bluuebunny/crossref_metadata_2025 huggingface dataset:
Poster Sessions CP10: Blood–Brain Barrier. Expression of vitamin C transporter SVCT2 in hypothalamic glial cells. An immunohistochemical and kinetic analysis
Authors: García M. A. , Montecinos H. , Balmaceda‐Aguilera C. , Millán C. , Nualart F.
Published: July 2010

Kinetic analysis of vitamin C uptake has demonstrated that specialized cells take up ascorbic acid (AA), the reduced form of vitamin C, through sodium‐AA cotransporters. Recently, two different isoforms of sodium‐vitamin C cotransporters (SVCT 1, 2) that mediate high affinity Na+‐dependent l‐ascorbic acid have been cloned. SVCT2 was detected mainly in choroid plexus cells and neurons, however, there are no evidences of SVCT2 expression in glial cells. High concentrations of vitamin C has been demonstrated in brain hypothalamic area. The hypothalamic glial cells, known as alpha and beta tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid and the portal blood of the median eminence. Our hypothesis postulates that tanycytes take up reduced vitamin C from the portal blood and cerebrospinal fluid generating an high concentration of this vitamin in brain hypothalamic area. In situ immunohistochemical analyses demonstrated that SVCT2 transporter is selectively expressed in apical region of tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of SVCT2 isoform in these cells. Reduced vitamin C uptake was temperature and sodium dependent. Kinetic analysis showed an apparent Km of 20 μm and a Vmax of 45 pmol/min per million cells for the transport of ascorbic acid. The expression of SVCT2 was confirmed by immunoblots and RT–PCR. Tanycytes may perform a neuroprotective role concentrating the vitamin C in the hypothalamic area.

Acknowledgements:  Supported by Grands FONDECYT 1010843 and DIUC‐GIA 201.034.006‐1.4 from Concepción University.
Interleukin‐1 increases expression of cytosolic and membrane PGE synthase in mouse astrocytes and brain
Authors: O'Banion M. K. , Maida M. E. , Moore A. H.
Published: July 2010

Increased production of prostaglandin E2 (PGE2) accompanies inflammatory responses, both peripherally and in the brain. Previous studies have demonstrated marked up‐regulation of cyclooxygenase‐2 (COX‐2) expression by IL‐1β in mouse astrocytes and brain. However, selective increases in PGE2 production are dependent on expression of downstream PGE synthases. Therefore, we have investigated regulation of two PGE synthase isoforms in cultured murine astrocytes and in mouse brain treated with IL‐1β. In astrocytes exposed to 10 ng/mL IL‐1β we see an early and dramatic increase in cytosolic PGE synthase mRNA and protein expression at 4 and 8 h that largely subsides by 24 h. This pattern is similar to that observed for COX‐2 expression. Interestingly, increases in membrane PGE synthase levels also occur, but with a delayed time course. Similar increases in both isoforms are observed following intraparenchymal injection of 5 ng recombinant IL‐1β. More specifically, the in vivo increase in COX‐2 mRNA was accompanied by an increase in membrane PGE synthase mRNA levels 6 h following IL‐1β injection. Subsequently, protein expression of both the cytosolic and membrane PGE synthases was elevated at 24 h. Interestingly, administration of NS‐398, a COX‐2 specific inhibitor, attenuated IL‐1β‐induced changes in PGE synthase expression, suggesting regulation of these isoforms by a COX‐2‐dependent mechanism. Together these data suggest that up‐regulation of both PGE synthase isoforms contributes to cerebral inflammatory responses to IL‐1β.

Acknowledgements:  Supported by RO1 NS33553 to MKO.
Disruption of PPARβ results in distinct gender differences in mouse brain phospholipid
Authors: Rosenberger T. A. , Hovda J. T. , Peters J. M.
Published: July 2010

A homozygous PPARβ‐null mouse has been developed in which the ligand‐binding domain of the PPARβ receptor is disrupted. Analysis of brains from these animals show that female null mice have a 24 and 17% increase of plasmenylethanolamine and phosphatidylserine and a 9% decrease in the level of phosphatidylinositol when compared to controls. The phospholipid changes found in female null mice were associated with significant increases in esterified 18:1n‐9, 20:1n‐9, 20:4n‐6, and 22:5n‐3 in plasmenylethanolamine, 20:1n‐9 in phosphatidylinositol, and 18:0, 18:1n‐9, 18:3n‐6, 20:1n‐9, and 20:4n‐6 in phosphatidylserine. Increased esterified 18:1n‐9, 18:2n‐6, 18:3n‐6, and 20:1n‐9 were also found in the phosphatidylethanolamine fraction despite its cellular content remaining unchanged. Brain phospholipid content in male PPARβ‐null mice was not different than controls, but increased levels of 20:1n‐9 in the phosphatidylinositol and 18:1n‐9 in the phosphatidylserine fractions were observed. No changes were found in the content of brain cholesterol, triglycerides, and free fatty acid in either female or male PPARβ‐null mice. These data suggests that the peroxisomal proliferator‐activated receptor beta is involved in maintaining fatty acid and phospholipid levels in adult female mouse brain and provides strong evidence suggesting a role for PPARβ in peroxisomal acyl‐CoA utilization.
NMR study of 13C‐glucose and 13C‐acetate metabolism in the brain of rats with acute liver failure
Authors: Zwingmann C. , Chatauret N. , Leibfritz D. , Butterworth R. F.
Published: July 2010

