Regulation of neural cell adhesion molecule polysialylation state by cell-cell contact and protein kinase C delta

Post-translational modification of neural cell adhesion molecule (NCAM) with alpha2,8-linked polysialic acid, which regulates homophilic adhesion and/or signal transduction events, is crucial to synaptic plasticity in the developing and adult brain. Evidence from in vitro models has implicated polysialylation in the regulation of cell growth, migration, and differentiation. Here, using two in vitro models, we demonstrate that polysialylation is downregulated by cell-cell contact and correlated with a state of neuronal differentiation. Furthermore, we report a role for protein kinase C delta (PKCdelta) in the regulation of NCAM polysialylation. Pharmacological studies using the PKC activator, phorbol myristate acetate, and inhibitors, calphostin-C, and staurosporine, demonstrated PKC activity to be inversely related to NCAM polysialylation in the mouse neuro-2A cell line. Isoform-specific immunoblot studies indicated this effect to be mediated by the calcium-independent PKCdelta isozyme, as its expression was inversely related to NCAM polysialylation state in both neuro-2A and rat PC-12 cell lines. Isoform specificity was further confirmed using the PKCdelta-selective inhibitor rottlerin, which produced a marked increase in PSA expression (36.9+/-5.25 a.u. vs. 24.7+/-0.80 arbitrary units control) coupled with a neuritogenic response. Likewise, decreased expression of PKCdelta was seen in nerve growth factor (NGF)-differentiated PC-12 cells. These findings suggest that the neuronal differentiation process may involve inhibition of PKCdelta, resulting in enhanced morphological plasticity, as evidenced by activation of NCAM polysialylation.     

Regulation of gene expression in astrocytes by excitatory amino acids.

We have studied the effect of excitatory amino acids on the expression of mRNA for the immediate early genes c-fos, c-jun, jun-B, and NGF-1A in isolated cortical astrocytes. The expression of the different genes was induced by 100 microM kainate, quisqualate, AMPA and high concentrations of K+ (140 mM). NMDA did not induce the expression of any of the genes studied. The effect of quisqualate stimulation was not inhibited by the antagonist CNQX or by withdrawal of external Ca2+. In contrast the kainate effect was abolished by CNQX but not by the removal of external Ca2+. However, elevated K+ induced c-fos only when calcium was present in the external medium. These findings suggest that type-1 astrocytes lack NMDA receptors and that the induction of genes by quisqualate and kainate is in part independent of the presence of calcium in the external medium and may be mediated through second messenger pathways.     

Regulation of prodynorphin gene expression in the hippocampus by glucocorticoids.

The regulation of prodynorphin gene expression by glucocorticoids in the hippocampus was examined in rats that were adrenalectomized (ADX) either 7, 30, 60 and 90 days prior to sacrifice. Peptide levels in the hippocampus of ADX rats were determined by radioimmunoassay and immunocytochemistry. Prodynorphin (PDYN) mRNA was measured by Northern blot analysis and in situ hybridization. A time-dependent decrease in dynorphin A(1-8)(DYN) levels in the hippocampus (18% at 7 days; 44% at 30 days; 58% at 60 days) of ADX rats was found, which was accompanied by a comparable decrease in the abundance of PDYN mRNA. An in situ hybridization analysis revealed that both the number of positively hybridized cells and the number of silver grains per cell were decreased in the dentate gyrus after ADX. The administration of dexamethasone after surgery reversed the peptide and mRNA attenuation induced by ADX. ADX had no effect on the expression of proenkephalin mRNA or [Met5]-enkephalin immunoreactivity in the hippocampus. Examination of thionin-counterstained tissue showed that the dentate granule cell layer was intact. The decrement of DYN expression in this system is proposed to have resulted from the removal of glucocorticoid input and not dentate granule cell loss. This study provides the strong evidence for a differential susceptibility of these two opioid peptides in the hippocampus to the removal of glucocorticoids. In addition, these data provide support for a potentially selective, glucocorticoid-permissive component in PDYN gene expression.     

