic acid (ASA, aspirin) for their ability to protect DA neurons against either 6‐OHDA or MPP+ toxicity. Interestingly, all three nonselective COX inhibitors protected DA neurons in cultures against both 6‐OHDA and MPP+ (p < 0.05), despite the difference in PG induction by 6‐OHDA vs. MPP+. The selective COX‐2 inhibitor NS398 did protect DA neurons against 5 μm MPP+ (*p < 0.05), but failed to protect DA neurons against 5 μm 6‐OHDA (p < 0.05). Our results suggest that COX‐inhibitors may have neuroprotective benefits unrelated to inhibition of PG synthesis, and that 6‐OHDA and MPP+ have partially overlapping mechanisms of neurodegeneration possibly involving COX activity.

Acknowledgement:  Supported, in part, by the International Federation for Parkinson's disease, NY, NY.
Poster Sessions BP01: Energy Metabolism. NMR investigation of metabolic alterations in the brain of thiamine‐deficient rats
Authors: Navarro D. , Zwingmann C. , Hazell A. S. , Leibfritz D. , Butterworth R. F.
Published: July 2010

Thiamine deficiency (TD) results in region‐selective impairment of brain metabolism. Since thiamine is a cofactor for enzymes involved in glucose metabolism, 1H and 13C‐NMR was used to investigate metabolic fluxes through the major pathways of glucose metabolism in vulnerable (medial thalamus, MT; inferior colliculus, IC) and nonvulnerable brain structures of rats made thiamine deficient following treatment with the central thiamine antagonist pyrithiamine vs. pair‐fed controls. Symptomatic stages of TD resulted in decreased glutamate and GABA in MT an IC confirming previous biochemical studies. 13C‐isotopomer analysis revealed decreased de novo synthesis of [4–13C]glutamate (30%p < 0.02) and [2–13C]GABA (60%p < 0.01) in MT and IC consistent with decreased activities of pyruvate‐ and α‐ketoglutarate dehydrogenases. These changes were accompanied by decreased consumption of glucose and increased synthesis of lactate from [1–13C]glucose confirming decreased mitochondrial metabolism. Accumulation of glyceraldehyde‐3‐phosphate suggested inhibition of glucose flux through the thiamine‐deficient enzyme transketolase. Onset of symptoms of TD and significant cell death was accompanied by decreased neuronal marker molecules NAA and NAAG in MT. Focal lactate accumulation resulting from decreased activities of mitochondrial thiamine‐dependent enzymes appears to play a key role in the pathogenesis of selective neuronal cell death in TD. [funded by CIHR Canada].
13C‐[2,4]‐b‐hydroxybutyrate metabolism in human brain
Authors: Pan J. W. , De Graaf R. A. , Rothman D. L. , Hetherington H. P.
Published: July 2010

Although ketone metabolism is generally believed to be qualitatively similar to glucose consumption in the human brain, this has never been explicitly determined. We used 13C‐MR spectroscopy with infusions of 13C‐[2,4]‐b‐hydroxybutyrate (BHB, 200 mm, 100% enrichment) in overnight fasted adult subjects (n = 4) to examine the fate of this ketone. This was performed on a 2.1T Bruker spectrometer, volume size 6 × 4 × 6 cc3, previously determined to contain ∼ 56% gray, 39% white matter in normal adults. Results: The 2 h infusion increased plasma ketone levels from near negligible (baseline) to 2.25 ± 0.24 mm at steady state. Steady state 13C BHB levels were measured at 0.18 ± 0.04 mm. Similar to glucose, the 13C label appeared rapidly in the amino acid pools. The steady state fractional enrichment reached were: 13C‐4‐glutamate (4‐Glu), 6.78 ± 1.71%, 13C‐4‐glutamine (4‐Gln), 5.68 ± 1.84 and 13C‐3‐aspartate at 3.99 ± 0.57%. Discussion: The relatively low [BHB] of 0.18 ± 0.04 mm is an upper bound for tissue BHB, and is in agreement with the view that ketone consumption is largely regulated at the blood brain barrier in these nonfasted acutely hyperketonemic subjects. The general pattern of labeling of ketones is similar to that which has been previously seen with glucose. Steady state analysis of the ratio of 4‐Gln/4‐Glu suggests that ketones are predominantly used by neurons, and are consumed at a rate of 0.032 ± 0.009 mm/kg/min. This rate includes gray and white matter consumption. Dynamic modelling of the time courses of the brain 4‐Glu and 13C‐BHB, where gray matter ketone consumption is taken as three times greater than white matter (similar to that seen with glucose) gives an estimated upper bound for the flux of ketone oxidation in pure gray matter to be 0.041 ± 0.007 mm/kg/min.
Transgenic mice exhibiting oligodendrocyte‐specific expression of a mutant protein tyrosine phosphatase epsilon
Authors: Muja N. , Lovas G. , Romm E. , Machleder D. , Ranjan M. , Hudson L. D.
Published: July 2010

