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Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for molecules associated with metabolism, signaling and regulation in central nervous system mixed glial cell cultures

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Cytokines secreted by immune cells and activated glia play central roles in both the pathogenesis of and protection from damage to the central nervous system (CNS) in multiple sclerosis (MS).


We have used gene array analysis to identify the initial direct effects of cytokines on CNS glia by comparing changes in early gene expression in CNS glial cultures treated for 6 hours with cytokines typical of those secreted by Th1 and Th2 lymphocytes and monocyte/macrophages (M/M).


In two previous papers, we summarized effects of these cytokines on immune-related molecules, and on neural and glial related proteins, including neurotrophins, growth factors and structural proteins. In this paper, we present the effects of the cytokines on molecules involved in metabolism, signaling and regulatory mechanisms in CNS glia. Many of the changes in gene expression were similar to those seen in ischemic preconditioning and in early inflammatory lesions in experimental autoimmune encephalomyelitis (EAE), related to ion homeostasis, mitochondrial function, neurotransmission, vitamin D metabolism and a variety of transcription factors and signaling pathways. Among the most prominent changes, all three cytokine mixtures markedly downregulated the dopamine D3 receptor, while Th1 and Th2 cytokines downregulated neuropeptide Y receptor 5. An unexpected finding was the large number of changes related to lipid metabolism, including several suggesting a switch from diacylglycerol to phosphatidyl inositol mediated signaling pathways. Using QRT-PCR we validated the results for regulation of genes for iNOS, arginase and P glycoprotein/multi-drug resistance protein 1 (MDR1) seen at 6 hours with microarray.


Each of the three cytokine mixtures differentially regulated gene expression related to metabolism and signaling that may play roles in the pathogenesis of MS, most notably with regard to mitochondrial function and neurotransmitter signaling in glia.


Genomic analysis has been applied to investigate changes occurring in the central nervous system (CNS) in multiple sclerosis (MS). These include analyses of acute and chronic active lesions, lesions from patients at different stages of MS, and comparisons of normal appearing white matter (NAWM) and normal appearing gray matter (NAGM). Examination of changes in the lesions themselves showed numerous changes in genes related to immune and stress responses, as might be predicted from the pathologic changes in lesions [16]. Based on the premise that some of the earliest changes in the pathogenesis of MS lesions would be found in NAWM, where infiltration of immune cells is much less prominent [7, 8], Graumann and colleagues [9] analyzed genomic changes in NAWM from patients with secondary progressive MS (SPMS), and found evidence for changes characteristic of neuroprotective mechanisms initially identified in ischemic preconditioning associated with hypoxic insult. Dutta et al [10] examined NAGM and identified reduced expression of nuclear-encoded mitochondrial genes, as well as in genes related to ion homeostasis and neurotransmission. Several of the changes could be localized to neurons but since glia comprise a large proportion of the tissue samples, the relative contribution of neurons and glia to the changes in gene expression could not be quantitated. More recently the same group found upregulation of genes and proteins associated with ciliary neurotrophic factor (CNTF) and signaling pathways in normal cortical gray matter [11]. Subsequently, Mahad, et al [12] found decreased expression of mitochondrial Complex IV cytochrome oxidase subunits COX I and COX IV in type III MS lesions, suggesting that the hypoxia-like damage in this type of lesion may result from mitochondrial dysfunction. These findings suggest that a wide range of metabolic changes occur in both neurons and glia throughout the MS brain, independent of the local presence of systemic inflammatory cells, and that secretory products of immune cells and activated glia may play central roles in the pathogenesis of and protection from both white matter and gray matter damage in MS.

To dissect the underlying molecular changes that might occur in glial cells exposed to secreted products of immune cells, we are utilizing gene array analysis to compare the early effects of mixtures of cytokines typical of Th1 cells, monocyte/macrophages (M/M) or Th2 cells on gene transcription in cultures of mixed CNS glia from rat brain. We have initially focused on changes in gene expression at 6 hours of exposure of CNS glia to cytokines to identify some of the earliest primary responses that might occur in MS brains in response to cytokines, without the confound of changes in gene expression in the inflammatory cells, especially those regulated in the Th1 and Th2 cells. We are currently examining several of the changes in glial cell gene expression by quantitative real time-polymerase chain reaction (QRT-PCR) to analyze the duration of the effects, and find that some changes persist for as long as 5 days [13, 14]. In two previous papers, we summarized the effects of these cytokine mixtures on immune-related molecules [15] and on neural and glial related proteins, including neurotrophins, growth factors and structural proteins [16]. Each of the cytokine mixtures induced an unique and complex pattern of changes after 6 hours of incubation. In this third paper, we present the effects of the Th1, M/M and Th2 cytokine mixtures on early gene expression (6 hours) for molecules involved in metabolism, signaling and regulatory mechanisms in CNS glia. A number of the changes found are similar to those found in a gene array analysis of changes in rat spinal cord during the course of myelin basic protein (MBP)-induced experimental autoimmune encephalomyelitis (EAE) [17], including changes in ion homeostasis, mitochondrial function, neurotransmitter-related enzymes, and a variety of signaling pathways. An unexpected finding was the large number of changes in early gene expression related to lipid metabolism.

The culture system we have analyzed is devoid of neurons to enable identification of the responses of the several types of glia to the cytokines in the absence of cross talk with neuronal signaling. For example, although classically thought of as neuron specific, neurotransmitter receptors on glial cells are now known to play prominent roles in glial differentiation [1822], axonal/neuronal protection [2126], microglial activation [23] and impulse conduction along myelinated axons [24]. We are initiating studies on enriched neuronal cultures to identify the direct effects of the three cytokine mixtures on early gene expression in neurons for comparison with the changes found in glia, with the goal of identifying those cytokines most supportive of preventing damage and promoting normal axonal function.


The methodology has been described in detail in the prior papers [15, 16].

Mixed CNS glial cell cultures

Mixed CNS glial cell cultures were obtained from neonatal rat brain using a modification of the so-called "shake-off" technique [25, 26] as we described previously [27]. Following shakeoff of cells from the astroglial bed layer, the time for partial removal of microglia by adherence to plastic was 1 hour prior to plating on poly-lysine coated flasks. Cells were maintained in defined medium containing 2% fetal bovine serum for 6–8 days, then treated with the cytokines. The composition of cultures was examined by indirect immunofluorescence (IF) with antibodies to phenotypic markers for different cells types: glial acidic fibrillary protein (GFAP) for astrocytes [28] (Chemicon, Temecula, CA); galactolipids (GalL) for oligodendrocytes [28, 29]; A2B5 for oligodendrocyte precursors [30] (ATCC, Bethesda, MD); ED-1 for microglia [31] (Serotec, Raleigh, NC), Thy1.1 for fibroblasts [32] and in glial cultures some astrocytes [33]; anti-neurofilament heavy chain (NFh) for neurons [34] and anti-factor VIII for endothelial cells (Dako Corporation, Carpinteria, CA).

Cytokine mixtures

The Th1 cytokine mixture included the rat recombinant cytokines interleukin-2 (IL-2), interferon-γ (IFN-γ) (R&D Systems, Inc, Minneapolis), tumor necrosis factor-α (TNF-α; BD PharMigen, San Diego, CA) and mouse granulocyte-colony stimulating factor (G-CSF; PeproTech, Rocky Hill, NJ).

The M/M cytokine mixture included the rat recombinant cytokines IL-1α and IL-1β, IL-6, IL-12p40 (all from R&D Systems, Inc) and TNF-α. These cytokines would be considered proinflammatory products of M1 macrophages or microglia [35].

The Th2 cytokine mixture included the rat recombinant cytokines IL-4, IL-5, and IL-10 (all from R&D Systems, Inc), mouse G-CSF and purified porcine transforming growth factor-β1 (TGF-β1; R&D Systems, Inc.). In the cognate immune system, in some species, TGF-β1 is considered by some to be the product of so-called Th3 cells. TGF-β1 is also important in the development of another population of T-cells called regulatory T-cells (Treg cells) which are phenotypically characterized as CD4+/CD25 high+/Fox3 [36, 37]. These Treg cells may also secrete TGF-β1.

Cytokine mixtures contained 10 ng/ml of each of the constituent cytokines as is typically employed many in vitro studies of cytokine biology. For each experiment, four groups of three T75 flasks per group were incubated either with mixtures of Th1, Th2, M/M cytokines or additional medium (control) for 6 hours. Three sets of separate experiments consisting of control, Th1, M/M and Th2 stimulated cultures were performed.


As reported [15, 16], we examined the cytokine-induced effect on cell death in mixed CNS glial cell cultures by incubating cultures from 6 hours to 4 days with the cytokine mixtures. Cell death was determined by uptake of 0.4% trypan blue [38].

RNA extraction

Cultures were washed and frozen after 6 hours of incubation with cytokine mixtures or additional medium. RNA was extracted employing TRIzol (Gibco BRL, Grand Island, NY) followed by Qiagen RNeasy kits (Qiagen, Valencia, CA). The RNA was quantitated at A260 nm and the quality was assessed by at A260 nm/A280 nm. The 28S/18S ratio was assessed using a Bioanalyzer 2100 (Agilent Technologies, Wilmington, DE), and was > 1.7 for all samples.

Expression analysis

Biotin-labeled RNA fragments were prepared and hybridized to the Affymetrix rat RG-U34A microarray at 45°C for 16 hours, as previously described [15, 16]. Subsequent signal amplification was performed employing biotinylated anti-streptavidin antibody. The RG-U34A chip contains 7,985 genes. The control and three cytokine-incubated cultures from one experiment were analyzed with one gene chip for each sample and three separate experiments using different cultures were analyzed.

Data analysis

Data were analyzed by comparing the average of the replicates from each of the separate 3 sets of experiments. Affymetrix data were analyzed with dChip v1.2 to correct for background and calculate gene expression values [39]. We analyzed values from 3 separate experiments employing the t-test in GeneSpring comparing Th1, M/M and Th2 with control. Multiple testing analyses that compare all 7,985 genes at different levels of stringency using the Bonferoni and false discovery value (FDV) are statistically most rigorous, but at such high levels of stringency, there were very few changes that reached statistical significance. In order to increase sensitivity and allow identification of potentially important biologic changes, we employed a lower level of stringency [15, 16]. In these screening studies at a single time point, we have arbitrarily chosen to represent as probably significant those genes in which the mean expression was > 2 fold (upregulation) or < -2 fold (downregulation) compared to expression in controls (p < 0.2) [15]. We believe this is reasonable given that our experiments consisted of biological replicates that are prone to greater variability than experimental replicates. A similar p value was used in a gene array analysis of MS lesions [2]. The recent literature suggests that a 2-fold cut-off using the Affymetrix platform produces a low false positive rate [40].

Quantitative real time-polymerase chain reaction (QRT-PCR) expression analysis

Expression of message for iNOS was analyzed by QRT-PCR on an ABI 7500 Fast System, using ABI Taqman rat specific gene expression assays. RNA was extracted as above and reversed transcribed. Relative expression levels were calculated with GAPDH as the internal reference, using the delta-delta Ct method [41]. The values from the treated cultures were compared to those from control. Those ratios were averaged for the three experiments, then expressed as fold changes in the treated cultures relative to control for comparison with the gene array results. Each PCR value represents the average from 2–3 separate experiments.


Mixed CNS glial cell cultures

As in our earlier papers, cultures consisted of approximately 35% each of oligodendrocytes and astrocytes and 10% microglia. The remaining cells were glial cell precursors including A2B5 positive oligodendrocyte precursors. Endothelial cells and neurons were not present. Viability was > 98% in all cultures control and cytokine stimulated, at all time points examined (6 hours to 4 days) demonstrating the lack of cytotoxicity under these conditions.

Overview of cytokine effects on early gene expression

In the preceding papers we first described changes in CNS glia in genes for proteins predominantly associated with the immune system including major histocompatibility molecules, several adhesion and extracellular matrix molecules, cytokines and chemokines and their receptors and complement components [15]. Because of our interests in the effects of cytokines on the production of factors important in oligodendrocyte, axonal and neuronal function, in a second paper we compared the effects of the different cytokine mixtures on expression of genes for neurotrophins, growth factors, related receptors and structural proteins [16]. This third paper summarizes our findings for cytokine-induced changes in glial expression of genes for proteins associated with metabolism, signaling and regulation as well as neurotransmitters and ion channels. As noted, this is a series of screening experiments and therefore Tables 1 and 2 were prepared using the criteria of > 2 fold (increased expression) or < -2 fold (decreased expression) with a p value of < 0.2 for one or two replicates of the gene transcript [15, 16]. Unknown genes (ESTs) are not presented.

Table 1 Changes in early gene expression: neurotransmitters, ion channels and exchangers, apoptosis, mitochondria and glutathione metabolism
Table 2 Changes in early gene expression: gene regulation, signaling, cytoplasmic transport and metabolism

Neurotransmitters and receptors

All three cytokine mixtures had regulatory effects on message levels for a wide range of message levels for neurotransmitters and their receptors as well as on transporters involved with transmitters including glutamate, adrenergic, cholinergic, glycine, serotonergic, dopaminergic and purinergic systems (Table 1). The only adrenergic receptor affected was alpha 2 c-4, upregulated 2.5 fold (p < 0.05) by Th1 cytokines. Among cholinergic receptors, the largest change was for nicotinic cholinergic receptor alpha5, downregulated -2.3 fold (p < 0.05) by Th1. Dopaminergic receptors A3 and D1 were markedly downregulated -8 to -14 fold by Th1 and Th2 cytokines. Among several changes in glutamate receptors, Th1 upregulated ionotropic glutamate receptor delta 1 by 2.7 fold (p < 0.01), but markedly downregulated metabotropic glutamate receptor 7b by -9.5 fold (p < 0.01). Neuropeptide Y receptor 5 was downregulated by both Th1 and MM cytokine, -18 fold (p < 0.10) and -8 fold (p < 0.20), respectively, while the substance P precursor preprotachykinin A was downregulated -7 fold (p < 0.10) by Th2. For purinergic receptors, the most robust changes were -3 fold (p < 0.05) downregulation of P2X1by Th2, and upregulation of P2Y2 by MM and Th2, 3.5 fold and 2.4 fold, respectively, both p < 0.05.