Robust regeneration of CNS axons through a track depleted of CNS glia

Transected CNS axons do not regenerate spontaneously but may do so if given an appropriate environment through which to grow. Since molecules associated with CNS macroglia are thought to be inhibitory to axon regeneration, we have tested the hypothesis that removing these cell types from an area of brain will leave an environment more permissive for axon regeneration. Adult rats received unilateral knife cuts of the nigrostriatal tract and ethidium bromide (EB) was used to create a lesion devoid of astrocytes, oligodendrocytes, intact myelin sheaths, and NG2 immunoreactive cells from the site of the knife cut to the ipsilateral striatum (a distance of 6 mm). The regenerative response and the EB lesion environment was examined with immunostaining and electron microscopy at different timepoints following surgery. We report that large numbers of dopaminergic nigral axons regenerated for over 4 mm through EB lesions. At 4 days postlesion dopaminergic sprouting was maximal and the axon growth front had reached the striatum, but there was no additional growth into the striatum after 7 days. Regenerating axons did not leave the EB lesion to form terminals in the striatum, there was no recovery of function, and the end of axon growth correlated with increasing glial immunoreactivity around the EB lesion. We conclude that the removal of CNS glia promotes robust axon regeneration but that this becomes limited by the reappearance of nonpermissive CNS glia. These results suggest, first, that control of the glial reaction is likely to be an important feature in brain repair and, second, that reports of axon regeneration must be interpreted with caution since extensive regeneration can occur simply as a result of a major glia-depleting lesion, rather than as the result of some other specific intervention.     

Free radicals induce gene expression of NGF and bFGF in rat astrocyte culture.

Hydrogen peroxide (H2O2) is a type of active oxygen species produced mainly in blood by inflammation, ischemia or anoxia. Treatment of rat neonatal cortical astrocytes in culture with 0.2-1.0 mM H2O2 which is lethal for hippocampal neurons, increases nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) mRNA content in a time dependent manner. H2O2 also increases c-fos mRNA expression, which is probably involved in the gene regulation of both NGF and bFGF. Maximal induction was reached after 6 h of incubation (5.7-fold increase in NGF and 2.4-fold induction of bFGF mRNA). Hydrogen peroxide induced bFGF and NGF gene expression suggests that neurotrophic factors in astrocytes could be induced by lesion, consistent with their protective function in the CNS.     

Sexually dimorphic expression of the NGF receptor gene in the developing rat brain.

To define relations between trophic molecules and known sexually dimorphic traits in brain, we examined possible sex differences in nerve growth factor (NGF) and NGF receptor (NGF-R) gene expression in the rat cholinergic basal forebrain (BF)-hippocampal system. Hippocampal NGF mRNA levels did not differ between sexes; in contrast, BF NGF-R mRNA levels were greater in neonatal females than males, paralleling the known dimorphic development of cholinergic enzyme activity. Cerebellar NGF-R mRNA levels were also dimorphic in the neonate, suggesting that sex-specific influences may regulate trophic receptor gene expression in diverse brain systems.     

CNS中抗原递呈细胞调节T细胞对CNS抗原的应答

在中枢神经系统(CNS)炎症或损伤时,T细胞对CNS抗原的应答启动于外周免疫系统,而作用于CNS.CNS中小胶质细胞、星形胶质细胞和血管周围巨噬细胞可作为抗原递呈细胞,通过对Th1和Th2细胞的再刺激,分泌可溶性因子调节Th1和Th2细胞应答,以及与T细胞间相互作用,调控Th1细胞和Th2细胞间的平衡,从而促进或抑制T细胞应答,影响CNS炎症损伤的结局.     

CNS中抗原递呈细胞调节T细胞对CNS抗原的应答

在中枢神经系统 (CNS)炎症或损伤时 ,T细胞对CNS抗原的应答启动于外周免疫系统 ,而作用于CNS。CNS中小胶质细胞、星形胶质细胞和血管周围巨噬细胞可作为抗原递呈细胞 ,通过对Th1和Th2细胞的再刺激 ,分泌可溶性因子调节Th1和Th2细胞应答 ,以及与T细胞间相互作用 ,调控Th1细胞和Th2细胞间的平衡 ,从而促进或抑制T细胞应答 ,影响CNS炎症损伤的结局     

NGF rescues substance P expression but not neurofilament or tubulin gene expression in axotomized sensory neurons.