Reversible tyrosine phosphorylation is integral to oligodendrocyte differentiation, and one participant of the phosphorylation cycle, PTPε, is induced in developing oligodendrocytes (Ranjan and Hudson, 1996). To define the role of PTPε, we generated mice expressing a catalytically inactive, hemagglutinin‐epitope tagged PTPε (HA‐PTP ε) from the 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP) promoter. By competing with endogenous, normal PTPε for substrate binding, HA‐PTPε would behave in a dominant negative fashion when overexpressed in these mice. Transgene mRNA peaks at postnatal day 21, coincident with the maximal expression of myelin protein mRNAs. Immunohistochemical analyses using antibodies against the HA epitope tag demonstrate that HA‐PTPε is expressed in oligodendrocytes, but not in astrocytes and neurons. HA immunoreactivity was present in all myelinated brain structures including the corpus callosum, anterior commissure, and fornix, as well as in the subcortical, cerebellar, and spinal cord white matter. Gross differences in myelination or oligodendrocyte cell density in these brain regions were not detected using antibodies against CNP, myelin basic protein, and an oligodendrocyte marker, CC1. However, by EM axons of the optic nerve appear smaller and less extensively myelinated in transgenic mice than in wild‐type littermates. Studies are underway to determine the functional effects of transgene expression on conduction velocity, on the profile of expressed genes, and on potential phosphorylated protein targets of PTPε.
Glutamatergic effects of dynorphin peptides on cultured oligodendrocytes
Authors: Knapp P. E. , Rizzo J. P. , Alimova Y. , Singh I. , Goody R. J. , Hauser K. F.
Published: July 2010

Our previous work has shown that oligodendrocytes (OLs), in addition to neurons, can synthesize the endogenous opioid dynorphin A (dyn A). Opioid effects of dynorphin are preferentially mediated through kappa opioid receptors (KORs). Signalling through KORs appears to promote OL survival in culture since blockade of KORs with the specific antagonist nor‐binaltorphamine significantly reduces OL survival and exacerbates glutamate toxicity. Structure‐activity studies have shown that kappa ligand activity resides in the N‐terminus of dyn A derived‐peptides, while C‐terminal fragments exhibit glutamatergic effects on neurons through NMDA and/or AMPA/kainate receptors. The present study was undertaken to determine whether dynorphin peptides also have glutamatergic effects on OLs. Since OLs grown in vitro are thought to have an AMPA/kainate phenotype, OLs were treated with dyn A (1–17) or dyn A (2–17) in the presence or absence of CNQX. Survival of mature and immature OLs was assessed over 72 h using a sensitive repeated measures assay. Dyn A (1–17) had no effect on OL survival. Dyn A (2–17), a dyn A‐derived peptide with nominal opioid activity, significantly reduced the survival of both immature and mature OLs. The effect was completely reversible in mature OLs by addition of CNQX at 2 h prior to dyn A (2–17), but not by simultaneous addition of CNQX. Of great interest, CNQX was unable to reverse the toxicity of dyn A (2–17) on immature OLs, suggesting that dynorphin toxicity in this population is mediated via an alternate glutamate receptor type. CNQX alone had no effect on OL viability. Results suggest that elevated dynorphin levels in CNS disease and trauma may contribute to demyelinating pathologies.