Acute liver failure (ALF) is characterized by hepatic encephalopathy (HE) with brain edema as the main cause of death. The role of brain energy failure in ALF is still controversial. In the present study we investigated the association of encephalopathy with brain energy metabolism in rats with ALF induced by liver devascularization. Using [1–13]glucose and [2–13C]acetate combined with [1H, 13C]NMR spectroscopy anaerobic and aerobic carbon fluxes were studied in brain extracts from ALF rats. The anaplerotic flux of 13C‐label from glucose was found to be stimulated resulting in a 10‐fold increase of [13C]glutamine (Gln) starting at precoma stages of encephalopathy. In contrast, de novo synthesis of alanine and lactate increased to 320 and 210% in precoma, followed by a further elevation at coma stages to 615 and 425% of sham‐operated control values. Using [2–13C]acetate, the higher 13C‐enrichment of C‐2 in Gln, glutamate (Glu) and the corresponding C‐4 in GABA compared to C‐3 of either compound suggests cleavage of [2–13C]citrate and formation of [3–13C]oxaloacetate, which enters the glial TCA cycle. On the other hand, decreased synthesis of [4–13C]Glu and [2–13C]GABA was observed. This may result from decreased neuronal oxidative metabolism, since the ratio of [4–13C]Glu/[2–13C]GABA is unchanged and increased [2–13C]Glu and corresponding [4–13C]GABA suggests a stimulated flux of astrocytic label to neurons. Impaired neuronal energy metabolism and selective stimulation of lactate synthesis may contribute to the pathogenesis of HE in ALF.

Acknowledgements: Funded by CIHR Canada.
Poster Sessions CP11: Neurotoxicity and Neuroprotection. Secretory phospholipase A2 signalling partly overlaps glutamate‐mediated events
Authors: Kolko M. , Rodriguez de Turco E. B. , Diemer N. H. , Bazan N. G.
Published: July 2010

Here we explored the mechanisms of secretory phospholipase A2 (sPLA2) and glutamate (glu) in neuronal signalling and cell damage. Rats or primary neuronal cultures were treated with MK‐801 and injected with/exposed to sPLA2 or glu. MK‐801 partially inhibited sPLA2‐ and glu‐induced neuronal death as well as [3H]arachidonic acid release. The involvement of cytosolic PLA2 (cPLA2) and plateletactivating factor (PAF) in sPLA2 or glu signalling was explored by treating cells with the selective cPLA2 inhibitor, AACOCF3, PAF‐acetyl hydrolase (PAF‐AH) or the presynaptic PAF‐receptor antagonist, BN52021. AACOCF3 blocked sPLA2‐ and glu‐induced neuronal death by 26 and 77%, respectively. PAF‐AH ameliorated sPLA2 as well as glu neurotoxicity by 31 and 47%, whereas BN52021 inhibited sPLA2 induced neurotoxicity by 11% but did not significantly protect against glu‐induced neurotoxicity. Expression in neurons of early response genes in response to sPLA2 or glu was further examined. An up‐regulation of COX‐2, c‐fos, and c‐jun, but not COX‐1, was observed at earlier time points after rat striatal injection of glu as compared to sPLA2 injection. Moreover we treated neuronal cells with COX‐2 inhibitors and found that neuronal cell death after sPLA2 and glu exposure was inhibited by 35 and 33%, respectively. Thus sPLA2 activates a neuronal signalling cascade that includes activation of cPLA2, AA‐release, production of PAF and induction of COX‐2. Hence sPLA2 and glu signalling are overlapping, but not identical. Cytosolic PLA2 may primarily drive glutamatergic neurotransmission, whereas PAF plays a more crucial role in sPLA2 neuronal signalling.

Acknowledgements:  Supported by EPSCoR grant NSF/LEQSF(2001‐04)‐RII‐01 from the National Science Foundation.
Injury‐induced differential gene expression in laser captured hippocampal pyramidal and dentate granule cells
Authors: Lefebvre d'Hellencourt C. , Harry G. J.
Published: July 2010

Trimethyltin (TMT) is a neurotoxicant that produces a specific pattern of neuronal degeneration in the hippocampus. In mice, TMT induces a selective neuronal loss of dentate granule cells, astrocyte hypertrophy, and microglia activation. Previous studies in our laboratory demonstrated an early elevation in TNFα mRNA levels suggesting a role in initiation of the injury response. To investigate the role of TNF in this differential susceptibility of distinct neuronal populations, laser capture microdissection (LCM) was used to isolate cells in either the dentate gyrus (DG) or the CA pyramidal layer. RNA levels for TNFα, TNF receptor (TNFR) 1 and TNFR2 were measured in each distinct LCM region by real time RT–PCR. In control tissue, mRNA levels of TNFR2 and, to a lesser extent, TNFR1 were higher in cells of the DG as compared to the pyramidal cell layer. 24 h post‐TMT, the level of TNFα transcript was elevated. Consistent with the pattern of neuronal susceptibility, levels were significantly greater in the dentate region. While this differential pattern was evident at the time of neuronal necrosis, we examined differential effects at an earlier time point of 6 h. RNA from each region, as obtained by LCM, was amplified by a PCR‐based method, and hybridized to 1176 mouse cDNA arrays. Diverse categories of gene were differently express in the two regions including transcription factors (NEX‐1, LKLF), chemokines (MIP1α and β), G proteins (guanine nucleotide binding proteins) and protein kinase (PKCβ, PKCδ, KCIP1). Thus, prior to any evidence of neurodegeneration, significant alterations were detected in distinct cell regions. These results demonstrate the utility of applying these techniques to examine differential vulnerability of neurons in various models of neurodegeneration.
MDMA‐ and METH‐induced neurotoxicity in mice: sensitization and desensitization to psychostimulants effects
Authors: Ali S. F. , Itzhak Y.
Published: July 2010