Ion channels

Th1, M/M and Th2 cytokines had primarily downregulatory effects on expression of a very large number of genes for proteins that are components of ion channels including Na, K, Ca and Cl channels, both voltage-gated and non-voltage gated (Table 1). For example, Th1 and Th2 downregulated the voltage-gated alpha 1D L type Ca++ channel by -4 and -7 fold respectively, both p < 0.05. A large number of K+ channels were downregulated by Th2 cytokines, with fewer downregulated by Th1 or MM cytokines. The voltage-gated 1 alpha sodium channel was robustly downregulated by Th1 and MM cytokines, -9 fold (p < 0.05) and -7 fold (p < 0.10) respectively, while Th2 cytokines uniquely downregulated the 1 beta isoform, -2.5 fold, p < 0.01.

ATPase ion exchangers

In addition to the effects on ion channels shown in Table 1, there were effects on several ATPase ion exchangers. With the exception of upregulation of Ca++ATPase (plasma membrane 1) by M/M cytokines, several ATPase ion exchangers were downregulated by each of the cytokine mixtures (Table 1).


The cytokine mixtures induced up and down regulation of several genes for proteins involved in control of apoptosis (Table 1) including caspase 2, downregulated -3 fold, p < 0.05 by both MM and Th2 cytokines, and caspase 7, downregulated -3 fold by Th1 and MM cytokines.

Mitochondria and related proteins

There were cytokine mixture-induced changes in expression in genes of some mitochondrial proteins which are listed in Table 1, including a 4–6 fold (p < 0.05) upregulation of super oxide dismutase 2 (SOD2) by Th1 and MM, and an apparent -4 fold (p < 0.01) decrease for 16S mitochondrial ribosomal RNA.


The genes for several proteins involved in glutathione metabolism and secretion were regulated by the different types of mixtures of cytokines (Table 1). Subunits of glutathione S-transferase were generally downregulated, while both Th1 and MM upregulated P-glycoprotein(multi-drug resistance protein) by 5 fold, p < 0.10 and p < 0.05, respectively.

Transcription factors

Th1 cytokines markedly upregulated junB, CREB and the p105 subunit of NF κB. Both Th1 and M/M cytokines altered expression of genes for several other transcription factors, while the Th2 cytokines had minimal effects (Table 2). All three cytokines markedly upregulated message levels of junB (2–3 fold, p < 0.01) and downregulated hepatic nuclear factor alpha (-3 to -7 fold, p < 0.05.). Both Th1 and M/M cytokines upregulated the expression of the gene for NF kappa B p105 subunit and downregulated the aryl hydrocarbon receptor (Table 2).

Nuclear receptors

Th1 and M/M cytokines upregulated the gene for peroxisome proliferator activator receptor δ (PPAR δ) by 2 fold (p < 0.10), and downregulated the gene for PPAR γ by -5 fold (p < 0.01). Th2 cytokines downregulated the gene for PPAR γ but had no effect on PPAR δ (Table 2).


The cytokine mixtures had effects on the expression of genes for many signal transduction molecules, some of which are presented in Table 2. Among these are STAT1, 3 and 5, JAK2, homer and protein kinase 2 B. Of interest, only one gene in this category was affected by Th2 cytokines, stress activated protein kinase alpha 2, upregulated 3 fold (p < 0.05).

Cytoplasmic transport and degradation of proteins

Th1, M/M and Th2 cytokines had effects on the genes for several proteins involved in synthesis, degradation and intracellular transport of proteins including synucleins, proteasome subunits, ubiquitin conjugating enzymes and heat shock protein (HSP) 70 kDa (Table 2). For example, proteasome (macropain) alpha 6 was upregulated 10 fold (p < -.01) by Th1 cytokines, while the ubiquitin-conjugating enzyme E2D 2 was downregulated -4 to -6 fold (p < 0.05) by all three cytokine mixtures.

Lipid synthesis and signaling

As noted in Table 2, the genes for several proteins involved in lipid metabolism and signaling were regulated by the different cytokine mixtures. For example, fatty acid CoA ligase long chain 4 was downregulated -4 to -5 fold (p < 0.05 fold) by Th1 and Mm cytokines, while UDP-glucose ceramide glycosyl transferase was upregulated 3 fold (p < 0.05). With regard to lipid signaling, CDP diacylglycerol synthase was markedly upregulated 6–10 fold (p < 0.01) by all three cytokines, while EDG (endothelial sphingolipid GPCR) was downregulated -6 fold (p < 0.01) by MM cytokines.

Steroid and vitamin D related

Specific genes regulating proteins involved in sterol and vitamin D metabolism were also regulated by the cytokine mixtures (Table 2). Thus, 17-beta hydroxyl sterol reductase was downregulated -4 to -5 fold (p < 0.05) by Th1 and Th2 cytokines, and testosterone 6-beta hydroxylase was downregulated -7 fold (p < 0.05) by MM cytokines.

Miscellaneous proteins

There were a large number of genes for proteins not included in the above categories that are potentially of importance in understanding the pathogenesis of MS, as well as protective and reparative mechanisms. These are listed in Table 2. Of note, Th1 and MM cytokines upegulated nitric oxide synthase 2 (iNOS) by 15 fold (p < 0.05), while Th2 cytokines markedly downregulated genes for angiotensin receptor type 1b (-5 fold), beta tubulin 2b (-12 fold), and chapsyn-110 (-29 fold), all p < 0.05, but upregulated arginase and low density lipoprotein receptor 1 by 5 fold (p < 0.05).


We validated expression changes in three genes by QRT-PCR: iNOS, the enzyme that synthesizes NO from arginine; arginase, the enzyme that breaks down arginine, thus limiting production of NO; and P-glycoprotein (multi-drug resistance 1), an ABC transporter involved in regulation of glutathione levels. As noted in Table 3, we were able to confirm striking upregulation of the gene for iNOS by Th1 and M/M at 6 hours employing QRT-PCR. Although no effect on the gene for iNOS expression was observed at 6 hours in response to Th2 cytokines on microarray, we detected modest downregulation employing QRT-PCR. For arginase, we confirmed upregulation by Th2, with no change induced by Th1; however, in contrast to the array results, PCR indicated some upregulation of arginase by MM at 6 hours, rather than no change. For P glycoprotein, PCR showed upregulation by Th1 (in two of three analyses) and MM, as on the gene array, but also indicated a modest increase with Th2 rather than no change. The results for these three genes show relatively good agreement, and indicate that the arrays are not giving false positive results, but in some instances may give false negative results, suggesting that PCR may be more sensitive than the gene array.

Table 3 QRT-PCR validation of gene array results for cytokine-induced changes in gene expression at 6 hours


In our two preceding papers, we showed marked differential early effects of Th1 cytokines, M/M cytokines and Th2 cytokines on glial expression of a variety of genes, including those for immune-related molecules [15] and for neurotrophins, growth factors and structural proteins [16]. In addition, following the 6 hours of cytokine exposure used in these studies, we saw changes in expression of a large number of genes involved in signaling, regulation and metabolism. Some of these changes might be predicted from known effects of cytokines in vitro and in EAE or MS tissue, while other changes were unexpected. In contrast to the in vivo studies, our examination of an early 6 hour time point provides information about what might be some of the initial responses of glia per se to these cytokines.

Neurotransmitters and receptors

Glial cells have been reported to express different neurotransmitters and receptors as well as transporters for these transmitters [23, 4245]. With increasing evidence that both oligodendrocyte and neuronal/axonal damage may be caused by glutamate induced toxicity [4650], and that other glutamate receptors may be protective [51], the effects of cytokine mixtures on different GluR may influence and modify the effects of glutamate. AMPA, kainate and NMDA receptors may be important in oligodendrocyte toxicity in MS and EAE [5257] whereas upregulation of metabotropic GluRs (mGluRs) may provide protection [58]. The effects of upregulation of AMPA, NMDA and kainate GluR on neuronal death are well recognized [59]. It is of interest that Th1 and M/M upregulated GluRs associated with cell toxicity whereas all three cytokine mixtures markedly downregulated metabotropic mGluR7b. The group III mGluRs, including mGluR7, inhibit production of RANTES induced in astroglia by TNF-α or IFN-γ [60]. We previously reported upregulation of the gene for RANTES by Th1 and M/M cytokines [15].

The role of other transmitters in glial cell function is not as well understood. In addition to receptors for well established neurotransmitters and classically described ion channels (Ca++, K+ and Na+), we detected effects on genes for the purinergic P2X receptors, some of which serve as ligand gated ion channels [61], and the P2Y receptors which act as G protein coupled receptors when ligated by extracellularly released nucleotides, as occurs in inflammation and other stressful conditions within the CNS. The purinergic receptors modify the response of astrocytes to cytokines such as IL-1β and TNF-α and modify astrocyte functions including Ca++ influx [6264], as well as modulating transport of other ions [65]. The P2X7 receptor is found in resting and activated microglia in epileptic brain and several other neurologic diseases [66], and plays a role in microglial proliferation [61] and migration [23]. The P2X7 receptor is expressed in reactive astrocytes and microglia/macrophages in MS lesions [62], and is transiently upregulated by the M/M cytokine IL-1β in cultured fetal human astrocytes, resulting in increased iNOS activity [64]. We noted modest downregulation of the gene for the P2X7 receptor with M/M at 6 hours in our mixed glial cultures, suggesting that the mixture of cytokines or the presence of other glial cell types may modulate the glial responses to IL-1β, or that upregulation seen in vivo and in vitro in other studies may be a secondary response occurring at later time points.

The different cytokine mixtures had variable effects on a large number of receptors for several other transmitters including dopaminergic, serotonergic, cholinergic, adrenergic and melanocortin as well as the previously discussed purinergic receptors. The downregulation of the genes for the D1 and D3 dopamine receptors by the cytokine mixtures was especially striking. All three types of glia are known to express dopamine receptors, and D3 dopamine receptors may play roles in oligodendroglial differentiation and myelination [67], as well as protection of oligodendrocytes against glutamate oxidative stress and oxygen glucose deprivation [68]. Binding of neurotransmitters to their receptors on microglial cells seem to be important in microglial function [23].

It is also of interest that several of the neuronal nicotinic acetylcholine receptors are involved in downregulation of proinflammatory immune reactions in the systemic immune system, in particular the nicotinic α7 receptor [69, 70], which we found downregulated by both M/M and Th2 cytokine mixtures. Further, attenuation of cholinergic signaling with the acetylcholinesterase inhibitor physostigmine results in inhibition of CNS inflammation and development of EAE [71]. Conversely, we found that M/M cytokines upregulated the gene for the acetylcholinesterase T subunit, which could lead to increased acetylcholinesterase and a decreased "protective" cholinergic response.

Expression of genes for several transporters for glutamate and glycine were also observed along with changes in the genes for the R5 receptor for neuropeptide Y receptor 5 and preprotachykinin A (precursor of substance P). The role of such receptors and transporters in glial cells is not clear. Of interest, changes were found in activity of neurotransmitter-induced early genes 9, 10 and 12. It is not known if these cytokine mixtures have a similar effect on the same genes and their proteins in various subpopulations of neurons. If they do, this would have major implications on neuronal and axonal dysfunction in MS and other diseases characterized by CNS inflammation and/or microglial activation [72], as well as symptoms in patients with MS such as depression, memory loss, abnormalities in other cognitive functions, fatigue and pain.

Ion channels

We observed cytokine induced changes in gene expression for many ion channels including Na+, K+ and Ca++ channels. It is well established that glial cells have a wide variety of ion channels which are important in glial cell function [21, 7377] and that expression of some of these have been reported to be affected by cytokines in glial cells and neurons, as well as other types of cells [21, 7881]. Cytokine effects on ion channels and ion exchangers clearly are important in axonal and neuronal function and viability as well as likely contributing to symptomatology in patients with MS [8286]. Changes in genes for ion channels have been reported in the CNS in MS and EAE [2, 87].

There have been reports of inflammatory mediators such as inducible nitric oxide synthase (iNOS) inducing upregulation of certain Na channels in neurons [84, 88]. We observed significant effects on gene expression for a wide variety of ion channels in glial cells at 6 hours of incubation suggesting a direct effect of cytokines on expression of genes for some or all of these channels in glia. Changes in the distribution of ion channels could contribute to glial cell dysfunction. If similar changes were induced directly in neurons/axons, these changes could contribute to plasticity as well as to axonal and neuronal cell death. While such changes in neuronal ion channels and failure and reversal of ion exchangers, particularly Ca++ exchangers [89, 90], could result from failure of mitochondrial energy metabolism [10, 91], our results also raise the possibility of axonal dysfunction and ultimately death by direct effect of cytokines on expression of genes for ion channels and ATPase ion exchangers (see below). The cytokine mixtures also likely regulate ion channels on inflammatory cells such as lymphocytes, and ion channels are known to affect lymphocyte function [92].

ATPase ion exchangers

Th1 downregulated the genes for Na+/K+ ATPase, α4 polypeptide and Ca++ transporting ATPase. M/M upregulated the genes for Ca++ transporting ATPase and Ca++ ATPase, plasma membrane 1. Th2 likewise downregulated the genes for several ATPase ion transporters including Na+/K+ ATPase α4 polypeptide, Ca++ transporting ATPase, Ca++-pumping ATPase isoform 4, H+/K+-ATPase α2 gene, alternatively spliced and H+/K+ ATPase, nongastric, α polypeptide, nongastric; and the Na+/H+ ion exchanger (Table 1).


The possible role of oligodendrocyte cell death through apoptosis via caspases [93] or via other pathways to cell death [94] in MS lesions continues to be controversial, and it is likely that apoptosis as a mediator of oligodendrocyte death varies in different lesions [95]. Neuronal cell death by what appears to be apoptosis is also seen [96]. Up and downregulation of expression of various genes for proteins involved in apoptosis including caspases and Bcl X were noted. Th1 and M/M cytokines both induced upregulation of the genes for caspase 7, a downstream effector caspase involved in caspase-dependent apoptosis [97], and Bcl X, a protein which inhibits apoptosis [98, 99]. M/M cytokines downregulated the gene for caspase 2, a caspase implicated in oligodendrocyte cell death via the p75 receptor [93]. The gene for cytolysin (a constituent of lymphocyte toxic granules) was downregulated by both Th1 and Th2 cytokines, and Th2 and M/M cytokine mixtures both downregulate the gene for the protein programmed cell death 2.

Mutations in the gene for the protein huntingtin (Htt) result in Huntingon's chorea. Htt interacts with several proteins. One of these proteins is called htt-interacting protein (HIP-1). When HIP1 is bound to normal Htt, it inhibits apoptosis in certain neurons and Htt seems to be involved in endocytosis as well [100103]. In addition abnormal huntingtin interferes with normal ubiquitin-proteosome function and one could readily postulate that downregulation of proteins such as HIP-1 that interact with htt could also lead to abnormal protein aggregation such is seen in many degenerative diseases including Huntington's disease, where it is the htt that is qualitatively abnormal [104]. There was downregulation of the gene for HIP-1 by Th1 and M/M cytokines.