Acknowledgements: Supported by Natl. MS Soc. (PEK) & NIH DA 13559 (KFH).
Anti‐S‐nitrosocysteine antibodies in multiple sclerosis: emergence of a new marker of clinical activity
Authors: Boullerne A. I. , Schmidt S. , Brochet B. , Jensen M. A. , Petry K. G. , Arnason B. G. W.
Published: July 2010

Multiple sclerosis (MS) is characterized by inflammation within the CNS. This inflammatory response is associated with production of nitric oxide (NO) and NO‐related species that nitrosylate thiols. We postulated that MS patients would exhibit an antibody (Ab) response directed against proteins containing S‐nitrosocysteine (SNO‐cysteine) and showed that anti‐SNO‐cysteine Abs of the IgM isotype are often present in the sera of MS patients (Boullerne et al. 1995, J. Neuroimmunol. 60, 117–124; Boullerne et al. 2002, J. Neurosci.22, 123–132). A seemingly identical Ab response directed against SNO‐cysteine in experimental autoimmune encephalomyelitis (EAE) induced in Lewis rats with the MBP68‐84 peptide, peaked in serum one week before onset of clinical signs, suggesting a predictive role for clinical activity. In relapsing–remitting MS, we found elevated anti‐SNO‐cysteine Ab at times of relapse with normal values in most patients judged to be in remission or experiencing suspected but not definite attacks. The anti‐SNOcysteine Ab response returns to normal 2–3 months after a relapse. Anti‐SNO‐cysteine Ab levels are also elevated in progressive disease. Sixty per cent non treated secondary progressive MS (SPMS) were positive as were 50% of primary progressive MS. In treated SPMS patients anti‐SNOcysteine positivity was 45%. Anti‐SNOcysteine Ab levels correlated inversely with EDSS (Spearman coefficient r = −0.26, p = 0.01, n = 101), suggesting that the inflammatory component is reduced at high EDSS.

Acknowledgements:  This work is supported by NMSS, Berlex and the Alan Friend Multiple Sclerosis Research Fund (B.G.W.A.).
The mediterranean fever gene modifies the progression of disability in non‐Ashkenazi Jewish multiple sclerosis patients
Authors: Shinar Y. , Livneh A. , Vila Y. , Pinhasov A. , Zeitoun I. , Achiron A.
Published: July 2010

MS is an autoimmune, CNS demyelinating disease manifested in most patients with progressive disability. The progression rate varies between patients and may depend on modifier, immune related genes. The Mediterranean fever gene, expressed in peripheral blood leukocytes, is responsible for familial Mediterranean fever (FMF), a recessive, periodic autoinflammatory disease prevalent in Semitic populations, and less penetrant in Ashkenazi Jews. We related common, FMF associated MEFV mutations to the progression of disability in Jewish, relapsing remitting (RR) MS patients. The mutations 148Q, 694V, 695R and 726A were identified by enzymatic restriction of PCR‐amplified MEFV DNA. The progression to statuses 3 and 6 of the expanded disability status scale (EDSS) was analyzed on survival plots. 35% of 48 non‐Ashkenazi patients had one MEFV mutation. Compared to non‐carriers (n = 31) the heterozygous cohort (n = 17) represented with an increased fraction reaching both EDSS statuses (p < 0.05), and with a shorter median time to reach both EDSS = 3 (2 years in carriers vs. 10 years in non‐carriers, p < 0.01) and EDSS = 6 (6 vs. 23 years, respectively, p < 0.005). 17% of 71 Ashkenazi patients had one MEFV mutation. There was no significant difference in the fraction of disabled or in the progression of disability between Ashkenazi carrier patients and non‐carriers. The susceptibility of the non‐Ashkenazi group attributed, in part, to the detrimental non‐Ashkenazi 694V mutation. The results suggest phenotypic expression of one mutated MEFV gene in non‐ashkenazi patients, pertinent to the pathogenesis of disability.