Methylenedioxymethamphetamine (MDMA; ‘ecstasy’) and methamphetamine (METH)‐induced neurotoxicity, however, little is known about the behavioral consequences of exposure to neurotoxic doses of these substituted amphetamines. In the present study two behavioral paradigms were monitored after the exposure of mice to METH and MDMA. Swiss Webster mice were treated with METH (5 mg/kg × 3) which caused 50–65% depletion of striatal dopaminergic markers. This treatment resulted in long‐lasting sensitization to the psychomotor stimulating effect of METH (74 days after the initial exposure to METH). In the CPP paradigm, control saline pretreated mice acquired significant CPP following METH administration (0.5 mg/kg), while the METH pretreated mice acquired very low conditioned response. Following the extinction of CPP, the conditioned response to METH was completely reinstated in the control but not the METH group. The administration of MDMA (30 mg/kg × 2) resulted in 40–60% depletion of striatal and cortical dopaminergic and serotonergic markers, respectively. This treatment was associated with marked sensitization to the psychomotor stimulating effects of MDMA, METH and cocaine as determined at various intervals after the initial exposure to MDMA. These results suggest that amphetamines‐induced neurotoxicity is associated with two opposing behavioral outcomes: (1) sensitization to the psychomotor stimulating effects and (2) desensitization to the rewarding effects of related drugs. These consequences may be relevant to the psychopathology of METH and MDMA abuse.

Acknowledgements:  Supported by NIDA DA08584 and DA12867.
Estrogen attenuates oxidative stress‐induced apoptosis in C6 glial cells
Authors: Sur P. , Sribnick E. A. , Wingrave J. M. , Nowak M. F. , Ray S. K. , Banik N. L.
Published: July 2010

The neuroprotective effects of estrogen have been previously implicated in neurodegenerative diseases. In order to examine whether estrogen protects cells from calpain‐mediated death, we investigated the mechanism of estrogen's inhibitory effects of apoptotic death in C6 (rat glioma) cells following exposure to H2O2. Cells were preincubated with 4 μm estrogen for 2 h and then exposed to 100 μm H2O2 or 2 μm ionomycin for 24 h. Intracellular free calcium (Ca2+) was quantified using Fura‐2. Levels of cell death were measured by colorometric assay. Bax/Bcl‐2 ratio and calpain activity, as measured by calpain‐cleaved spectrin degradation, were monitored by Western blot. Apoptosis was evaluated morphologically by Wright staining. Treatment with H2O2 caused significant increases in intracellular [Ca2+] which were attenuated by preincubation with estrogen. H2O2 and ionomycin caused cell death in a dose‐dependent manner. Preincubation with estrogen increased viability in cells treated with H2O2 but not in cells treated with ionomycin. Western blotting showed an increase in Bax/Bcl‐2 ratio and calpain activity following treatment with H2O2, and estrogen pretreatment decreased levels of both. Cell morphology indicated apoptosis in cells treated with H2O2, and pretreatment with estrogen reduced apoptosis. These results indicate a role for estrogen in preventing apoptosis in C6 glial cells exposed to H2O2. Our results suggest that estrogen may have a protective role in minimizing glial cell death in neurological diseases.

Acknowledgements:  Supported in part by grants from NIH‐NINDS and South Carolina Electric and Gas.
Mechanism of the protective effect of hypothermia on ammonia toxicity in astrocytes
Authors: Zwingmann C. , Leibfritz D. , Butterworth R. F.
Published: July 2010

Ammonia is a key factor in the pathogenesis of hepatic encephalopathy (HE). Acute ammonia treatment causes energy failure of astrocytes, which are able to compensate partly by increased anaerobic metabolism as a means of making up for the energetic shortfall. As hypothermia offers protection from severe encephalopathy and lactate accumulation in liver failure, we investigated the mechanism by which hypothermia protects against ammonia toxicity by multinuclear NMR spectroscopy. 12 h exposure to 5 mm NH4CL decreased the phosphocreatine (PCr)/creatine (Cr) and ATP/ADP ratios to 65 and 76% of control, increased synthesis and release of glutamine to 200–250% and led to a significant stimulation of glycolytic activity reflected by increased uptake and consumption of glucose and accumulation of de novo synthesized intra‐ and extracellular lactate to 161 and 230% of control. The protective effect of mild hypothermia was evident from inhibiton of lactate accumulation and restoration of ammonia‐induced depletion of PCr/Cr. Moderate hypothermia led to an increase of PCr/Cr ratio and inhibited lactate synthesis to 14% of normothermic control, but did not prevent the ATP decrease. While hypothermia inhibited glycolytic flux, intracellular glutamine remained elevated. The results suggest that hypothermia‐induced protection against ammonia toxicity results from reduction of cellular energy demand leading to inhibition of anaerobic glucose metabolism and a compensatory stimulation of mitochondrial energy production.

Acknowledgements:  Funded by CIHR Canada.
Trimethyltin induced hippocampal neurodegeneration elevates cyclins A and B
Authors: McPherson C. A. , Wine R. N. , Kubik J. A. , Harry G. J.
Published: July 2010

In certain pathologic states, the response of microglia and associated cytokine production may become spatially and temporally dysregulated contributing to neurodegeneration. Often these responses have been contributed to proliferation of microglia cells. To examine cell cycle related genes and proteins in a model of acute neurodegeneration, the hippocampal toxicant, trimethyltin (TMT; 2.0 mg/kg), was administered to 21 day old CD‐1 male mice and the level of cell cycle genes and cellular localization of proteins examined. Neurodegeneration was characterized by neuronal necrosis in the dentate followed by microglia reactivity and activation. mRNA levels for cyclins A2 and B1 were significantly elevated 72 h post‐TMT. Western blot analysis indicated minimal increase in whole hippocampal protein levels for cyclin A or B however, immunohistochemical examination of cellular localization demonstrated increased staining for cyclin A primarily in neurons in both the dentate and pyramidal cell layers as early at 24 h post‐TMT. Immunohistochemisttry for cyclin B1 showed a peak level of staining at 24 and 48 h in both the dentate and pyramidal cell layers with prominent staining both around the neuronal cell body and in fibrous processes. The staining pattern for each cyclin suggested the involvement of both neurons and microglia. BrdU positive cells were demonstrated predominately in the dentate region and appeared to be primarily neurons with minimal contribution from microglia cells suggesting that the alteration in cell cycle genes is not due to an injury‐induced proliferative response of microglia cells in the hippocampus.
Dehydroevodiamine.HCl protects neuron from cytotoxicity induced by Aβ or oxidative stress: its antioxidant
Authors: Choi S. H. , Park C. H. , Seo J. H. , Suh Y‐H.
Published: July 2010