Changes in expression of mitochondrial protein genes, including genes associated with some apoptotic pathways, were noted (see below).

Mitochondria and related proteins

Changes in mitochondrial related genes have been noted in MS cortical gray matter in patients with long-standing chronic MS [10], and failure in mitochondrial associated energy metabolism may be important in axonal and neuronal degeneration and cell death [89, 91]. Most of the detected changes were reduced expression of genes, particularly components of complex I, III and IV. Decreased expression of COX subunits I and IV (Complex IV) has been detected in oligodendroglia, astroglia and axons, but not in microglia, in acute Type III MS lesions [12]. In our CNS glial cultures, we found predominately downregulation of genes associated with mitochondria. Th1 cytokines upregulated genes for hexokinase II and superoxide dismutase 2 (Mn++ SOD2), and downregulated genes for NADH dehydrogenase (Complex I), COX VIa and COX VIc (Complex IV), ferredoxin 1, and perhaps for 16s ribosomal RNA, which is a component of the large subunit of the mammalian mitochondrial ribosome, responsible for synthesis of 13 proteins localized in the inner mitochondrial membrane [105]. This latter finding has yet to be confirmed. M/M cytokines upregulated genes for SOD2 and down regulated the genes for NADH dehydrogenase and creatine kinases. Th2 cytokines down regulated genes for NADH dehydrogenase and 16S ribosomal RNA. Th1 down regulated the gene for SOD3, an extracellular Cu++/Zn++ SOD. None of the cytokine mixtures had an effect on the gene for the mitochondrial protein Cu++/Zn++ SOD1; some familial forms of amyotrophic lateral sclerosis (ALS) are associated with mutations in this gene [106]. One could postulate that inflammatory cytokines, perhaps products of activated microglia, at first stimulate transcription of genes for some mitochondrial enzymes, but decreased expression of the 16S subunit of the mitochondrial ribosome could lead to ongoing downregulation of genes and their proteins critical for mitochondrial function in oligodendrocytes and neurons.


We observed effects on the genes for several proteins involved in glutathione metabolism and secretion. Glutathione serves a protective function by reducing the effect of free radicals produced via oxidative stress [107, 108], and the cytokine mixtures had significant effects on genes for several proteins involved in synthesis, regulation and release of glutathione. Th1 upregulated the gene for P-glycoprotein/multidrug resistance protein 1/MDR1 (P-glycoprotein/ABCB1), as did M/M cytokines. MDR1, which in addition to inhibiting the therapeutic effects of drugs, has effects on vascular structures and influences secretion of glutathione by cells such as astrocytes [109111]. In addition to astrocytes, it has been detected in microglia, oligodendrocytes, endothelial cells and neurons [112, 113]. Glutathione is more abundant in astrocytes than in other brain cell types, which may contribute to the relative resistance of astrocytes to ischemia and other pathologic processes that involve oxidative stress. Changes in glutathione may also contribute to the relative vulnerability of oligodendrocytes and precursors at different stages of maturation to oxidative stress [107, 114116]. Glutathione also modulates prostaglandin metabolism [117]. We describe cytokine modulation of expression of several genes associated with prostaglandin metabolism [15], and prostglandin D synthase has been reported to be upregulated in MS lesions [2].

Transcription factors

Some genes for transcription factors were upregulated, quite predictably, such as NF-κB p105 in the presence of IL-1 or TNF-α (Th1 and M/M cytokines). Cyclic AMP response element binding protein 1 (CREB) was upregulated in response to Th1 cytokines.

The upregulation of the gene for CREB by Th1 cytokines may result from the effect of TNF-α[118], although M/M cytokines which also contain this cytokine did not appear to have the same effect.

Jagged 1 is a transcription factor reported to be upregulated in astrocytes by TGF-β and through Hes and notch1 leads to inhibition of myelination [119], although its presence may not be necessary to inhibit myelination [120]. Given the report of upregulation of jagged 1 by TGF-β in astrocytes [119], an unexpected finding in our experiments was upregulation of jagged 1 by Th1 and M/M cytokine mixtures which do not contain TGF-β and the failure of the Th2 cytokine mixture, which contains this cytokine/growth factor, to upregulate jagged 1. These differences could relate to differences in the target tissues (single cell types versus mixtures of different cell types), species and/or effects of a single cytokine versus mixtures of cytokines, effects of some of the induced proteins and their influence on downstream signaling. At 6 hours none of the cytokine mixtures had any discernable effect on expression of notch1 or Hes.

In addition we observed effects on gene expression of other transcription factors including hepatic nuclear factors (HNF), POU and elongation initiation factor 5, important in initial stress responses.

Nuclear receptors

PPARs are nuclear receptors originally associated with lipid metabolism but subsequently found to also be involved in cellular differentiation. Th1 and M/M cytokines both upregulated the gene for PPAR δ and down regulated the gene for PPAR γ, whereas Th2 cytokines down regulated the gene for PPAR γ and had no effect on the gene for PPAR δ. TNF-α, a component of both the Th1 and M/M cytokine mixtures, has been reported to down regulate the gene for PPAR δ [121]. This is another potential example of differences in the effects of single cytokines versus mixtures of cytokines. Ligation of PPAR γ results in down regulation of inflammatory responses and can inhibit EAE [122, 123] and has lead to studies to evaluate such agents, which are used in the treatment of diabetes and hyperlipidemia, as therapy for MS. Activation of PPAR δ with different ligands than those that react with PPAR γ causes activation and accelerates differentiation of oligodendrocytes in vitro [124]. How the differential regulation of the PPARs affects inflammation and the potential for favorably influencing remyelination through these receptors is not as yet clear.


Th1 and M/M cytokines markedly upregulated Janus kinase 2 (JAK) as well as several members of the STAT family, in keeping with the well known activation of the JAK/STAT pathway by inflammatory cytokines. Studies in brain ischemia indicate that increased signaling via the JAK/STAT pathway occurs predominantly in microglia rather than astroglia or neurons [125]. We found a 10 fold increase in STAT 1 with Th1 cytokines, consistent with an increase in STAT 1 identified by gene array analysis in both chronic and active MS lesions [126].

The gene for homer, a key protein in Group I metabotropic GluR signaling, was upregulated by Th1 and Th2 cytokines [127130]. In addition there were effects on expression for genes of protein kinase C, protein kinase A, protein tyrosine kinase 2B, CaM kinase II, Rho family GTPase, receptor serine threonine kinase, lyn protein non-receptor kinase and fyn-related kinase. Fyn, the only src family kinase, is upregulated during oligodendrocyte differentiation [131] and signals through Rho family GTPases to regulate their morphologic maturation [132].

Cytoplasmic transport and degradation of proteins

Th1 cytokines upregulated the genes for α synuclein and for several proteasome proteins and ubiquitin-like protein (NEDD 6), and down regulated the genes for proteasome subunit R-RING (although as noted upegulated other transcripts of the R-RING subunit), β synuclein (SYN 2), receptor serine threonine kinases, ubiquitin ligase (NEDD 4), and ubiquitin conjugating enzyme E2D 2. M/M cytokines upregulated the genes for NEDD 6 and SYN2, and downregulated the genes for ubiquitin-conjugating enzyme E2D 2, NEDD 4 and proteasome subunit R-RING. Th2 cytokines downregulated the genes for ubiquitin conjugating enzyme E2D 2 and similar to ubiquitin-conjugating enzyme E2D 2. If changes in the expression of these genes result in changes in the level of these proteins, it would imply that inflammation could contribute to the changes in these proteins seen in sporadic forms of several degenerative disorders where synucleins and ubiquitin aggregation have been described [133, 134]. The synucleins, considered neuronal proteins, are involved in synaptic function and have chaperone functions as well [135, 136]. Th1 cytokines upregulated α-synuclein, which has been detected transiently in rat oligodendrocytes in vitro [137] and in inclusions in glial cells in some CNS diseases including multisystem atrophy (MSA) [138]. Proteasomes are involved in transport of protein degradation products as well as in transport of MHC proteins and antigen within antigen presenting cells (APC).

Heat shock proteins (HSP) are upregulated in response to several types of cell stress stimuli [139]. One of several functions of HSP is acting as chaperones to help in normal transport of other proteins within the cytoplasm of many cell types. M/M cytokines upregulated the gene for heat shock protein (HSP) 70 kDa. Interestingly upregulation of the gene for α/β crystalline which also serves as a stress response protein has been reported to be increased in MS lesions [2].

Lipid synthesis

Th1 cytokines altered gene expression for several enzymes involved in synthesis of fatty acids and phospholipids (Table 2). Both Th1 and M/M cytokines downregulated message of the gene for HMGCoA reductase, the principal regulatory enzyme for cholesterol and other isoprenoids. Interestingly statins, which are inhibitors of this enzyme, are being tested as treatment for MS [139142] because they inhibit experimental autoimmune encephalomyelitis (EAE), an animal model for MS. The mechanisms include decreased farnesylation causing a Th1 to Th2 shift and monocyte/macrophage inflammation [141, 143145], and perhaps alteration of other signaling pathways [16].

UDP-glucose:ceramide glycosyltransferase is upregulated in the presence of TNF-α (Th1 and M/M cytokines). This enzyme is involved in ceramide metabolism as part of both ceramide induced cell death via TNF-R type I signaling pathways, as well as catalyzing the initial step in ganglioside synthesis. Th1 cytokines also downregulated the gene for UDP-galactose ceramide galactosyltransferase (member 8 of the UDP-glucuronosyl transferase family). This enzyme is markedly upregulated during differentiation of oligodendroglia and synthesizes galactocerebroside, the major glycolipid of myelin and precursor to sulfatide. An early response to inflammatory cytokines has not been previously reported for the gene or the protein.

Notably Th1 cytokines upregulated the gene for phospholipid scramblase, which translocates phospholipids from one surface of the plasma membrane to the other.

In our initial paper we reported that Th1 and M/M cytokines induced robust upregulation of genes for ABC transporter 1, which among its several functions, translocates phosphatidyl choline and cholesterol to the outer membrane leaflet in astrocytes and neurons [146], and for ABC transporter 2, active in oligodendrocytes during myelination [147]. The ABC transporters are also important in intraceullar transport of other proteins including peptide epitopes with MHC class I molecules [15].

Lipid signaling

Th1 cytokines downregulated the gene for diacyl glycerol kinase beta, which phosphorylates diacyglycerol to produce phosphatidic acid, leading to termination of diacylglyceryol signaling via PKC, Ras GTPase and other signaling pathways.

IL-2, one of the components of the Th1 mixture, upregulates diacylglycerol kinase in myelin [148], raising the possibility that oligodendroglia are upregulating this gene in response to the Th1 cytokines. In addition, the gene for CDP-diacylglycerol synthase, the enzyme that synthesizes phosphatidyl inositol from phosphatidic acid, is robustly upregulated by both Th1 and M/M cytokines, suggesting a switch from diacylglycerol to phosphatidyl inositol mediated signaling pathways. Conversely, the gene for myo-inositol monophosphatase was downregulated by M/M cytokines; this enzyme, the key enzyme inhibited by lithium, generates free inositol from inositol-3-phosphate derived from glucose-6-phosphate, and regulates levels of inositol available for synthesis of phosphatidyl inositol and its multi-phosphorylated derivatives critical for intracellular signaling and trafficking as well as calcium homeostasis. It is of note that lithium is currently being evaluated as a treatment of amyotrophic lateral sclerosis based on its effects on inositol pathways [149152].

Sphingosine-1-phosphate plays a key role in cell survival and inflammatory responses [153]; the gene for one of its receptors, EDG (endothelial sphingolipid GPCR) was down regulated by both M/M and Th2 cytokines. There has been a phase II trial in patients with MS of an oral agent called FTY72, which binds to the EDG (S1P) receptor (endothelial differentiation sphingolipid G-protein coupled receptor) [154]. A large Phase III study is underway. Inhibition of this receptor both blocks emigration from and favors homing of lymphocytes to secondary lymph structures, ostensibly without affecting T-cell viability or inhibiting memory T-cells. In experimental animals other inflammatory cells, such as monocytes and mature dendritic cells, are also affected and the protein is also found on endothelial cells. The drug has also been used in studies of treatment of other immune disorders [155160] The S1P receptor EDG is also found in the CNS on glial cells [161163]. The roles of S1P and its G-coupled receptor in the normal CNS are not known. It has recently been shown that activation of S1P results in changes in glial cells in vitro [164, 165]. If FTY720 gains access to the CNS there is the potential to modulate the activity of S1P with uncertain consequences for the patient.

Steroid and vitamin D related

Several genes coding for enzymes involved in steroid metabolism were downregulated by each of the three cytokine mixtures, including the gene for testosterone 6-beta-hydroxylase, markedly downregulated by M/M cytokines. Of note, Th1 cytokines upregulated the gene for vitamin D3 25-hydroxylase, the enzyme catalyzing the first step in activation of dehydrocholesterol to the active hormone, 1, 25-hydroxy vitamin D3. However, both M/M and Th2 cytokines down regulated 25-OH vitamin D3 24-hydroxylase, a key step in the inactivation of the active form of vitamin D3 [166]. Both are mitochondrial enzymes and members of cytochrome p450 family. In several studies vitamin D3 dietary supplementation prevented the onset and progression of EAE. In MBP-induced EAE in mice, the treated animals showed marked decreases in chemokines, iNOS and CD11b+ recruitment into the CNS, perhaps due to activated T cell apoptosis [167]. One large study found that vitamin D3 supplementation reduced the risk of developing MS [168], while four smaller studies suggested a reduction in exacerbations (reviewed in Brown, 2006) [169]. Our findings suggest that both M/M and Th2 cytokines might act to attenuate the effects of the active forms of vitamin D3.

Miscellaneous proteins

The classically proinflammatory Th1 and M/M cytokines markedly upregulated the gene for iNOS, a critical protein in generation of NO, which gives rise to related reactive oxygen species such as peroxynitrite [170]. Increases in iNOS have been reported in the CNS in EAE and in MS [171174]. There is evidence that NO could directly or indirectly, by forming peroxynitrite, damage oligodendrocytes, myelin and neurons/axons [175]. Reactive nitrogen species can also influence neuronal Na channels and thus cause damage, especially with rapid firing bare axons [171, 176]. It has also been suggested that NO could have an immumodulatory effect on inflammatory cells. NO production in inflammatory cells and in glial cells is induced by iNOS. As described in Results, employing QRT-PCR we confirmed the upregulation of expression of the gene for iNOS by Th1 and M/M cytokine mixtures and also found modest downregulation of the gene in response to Th2 cytokines (Table 3).