Acknowledgements:  Granted by the Israeli Ministry of Science (#6279).
Manipulation of brain iron status in two mutant mouse models: effects on myelin
Authors: Connor J. R. , Thompson K. , Ortiz E. , Pasquini J. M.
Published: July 2010

A number of observations point to iron as an essential factor in myelination and oligodendrocyte biology. However, the specific role of iron in myelin production and maintenance is not known. This role could be as an essential co‐factor, as a transcriptional or translational regulator or a metabolic fuel. We introduce two animal models each with a unique defect in iron metabolism to begin to dissect the role of iron in myelin production. One of the animal models, the hypotransferrinemic (hpx) mouse, lacks the ability to make transferrin. This animal represents a model of compromised ability to mobilize iron within oligodendrocytes. The other animal model is a heterozygote for a null mutation in H‐ferritin the iron storage protein. Thus these oligodendrocytes have limited capacity to store iron. In the hpx mice, the total myelin protein concentration is decreased by 20% (p < 0.05) and the galactolipid content is decreased by 21% (p < 0.05), whereas the phospholipid content was unchanged. CNPase and PLP were significantly higher in the wild type controls compared to the hpx mice, but MBP18 and MBP17 were higher in the hpx mice. The decrease in H‐ferritin expression was associated with a 22% decrease in myelin proteins with only the decrease in PLP reaching statistical significance among the individual proteins. The galactolipids decreased by 20% but there was no significant difference in the phospholipids. These data demonstrate that compromised iron status has a selective effect on galactolipids but not phospholipids in myelin. The protein content of myelin is also altered, but there are specific changes in myelin protein content based on the type of change in iron metabolism.
Structure and regional distribution of low molecular weight myelin proteolipids
Authors: Bizzozero O. A. , Malkoski S. P. , Mobarak C. D. , Evans J. E. , Dejesus G.
Published: July 2010

We have investigated the structure of the native PLP, DM‐20 and several low molecular weight proteolipids (LMWPs) by mass spectrometry (MS). The various proteolipid species were isolated from bovine spinal cord by size‐exclusion and ion‐exchange chromatography. Matrix‐assisted laser desorption ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS) of PLP and DM‐20 revealed molecular masses of 31.6 and 27.2 kDa, respectively, which is consistent with the presence of six and four molecules of thioester‐bound fatty acids. Electrospray ionization‐MS analysis of the deacylated proteins produced the predicted molecular masses of the apoproteins, demonstrating that palmitoylation is the major modification of PLP, and that the majority of PLP and DM‐20 molecules in the CNS are fully acylated. A series of proteolipids with molecular masses from 12 to 18 kDa were also isolated and subjected to amino acid analysis, fatty acid analysis, N‐ and C‐terminal sequencing, tryptic digestion and peptide mapping by MALDI‐TOF‐MS. The results showed that these polypeptides correspond to the N‐terminal region (residues 1‐105/112) and C‐terminal region (residues 113/131‐276) of PLP, and they appear to be generated by natural proteolytic cleavage within the 50 amino acid‐long cytoplasmic domain. These LMWPs are derived from myelin, and are not generated during their isolation or during the postmortem period. Moreover, they are present in other species and are unevenly distributed across the CNS. It is tempting to speculate that these minor proteolipids may play a role in myelin formation.

Acknowledgements:  Supported by PHHS grant NS 38325.
Adenovirus infection induces microglial activation: involvement of mitogen‐activated protein kinase pathways
Authors: Bhat N. R. , Fan F.
Published: July 2010