We previously found that DHED has memory enhancing and neuroprotective activities using various in vitro and in vivo models. In the present study, we further investigated the effects of DHED on neurotoxicities induced by glutamate (Glu), hydrogen peroxide (H2O2) and amyloid beta peptide (Aβ) to its exact neuroprotective mechanism in a primary cultured neuron of rat. In addition, we assayed binding ability of DHED on N‐methyl‐D‐Aspartate (NMDA) receptor. A pretreatment of DHED (0.5, 1, 2, 4 μm) reduced significantly cell death induced by Glu (1 mm), H2O2 (150 μm) and Aβ (10 μm) in a dose‐dependent manner. In the H2O2 treated neuron, the production of reactive oxygen species (ROS) and malondialdehyde (MDA) were decreased by the pretreatment of DHED (0.5, 1, 2, 4 μm). Binding of MK‐801 to NMDA receptor was antagonized dose‐dependently by DHED (IC50; 32.9 μm). These results suggest that DHED attenuate neuronal dysfunction and death in brain disorders related to excitotoxicity and Ab through antagonizing NMDA receptor and oxidative stress. Therefore, these studies provide direct evidence that DHED might exert therapeutic action on pathological and behavioral changes in various brain diseases including Alzheimer's disease and stroke through multineuroprotective mechanisms.
Dipyridamole protects cultured rat embryonic cortical neurons from neurotoxic insult
Authors: Blake A. D.
Published: July 2010

The effects of a clinically useful cardiovascular agent, dipyridamole, were examined in a rodent tissue culture model of neural protection. Dipyridamole effectively protected rat embryonic day 18 (E18) cortical neurons from either trophic deprivation or endogenous glutathione depletion by l‐buthionine (R,S) sulfoximine (BSO). Trophic deprivation was associated with an increase in intracellular oxidative stress, as determined by the increased fluorescence of dichloro, dihydrofluorescein (H2DCFDA). Dipyridamole's neural protection was time and concentration‐dependent (EC50 = 342 nm), and its continuous presence in the culture medium was required. Dipyridamole or exogenously added glutathione markedly decreased trophic deprivation induced H2DCFDA fluorescence, indicating a reduction in neuronal oxidative stress. These results demonstrate that dipyridamole protects primary neuronal cultures against either trophic or chemically mediated insults, and suggest that dipyridamole has a potent antioxidant ability that compensates for glutathione depletion in primary neuronal cells.
Quantitative assessment of gene expression following acute spinal cord injury
Authors: Earnhardt J. N. , McCray B. , Dobrzeniecka M. , Keeler B. E. , Tabernilla A. V. C. , Nick H. S. , Anderson D. K. , Evelyn F. , William L.
Published: July 2010

A unique gene expression pattern/transcriptome characterizes the presentation of every disease state. Strategies for identification of gene families with altered expression patterns offer an information rich approach to initially assess the relevance of gene expression to disease pathology. To identify important candidate genes critical to the biochemical mechanisms mediating spinal cord injury (SCI), we have selected gene families that may have a relevant impact on SCI for analysis by real time PCR. Using this quantitative, fluorescence‐based method for the real time detection of PCR, we have evaluated the expression of 80 known genes at time points up to 24 h post SCI. These families/groups include the TGF‐β ligand, receptor and Smad superfamily; matrix metalloproteinases; and genes involved in angiogenesis and wound healing; free radical metabolism; cell death; as well as miscellaneous growth and regulatory factors. The gene expression patterns were evaluated in samples from 12 and 24 h post injury or sham controls using a rat contusion model. 36 genes had a two or greater cycle number difference between at least one injury time point and control. 10 genes exhibiting the largest change with a four or greater cycle number difference compared to controls were also analyzed at 2 and 6 h post injury. Additional real time PCR studies were performed on 4 of these 10 genes at all time points directly relating changes in cycle number with fold induction. The ability to rapidly and directly quantitate gene expression has provided a powerful approach to elucidate genes potentially relevant to cellular events in SCI.
A study of juvenile rat spinal cord injury
Authors: Wingrave J. M. , Matzelle D. D. , Wilford G. G. , Hogan E. L. , Banik N. L.
Published: July 2010

Greater than 5% of all spinal cord injuries (SCI) in the US occur in people younger than 16, although a minority, children will require extended attention during their lifetime. While facing increased mortality in the initial 24 h after trauma, children with incomplete injuries seem to have a greater capacity for recovery of function compared to adults suggesting that there is a difference in injury tolerance in the young over the adult. Knowledge of the factors involved in this difference would not only increase understanding of SCI, but also potentiate new avenues for SCI treatment. Yet there has not been a model for the study of youth SCI. For these reasons, we developed a model of SCI in juvenile rats equivalent to an adult injury of 25 g cm force (GCF). To do so, we recorded spinal cord masses of Sprague–Dawley rats at 21, 30, 45, and 60 days of age, compared them to adult cord masses, and assembled a conversion factor that provides youth injuries comparable to adult. To investigate the pathophysiology in juvenile SCI, two cord segments, 1 cm long, were removed from animals 24 h following injury. One segment was centered at the impact site, the other immediately caudal. After homogenization, the samples were assayed by Western blot analysis for calpain content and degradation of 68K Neuro‐Filament Protein (68K NFP), a neuronal structural protein. mCalpain expression, a neutral protease previously implicated in secondary SCI, was reduced in juvenile animals relative to adult cohorts. The degradation of 68K NFP was also found to be reduced in juvenile animals. From these analyses, it seems plausible that calpain expression and pathogenic activity is abated in the setting of young rat SCI.