Galanin is a peptide in the CNS and PNS which is upregulated in response to injury [177, 178]. While originally described in various neurons it has been demonstrated in glia as well.[179, 180] and has a positive effect on neurite growth, cell survival and regeneration [181183] as well as involved in interactions with hormones [184, 185], pain signaling pathways [186, 187] and other CNS functions [188, 189]. Galanin receptors are also co-localized with cholinergic receptors in astroglia [190]. During oligodendrocyte differentiation, the gene for galanin is markedly downregulated [191]. Therefore upregulation by Th1 and M/M cytokines may represent an early attempt of oligodendroglia to return to a less differentiated state, one capable of proliferation.

The alpha-fetoprotein that is increased in the serum of women in the last trimester of pregnancy has been shown to have immunosuppressive effects in EAE as well as in experimental autoimmune myasthenia gravis (EAMG) [192195]. It also can suppress autoreactivity in vitro to two respective autoantigens, MBP and acetylcholine receptor (AChR). This has lead to the suggestion that it may be one of several factors responsible for inhibition of disease activity during the third trimester of pregnancy in patients with MS as well as MG. It is of some interest that expression of the gene for this potentially immunosuppressive protein is downregulated by Th1 and M/M cytokines.

Nestin, an intermediate filament protein, is a marker of early neuronal cell development. It is also a marker of other progenitor cells, particularly glial cells in the CNS, and may be involved in cell proliferation [196199]. The proinflammatory Th1 cytokine mixture down regulated the gene for nestin, which would be compatible with an inhibitory effect of such cytokines on neuronal and glial cell precursors.

VIPR2 binds VIP, a peptide shown to induce release of cytokines and other factors from glial cells [200, 201]. Downregulation of this protein by Th1 cytokines secreted by infiltrating inflammatory cells or endogenous glia would inhibit the release of both cytokines and growth factors by glial cells.

We detected M/M induced upregulation of a gene transcript for angiotensin receptor 2 (ATR2), whereas Th1 cytokines down regulated expression of the same transcript and Th2 cytokines down regulated a different ATR2 transcript. M/M cytokines upregulated the gene for ATR 1. ATR 2 is expressed by endothelial cells as well as glial cells. ATR1 is also expressed by endothelial cells as well as other cells within the CNS. Angiotensin and ATR are involved with interactions with VEGF and other molecules and may be involved in CNS cell death via apoptosis [202204]. Increased expression of the gene for angiotensin, the ligand for angiotensin receptors, has been described in studies of MS brain tissue [205, 206].

Two unexpected and novel findings were the marked decreases in expression of the genes for chapsyn-110 and beta tubulin by Th2 cytokines. Chapsyn-110 is a member of the PSD95/SAP90 protein family. The protein is found in postsynaptic densities in somatic/dendritic neuronal processes, and interacts with the C-terminus of subunits of the NMDA GluR and shaker-type potassium channel [207, 208]. The protein is linked indirectly to microtubules and involved in clustering of the receptors and ion channels; its presence and function in glia have not been previously reported. The marked down regulation of chapsyn-110 along with that of beta tubulin in glia may lead to potentially neuroprotective disruption of signaling through NMDA receptors and potassium channels in these cells.

Ceruloplasmin is a metal binding protein which is increased in response to inflammatory signals. In the brain ceruloplasmin is important as a binder of iron, and in the absence of ceruloplasmin (aceruloplasminemia), iron is able to induce tissue injury by increasing lipid peroxidation [209211]. M/M cytokines upregulated the expression of the gene for ceruloplasmin whereas Th1 and Th2 cytokines had no effect. Effects on genes for iron binding proteins if resulting in sufficient increase in protein would down regulate free iron induced lipid peroxidation, whereas a reduction or even a failure of increase in such proteins could result in cell damage or even death.

Caveolins are a group of proteins that are important in the structure of cell membranes including neurons and myelin. They are components of the so called "lipid rafts", important constituents of plasma membranes. Caveolins 1, 2 and 3 are upregulated in spinal cord of rat with EAE with caveolin 3 being expressed by astrocytes [212], although at 6 hours in vitro Th1 cytokines down regulated the expression of the gene for caveolin 3.

Arginase 1 is involved in synthesis of polyamines which have been shown to improve axonal regeneration on myelin substrates [213]. Th2 upregulated the gene for this protein, which would favor axonal regeneration. Th2 cytokines, particularly IL-4, stimulate production of arginase by macrophages, and there is an inverse relationship between production of iNOS induced by Th1 cytokines and arginase induced by Th2 cytokines in these cells [214216]. By inhibiting production of nitric oxide, arginase may also play a neuroprotective role for motor neurons deprived of trophic factors [217]. Recently, loss of arginase 1 was shown to increase proliferation of neural stem cells [218]. One could postulate that the microglia may be the glial cells upregulating the gene for arginase in our system.


In this paper and the preceding two [15, 16], we have identified responses to cytokines that would be predicted from analysis of MS tissue, others identified following treatment of individual glial types in culture, and yet others that have not been previously reported. Among the genes predicted from analysis of MS plaques are those related to hypoxic/ischemic responses, inflammatory responses and neuroprotective responses. Most strikingly, our finding that transcription of these genes in glia is changed within 6 hours of exposure to the cytokines implicates the glia as primary responders in the amplification or suppression of damage in white matter. In this paper, we report early changes in a wide variety of genes related to neurotransmitter signaling and ion homeostasis in glial cells. The most striking changes were the decreases induced by Th1 cytokines in dopaminergic receptors, metabotropic glutamate receptor 7b, and a receptor for neuropeptide Y. Identification of which glial type is responding and whether these changes result in long-lasting changes in gene expression, function, and interaction with neurons promise to be informative. With regard to changes in mitochondrial enzymes, the pattern of changes with Th1 cytokines was quite distinct from that seen with M/M cytokines, while Th2 cytokines induced only a few more modest changes. With Th1 cytokines, marked downregulation of the COX VI subunit was seen; this differs from the decrease in the COX IV subunit reported in MS tissue, and may provide a clue to the very earliest changes occurring in mitochondrial function in glia exposed to proinflammatory cytokines, as may the very early downregulation of the 16s mitochondrial ribosomal RNA, which would effect all of the 13 mitochondrial encoded genes. Upregulation by Th1 of genes for transcription factors such as junB, NF-κB and CREB might be predicted, while the decreases in HNF3 and 4 and the increase in the genes for the fox-1 homolog and jagged 1 by Th1 cytokines in glia have not been previously reported. Again, the many changes seen in expression of genes for proteasome, ubiquitin and synuclein proteins with Th1 cytokines might be anticipated, but stand in contrast to the relatively few changes seen in response to M/M and Th2 cytokines. Finally, lipid synthesis and signaling pathways have not been extensively explored in glia in response to cytokines; most notably, decreases by Th1 at 6 hours in the genes coding for synthesis of galactocerebroside implicate changes in oligodenroglial function, since the lipid serves as the precursor for sulfatide, shown to be critical for maintaining normal architecture and function at the nodes. The decrease in the gene for diacyl glycerol kinase and increase in CDP-diglyceride synthase suggests an early switch in signaling pathways within glia.

Table 4 summarizes the largest changes seen with each of the three cytokine mixtures, with the 12 most upregulated genes arranged in order from highest to lowest, and the 12 downregulated genes from most downregulated to least downregulated. While the magnitude of change in gene expression does not necessarily reflect the extent of biological relevance, the summary illustrates a number of changes in common between Th1 and M/M cytokines, as predicted by their predominance of proinflammatory cytokines. Very few genes were upregulated by Th2 cytokines in the categories analyzed in this study, only the 12 genes shown in the table.

Table 4 Summary of most upregulated and downregulated gene expression


It has been reported that certain MS lesions have features characteristic of ischemic or hypoxic injury to oligodendrocytes [12, 219] although inflammatory cells, particularly macrophages, are present in the lesions. Studies of normal appearing white matter in MS, employing gene array technology, have also shown changes in patterns of gene regulation consistent with ischemia and the response to ischemia [9]. It has also been suggested that local angiogenesis occurs in EAE and in MS [220]. We have identified early effects of these cytokine mixtures on molecules that are important in vascular pathology and angiogenesis as well as upregulated in ischemia and hypoxia. Obviously this includes a vast number of genes and gene products involved in transcription, cell signaling, mitochondrial function and apoptosis along with many others.

In addition to the changes in genes for proteins associated with apoptosis and mitochondria, in our current and prior studies [15, 16], we found that Th1 cytokines upregulated other genes reported to be regulated in the CNS in ischemia including adhesion molecules (ICAM-1, VCAM), cytokines and chemokines and their receptors (IL-1β, MCP-1), death and survival proteins (Bcl-X), proteases and inhibitors (MMP-9) and growth factors (FGF 1 and NGFRp75). Th1 cytokines did not affect the gene for e-selectin but upregulated the gene for its ligand. Among other genes for proteins regulated in CNS ischemia, Th1 cytokines down regulated genes for neurotrophins and their receptors (BDNF, NT3 and trkB), and cytokines, chemokines and their relevant receptors (several related to TGF-β). M/M cytokines upregulated genes for cell adhesion molecules (ICAM-1, VCAM), HSP 70, cytokines, chemokines and receptors (IL-1β, IL-1R type 1, IL-6, MCP-1), FGF 5 and 10, and MMPs and inhibitors (MMP9, TIMP-1) and downregulated genes of interest for response to ischemia, including TGF-β3, NT3, and FGF2. Th2 cytokines upregulated ischemia related genes for growth factors (BDNF, FGF 10 and 14, FGF-R1), cytokines and chemokines and receptors (IL-6, IL-1R type I and TGF-βR2) and down regulated genes for IL-1R type I, and NT3. Differential expression of many of these genes were reported in the NAWM of some patients with MS [9].

As previously reported [15, 16], Th1, M/M and Th2 cytokines had varying effects on the genes for molecules that are involved in altering in the cells of the blood brain barrier including several adhesion molecules and MMPs although our cultures do not contain endothelial cells. Some of these molecules are undoubtedly important in glial cells as well. In a previous study, we detected upregulation of the gene for VEGF [16]. Upregulation of VEGF could contribute to endothelial cell proliferation seen in some MS lesions producing local hypoxia and oligodendroglial death. The function of VEGF in glial cells as well as other non-glial non-neuronal cells, such as pericytes, which conceivably might be in our cultures is not known. Since inflammatory cytokines were able to upregulate the gene for VEGF as well as other genes that are associated with ischemia and the response to ischemia, our data suggests that cytokine release secondary to inflammation can lead to changes compatible with hypoxia and perhaps to induction of hypoxia itself.

We recognize the limitations of microarray analysis as well as gene expression studies since post-transcriptional and post-translational changes are not detected. In addition proteins such as receptors may be present in sufficient amount to be ligated and involved in a biologic process without requiring additional protein in the short run and thus no upregulation of gene for that protein. Nevertheless as a screening technique to obtain an overview of proteins that may be important in a particular process as well as the complexities of the effect of a mixture of factors on a mixture of cells, we believe that this is a promising approach. In addition microarray technology allows discovery of unexpected findings in complex experiments. Such findings may turn out to be both interesting and important.


  1. 1.

    Whitney LW, Becker KG, Tresser NJ, Caballero-Ramos CI, Munson PJ, Prabhu VV, Trent JM, McFarland HF, Biddison WE: Analysis of gene expression in multiple sclerosis lesions using cDNA microarrays. Ann Neurol. 1999, 46: 425-428. 10.1002/1531-8249(199909)46:3<425::AID-ANA22>3.0.CO;2-O.

  2. 2.

    Lock C, Hermans G, Pedotti R, Brendolan A, Schadt E, Garren H, Langer-Gould A, Strober S, Cannella B, Allard J, et al: Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med. 2002, 8: 500-508. 10.1038/nm0502-500.

  3. 3.

    Tajouri L, Mellick AS, Ashton KJ, Tannenberg AE, Nagra RM, Tourtellotte WW, Griffiths LR: Quantitative and qualitative changes in gene expression patterns characterize the activity of plaques in multiple sclerosis. Brain Res Mol Brain Res. 2003, 119: 170-183. 10.1016/j.molbrainres.2003.09.008.

  4. 4.

    Mycko MP, Papoian R, Boschert U, Raine CS, Selmaj KW: cDNA microarray analysis in multiple sclerosis lesions: detection of genes associated with disease activity. Brain. 2003, 126: 1048-1057. 10.1093/brain/awg107.

  5. 5.

    Mycko MP, Papoian R, Boschert U, Raine CS, Selmaj KW: Microarray gene expression profiling of chronic active and inactive lesions in multiple sclerosis. Clin Neurol Neurosurg. 2004, 106: 223-229. 10.1016/j.clineuro.2004.02.019.

  6. 6.

    Lindberg RL, De Groot CJ, Certa U, Ravid R, Hoffmann F, Kappos L, Leppert D: Multiple sclerosis as a generalized CNS disease – comparative microarray analysis of normal appearing white matter and lesions in secondary progressive MS. J Neuroimmunol. 2004, 152: 154-167. 10.1016/j.jneuroim.2004.03.011.

  7. 7.

    Allen IV, McQuaid S, Mirakhur M, Nevin G: Pathological abnormalities in the normal-appearing white matter in multiple sclerosis. Neurol Sci. 2001, 22: 141-144. 10.1007/s100720170012.

  8. 8.

    Allen IV, McKeown SR: A histological, histochemical and biochemical study of the macroscopically normal white matter in multiple sclerosis. J Neurol Sci. 1979, 41: 81-91. 10.1016/0022-510X(79)90142-4.

  9. 9.

    Graumann U, Reynolds R, Steck AJ, Schaeren-Wiemers N: Molecular changes in normal appearing white matter in multiple sclerosis are characteristic of neuroprotective me chanisms against hypoxic insult. Brain Pathol. 2003, 13: 554-573.

  10. 10.

    Dutta R, McDonough J, Yin X, Peterson J, Chang A, Torres T, Gudz T, Macklin WB, Lewis DA, Fox RJ, et al: Mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients. Ann Neurol. 2006, 57: 478-489. 10.1002/ana.20736.

  11. 11.

    Dutta R, McDonough J, Chang A, Swamy L, Siu A, Kidd GJ, Rudick R, Mirnics K, Trapp BD: Activation of the ciliary neurotrophic factor (CNTF) signalling pathway in cortical neurons of multiple sclerosis patients. Brain. 2007, 130: 2566-2576. 10.1093/brain/awm206.