Non‐replicating adenovirus (Ad) vectors represent effective tools for long‐term gene expression in the CNS, but they also elicit inflammation. The cellular and molecular mechanisms of such a response are not well‐understood. We show here that infection with Ad causes an activation of microglial cells, the key cells involved in inflammatory and immune‐regulatory functions in the brain. Exposure of cultured rat brain microglia to Ad resulted in an induced production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) and the pro‐inflammatory cytokine, TNFα. The roles of signal transduction pathways believed to be involved in microglial activation, in particular, mitogen‐activated protein kinases (MAPKs) and nuclear factor κB (NFκB) were explored by determining their activation in response to Ad infection and by testing the effects of specific pharmacological inhibitors. It was found that Ad strongly activates extracellular signal‐regulated kinase (ERK) and to a lesser extent, p38 MAPK but not NFκB. The kinase inhibitor i.e. PD98059, specific for the ERK pathway, potently inhibits and, in combination with the p38 MAPK inhibitor, SB203580, almost completely abolishes Ad‐induced expression of iNOS and TNFα. The results suggest that Ad uses cellular signal transduction machinery, in particular, the ERK pathway to elicit microglial activation and that increased production by these cells of inflammatory mediators may primarily contribute to CNS inflammatory responses commonly seen in models of gene therapy using Ad vectors.

Acknowledgements:  Supported by NINDS grant # NS41035‐01.
Inhibition of interferon‐gamma‐induced nitric oxide production in BV‐2 microglial cells by polyphenolic compounds
Authors: Zheng Q. , Binek J. , Sun G. Y.
Published: July 2010

Microglial cells are known to play an important role in mediating the host defense functions in the brain. These cells respond readily to pro‐inflammatory cytokines, which stimulate signalling pathways and induction of many genes, including the inducible nitric oxide synthase (iNOS). In the immortalized BV‐2 microglial cells, our studies have demonstrated that interferon gamma (IFNγ) alone could stimulate the induction of iNOS through activation of the nuclear factor‐kappa B (NF‐κB) pathway. Increased NO production in the brain has been implicated in enhanced oxidative stress associated with many neurodegenerative diseases. Many polyphenolic compounds in vegetables and fruits, including resveratrol, catechin, quercetin, genistein are regarded as phytoestrogens and are known to possess potent antioxidant properties. However, whether these compounds also offer anti‐inflammatory effects and inhibit cytokine‐induced NO production in microglial cells have not been examined in detail. The major goal for this project is to use BV‐2 microglial cells as cell model to investigate the effects of these polyphenolic compounds on IFNγ induction of NO. Results show that all the polyphenolic compounds tested could inhibit NO production albeit to different extents, with quercetin having the lowest IC50 and catechin the highest IC50. Surprising, daidzin, a non‐tyrosine kinase analog of genistein, also inhibited IFNγ‐induced NO production, suggesting that the inhibitory effects may not be due entirely to their action on the tyrosine kinase pathway. These results lead to the conclusion that polyphenolic compounds tested not only possess antioxidative but also anti‐inflammatory properties.

Acknowledgements:  Supported by 5 P01 ES10535 from NIH.
Double‐stranded RNA induces nitric oxide synthase in human astrocytes
Authors: Auch C. J. , Dasgupta S. , Jana M. , Liu X. , Pahan K.
Published: July 2010

Environmental factor(s), such as viral infection, has been implicated as one of the potential triggering events leading to demyelination in MS. This study underlines the importance of double‐stranded RNA (dsRNA), the active component of a viral infection that stimulates antiviral responses in infected cells, in inducing the expression of inducible nitric oxide synthase (iNOS) in human astroglia. Treatment of human U373MG astroglial cells with dsRNA in the form of synthetic polyinosinic‐polycytidylic acid (poly IC) induces the production of NO and the expression of iNOS in a dose‐dependent manner. Similar to human U373MG astroglial cells, poly IC also induces the expression of iNOS in human primary astroglia. Induction of human iNOS promoter‐derived luciferase activity by poly IC suggests that poly IC induces the transcription of iNOS. As activation of both NF‐κB and C/EBPb is necessary for the transcription of iNOS in human astroglia, we investigated the effect of poly IC on the activation of both NF‐κB and C/EBPb. Poly IC induced the activation of both NF‐κB and C/EBPb. Furthermore, inhibition of poly IC‐induced production of NO by Dp65, a dominant‐negative mutant of p65, and DC/EBPb, a dominant‐negative mutant of C/EBPb, suggests that poly IC induces the expression of iNOS in human astroglial cells through the activation of NF‐κB and C/EBPb. This study delineates a novel role of dsRNA in inducing the expression of iNOS in human astroglia which may participate in virus‐induced neurological abnormalities.