Acknowledgements:  Supported by grants from NIH‐NINDS.
Interleukin 1 dependent cell loss after spinal cord injury (SCI)
Authors: Nesic O. , Svrakic N. M. , Xu G.‐Y. , McAdoo D. , Westlund K. N. , Hulsebosch C. E. , Perez‐Polo R. J.
Published: July 2010

We hypothesized that blocking receptors for pro‐inflammatory cytokine, interleukin‐1 (IL‐1) would ameliorate SCI‐dependent cell loss and resulting motor dysfunction. We found that chronic administration of recombinant IL‐1 receptor antagonist (rIL‐1ra) reduced SCI‐induced increases in cell death markers and caspase‐3 activity, suggesting that IL‐1ra prevents apoptosis in injured spinal cords. We also found that IL‐1ra significantly improved locomotor recovery. To answer the question how is the inhibition of IL‐1 receptors preventing cell death in injured spinal cord and improving motor recovery, we analyzed transcriptional changes in injured vs. injured spinal cord treated with IL‐1ra, via DNA microarrays. We found a large number of mRNAs whose expression levels were significantly changed after SCI and reversed in the presence of IL‐1ra. For example, we found that iNOS and COX‐2 mRNA are significantly up‐regulated after injury and down‐regulated in IL‐1ra‐treated spinal cords. If iNOS and COX‐2 proteins are synthesized, it is very likely that their activity will produce reactive oxygen species (ROS), which will cause cell death via different converging apoptotic pathways. We also found that SCI induces IL‐1 dependent up‐regulation of pro‐apoptotic cathepsins and cell cycle regulators, a novel finding. Therefore, we hypothesize that the anti‐inflammatory IL‐1ra could be therapeutic and alleviate secondary damage after SCI.

Acknowledgements:  Supported in part by grant NS39161 from NINDS and Mission Connect Foundation.
Inhibition of peroxisomal functions and production of excessive oxidative stress by psychosine in rat C6 glial cells
Authors: Khan M. , Giri S. , Singh I. , Singh A. K.
Published: July 2010

The primary defect of abnormality in the metabolism of psychosine in Krabbe disease results in the loss of oligodendrocytes, increased oxidative stress, inflammatory reactions and profound demyelination. To understand the basis of toxicity of psychosine in the CNS of Krabbe disease, we examined the effects of psychosine on the peroxisomal β‐oxidation of very long chain fatty acids (VLCFA), plasmalogen biosynthesis and on the production of reactive oxygen/nitrogen species. Rat C6 glial cells were treated with psychosine (2–10 mm) in absence and presence of cytokines (LPS + IFN‐γ, or TNF‐α + IL‐1β) for 24 and 72 h. Psychosine inhibited peroxisomal b‐oxidation of VLCFA (30–40% decrease compared to control) and induced the accumulation of VLCFA measured as levels of 26 : 0 (100% increase compared to control). Furthermore, psychosine treatment decreased the levels of plasmalogens (35–50% decrease compared to control). Plasmalogens levels were also drastically low in Krabbe brain than normal control brain. Psychosine also potentiated the known cytokine‐induced inhibition of peroxisomal β‐oxidation, accumulation of VLCFA and NO production. We also measured overall oxidative stress using dichlorofluorescin diacetate in psychosine‐treated cells and found 1–2 fold increase in oxidative stress production. Both psychosine‐mediated loss of peroxisomal functions as well as ROS production could be inhibited by antioxidant, N‐acetylcysteine. Our results suggest that inhibition of peroxisomal functions and increased free radical production by psychosine may be responsible, at least in part, for the loss of oligodendrocytes and break down of myelin in Krabbe brain.

Acknowledgements:  Supported by NIH grants NS‐40144, NS‐40810, NS‐22576, NS‐34741 and NS‐37766.
Nitric oxide – mediated activation of ERK and JNK during hypoxia in neuronal nuclei of newborn pigs
Authors: Miahra O. P. , Ashraf Q. M.
Published: July 2010

The extracellular signal‐regulated kinase (ERK) and c‐Jun N‐Terminal kinase (JNK) phosphorylate antiapoptotic proteins thereby may regulate hypoxia‐induced programmed cell death. The present study tests the hypotheses that hypoxia activates ERK and JNK in neuronal nuclei of newborn pigs and the hypoxa‐induced activation of ERK and JNK is mediated by nitric oxide (NO). Activated ERK and JNK were assessed by determining phosphorylated ERK and JNK by immunoblotting in 6 normoxic (Nx), 10 hypoxic (Hx) and 5 N‐nitro‐l‐arginine (a NOS inhibitor)‐treated (40 mg/kg) hypoxic (NNLA‐HX) 3–5‐day‐old‐piglets. Animals were exposed to an FiO2 of 0.05–0.07 for 60 min Neuronal nuclei were isolated and nuclear proteins were immunoblotted with antiphosphorylated ERK and JNK. Protein bands were detected, analyzed and expressed as (OD × mm2). Tissue hypoxia was documented by ATP levels. ATP levels were 4.45 ± 0.57, 1.16 ± 0.33 and 1.10 ± 0.25 µm/g br in Nx, Hx (p < 0.001 vs. Nx) and NNLA‐Hx (p < 0.001 vs. Nx) groups, respectively. Phosphorylated ERK density was 170.5 ± 53.7 in Nx vs. 419.6 ± 63.9 in the Hx group (p < 0.001) and 270.0 ± 28.7 in the NNLA‐Hx group (p < 0.002 vs. Hx). Density of phosphorylated JNK was 172.8 ± 42.8 in the Nx vs. 364.6 ± 60.1 in the Hx (p < 0.002 vs. Nx) and 254.8 ± 24.8 in the NNLA‐Hx group (p < 0.002 vs. Hx). We data demonstrate increased activation of ERK and JNK following hypoxia. NNLA,a NOS inhibitor, decreased the hypoxia‐induced activation of ERK and JNK. We conclude that the hypoxia‐induced activation of ERK and JNK is NO‐mediated.