  12. 12.

    Mahad D, Ziabreva I, Lassmann H, Turnbull D: Mitochondrial defects in acute multiple sclerosis lesions. Brain. 2008, 131: 1722-1735. 10.1093/brain/awn105.

  13. 13.

    Lisak R, Studzinski D, Bealmear B, Nedelkoska L, Benjamins J: Kinetics of gene expression in central nervous system glial cells induced by Th1, Th2 and monocyte/macrophage cytokines. Mult Scler. 2005, 11 (Suppl 2): S136-S137.

  14. 14.

    Lisak R, Studzinski D, Bealmear B, Nedelkoska L, Benjamins J: Regulation of genes for neurotrophins and their receptors in central nervous system mixed glial cell cultures by mixtures of cytokines. Mult Scler. 2007, 13: S219.

  15. 15.

    Lisak R, Benjamins J, Bealmear B, Yao B, Land S, Skundric DS: Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for mmune-related molecules by central nervous system mixed glial cell cultures. Mult Scler. 2006, 12: 149-168. 10.1191/135248506ms1251oa.

  16. 16.

    Lisak RP, Benjamins JA, Bealmear B, Nedelkoska L, Yao B, Land S, Studzinski D: Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for glial and neural-related molecules in central nervous system mixed glial cell cultures: neurotrophins, growth factors and structural proteins. J Neuroinflammation. 2007, 4: 30-10.1186/1742-2094-4-30.

  17. 17.

    Nicot A, Ratnakar PV, Ron Y, Chen CC, Elkabes S: Regulation of gene expression in experimental autoimmune encephalomyelitis indicates early neuronal dysfunction. Brain. 2003, 126: 398-412. 10.1093/brain/awg041.

  18. 18.

    Gallo V, Patneau DK, Mayer ML, Vaccarino FM: Excitatory amino acid receptors in glial progenitor cells: molecular and functional properties. Glia. 1994, 11: 94-101. 10.1002/glia.440110204.

  19. 19.

    Gallo V, Zhou JM, McBain CJ, Wright P, Knutson PL, Armstrong RC: Oligodendrocyte progenitor cell proliferation and lineage progression are regulated by glutamate receptor-mediated K+ channel block. J Neurosci. 1996, 16: 2659-2670.

  20. 20.

    Gallo V, Ghiani CA: Glutamate receptors in glia: new cells, new inputs and new functions. Trends Pharmacol Sci. 2000, 21: 252-258. 10.1016/S0165-6147(00)01494-2.

  21. 21.

    Soliven B: Calcium signalling in cells of oligodendroglial lineage. Microsc Res Tech. 2001, 52: 672-679. 10.1002/jemt.1051.

  22. 22.

    Kim WT, Rioult MG, Cornell-Bell AH: Glutamate-induced calcium signaling in astrocytes. Glia. 1994, 11: 173-184. 10.1002/glia.440110211.

  23. 23.

    Pocock JM, Kettenmann H: Neurotransmitter receptors on microglia. Trends Neurosci. 2007, 30: 527-535. 10.1016/j.tins.2007.07.007.

  24. 24.

    Fields RD, Stevens B: ATP: an extracellular signaling molecule between neurons and glia. Trends Neurosci. 2000, 23: 625-633. 10.1016/S0166-2236(00)01674-X.

  25. 25.

    McCarthy KD, de Vellis J: Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 1980, 85: 890-902. 10.1083/jcb.85.3.890.

  26. 26.

    Dyer CA, Benjamins JA: Antibody to galactocerebroside alters organization of oligodendroglial membrane sheets in culture. J Neurosci. 1988, 8: 4307-4318.

  27. 27.

    Lisak RP, Bealmear B, Nedelkoska L, Benjamins JA: Secretory products of central nervous system glial cells induce Schwann cell proliferation and protect from cytokine-mediated death. J Neurosci Res. 2006, 83: 1425-1431. 10.1002/jnr.20851.

  28. 28.

    Raff MC, Mirsky R, Fields KL, Lisak RP, Dorfman SH, Silberberg DH, Gregson NA, Leibowitz S, Kennedy MC: Galactocerebroside is a specific cell-surface antigenic marker for oligodendrocytes in culture. Nature. 1978, 274: 813-816.

  29. 29.

    Ranchst B, Clapshaw PA, Price J, Noble M, Seifert W: Development of oligodendrocytes with a monoclonal antibody against galactocerebroside. Proc Natl Acad Sci USA. 1982, 79: 2709-2713. 10.1073/pnas.79.8.2709.

  30. 30.

    Eisenbarth GS, Walsh FS, Nirenberg M: Monoclonal antibody to a plasma membrane antigen of neurons. Proc Natl Acad Sci USA. 1979, 76: 4913-4917. 10.1073/pnas.76.10.4913.

  31. 31.

    Dijkstra CD, Van Vliet E, Dopp EA, Lelij van der AA, Kraal G: Marginal zone macrophages identified by a monoclonal antibody: characterization of immuno- and enzyme-histochemical properties and functional capacities. Immunology. 1985, 55: 23-30.

  32. 32.

    Mirsky R, Thompson EJ: Thy 1 (theta) antigen on the surface of morphologically distinct brain cell types. Cell. 1975, 4: 95-101. 10.1016/0092-8674(75)90114-2.

  33. 33.

    Pruss RM: Thy-1 antigen on astrocytes in long-term cultures of rat central nervous system. Nature. 1979, 280: 688-690. 10.1038/280688a0.

  34. 34.

    Sternberger LA, Harwell LW, Sternberger NH: Neurotypy: regional individuality in rat brain detected by immunocytochemistry with monoclonal antibodies. Proc Natl Acad Sci USA. 1982, 79: 1326-1330. 10.1073/pnas.79.4.1326.

  35. 35.

    Kim HJ, Ifergan I, Antel JP, Seguin R, Duddy M, Lapierre Y, Jalili F, Bar-Or A: Type 2 monocyte and microglia differentiation mediated by glatiramer acetate therapy in patients with multiple sclerosis. J Immunol. 2004, 172: 7144-7153.

  36. 36.

    Liu Y, Teige I, Birnir B, Issazadeh-Navikas S: Neuron-mediated generation of regulatory T cells from encephalitogenic T cells suppresses EAE. Nat Med. 2006, 12: 518-525. 10.1038/nm1402.

  37. 37.

    Baecher-Allan C, Wolf E, Hafler DA: Functional analysis of highly defined, FACS-isolated populations of human regulatory CD4+ CD25+ T cells. Clin Immunol. 2005, 115: 10-18. 10.1016/j.clim.2005.02.018.

  38. 38.

    Beg AA, Baltimore D: An essential role for NF-kappaB in preventing TNF-alpha-induced cell death [see comments]. Science. 1996, 274: 782-784. 10.1126/science.274.5288.782.

  39. 39.

    Li C, Wong WH: Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci USA. 2001, 98: 31-36. 10.1073/pnas.011404098.

  40. 40.

    Yao B, Rakhade SN, Li Q, Ahmed S, Krauss R, Draghici S, Loeb JA: Accuracy of cDNA microarray methods to detect small gene expression changes induced by neuregulin on breast epithelial cells. BMC Bioinformatics. 2004, 5: 99-10.1186/1471-2105-5-99.

  41. 41.

    Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25: 402-408. 10.1006/meth.2001.1262.

  42. 42.

    Parpura V, Basarsky TA, Liu F, Jeftinija K, Jeftinija S, Haydon PG: Glutamate-mediated astrocyte-neuron signalling. Nature. 1994, 369: 744-747. 10.1038/369744a0.

  43. 43.

    Lehre KP, Levy LM, Ottersen OP, Storm-Mathisen J, Danbolt NC: Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations. J Neurosci. 1995, 15: 1835-1853.

  44. 44.

    Porter JT, McCarthy KD: Astrocytic neurotransmitter receptors in situ and in vivo. Prog Neurobiol. 1997, 51: 439-455. 10.1016/S0301-0082(96)00068-8.

  45. 45.

    Biber K, Laurie DJ, Berthele A, Sommer B, Tolle TR, Gebicke-Harter PJ, van Calker D, Boddeke HW: Expression and signaling of group I metabotropic glutamate receptors in astrocytes and microglia. J Neurochem. 1999, 72: 1671-1680. 10.1046/j.1471-4159.1999.721671.x.

  46. 46.

    Pitt D, Nagelmeier IE, Wilson HC, Raine CS: Glutamate uptake by oligodendrocytes: Implications for excitotoxicity in multiple sclerosis. Neurology. 2003, 61: 1113-1120.

  47. 47.

    Pitt D, Werner P, Raine CS: Glutamate excitotoxicity in a model of multiple sclerosis. Nat Med. 2000, 6: 67-70. 10.1038/71555.

  48. 48.

    Werner P, Pitt D, Raine CS: Glutamate excitotoxicity – a mechanism for axonal damage and oligodendrocyte death in Multiple Sclerosis?. J Neural Transm Suppl. 2000, 60: 375-385.

  49. 49.

    Werner P, Pitt D, Raine CS: Multiple sclerosis: altered glutamate homeostasis in lesions correlates with oligodendrocyte and axonal damage. Ann Neurol. 2001, 50: 169-180. 10.1002/ana.1077.

  50. 50.

    Takahashi JL, Giuliani F, Power C, Imai Y, Yong VW: Interleukin-1beta promotes oligodendrocyte death through glutamate excitotoxicity. Ann Neurol. 2003, 53: 588-595. 10.1002/ana.10519.

  51. 51.

    Flor PJ, Battaglia G, Nicoletti F, Gasparini F, Bruno V: Neuroprotective activity of metabotropic glutamate receptor ligands. Adv Exp Med Biol. 2002, 513: 197-223.

  52. 52.

    Matute C, Sanchez-Gomez MV, Martinez-Millan L, Miledi R: Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes. Proc Natl Acad Sci USA. 1997, 94: 8830-8835. 10.1073/pnas.94.16.8830.

  53. 53.

    Matute C, Alberdi E, Domercq M, Perez-Cerda F, Perez-Samartin A, Sanchez-Gomez MV: The link between excitotoxic oligodendroglial death and demyelinating diseases. Trends Neurosci. 2001, 24: 224-230. 10.1016/S0166-2236(00)01746-X.

  54. 54.

    Matute C, Perez-Cerda F: Multiple sclerosis: novel perspectives on newly forming lesions. Trends Neurosci. 2005, 28: 173-175. 10.1016/j.tins.2005.01.006.

  55. 55.

    Alberdi E, Sanchez-Gomez MV, Torre I, Domercq M, Perez-Samartin A, Perez-Cerda F, Matute C: Activation of kainate receptors sensitizes oligodendrocytes to complement attack. J Neurosci. 2006, 26: 3220-3228. 10.1523/JNEUROSCI.3780-05.2006.

  56. 56.

    Sanchez-Gomez MV, Matute C: AMPA and kainate receptors each mediate excitotoxicity in oligodendroglial cultures. Neurobiol Dis. 1999, 6: 475-485. 10.1006/nbdi.1999.0264.

  57. 57.

    McDonald JW, Althomsons SP, Hyrc KL, Choi DW, Goldberg MP: Oligodendrocytes from forebrain are highly vulnerable to AMPA/kainate receptor-mediated excitotoxicity. Nat Med. 1998, 4: 291-297. 10.1038/nm0398-291.

  58. 58.

    Geurts JJ, Wolswijk G, Bo L, Valk Van Der P, Polman CH, Troost D, Aronica E: Altered expression patterns of group I and II metabotropic glutamate receptors in multiple sclerosis. Brain. 2003, 126: 1755-1766. 10.1093/brain/awg179.

  59. 59.

    Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA, Nicotera P: Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function. Neuron. 1995, 15: 961-973. 10.1016/0896-6273(95)90186-8.

  60. 60.

    Castiglione M, Mangano K, Busceti CL, Nicoletti FR, et al: Activation of group III metabotropic glutamate receptors inhibits the production of RANTES in glial cell cultures. J Neurosci. 2002, 22: 5403-5411.

  61. 61.

    Abbracchio MP, Burnstock G: Purinergic signalling: pathophysiological roles. Jpn J Pharmacol. 1998, 78: 113-145. 10.1254/jjp.78.113.

  62. 62.

    Bianco F, Ceruti S, Colombo A, Fumagalli M, Ferrari D, Pizzirani C, Matteoli M, Di Virgilio F, Abbracchio MP, Verderio C: A role for P2X7 in microglial proliferation. J Neurochem. 2006, 99: 745-758. 10.1111/j.1471-4159.2006.04101.x.

  63. 63.

    Yiangou Y, Facer P, Durrenberger P, Chessell IP, Naylor A, Bountra C, Banati RR, Anand P: COX-2, CB2 and P2X7-immunoreactivities are increased in activated microglial cells/macrophages of multiple sclerosis and amyotrophic lateral sclerosis spinal cord. BMC Neurol. 2006, 6: 12-10.1186/1471-2377-6-12.

  64. 64.

    Narcisse L, Scemes E, Zhao Y, Lee SC, Brosnan CF: The cytokine IL-1beta transiently enhances P2X7 receptor expression and function in human astrocytes. Glia. 2005, 49: 245-258. 10.1002/glia.20110.

  65. 65.

    Wang Y, Roman R, Lidofsky SD, Fitz JG: Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation. Proc Natl Acad Sci USA. 1996, 93: 12020-12025. 10.1073/pnas.93.21.12020.

  66. 66.

    Rappold PM, Lynd-Balta E, Joseph SA: P2X7 receptor immunoreactive profile confined to resting and activated microglia in the epileptic brain. Brain Res. 2006, 1089: 171-178. 10.1016/j.brainres.2006.03.040.

  67. 67.

    Bongarzone ER, Howard SG, Schonmann V, Campagnoni AT: Identification of the dopamine D3 receptor in oligodendrocyte precursors: potential role in regulating differentiation and myelin formation. J Neurosci. 1998, 18: 5344-5353.

  68. 68.

    Rosin C, Colombo S, Calver AA, Bates TE, Skaper SD: Dopamine D2 and D3 receptor agonists limit oligodendrocyte injury caused by glutamate oxidative stress and oxygen/glucose deprivation. Glia. 2005, 52: 336-343. 10.1002/glia.20250.

  69. 69.

    Wang H, Yu M, Ochani M, Amella CA, Tanovic M, Susarla S, Li JH, Yang H, Ulloa L, Al-Abed Y, et al: Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature. 2003, 421: 384-388. 10.1038/nature01339.

  70. 70.