Acknowledgements:  This study was supported by NIH grants (NS39940 and AG19487).
Effects of calpain inhibition on IkBα degradation following PBMC activation: identification of calpain cleavage sites
Authors: Rocchini A. , Schaecher K. , Goust J. M. , Banik N. L.
Published: July 2010

We previously demonstrated calpain‐mediated activation of PBMCs. In order to examine whether calpain plays a role in the degradation of IkBα and NFkβ translocation in the mechanism of T cell activation, human PBMCs were activated using anti‐CD3/CD28 (HIT3A/CD28.2). Degradation of IkBα was monitored by Western blot, degradation products were sequenced, and calpain‐cleaved sites were determined. The activated cells degraded IkBα, the inhibitor of NFkβ, relative to non‐activated cells. IkBα degradation was progressive and inhibited by calpain inhibitor calpeptin in a dose and time dependent manner. Further, calpain inhibition increased IkBα levels above those observed in non‐activated negative controls. Recombinant IkBα was incubated with purified porcine mcalpain to initiate the degradation of IkBα. The degradation products were separated via SDS–PAGE and visualized using Coomassie Stain. The stained degradation products were isolated and subjected to sequencing. Calpain cleaved the recombinant IkBα most effectively when incubated with IkBα at a ratio of 1 : 1 for 20 min in the presence of 0.1% Triton‐X100. After incubation, several degradation products were detected, including a peptide corresponding to a cleavage of IkBα between amino acids 50 and 51 (glutamine and glutamic acid). The liberated fragment included the entire Signal Response Domain (SRD), a region containing key serine and threonine residues necessary for phosphorylation by the IKKinase complex and sites required for ubiquitination. These results show that calpain plays an important role in IkBα degradation, a crucial event in T cell activation and is involved in the normal turnover of IkBα.

Acknowledgements:  Supported by grants from NIH‐NINDS.
LPS and IL‐1α increase endogenous morphine production in mollusk Mytilus edulis pedal ganglia
Authors: Zhu W. , Cadet P. , Stefano G. B.
Published: July 2010

We have found that morphine and its metabolite, morphine‐6‐glucuronide, are endogenously produced in the ganglionic tissue of the marine mollusk Mytilus edulis. Stress, such as cold and starvation, appears to regulate the production of these opiate alkaloids. In order to determine which signals can trigger the release and possible synthesis of morphine in the mussel, we incubated dissected ganglia with lipopolysaccharide (LPS) and interleukin one alpha (IL‐1α). Using solid phase extraction, HPLC, RIA and mass spectrometry, we have found both compounds are able to increase morphine production at the micromole level. At 24 h post incubation, morphine reaches its highest level for LPS (42.63 ± 3.81 ng/mL) and IL‐1α (31.57 ± 6.24 ng/mL) respectly. These results are comparable to the data found in rats.
Complement inhibition ameliorates lupus cerebritis in NZB/W and MRL/LPR mice
Authors: Alexander J. J. , Bao L. , Anderson R. , Quigg R. J.
Published: July 2010

Systemic lupus erythematosus is a multigenic autoimmune disease in which multiple organs are involved including CNS. To examine the role of complement in the pathogenesis of lupus cerebritis, MRL mice with Fas deficiency (MRL/lpr) and NZB/W mice were studied. In these animals, Crry, a protein that inhibits both alternative and classical C3 convertases of the complement system, was overexpressed in CNS as a soluble protein, they were sacrificed at 15 and 24 weeks, respectively, and brains studied. Marked deposition of the pivotal complement component C3, and the terminal component C9, in brains of both mouse strains were decreased in those animals overexpressing Crry. RNA expression of the inflammatory cytokine, TNF‐a and inducible nitric oxide synthase were up‐regulated in lupus mice; these increases were abrogated in the presence of Crry. Programmed cell death is associated with the pathology of autoimmune diseases. Therefore, apoptosis was studied using TUNEL staining and examination of DNA laddering. A large number of TUNEL‐stained cells were present in lupus mouse brains and were reduced by complement inhibition. DNA laddering was seen to increase with the progression of the disease in both models and was markedly reduced by Crry. Further, caspase‐3 expression was reduced in these mice as compared to their lupus counterparts. Our results clearly demonstrate that apoptosis in these lupus models occurred by a Fas‐independent mechanism, as it occurred in MRL/lpr mice. Further, complement inhibition led to a significant amelioration of neurodegeneration in lupus, as indicated by measures of the extent of apoptosis. This reversal of disease suggests complement inhibition may be a therapeutic avenue for lupus cerebritis.
Poster Sessions BP06: Neurogenesis, Stem Cells and Apoptosis ‘DECOY’ oligonucleotides' effects on NF‐κB regulation of bcl‐x gene expression in rat hippocampus
Authors: Qiu J. , Grafe M. R. , Martin S. S. , Kent T. A. , Rassin D. K. , Perez‐Polo J. R.
Published: July 2010