Acknowledgements:  Supported by NIH‐HD‐38079.
Novel mediators of ischemic preconditioning induced neuroprotection
Authors: Dhodda V. K. , Sailor K. , Dempsey R. J. , Vemuganti R. L. R.
Published: July 2010

A brief ischemic episode induces protection against a subsequent severe ischemic insult. This phenomenon, known as ischemic preconditioning (PC) might be mediated by the activation of several genes of the second messenger signalling pathways. A 60‐min transient middle cerebral artery occlusion (MCAO) in adult, SHR rats results in an infarct encompassing cerebral cortex and striatum by 24 h reperfusion. Present study observed that a 10‐min PC, 3 days before the 60 min transient MCAO, results in a 30% reduction in the infarct volume. To understand the molecular mechanisms of the PC induced neuroprotection, we analyzed the mRNA expression profiles of rat cerebral cortex at 6, 24 and 72 h after a 10‐min transient MCAO by using Affymetrix GeneChip and real‐time PCR. Expression of the members of TGF‐β signalling (TGF‐β1, p 38 MAP kinase, Smad 1 and Smad 7) is observed to be up‐regulated after PC. This pathway plays an essential role in tissue repair and immune homeostasis, but not hitherto implicated in PC induced neuroprotection. TGF‐β1 enhances Smad transcriptional activity through activation of P38 gene expression to induce neuroprotection. Consistent with previous studies, expression of several neuroprotective genes including HSP70, HSP27, HO‐1, MT‐1/MT‐2 and IL‐6, were also observed to be up‐regulated after PC. These studies shows that TGF‐β1/p38 MAP‐kinase signalling pathway is a promising target for developing drugs to limit ischemic neuronal damage.

Acknowledgements:  Funded by AHA.
PARP‐1 activation causes mitochondrial dysfunction
Authors: Swanson R. A. , Ying W. , Alano C. C. , Chen Y.
Published: July 2010

When activated by DNA damage, PARP1 consumes NAD+ to form ADP‐ribose polymers on acceptor proteins. Excessive PARP1 activation during oxidative DNA damage is known to cause NAD+ depletion and cell death, but the intervening steps are not well understood. Sustained inhibition of the NAD+‐dependent step in glycolysis is a potential consequence of NAD+ depletion. If this occurs, it should be possible to rescue cells from PARP1‐mediated cell death by supplying alternative substrates for the mitochondrial tricarboxylic acid cycle. PARP1 was activated in cortical astrocyte and astrocyte‐neuron cocultures with the DNA alkylating agent, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). Studies using the 2‐deoxyglucose method confirmed a sustained reduction in glycolytic flux, and studies with the JC‐1 and TMRM indicate mitochondrial membrane depolarization. The addition of 1 mm of a‐ketoglutarate or 1 mm pyruvate after washout of MNNG reduced cell death from 60 to 80% to near control levels, while addition of PARP inhibitors or excess glucose after MNNG washout had negligible effect. Cytoprotective effects remained significant with substrate delivery delayed up to 3 h after MNNG washout. The findings suggest that impaired glycolytic flux is a major factor in PARP1‐mediated cell death, and suggest that supply of alternative mitochondrial substrates may be a promising strategy for delayed treatment of PARP1‐mediated cell death in ischemia and other disorders.
Role of oxidative stress in the ammonia‐induced mitochondrial permeability transition in cultured astrocytes
Authors: Jayakumar A. R. , Rama Rao K. V. , Bai G. , Norenberg M. D.
Published: July 2010

Ammonia is the principal neurotoxin implicated in the pathogenesis of hepatic encephalopathy (HE). The mechanisms of ammonia toxicity are unclear, but appear to involve oxidative stress and mitochondrial dysfunction. Astrocytes appear to be cell mainly affected. We have previously shown that ammonia increases the production of reactive oxygen species (ROS), and that it also induces the mitochondrial permeability transition (MPT) in cultured astrocytes. Since ROS is a factor commonly implicated in the induction of the MPT, we investigated the role of oxidative stress in the induction of the MPT by ammonia. We therefore examined the effects of various antioxidants on the induction by ammonia of the MPT in cultured astrocytes. Astrocytes were subjected to NH4Cl (5 mm) treatment for two days alone or with various antioxidants. The MPT was assessed by 2‐deoxyglucose (2‐DG) permeability into mitochondria, and by changes in the mitochondrial membrane potential (ΔΨm) by confocal microscopy using the potentiometric dye JC‐1. Astrocytes treated with ammonia significantly increased 2‐DG permeability (90%, p < 0.01) and concomitantly dissipated the ΔΨm (60%, p < 0.01), consistent with the induction of the MPT. The antioxidants superoxide dismutase (by 50%, p < 0.05), vitamin E (by 80%, p < 0.01), and the spin trapping agent PBN (by 80%, p < 0.01), all significantly blocked the increase in 2‐DG permeability and the dissipation of the ΔΨm produced by ammonia. These data demonstrate the induction of the MPT by ammonia and suggest the involvement of ROS in this process. Increased oxidative stress and subsequent induction of the MPT by ammonia in astrocytes may contribute to the pathogenesis of HE.
cAMP pretreatment increases the number of dopamine neurons in intrastriatal grafts
Authors: McGuire S. O. , Counts S. E. , Lum H. , Sortwell C. E. , Daley B. F. , Collier T. J.
Published: July 2010