    Shytle RD, Mori T, Townsend K, Vendrame M, Sun N, Zeng J, Ehrhart J, Silver AA, Sanberg PR, Tan J: Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors. J Neurochem. 2004, 89: 337-343. 10.1046/j.1471-4159.2004.02347.x.

  71. 71.

    Nizri E, Hamra-Amitay Y, Sicsic C, Lavon I, Brenner T: Anti-inflammatory properties of cholinergic up-regulation: A new role for acetylcholinesterase inhibitors. Neuropharmacology. 2006, 50: 540-547. 10.1016/j.neuropharm.2005.10.013.

  72. 72.

    Burghaus L, Schutz U, Krempel U, Lindstrom J, Schroder H: Loss of nicotinic acetylcholine receptor subunits alpha4 and alpha7 in the cerebral cortex of Parkinson patients. Parkinsonism Relat Disord. 2003, 9: 243-246.

  73. 73.

    Soliven B, Szuchet S, Arnason BG, Nelson DJ: Expression and modulation of K+ currents in oligodendrocytes: possible role in myelinogenesis. Dev Neurosci. 1989, 11: 118-131. 10.1159/000111893.

  74. 74.

    Black JA, Westenbroek R, Minturn JE, Ransom BR, Catterall WA, Waxman SG: soform-specific expression of sodium channels in astrocytes in vitro: immunocytochemical observations. Glia. 1995, 14: I133-144. 10.1002/glia.440140208.

  75. 75.

    Visentin S, Levi G: Protein kinase C involvement in the resting and interferon-gamma-induced K+ channel profile of microglial cells. J Neurosci Res. 1997, 47: 233-241. 10.1002/(SICI)1097-4547(19970201)47:3<233::AID-JNR1>3.0.CO;2-J.

  76. 76.

    Attali B, Wang N, Kolot A, Sobko A, Cherepanov V, Soliven B: Characterization of delayed rectifier Kv channels in oligodendrocytes and progenitor cells. J Neurosci. 1997, 17: 8234-8245.

  77. 77.

    Verkhratsky A, Steinhauser C: Ion channels in glial cells. Brain Res Brain Res Rev. 2000, 32: 380-412. 10.1016/S0165-0173(99)00093-4.

  78. 78.

    Soliven B, Szuchet S, Nelson D: Tumor necrosis factor inhibits K+current expression in cultured oligodendrocytes. J Membr Biol. 1991, 124: 127-137. 10.1007/BF01870457.

  79. 79.

    Cameron JS, Lhuillier L, Subramony P, Dryer SE: Developmental regulation of neuronal K+ channels by target-derived TGF beta in vivo and in vitro. Neuron. 1998, 21: 1045-1053. 10.1016/S0896-6273(00)80622-4.

  80. 80.

    Iwagaki H, Fuchimoto S, Miyake M, Aoki H, Orita K: Interferon-gamma activates the voltage-gated calcium channel in RPMI 4788 cells. Biochem Biophys Res Commun. 1988, 153: 1276-1281. 10.1016/S0006-291X(88)81366-4.

  81. 81.

    Norenberg W, Gebicke-Haerter PJ, Illes P: Inflammatory stimuli induce a new K+ outward current in cultured rat microglia. Neurosci Lett. 1992, 147: 171-174. 10.1016/0304-3940(92)90587-W.

  82. 82.

    Smith KJ, McDonald WI: The pathophysiology of multiple sclerosis: the mechanisms underlying the production of symptoms and the natural history of the disease. Philos Trans R Soc Lond B Biol Sci. 1999, 354: 1649-1673. 10.1098/rstb.1999.0510.

  83. 83.

    Smith KJ, Hall SM: Factors directly affecting impulse transmission in inflammatory demyelinating disease: recent advances in our understanding. Curr Opin Neurol. 2001, 14: 289-298. 10.1097/00019052-200106000-00005.

  84. 84.

    Smith KJ, Kapoor R, Hall SM, Davies M: Electrically active axons degenerate when exposed to nitric oxide. Ann Neurol. 2001, 49: 470-476. 10.1002/ana.96.

  85. 85.

    Waxman SG: Nitric oxide and the axonal death cascade. Ann Neurol. 2003, 53: 150-153. 10.1002/ana.10397.

  86. 86.

    Craner MJ, Lo AC, Black JA, Waxman SG: Abnormal sodium channel distribution in optic nerve axons in a model of inflammatory demyelination. Brain. 2003, 126: 1552-1561. 10.1093/brain/awg153.

  87. 87.

    Carmody RJ, Hilliard B, Maguschak K, Chodosh LA, Chen YH: Genomic scale profiling of autoimmune inflammation in the central nervous system: the nervous response to inflammation. J Neuroimmunol. 2002, 133: 95-107. 10.1016/S0165-5728(02)00366-1.

  88. 88.

    Kapoor R, Davies M, Blaker PA, Hall SM, Smith KJ: Blockers of sodium and calcium entry protect axons from nitric oxide-mediated degeneration. Ann Neurol. 2003, 53: 174-180. 10.1002/ana.10443.

  89. 89.

    Stys PK: General mechanisms of axonal damage and its prevention. J Neurol Sci. 2005, 233: 3-13. 10.1016/j.jns.2005.03.031.

  90. 90.

    Stys PK, Waxman SG, Ransom BR: Na(+)-Ca2+ exchanger mediates Ca2+ influx during anoxia in mammalian central nervous system white matter. Ann Neurol. 1991, 30: 375-380. 10.1002/ana.410300309.

  91. 91.

    Kalman B, Leist TP: A mitochondrial component of neurodegeneration in multiple sclerosis. Neuromolecular Med. 2003, 3: 147-158. 10.1385/NMM:3:3:147.

  92. 92.

    Wulff H, Calabresi PA, Allie R, Yun S, Pennington M, Beeton C, Chandy KG: The voltage-gated Kv1.3 K(+) channel in effector memory T cells as new target for MS. J Clin Invest. 2003, 111: 1703-1713.

  93. 93.

    Gu C, Casaccia-Bonnefil P, Srinivasan A, Chao MV: Oligodendrocyte apoptosis mediated by caspase activation. J Neurosci. 1999, 19: 3043-3049.

  94. 94.

    Bredesen DE, Mehlen P, Rabizadeh S: Apoptosis and dependence receptors: a molecular basis for cellular addiction. Physiol Rev. 2004, 84: 411-430. 10.1152/physrev.00027.2003.

  95. 95.

    Lucchinetti C, Bruck W, Parisi J, Scheithauer B, Rodriguez M, Lassmann H: Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination [see comments]. Ann Neurol. 2000, 47: 707-717. 10.1002/1531-8249(200006)47:6<707::AID-ANA3>3.0.CO;2-Q.

  96. 96.

    Peterson JW, Bo L, Mork S, Chang A, Trapp BD: Transected neurites, apoptotic neurons, and reduced inflammation in cortical multiple sclerosis lesions. Ann Neurol. 2001, 50: 389-400. 10.1002/ana.1123.

  97. 97.

    Tomita Y, Bilim V, Hara N, Kasahara T, Takahashi K: Role of IRF-1 and caspase-7 in IFN-gamma enhancement of Fas-mediated apoptosis in ACHN renal cell carcinoma cells. Int J Cancer. 2003, 104: 400-408. 10.1002/ijc.10956.

  98. 98.

    Gross A, McDonnell JM, Korsmeyer SJ: BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 1999, 13: 1899-1911. 10.1101/gad.13.15.1899.

  99. 99.

    Gross A, Yin XM, Wang K, Wei MC, Jockel J, Milliman C, Erdjument-Bromage H, Tempst P, Korsmeyer SJ: Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. J Biol Chem. 1999, 274: 1156-1163. 10.1074/jbc.274.2.1156.

  100. 100.

    Rigamonti D, Sipione S, Goffredo D, Zuccato C, Fossale E, Cattaneo E: Huntingtin's neuroprotective activity occurs via inhibition of procaspase-9 processing. J Biol Chem. 2001, 276: 14545-14548. 10.1074/jbc.C100044200.

  101. 101.

    Gervais FG, Singaraja R, Xanthoudakis S, Gutekunst CA, Leavitt BR, Metzler M, Hackam AS, Tam J, Vaillancourt JP, Houtzager V, et al: Recruitment and activation of caspase-8 by the Huntingtin-interacting protein Hip-1 and a novel partner Hippi. Nat Cell Biol. 2002, 4: 95-105. 10.1038/ncb735.

  102. 102.

    Engqvist-Goldstein AE, Warren RA, Kessels MM, Keen JH, Heuser J, Drubin DG: The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro. J Cell Biol. 2001, 154: 1209-1223. 10.1083/jcb.200106089.

  103. 103.

    Harjes P, Wanker EE: The hunt for huntingtin function: interaction partners tell many different stories. Trends Biochem Sci. 2003, 28: 425-433. 10.1016/S0968-0004(03)00168-3.

  104. 104.

    Bence NF, Sampat RM, Kopito RR: Impairment of the ubiquitin-proteasome system by protein aggregation. Science. 2001, 292: 1552-1555. 10.1126/science.292.5521.1552.

  105. 105.

    Sharma MR, Koc EC, Datta PP, Booth TM, Spremulli LL, Agrawal RK: Structure of the mammalian mitochondrial ribosome reveals an expanded functional role for its component proteins. Cell. 2003, 115: 97-108. 10.1016/S0092-8674(03)00762-1.

  106. 106.

    Kunst CB, Mezey E, Brownstein MJ, Patterson D: Mutations in SOD1 associated with amyotrophic lateral sclerosis cause novel protein interactions. Nat Genet. 1997, 15: 91-94. 10.1038/ng0197-91.

  107. 107.

    Dringen R, Kussmaul L, Gutterer JM, Hirrlinger J, Hamprecht B: The glutathione system of peroxide detoxification is less efficient in neurons than in astroglial cells. J Neurochem. 1999, 72: 2523-2530. 10.1046/j.1471-4159.1999.0722523.x.

  108. 108.

    Baud O, Greene AE, Li J, Wang H, Volpe JJ, Rosenberg PA: Glutathione peroxidase-catalase cooperativity is required for resistance to hydrogen peroxide by mature rat oligodendrocytes. J Neurosci. 2004, 24: 1531-1540. 10.1523/JNEUROSCI.3989-03.2004.

  109. 109.

    Hirrlinger J, Konig J, Dringen R: Expression of mRNAs of multidrug resistance proteins (Mrps) in cultured rat astrocytes, oligodendrocytes, microglial cells and neurones. J Neurochem. 2002, 82: 716-719. 10.1046/j.1471-4159.2002.01082.x.

  110. 110.

    Hirrlinger J, Konig J, Keppler D, Lindenau J, Schulz JB, Dringen R: The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress. J Neurochem. 2001, 76: 627-636. 10.1046/j.1471-4159.2001.00101.x.

  111. 111.

    Volk H, Potschka H, Loscher W: Immunohistochemical localization of P-glycoprotein in rat brain and detection of its increased expression by seizures are sensitive to fixation and staining variables. J Histochem Cytochem. 2005, 53: 517-531. 10.1369/jhc.4A6451.2005.

  112. 112.

    Volk HA, Burkhardt K, Potschka H, Chen J, Becker A, Loscher W: Neuronal expression of the drug efflux transporter P-glycoprotein in the rat hippocampus after limbic seizures. Neuroscience. 2004, 123: 751-759. 10.1016/j.neuroscience.2003.10.012.

  113. 113.

    Volk HA, Loscher W: Multidrug resistance in epilepsy: rats with drug-resistant seizures exhibit enhanced brain expression of P-glycoprotein compared with rats with drug-responsive seizures. Brain. 2005, 128: 1358-1368. 10.1093/brain/awh437.

  114. 114.

    Juurlink BH: Response of glial cells to ischemia: roles of reactive oxygen species and glutathione. Neurosci Biobehav Rev. 1997, 21: 151-166. 10.1016/S0149-7634(96)00005-X.

  115. 115.

    Barker JE, Bolanos JP, Land JM, Clark JB, Heales SJ: Glutathione protects astrocytes from peroxynitrite-mediated mitochondrial damage: implications for neuronal/astrocytic trafficking and neurodegeneration. Dev Neurosci. 1996, 18: 391-396. 10.1159/000111432.

  116. 116.

    Back SA, Gan X, Li Y, Rosenberg PA, Volpe JJ: Maturation-dependent vulnerability of oligodendrocytes to oxidative stress-induced death caused by glutathione depletion. J Neurosci. 1998, 18: 6241-6253.

  117. 117.

    Margalit A, Hauser SD, Zweifel BS, Anderson MA, Isakson PC: Regulation of prostaglandin biosynthesis in vivo by glutathione. Am J Physiol. 1998, 274: R294-302.

  118. 118.

    Götschel F, Kern C, Lang S, Sparna T, Markmann C, Schwager J, McNelly S, von Weizsäcker F, Laufer S, Hecht A, Merfort I: Inhibition of GSK3 differentially modulates NF-kappaB, CREB, AP-1 and beta-catenin signaling in hepatocytes, but fails to promote TNF-alpha-induced apoptosis. Exp Cell Res. 2008, 314: 1351-66. 10.1016/j.yexcr.2007.12.015.

  119. 119.

    John GR, Shankar SL, Shafit-Zagardo B, Massimi A, Lee SC, Raine CS, Brosnan CF: Multiple sclerosis: Re-expression of a developmental pathway that restricts oligodendrocyte maturation. Nat Med. 2002, 8: 1115-1121. 10.1038/nm781.

  120. 120.

    Stidworthy MF, Genoud S, Li WW, Leone DP, Mantei N, Suter U, Franklin RJ: Notch1 and Jagged1 are expressed after CNS demyelination, but are not a major rate-determining factor during remyelination. Brain. 2004, 127: 1928-1941. 10.1093/brain/awh217.

  121. 121.

    Cimini A, Bernardo A, Cifone G, Di Muzio L, Di Loreto S: TNFalpha downregulates PPARdelta expression in oligodendrocyte progenitor cells: implications for demyelinating diseases. Glia. 2003, 41: 3-14. 10.1002/glia.10143.

  122. 122.

    Feinstein DL, Galea E, Gavrilyuk V, Brosnan CF, Whitacre CC, Dumitrescu-Ozimek L, Landreth GE, Pershadsingh HA, Weinberg G, Heneka MT: Peroxisome proliferator-activated receptor-gamma agonists prevent experimental autoimmune encephalomyelitis. Ann Neurol. 2002, 51: 694-702. 10.1002/ana.10206.

  123. 123.