A reduction in the supply of retrogradely transported NGF has been proposed as a possible signal for the axotomy response in dorsal root ganglion (DRG) neurons. Components of the axotomy response that have previously been well characterized in axotomized DRG cells include changes in cytoskeletal gene expression and changes in the expression of neurotransmitters/neuromodulators such as substance P. In this study, we examined the role of NGF in the axotomy response by examining protein synthesis and mRNA levels of the low-MW neurofilament protein (NF-L) and beta-tubulin in DRG cells at 1, 7, and 12 d after axotomy with and without continuous administration of exogenous NGF. We also examined substance P levels in the DRG by immunocytochemistry under the same experimental conditions. Sciatic nerves of adult male rats were unilaterally transected at the midthigh level, and the proximal nerve stumps were placed into Silastic tubes connected to osmotic minipumps that were filled with biologically active NGF. NGF (0.5 mg/ml in saline) was continuously infused (0.5 microliter/hr) onto the proximal stumps of transected sciatic nerves for 1-12 d. Control animals were prepared in an identical fashion except that the nerves were treated with saline alone. At death, DRGs were removed from the animals; the L4 experimental DRGs (axotomized) and contralateral L4 DRGs (uninjured) were used immediately for protein synthesis experiments, while the experimental and contralateral L5 DRGs were fixed in 4% paraformaldehyde and subsequently used for in situ hybridization and immunocytochemistry. From another set of experimental animals, the L4 and L5 DRGs were harvested and used for total RNA isolation and RNA blotting experiments. Immunocytochemical studies using a polyclonal antibody to substance P showed that the immunodetectable levels of this peptide decreased to undetectable levels in DRG neurons after axotomy and saline administration. However, in axotomized neurons treated with NGF, the level of immunodetectable substance P did not decrease, but instead, increased over even that present in normal DRG neurons. Pulse labeling of DRGs with 35S-methionine:cysteine followed by 2-dimensional (2D) gel electrophoresis and fluorography revealed that the synthesis of neurofilament (NF) proteins was decreased, while that of tubulin was increased, 12 d after sciatic nerve transection. NGF administration to axotomized neurons did not alter this pattern. Quantitative analysis of in situ hybridizations of DRG neurons and RNA blot analysis with cDNA probes specific for NF-L and beta-tubulin mRNAs showed that NGF treatment of axotomized DRGs did not significantly affect cytoskeletal gene expression at the mRNA level.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of neurofilament gene expression by thyroid hormone in the developing rat brain.

The role of thyroid hormone (TH) in the expression of neurofilament (NF) genes during the first 3-4 postnatal weeks of rat brain development has been examined. I.p. administration of TH to 2-day-old hypothyroid rats resulted in a 2-fold increase in cerebral NF-M and NF-L mRNAs, while administration to 15-day-old hypothyroid rats led to a 1.5- to 3-fold increase in NF-H mRNA within 2-4 h of hormone injection. Comparison of the level of these mRNAs in cerebra from 5, 10, 15 and 20-day-old normal and hypothyroid rats by Northern blot analysis revealed that hypothyroidism declined the expression of all three mRNAs by 50-70% at all ages examined. Western blot analysis of total protein and cytoskeletal proteins isolated from cerebras of 5, 10, 15, 20 and 25-day-old normal and hypothyroid rats demonstrated an even greater reduction (60-90%) in the expression of NF proteins in the hypothyroid cerebra during the period examined. The overall results show that TH plays an important role in regulating the expression of all three NF genes during rat brain development.     

Regulation of dynorphin gene expression by kappa-opioid agonist treatment

The effects of K-opioid agonist treatment on prodynorphin mRNA expression in the rat brain were studied. Rats were treated with the selective kappa-opioid agonist U-69593 or vehicle for 5 days and prodynorphin mRNA was measured on day 8 (3 days after the last injection) or 22 (17 days after the last injection). On day 8 prodynorphin mRNA was increased in the hypothalamus and decreased in the striatum, frontal cortex, and hippocampus of rats treated with U-69593. On day 22, prodynorphin mRNA was increased in the hypothalamus, frontal cortex and striatum of U-69593 treated rats. These findings suggests that kappa-opioid receptor agonist treatment has long-term, continually changing effects on prodynorphin mRNA expression.     

Motile membrane protrusions regulate cell-cell adhesion and migration of olfactory ensheathing glia

Olfactory ensheathing cells (OECs) are candidates for therapeutic approaches for neural regeneration due to their ability to assist axon regrowth in central nervous system lesion models. However, little is understood about the processes and mechanisms underlying migration of these cells. We report here that novel lamellipodial protrusions, termed lamellipodial waves, are integral to OEC migration. Time-lapse imaging of migrating OECs revealed that these highly dynamic waves progress along the shaft of the cells and are crucial for mediating cell-cell adhesion. Without these waves, cell-cell adhesion does not occur and migrational rates decline. The activity of waves is modulated by both glial cell line-derived neurotrophic factor and inhibitors of the JNK and SRC kinases. Furthermore, the activity of lamellipodial waves can be modulated by Mek1, independently of leading edge activity. The ability to selectively regulate cell migration via lamellipodial waves has implications for manipulating the migratory behavior of OECs during neural repair.     