The NF‐κB transcription factor regulates bcl‐x gene expression, which may determine hypoxia‐induced neuronal apoptosis. We examined hypoxia‐induced NF‐κB and Bcl‐xL changes in rat hippocampus. We showed differential hypoxia‐induced NF‐κB binding to the bcl‐x promoter CS4 and IgG–κB enhancer sequences by rat hippocampal nuclear extracts. The differential NF‐κB binding to these two promoter sequences was also determined in a contused spinal cord injury model and in vitro studies with LPS‐treated Hela cells. There was tissue‐, gene promoter‐specific and time‐dependent regulation of bcl‐x gene expression by NF‐κB in support of the hypothesis that NF‐κB has tissue‐ and gene‐specific regulatory effects and that these effects may account for both the pro‐ and anti‐apoptotic roles assigned to NF‐κB in different apoptotic processes. We applied ‘decoy’ oligonucleotides with sequences specific to different promoters to the rat hippocampus and measured ‘decoy’ inhibitory effects on nuclear NF‐κB binding. The IgG‐κB enhancer sequence ‘decoy’ showed stronger inhibition on nuclear NF‐κB c‐Rel/p50 binding to the bcl‐x gene promoter CS4 sequence when compared to NF‐κB p50/p50 binding to the same sequence. This result suggests that the ‘decoy’ approach has the potential to selectively manipulate NF‐κB regulation of gene expression in response to hypoxia.

Acknowledgements:  Supported by NINDS NS‐39161, Shriner Grant 8710 and a Grant from the Sealy Center on Aging to J. Qiu.
Cyclic GMP is more effective than cyclic AMP or GDNF in protecting mature oligodendrocytes from cell death
Authors: Benjamins J. A. , Nedelkoska L.
Published: July 2010

During immune‐mediated or ischemic attack, oligodendrocytes (OLs) receive a barrage of signals, both protective and injurious. The specific ligands at the site of damage in vivo vary widely in type and concentration. Using cultured mature mouse OLs, we have focused on four models of cell death which represent common pathways initiated by a number of ligands. These include (1) staurosporine, which induces peroxynitrite formation and subsequent release of cytochrome c; (2) thapsigargin, which disrupts Ca++ homeostasis in the endoplasmic reticulum; (3) kainate, which activates non‐NMDA receptors; and (4) SNAP and NOC‐18, which release NO. GDNF protects only against cell death induced by staurosporine, and acts via PI‐3‐kinase but not MAP kinase to protect. Elevation of cyclic AMP by addition of 8Br‐cyclic AMP or forskolin protects against staurosporine, but does not increase the protective effect of GDNF when added in combination. In addition, elevation of cyclic AMP protects against cell death initiated by thapsigargin, but not kainate or SNAP. Inhibition of protein kinase C with calphostin protects only against kainate. Of the possible protective agents tested, 8Br‐cyclic GMP is the only one which protects OLs in all four models of cell death. These results highlight the importance of signaling via cyclic GMP in survival pathways in mature OLs.

Acknowledgements:  Supported by grant NS13143 from NIH and grant RG 3030A/6 from the National Multiple Sclerosis Society.