    Natarajan C, Bright JJ: Peroxisome proliferator-activated receptor-gamma agonists inhibit experimental allergic encephalomyelitis by blocking IL-12 production, IL-12 signaling and Th1 differentiation. Genes Immun. 2002, 3: 59-70. 10.1038/sj.gene.6363832.

  124. 124.

    Saluja I, Granneman JG, Skoff RP: PPAR delta agonists stimulate oligodendrocyte differentiation in tissue culture. Glia. 2001, 33: 191-204. 10.1002/1098-1136(200103)33:3<191::AID-GLIA1018>3.0.CO;2-M.

  125. 125.

    Satriotomo I, Bowen KK, Vemuganti R: JAK2 and STAT3 activation contributes to neuronal damage following transient focal cerebral ischemia. J Neurochem. 2006, 98: 1353-1368. 10.1111/j.1471-4159.2006.04051.x.

  126. 126.

    Tajouri L, Mellick AS, Tourtellotte A, Nagra RM, Griffiths LR: An examination of MS candidate genes identified as differentially regulated in multiple sclerosis plaque tissue, using absolute and comparative real-time Q-PCR analysis. Brain Res Brain Res Protoc. 2005, 15: 79-91. 10.1016/j.brainresprot.2005.04.003.

  127. 127.

    Xiao B, Tu JC, Worley PF: Homer: a link between neural activity and glutamate receptor function. Curr Opin Neurobiol. 2000, 10: 370-374. 10.1016/S0959-4388(00)00087-8.

  128. 128.

    Rong R, Ahn JY, Huang H, Nagata E, Kalman D, Kapp JA, Tu J, Worley PF, Snyder SH, Ye K: PI3 kinase enhancer-Homer complex couples mGluRI to PI3 kinase, preventing neuronal apoptosis. Nat Neurosci. 2003, 6: 1153-1161. 10.1038/nn1134.

  129. 129.

    Roche KW, Tu JC, Petralia RS, Xiao B, Wenthold RJ, Worley PF: Homer 1b regulates the trafficking of group I metabotropic glutamate receptors. J Biol Chem. 1999, 274: 25953-25957. 10.1074/jbc.274.36.25953.

  130. 130.

    Ango F, Robbe D, Tu JC, Xiao B, Worley PF, Pin JP, Bockaert J, Fagni L: Homer-dependent cell surface expression of metabotropic glutamate receptor type 5 in neurons. Mol Cell Neurosci. 2002, 20: 323-329. 10.1006/mcne.2002.1100.

  131. 131.

    Osterhout DJ, Wolven A, Wolf RM, Resh MD, Chao MV: Morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase. J Cell Biol. 1999, 145: 1209-1218. 10.1083/jcb.145.6.1209.

  132. 132.

    Liang X, Draghi NA, Resh MD: Signaling from integrins to Fyn to Rho family GTPases regulates morphologic differentiation of oligodendrocytes. J Neurosci. 2004, 24: 7140-7149. 10.1523/JNEUROSCI.5319-03.2004.

  133. 133.

    Galvin JE, Lee VM, Trojanowski JQ: Synucleinopathies: clinical and pathological implications. Arch Neurol. 2001, 58: 186-190. 10.1001/archneur.58.2.186.

  134. 134.

    Cooper AA, Gitler AD, Cashikar A, Haynes CM, Hill KJ, Bhullar B, Liu K, Xu K, Strathearn KE, Liu F, et al: {alpha}-Synuclein Blocks ER-Golgi Traffic and Rab1 Rescues Neuron Loss in Parkinson's Models. Science. 2006, 313: 324-328. 10.1126/science.1129462.

  135. 135.

    Kaplan B, Ratner V, Haas E: Alpha-synuclein: its biological function and role in neurodegenerative diseases. J Mol Neurosci. 2003, 20: 83-92. 10.1385/JMN:20:2:83.

  136. 136.

    Lee D, Paik SR, Choi KY: Beta-synuclein exhibits chaperone activity more efficiently than alpha-synuclein. FEBS Lett. 2004, 576: 256-260. 10.1016/j.febslet.2004.08.075.

  137. 137.

    Richter-Landsberg C, Gorath M, Trojanowski JQ, Lee VM: alpha-synuclein is developmentally expressed in cultured rat brain oligodendrocytes. J Neurosci Res. 2000, 62: 9-14. 10.1002/1097-4547(20001001)62:1<9::AID-JNR2>3.0.CO;2-U.

  138. 138.

    Piao YS, Mori F, Hayashi S, Tanji K, Yoshimoto M, Kakita A, Wakabayashi K, Takahashi H: Alpha-synuclein pathology affecting Bergmann glia of the cerebellum in patients with alpha-synucleinopathies. Acta Neuropathol. 2003, 105 (4): 403-409.

  139. 139.

    Brosnan CF, Battistini L, Gao YL, Raine CS, Aquino DA: Heat shock proteins and multiple sclerosis: a review. J Neuropathol Exp Neurol. 1996, 55: 389-402. 10.1097/00005072-199604000-00001.

  140. 140.

    Stuve O, Prod'homme T, Slavin A, Youssef S, Dunn S, Steinman L, Zamvil SS: Statins and their potential targets in multiple sclerosis therapy. Expert Opin Ther Targets. 2003, 7: 613-622.

  141. 141.

    Neuhaus O, Stuve O, Archelos JJ, Hartung HP: Putative mechanisms of action of statins in multiple sclerosis – comparison to interferon-beta and glatiramer acetate. J Neurol Sci. 2005, 233: 173-177. 10.1016/j.jns.2005.03.030.

  142. 142.

    Weber MS, Youssef S, Dunn SE, Prod'homme T, Neuhaus O, Stuve O, Greenwood J, Steinman L, Zamvil SS: Statins in the treatment of central nervous system autoimmune disease. J Neuroimmunol. 2006, 178: 140-148. 10.1016/j.jneuroim.2006.06.006.

  143. 143.

    Youssef S, Stuve O, Patarroyo JC, Ruiz PJ, Radosevich JL, Hur EM, Bravo M, Mitchell DJ, Sobel RA, Steinman L, Zamvil SS: The HMG-CoA reductase inhibitor, atorvastatin, promotes a Th2 bias and reverses paralysis in central nervous system autoimmune disease. Nature. 2002, 420: 78-84. 10.1038/nature01158.

  144. 144.

    Nath N, Giri S, Prasad R, Singh AK, Singh I: Potential targets of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor for multiple sclerosis therapy. J Immunol. 2004, 172: 1273-1286.

  145. 145.

    Greenwood J, Walters CE, Pryce G, Kanuga N, Beraud E, Baker D, Adamson P: Lovastatin inhibits brain endothelial cell Rho-mediated lymphocyte migration and attenuates experimental autoimmune encephalomyelitis. Faseb J. 2003, 17: 905-907.

  146. 146.

    Gong JS, Kobayashi M, Hayashi H, Zou K, Sawamura N, Fujita SC, Yanagisawa K, Michikawa M: Apolipoprotein E (ApoE) isoform-dependent lipid release from astrocytes prepared from human ApoE3 and ApoE4 knock-in mice. J Biol Chem. 2002, 277: 29919-29926. 10.1074/jbc.M203934200.

  147. 147.

    Tanaka Y, Yamada K, Zhou CJ, Ban N, Shioda S, Inagaki N: Temporal and spatial profiles of ABCA2-expressing oligodendrocytes in the developing rat brain. J Comp Neurol. 2003, 455: 353-367. 10.1002/cne.10493.

  148. 148.

    Chakraborty G, Reddy R, Drivas A, Ledeen RW: Interleukin-2 receptors and interleukin-2-mediated signaling in myelin: activation of diacylglycerol kinase and phosphatidylinositol 3-kinase. Neuroscience. 2003, 122: 967-973. 10.1016/j.neuroscience.2003.09.003.

  149. 149.

    Sarkar S, Floto RA, Berger Z, Imarisio S, Cordenier A, Pasco M, Cook LJ, Rubinsztein DC: Lithium induces autophagy by inhibiting inositol monophosphatase. J Cell Biol. 2005, 170: 1101-1111. 10.1083/jcb.200504035.

  150. 150.

    Bedlack RS, Maragakis N, Heiman-Patterson T: Lithium may slow progression of amyotrophic lateral sclerosis, but further study is needed. Proc Natl Acad Sci USA. 2008, 105: E17-10.1073/pnas.0801762105. author reply E18

  151. 151.

    Fornai F, Longone P, Ferrucci M, Lenzi P, Isidoro C, Ruggieri S, Paparelli A: he multiple roles of lithium. Autophagy. 2008, 4: T527-530.

  152. 152.

    Fornai F, Longone P, Cafaro L, Kastsiuchenka O, Ferrucci M, Manca ML, Lazzeri G, Spalloni A, Bellio N, Lenzi P, et al: Lithium delays progression of amyotrophic lateral sclerosis. Proc Natl Acad Sci USA. 2008, 105: 2052-2057. 10.1073/pnas.0708022105.

  153. 153.

    Chalfant CE, Spiegel S: Sphingosine 1-phosphate and ceramide 1-phosphate: expanding roles in cell signaling. J Cell Sci. 2005, 118: 4605-4612. 10.1242/jcs.02637.

  154. 154.

    Kappos L, Antel J, Comi G, Montalban X, O'Connor P, Polman CH, Haas T, Korn AA, Karlsson G, Radue EW: Oral fingolimod (FTY720) for relapsing multiple sclerosis. N Engl J Med. 2006, 355: 1124-1140. 10.1056/NEJMoa052643.

  155. 155.

    Singer II, Tian M, Wickham LA, Lin J, Matheravidathu SS, Forrest MJ, Mandala S, Quackenbush EJ: Sphingosine-1-phosphate agonists increase macrophage homing, lymphocyte contacts, and endothelial junctional complex formation in murine lymph nodes. J Immunol. 2005, 175: 7151-7161.

  156. 156.

    Yopp AC, Ochando JC, Mao M, Ledgerwood L, Ding Y, Bromberg JS: Sphingosine 1-phosphate receptors regulate chemokine-driven transendothelial migration of lymph node but not splenic T cells. J Immunol. 2005, 175: 2913-2924.

  157. 157.

    Lee MJ, Van Brocklyn JR, Thangada S, Liu CH, Hand AR, Menzeleev R, Spiegel S, Hla T: Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1. Science. 1998, 279: 1552-1555. 10.1126/science.279.5356.1552.

  158. 158.

    Hla T: Signaling and biological actions of sphingosine 1-phosphate. Pharmacol Res. 2003, 47: 401-407. 10.1016/S1043-6618(03)00046-X.

  159. 159.

    Czeloth N, Bernhardt G, Hofmann F, Genth H, Forster R: Sphingosine-1-phosphate mediates migration of mature dendritic cells. J Immunol. 2005, 175: 2960-2967.

  160. 160.

    Cyster JG: Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs. Annu Rev Immunol. 2005, 23: 127-159. 10.1146/annurev.immunol.23.021704.115628.

  161. 161.

    Toman RE, Spiegel S: Lysophospholipid receptors in the nervous system. Neurochem Res. 2002, 27: 619-627. 10.1023/A:1020219915922.

  162. 162.

    Hida H, Nagano S, Takeda M, Soliven B: Regulation of mitogen-activated protein kinases by sphingolipid products in oligodendrocytes. J Neurosci. 1999, 19: 7458-7467.

  163. 163.

    Pebay A, Toutant M, Premont J, Calvo CF, Venance L, Cordier J, Glowinski J, Tence M: Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades. Eur J Neurosci. 2001, 13: 2067-2076. 10.1046/j.0953-816x.2001.01585.x.

  164. 164.

    Mullershausen F, Craveiro LM, Shin Y, Cortes-Cros M, Bassilana F, Osinde M, Wishart WL, Guerini D, Thallmair M, Schwab ME, et al: Phosphorylated FTY720 promotes astrocyte migration through sphingosine-1-phosphate receptors. J Neurochem. 2007, 102: 1151-1161. 10.1111/j.1471-4159.2007.04629.x.

  165. 165.

    Miron VE, Jung CG, Kim HJ, Kennedy TE, Soliven B, Antel JP: FTY720 modulates human oligodendrocyte progenitor process extension and survival. Ann Neurol. 2008, 63: 61-71. 10.1002/ana.21227.

  166. 166.

    Ebert R, Schutze N, Adamski J, Jakob F: Vitamin D signaling is modulated on multiple levels in health and disease. Mol Cell Endocrinol. 2006, 248: 149-159. 10.1016/j.mce.2005.11.039.

  167. 167.

    Spach KM, Pedersen LB, Nashold FE, Kayo T, Yandell BS, Prolla TA, Hayes CE: Gene expression analysis suggests that 1,25-dihydroxyvitamin D3 reverses experimental autoimmune encephalomyelitis by stimulating inflammatory cell apoptosis. Physiol Genomics. 2004, 18: 141-151. 10.1152/physiolgenomics.00003.2004.

  168. 168.

    Munger KL, Zhang SM, O'Reilly E, Hernan MA, Olek MJ, Willett WC, Ascherio A: Vitamin D intake and incidence of multiple sclerosis. Neurology. 2004, 62: 60-65.

  169. 169.

    Brown SJ: The role of vitamin D in multiple sclerosis. Ann Pharmacother. 2006, 40: 1158-1161. 10.1345/aph.1G513.

  170. 170.

    Brosnan CF, Battistini L, Raine CS, Dickson DW, Casadevall A, Lee SC: Reactive nitrogen intermediates in human neuropathology: an overview. Dev Neurosci. 1994, 16: 152-161. 10.1159/000112102.

  171. 171.

    Smith KJ, Lassmann H: The role of nitric oxide in multiple sclerosis. Lancet Neurol. 2002, 1: 232-241. 10.1016/S1474-4422(02)00102-3.

  172. 172.

    Zhang J, Cross AH, McCarthy TJ, Welch MJ: Measurement of upregulation of inducible nitric oxide synthase in the experimental autoimmune encephalomyelitis model using a positron emitting radiopharmaceutical. Nitric Oxide. 1997, 1: 263-267. 10.1006/niox.1997.0120.

  173. 173.

    Bagasra O, Michaels FH, Zheng YM, Bobroski LE, Spitsin SV, Fu ZF, Tawadros R, Koprowski H: Activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis. Proc Natl Acad Sci USA. 1995, 92: 12041-12045. 10.1073/pnas.92.26.12041.

  174. 174.

    Hill KE, Zollinger LV, Watt HE, Carlson NG, Rose JW: Inducible nitric oxide synthase in chronic active multiple sclerosis plaques: distribution, cellular expression and association with myelin damage. J Neuroimmunol. 2004, 151: 171-179. 10.1016/j.jneuroim.2004.02.005.