The effect of S-adenosylmethionine on CNS gene expression studied by cDNA microarray analysis

High homocysteine (Hcy) together with low S-adenosylmethionine (SAM) levels are often observed in Alzheimer disease (AD), and this could be a sign of alteration of SAM/Hcy metabolism. It has already been shown that DNA methylation is involved in amyloid-beta-protein precursor (AbetaPP) processing and amyloid-beta(Abeta) production through the regulation of Presenilin 1 (PS1) expression and that exogenous SAM can silence the gene reducing Abeta. To investigate whether SAM administration globally influenced gene expression in the brain, we analysed 588 genes of the central nervous system in SK-N-BE neuroblastoma cells, with cDNA probes derived from untreated (DM; Differentiation Medium) or SAM treated (DM+SAM) cultures. In these conditions only seven genes were modulated by SAM treatment (and therefore by DNA methylation); three were up-regulated and four down-regulated, showing low levels of modulation.     

Regulation of high-affinity glutamate uptake activity in Bergmann glia cells by glutamate

The effect of glutamate receptor activation on the high-affinity sodium-dependent glutamate transport expressed in chick Bergmann glia cells was examined. Pre-exposure to glutamate produced a time- and dose-dependent decrease in 3H-labeled D-aspartate uptake. This effect could not be reproduced by selective glutamate receptor agonists. Furthermore, it was insensitive to both ionotropic and metabotropic glutamate receptor antagonists. Replacement of extracellular sodium ions with choline in the preincubation media, abolished the reduction of the uptake. When the cells were pre-exposed to competitive transportable inhibitors of the transporter, such as D-aspartate, DL-threo-hydroxyaspartate (DL-THA), and aspartate-beta-hydroxamate (ABH), the glutamate effect was mimicked. From saturation experiments, it was found that the reduction on the uptake, after glutamate treatment, is related to an increase in K(m). Interestingly, the effect is blocked by staurosporine, a Ca(2+)/diacylglycerol-dependent protein kinase (PKC) inhibitor. The present findings suggest that glutamate regulates its transport in a non-receptor fashion, a phenomena that is most probably linked to changes induced by the translocation process of the substrate through the transporter.     

Regulation of RPTPbeta/phosphacan expression and glycosaminoglycan epitopes in injured brain and cytokine-treated glia

Several chondroitin sulfate proteoglycans (CSPGs) are upregulated after CNS injury and are thought to limit axonal regeneration in the adult mammalian CNS. Therefore, we examined the expression of the CSPG, receptor protein tyrosine phosphatase beta (RPTPbeta)/phosphacan, after a knife lesion to the cerebral cortex and after treatment of glial cultures with regulatory factors. The three splice variants of this CSPG gene, the secreted isoform, phosphacan, and the two transmembrane isoforms, the long and short RPTPbeta, were examined. Western blot and immunostaining analysis of injured and uninjured tissue revealed a transient decrease of phosphacan protein levels, but not of short RPTPbeta, in the injured tissue from 1 to 7 days postlesion (dpl). By real time RT-PCR, we show that phosphacan and long RPTPbeta mRNA levels are transiently down-regulated at 2 dpl, unlike those of short RPTPbeta which increased after 4 dpl. In contrast to the core glycoprotein, the phosphacan chondroitin sulfate (CS) glycosaminoglycan epitope DSD-1 was up-regulated after 7 dpl. Phosphacan was expressed by cultivated astrocytes and oligodendrocyte precursors but was more glycanated in oligodendrocyte precursors, which produce more of DSD-1 epitope than astrocytes. Epidermal growth factor/transforming growth factor alpha strongly increased the astrocytic expression of long RPTPbeta and phosphacan and slightly the short RPTPbeta protein levels, while interferon gamma and tumor necrosis factor alpha reduced astrocytic levels of phosphacan, but not of the receptor forms. Examining the effects of phosphacan on axon growth from rat E17 cortical neurons, we found that phosphacan stimulates outgrowth in a largely CS dependent manner, while it blocks the outgrowth-promoting effects of laminin through an interaction that is not affected by removal of the CS chains. These results demonstrate complex injury-induced modifications in phosphacan expression and glycanation that may well influence axonal regeneration and repair processes in the damaged CNS.