Neuroprotective signalling pathways are under investigation in order to improve the survival of transplanted embryonic dopamine (DA) neurons as a potential therapy for Parkinson's disease. Since cyclic AMP (cAMP)‐induced activation of cAMP‐response element binding protein (CREB) is associated with neuroprotection, we investigated whether cAMP could be used to enhance DA neuron survival and phenotype in a grafting paradigm. We previously reported that dibutyryl (db) cAMP increased the number of tyrosine hydroxylase positive (TH+) neurons in mesencephalic cultures. However, addition of dbcAMP to cell suspensions immediately prior to grafting did not increase the number of DA neurons in intrastriatal grafts. To increase the duration of cAMP exposure, we adapted a rotary, reaggregate culture system which allows protracted culture of embryonic mesencephalic cells without loss of viability. Cell suspensions were prepared and allowed to reaggregate for 24 h. Cultures were then incubated ± dbcAMP for an additional 72 h. The spheres were harvested and transplanted bilaterally into the intact striatum of male Fischer 344 rats. Grafts from dbcAMP‐pretreated spheres contained three times more TH+ cells than grafts from spheres not exposed to dbcAMP. However, due to a high degree of variance, this increase did not reach statistical significance (p = 0.10). Protein harvested from an aliquot of these spheres was subjected to Western blot analysis which revealed that treatment of reaggregate spheres with dbcAMP for 72 h increased TH expression by 78% and increased Bcl2 expression by 47%. Mechanisms underlying these results are currently under investigation.
DNA polymorphism in the human presenilin‐2 promoter: altered gene activity and potential contribution to the risk for AD
Authors: Lukiw W. J. , Riazanskaya N. , Grigorenko A. , Korovaitseva G. , Dvoryanchikov G. , Molyaka Y. , Nicolaou M. , Farrer L. , Bazan N. G. , Rogaev E. I.
Published: July 2010

We analyzed the presenilin‐2 (PS2) promoter for polymorphisms in Alzheimer's disease (AD). Multiple elements responsive to pro‐inflammatory (PI) and hypoxia signalling were identified. In cultured human brain cells these factors induced the expression of the endogenous PS2 gene. DNA sequence analysis revealed an adenine (A) nucleotide deletion in the PS2 promoter in several human populations. Examination of cohorts of AD patients revealed no significant differences in the frequency of this polymorphism, however, regression analysis of polymorphic allele frequency suggested an association (p = 0.012) of the –A/–A mutant genotype with an increased risk of AD in apoE4 noncarriers and a tendency for an association with early onset AD. Gel shift assay showed that this mutation negates binding of interferon IRF2 repressor in nuclear extracts from aged human brain while creating a C/EBPβ regulatory element responsive to PI induction. Reporter assay in PS2 mutant promoter‐transfected human neural cells shows that mutant PS2 exhibits a 1.8‐fold higher level of basal expression that is synergistically induced 3.2‐fold over wild‐type PS2 by [IL‐1β + Aβ42]. These results suggest that under PI and oxygen stress conditions this PS2 promoter polymorphism enhances PS2 expression and may play a role in the induction and/or proliferation of a PI response in aging and in AD brain.
Promoter analysis of the CMT1A‐disease gene PMP22 in vivo
Authors: Maier M. , Berger P. , Nave K. A. , Suter U.
Published: July 2010

The peripheral myelin protein 22 (PMP22) is highly expressed in myelinating Schwann Cells and in neurons of the peripheral nervous system where it accounts for 2–5% of total protein in the myelin sheat. Minor changes in PMP22 gene dosage have profound effects on the development and maintenance of peripheral nerves. This is evident from the genetic disease mechanisms in Charcot–Marie tooth disease type 1 A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP) as well as transgenic animals with altered PMP22 gene dosage. Thus, regulation of PMP22 is a crucial aspect in understanding the function of this protein in health and disease. In this study, we describe important regulatory elements of PMP22 by the generation of transgenic mice containing 10 kb of 5′‐flanking region of the PMP22 gene, including the two previously identified alternative promoters, fused to a lacZ reporter gene. We show that this part of the PMP22 gene contains the necessary information to mirror the endogenous expression pattern in peripheral nerves during development, regeneration, and in mouse models of demyelination due to genetic lesions. Transgene expression is strongly regulated during myelination, demyelination and remyelination in Schwann cells demonstrating the crucial influence of neuron–Schwann cell interactions in the regulation of PMP22. These results provide the crucial basis for the ongoing further dissection of the elements that direct the temporal and spatial regulation of the PMP22 gene and to elucidate the molecular basis of the master program regulating peripheral nerve myelination.
Apoptosis of neurons in rats with experimental allergic encephalomyelitis
Authors: Guyton K. , Wingrave M. , Rocchini A. , Scheacher K. , Ray S. , Banik N.
Published: July 2010