  175. 175.

    Boullerne AI, Benjamins JA: Nitric oxide synthase expression and nitric oxide toxicity in oligodendrocytes. Antioxid Redox Signal. 2006, 8: 967-980. 10.1089/ars.2006.8.967. Review

  176. 176.

    Waxman SG, Craner MJ, Black JA: Na+ channel expression along axons in multiple sclerosis and its models. Trends Pharmacol Sci. 2004, 25: 584-59. 10.1016/

  177. 177.

    Hokfelt T, Wiesenfeld-Hallin Z, Villar M, Melander T: Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy. Neurosci Lett. 1987, 83: 217-220. 10.1016/0304-3940(87)90088-7.

  178. 178.

    Kashiba H, Senba E, Kawai Y, Ueda Y, Tohyama M: Axonal blockade induces the expression of vasoactive intestinal polypeptide and galanin in rat dorsal root ganglion neurons. Brain Res. 1992, 577: 19-28. 10.1016/0006-8993(92)90532-E.

  179. 179.

    Shen PJ, Larm JA, Gundlach AL: Expression and plasticity of galanin systems in cortical neurons, oligodendrocyte progenitors and proliferative zones in normal brain and after spreading depression. Eur J Neurosci. 2003, 18: 1362-1376. 10.1046/j.1460-9568.2003.02860.x.

  180. 180.

    Ubink R, Calza L, Hokfelt T: 'Neuro'-peptides in glia: focus on NPY and galanin. Trends Neurosci. 2003, 26: 604-609. 10.1016/j.tins.2003.09.003.

  181. 181.

    Holmes FE, Mahoney S, King VR, Bacon A, Kerr NC, Pachnis V, Curtis R, Priestley JV, Wynick D: Targeted disruption of the galanin gene reduces the number of sensory neurons and their regenerative capacity. Proc Natl Acad Sci USA. 2000, 97: 11563-11568. 10.1073/pnas.210221897.

  182. 182.

    Mahoney SA, Hosking R, Farrant S, Holmes FE, Jacoby AS, Shine J, Iismaa TP, Scott MK, Schmidt R, Wynick D: The second galanin receptor GalR2 plays a key role in neurite outgrowth from adult sensory neurons. J Neurosci. 2003, 23: 416-421.

  183. 183.

    O'Meara G, Coumis U, Ma SY, Kehr J, Mahoney S, Bacon A, Allen SJ, Holmes F, Kahl U, Wang FH, et al: Galanin regulates the postnatal survival of a subset of basal forebrain cholinergic neurons. Proc Natl Acad Sci USA. 2000, 97: 11569-11574. 10.1073/pnas.210254597.

  184. 184.

    Sahu A, Xu B, Kalra SP: Role of galanin in stimulation of pituitary luteinizing hormone secretion as revealed by a specific receptor antagonist, galantide. Endocrinology. 1994, 134: 529-536. 10.1210/en.134.2.529.

  185. 185.

    Wynick D, Small CJ, Bacon A, Holmes FE, Norman M, Ormandy CJ, Kilic E, Kerr NC, Ghatei M, Talamantes F, et al: Galanin regulates prolactin release and lactotroph proliferation. Proc Natl Acad Sci USA. 1998, 95: 12671-12676. 10.1073/pnas.95.21.12671.

  186. 186.

    Holmes FE, Bacon A, Pope RJ, Vanderplank PA, Kerr NC, Sukumaran M, Pachnis V, Wynick D: Transgenic overexpression of galanin in the dorsal root ganglia modulates pain-related behavior. Proc Natl Acad Sci USA. 2003, 100: 6180-6185. 10.1073/pnas.0937087100.

  187. 187.

    Wiesenfeld-Hallin Z, Xu XJ, Villar MJ, Hokfelt T: Intrathecal galanin potentiates the spinal analgesic effect of morphine: electrophysiological and behavioural studies. Neurosci Lett. 1990, 109: 217-221. 10.1016/0304-3940(90)90566-R.

  188. 188.

    Nordstrom O, Melander T, Hokfelt T, Bartfai T, Goldstein M: Evidence for an inhibitory effect of the peptide galanin on dopamine release from the rat median eminence. Neurosci Lett. 1987, 73: 21-26. 10.1016/0304-3940(87)90024-3.

  189. 189.

    Sundstrom E, Archer T, Melander T, Hokfelt T: Galanin impairs acquisition but not retrieval of spatial memory in rats studied in the Morris swim maze. Neurosci Lett. 1988, 88: 331-335. 10.1016/0304-3940(88)90233-9.

  190. 190.

    Hosli E, Ledergerber M, Kofler A, Hosli L: Evidence for the existence of galanin receptors on cultured astrocytes of rat CNS: colocalization with cholinergic receptors. J Chem Neuroanat. 1997, 13: 95-103. 10.1016/S0891-0618(97)00024-0.

  191. 191.

    Dugas JC, Tai YC, Speed TP, Ngai J, Barres BA: Functional genomic analysis of oligodendrocyte differentiation. J Neurosci. 2006, 26: 10967-10983. 10.1523/JNEUROSCI.2572-06.2006.

  192. 192.

    Brenner T, Abramsky O: Immunosuppression of experimental autoimmune myasthenia gravis by alpha-fetoprotein rich formation. Immunol Lett. 1981, 3: 163-167. 10.1016/0165-2478(81)90121-8.

  193. 193.

    Lubetzki-Korn I, Hirayama M, Silberberg DH, Schreiber AD, Eccleston PA, Pleasure D, Brenner T, Abramsky O: Human alpha-fetoprotein-rich fraction inhibits galactocerebroside antibody-mediated lysis of oligodendrocytes in vitro. Ann Neurol. 1984, 15: 171-180. 10.1002/ana.410150210.

  194. 194.

    Evron S, Brenner T, Abramsky O: Suppressive effect of pregnancy on the development of experimental allergic encephalomyelitis in rabbits. Am J Reprod Immunol. 1984, 5: 109-113.

  195. 195.

    Brenner T, Evron S, Soffer D, Abramsky O: Treatment of experimental allergic encephalomyelitis in rabbits with alpha-fetoprotein. Isr J Med Sci. 1985, 21: 945-949.

  196. 196.

    Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A, Freeman TB, Saporta S, Janssen W, Patel N, et al: Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 2000, 164: 247-256. 10.1006/exnr.2000.7389.

  197. 197.

    Gallo V, Armstrong RC: Developmental and growth factor-induced regulation of nestin in oligodendrocyte lineage cells. J Neurosci. 1995, 15: 394-406.

  198. 198.

    Filippov V, Kronenberg G, Pivneva T, Reuter K, Steiner B, Wang LP, Yamaguchi M, Kettenmann H, Kempermann G: Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes. Mol Cell Neurosci. 2003, 23: 373-382. 10.1016/S1044-7431(03)00060-5.

  199. 199.

    Almazan G, Vela JM, Molina-Holgado E, Guaza C: Re-evaluation of nestin as a marker of oligodendrocyte lineage cells. Microsc Res Tech. 2001, 52: 753-765. 10.1002/jemt.1060.

  200. 200.

    Brenneman DE, Phillips TM, Hauser J, Hill JM, Spong CY, Gozes I: Complex array of cytokines released by vasoactive intestinal peptide. Neuropeptides. 2003, 37: 111-119. 10.1016/S0143-4179(03)00022-2.

  201. 201.

    Gozes I, Brenneman DE: A new concept in the pharmacology of neuroprotection. J Mol Neurosci. 2000, 14: 61-68. 10.1385/JMN:14:1-2:061.

  202. 202.

    Wu L, Iwai M, Li Z, Shiuchi T, Min LJ, Cui TX, Li JM, Okumura M, Nahmias C, Horiuchi M: Regulation of inhibitory protein-kappaB and monocyte chemoattractant protein-1 by angiotensin II type 2 receptor-activated Src homology protein tyrosine phosphatase-1 in fetal vascular smooth muscle cells. Mol Endocrinol. 2004, 18: 666-678. 10.1210/me.2003-0053.

  203. 203.

    Zhang X, Lassila M, Cooper ME, Cao Z: Retinal expression of vascular endothelial growth factor is mediated by angiotensin type 1 and type 2 receptors. Hypertension. 2004, 43: 276-281. 10.1161/01.HYP.0000113628.94574.0f.

  204. 204.

    Sarlos S, Rizkalla B, Moravski CJ, Cao Z, Cooper ME, Wilkinson-Berka JL: Retinal angiogenesis is mediated by an interaction between the angiotensin type 2 receptor, VEGF, and angiopoietin. Am J Pathol. 2003, 163: 879-887.

  205. 205.

    Steinman L, Zamvil S: Transcriptional analysis of targets in multiple sclerosis. Nat Rev Immunol. 2003, 3: 483-492. 10.1038/nri1108.

  206. 206.

    Steinman L: Gene microarrays and experimental demyelinating disease: a tool to enhance serendipity. Brain. 2001, 124: 1897-1899. 10.1093/brain/124.10.1897.

  207. 207.

    Passafaro M, Sala C, Niethammer M, Sheng M: Microtubule binding by CRIPT and its potential role in the synaptic clustering of PSD-95. Nat Neurosci. 1999, 2: 1063-1069. 10.1038/15990.

  208. 208.

    Fukaya M, Watanabe M: Improved immunohistochemical detection of postsynaptically located PSD-95/SAP90 protein family by protease section pretreatment: a study in the adult mouse brain. J Comp Neurol. 2000, 426: 572-586. 10.1002/1096-9861(20001030)426:4<572::AID-CNE6>3.0.CO;2-9.

  209. 209.

    Klomp LW, Gitlin JD: Expression of the ceruloplasmin gene in the human retina and brain: implications for a pathogenic model in aceruloplasminemia. Hum Mol Genet. 1996, 5: 1989-1996. 10.1093/hmg/5.12.1989.

  210. 210.

    Yoshida K, Kaneko K, Miyajima H, Tokuda T, Nakamura A, Kato M, Ikeda S: Increased lipid peroxidation in the brains of aceruloplasminemia patients. J Neurol Sci. 2000, 175: 91-95. 10.1016/S0022-510X(00)00295-1.

  211. 211.

    Patel BN, Dunn RJ, Jeong SY, Zhu Q, Julien JP, David S: Ceruloplasmin regulates iron levels in the CNS and prevents free radical injury. J Neurosci. 2002, 22: 6578-6586.

  212. 212.

    Shin T, Kim H, Jin JK, Moon C, Ahn M, Tanuma N, Matsumoto Y: Expression of caveolin-1, -2, and -3 in the spinal cords of Lewis rats with experimental autoimmune encephalomyelitis. J Neuroimmunol. 2005, 165: 11-20. 10.1016/j.jneuroim.2005.03.019.

  213. 213.

    Cai D, Deng K, Mellado W, Lee J, Ratan RR, Filbin MT: Arginase 1 and polyamines act downstream from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro. Neuron. 2002, 35: 711-719. 10.1016/S0896-6273(02)00826-7.

  214. 214.

    Hesse M, Modolell M, La Flamme AC, Schito M, Fuentes JM, Cheever AW, Pearce EJ, Wynn TA: Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism. J Immunol. 2001, 167: 6533-6544.

  215. 215.

    Louis CA, Mody V, Henry WL, Reichner JS, Albina JE: Regulation of arginase isoforms I and II by IL-4 in cultured murine peritoneal macrophages. Am J Physiol. 1999, 276: R237-242.

  216. 216.

    Mori M, Gotoh T: Regulation of nitric oxide production by arginine metabolic enzymes. Biochem Biophys Res Commun. 2000, 275: 715-719. 10.1006/bbrc.2000.3169.

  217. 217.

    Estevez AG, Sahawneh MA, Lange PS, Bae N, Egea M, Ratan RR: Arginase 1 regulation of nitric oxide production is key to survival of trophic factor-deprived motor neurons. J Neurosci. 2006, 26: 8512-8516. 10.1523/JNEUROSCI.0728-06.2006.

  218. 218.

    Becker-Catania SG, Gregory TL, Yang Y, Gau CL, de Vellis J, Cederbaum SD, Iyer RK: Loss of arginase I results in increased proliferation of neural stem cells. J Neurosci Res. 2006, 84: 735-746. 10.1002/jnr.20964.

  219. 219.

    Lassmann H: Hypoxia-like tissue injury as a component of multiple sclerosis lesions. J Neurol Sci. 2003, 206: 187-191. 10.1016/S0022-510X(02)00421-5.

  220. 220.

    Kirk SL, Karlik SJ: VEGF and vascular changes in chronic neuroinflammation. J Autoimmun. 2003, 21: 353-363. 10.1016/S0896-8411(03)00139-2.

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This study was funded by Teva Neuroscience, the Parker Webber Chair in Neurology Endowment and the Mary Parker Neuroscience Fund of the Detroit Medical Center/Wayne State University School of Medicine.

We wish to thank Joshua Adler, Steven Douglas, Paula Dore-Duffy, James Garbern, Alexander Gow, John Kamholz, Jeffrey Loeb, Kenneth Maiese, and Raymond Mattingly for helpful discussions.

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Correspondence to Joyce A Benjamins.

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Competing interests

RPL has served as a consultant to Teva Neuroscience as well as on the speakers' bureau for Teva Neuroscience. He has also served as a consultant to Genentech, Biogen/Idec, EMD Serono and MediciNova. He has had research funding from Teva Neuroscience, Biogen/Idec, EMD Serono, Bayer Health, Glaxo Smith Kline, BioMS, Abbott, Novartis and Accorda. The other authors declare that they have no competing interests. None of the authors hold stocks or shares in any pharmaceutical company or hold or are applying for any patents relating to the contents of the manuscript.

Authors' contributions

RPL and JAB were involved in the conception, design, acquisition of data, analysis and interpretation of data, and the drafting of the manuscript. BB and LN performed the tissue culture experiments and BB performed the indirect immunofluorescence experiments. The QRT-PCR was done by DS and for some of the arginase analyses by ER. The gene array procedures were carried out under the supervision of SL and the biometric analysis was carried out by BY. All authors read and approved the final version.

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Lisak, R.P., Benjamins, J.A., Bealmear, B. et al. Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for molecules associated with metabolism, signaling and regulation in central nervous system mixed glial cell cultures. J Neuroinflammation 6, 4 (2009).

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  • Multiple Sclerosis
  • Amyotrophic Lateral Sclerosis
  • Experimental Autoimmune Encephalomyelitis
  • Arginase
  • Multiple Sclerosis Lesion