Experimental allergic encephalomyelitis (EAE) is a rat model of multiple sclerosis (MS). In MS and EAE, calpain expression is increased in cells of the central nervous system including astrocytes, oligodendrocytes and microglia; as well as infiltrating macrophages and T cells. However, calpain expression in neurons has not previously been reported in the EAE model. Since degeneration of neurons has been demonstrated in progressive cases of MS, and calpain has been implicated in cell death in EAE, it is hypothesized that neurons expressing increased levels of calpain may undergo apoptosis. To test the hypothesis, spinal cords were collected from control and EAE rats from 4 to 12 days after challenge with guinea‐pig myelin and complete Freund's adjuvant. Apoptosis in neurons was determined by combined TUNEL and immunoflourescent staining using a neuron‐specific marker (NeuN). Calpain expression in neurons was determined using double‐immunoflourescent staining with antibodies specific for calpain and NeuN. At 8 and 9 days postchallenge, neither apoptosis nor increased calpain expression were detected in neurons in control or 9 day EAE spinal cord sections. In contrast, at 11 days postinduction neurons in acute EAE were TUNEL‐positive, indicating that cells underwent apoptosis. Further studies are in progress to determine if and when oligodendrocytes in EAE rats are apoptotic. By correlating cell‐specific increases in calpain expression with apoptosis in EAE, a better understanding of the role of calpain in EAE and MS might be achieved. This increased knowledge could suggest a potential benefit of calpain inhibitors in the treatment of EAE and MS.

Acknowledgements:  Supported in part by grants from NIH‐NINDS.
Elevation of cyclin D1 following trimethyltin induced hippocampal neurodegeneration
Authors: Wine R. N. , McPherson C. A. , Harry G. J.
Published: July 2010

Previous work has suggested that a major contributor to neuronal cell death is the aberrant induction of the cell cycle process, as indicated by an up‐regulation of cyclin D. In order to examine the temporal and spatial relationship of cyclin D in a model of acute neurodegeneration, the hippocampal toxicant, trimethyltin (TMT; 2.0 mg/kg), was administered to 21‐day old CD−1 male mice and the level and cellular localization of cyclin D1 examined. Within 24 h following TMT, dentate granule cells of the hippocampus showed evidence of neuronal necrosis resulting in severe cell loss over a 3‐day period. The pyramidal cell layer was spared with only sparse punctate neuronal necrosis. Microglia response was seen at 72 h with ameboid microglia present in the dentate and ramified microglia present in the pyramidal cell layer, contributing to the elevation seen in TNF‐alpha mRNA levels. A transient elevation was seen in mRNA levels for cyclin D1 over 48–72 h post‐TMT. Immunohistochemistry demonstrated a transient increase in staining for cyclin D1 in CA1 pyramidal neurons as early as 24 h. Punctate staining occurred in neurons throughout the dentate at 48 h. BrdU positive cells were present along the inner blades of the dentate in control animals. Following TMT exposure, an increase was seen in both the number of neurons stained and a diffusion of the staining pattern into the full dentate region. Thus, in TMT‐induced neurodegeneration, cyclin D1 is not expressed in the vulnerable neurons but rather in neurons spared from degeneration. This expression pattern appears to not be linked to an increase in the cellular processes for proliferation as the majority of BrdU positive cells were present in the region of neuronal damage.
Symposium 1: Regulation of Neural Development by BMP and Activin Family Members. BMP regulation of stem cell differentiation
Authors: Kessler J. A. , Gomes W. , Guha U. ,   N. Israsena
Published: July 2010

The effects of BMP family members on stem cell lineage commitment depend upon the developmental age of the stem cell. BMP4 promotes apoptosis of early ventricular zone (VZ) stem cells, neuronal differentiation of later stage VZ cells, and astroglial differentiation of subventricular zone (SVZ) cells. BMP4 inhibits oligodendroglial lineage commitment at all stages of development. The effects of BMP4 in promoting commitment to a specific lineage reflect active suppression of alternate lineages by transcriptional inhibitors including ID and HEY family members and others. For example, BMP mediated increases in ID expression in SVZ stem cells suppress both oligodendroglial and neuronal differentiation. Similarly HEY 1 expression in SVZ cells suppresses neuronal differentiation, whereas HEYL expression by VZ cells inhibits glial differentiation and promotes neurogenesis. The differing effects of the BMPs on VZ and SVZ stem cells reflect also differences in the complement of transcription factors that are expressed. For example, VZ stem cells express high levels of neurogenin and HEY L whereas SVZ stem cells express lower levels of these factors but higher levels of HEY1. Thus lineage commitment by stem cells reflects interplay among stimulatory and inhibitory transcription factors, and responses to the BMPs depend upon the repertoire of transcription factors already expressed by the cell.
Dorsally derived BMP4 inhibits the induction of spinal cord oligodendrocyte precursors
Authors: Miller R. H. , Wang R‐Z. N. , Geertman R. , Maier C. E.
Published: July 2010

During development oligodendrocyte precursors arise in a distinct domain of the ventral ventricular zone in the spinal cord that they share with motor neurons. The localized appearance of oligodendrocyte and motor neuron precursors is the result of local inductive signals including sonic hedgehog (Shh). Previous studies suggested that inhibitory signals from dorsal spinal cord act to sharpen the boundaries of the Shh induced region. Here we show that the dorsal spinal cord contains BMP4 during the developmental period when oligodendrocyte precursors first appear. In dissociated cultures of embryonic spinal cord cells, BMP4 competitively blocks the induction of oligodendrocyte precursors by Shh. Similarly, in embryonic slice preparations addition of BMP4 inhibited the appearance of oligodendrocyte precursors in the ventral spinal cord while addition of Shh enhanced their appearance. In vivo, transplantation of a BMP4 coated bead adjacent to the dorsal spinal cord inhibited ventral oligodendrogenesis while transplantation of a Shh coated bead enhanced ventral oligodendrogenesis. These data suggest that the initial localization of oligodendrocytes in the ventral spinal cord reflects the neutralization of dorsally‐derived BMP4 inhibition by locally supplied Shh.