Attenuation of microglial RANTES by NEMO-binding domain peptide inhibits the infiltration of CD8(+) T cells in the nigra of hemiparkinsonian monkey.

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Despite intense investigations, little is known about its pathological mediators. Here, we report the marked upregulation of RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin, chemokines that are involved in T cell trafficking, in the serum of hemiparkinsonian monkeys. Interestingly, 1-methyl-4-phenylpyridinium (MPP(+)), a Parkinsonian toxin, increased the expression of RANTES and eotaxin in mouse microglial cells. The presence of NF-κB binding sites in promoters of RANTES and eotaxin and down-regulation of these genes by NEMO-binding domain (NBD) peptide, selective inhibitor of induced NF-κB activation, in MPP(+)-stimulated microglial cells suggest that the activation of NF-κB plays an important role in the upregulation of these two chemokines. Consistently, serum enzyme-linked immuno assay (ELISA) and nigral immunohistochemistry further confirmed that these chemokines were strongly upregulated in MPTP-induced hemiparkinsonian monkeys and that treatment with NBD peptides effectively inhibited the level of these chemokines. Furthermore, the microglial upregulation of RANTES in the nigra of hemiparkinsonian monkeys could be involved in the altered adaptive immune response in the brain as we observed greater infiltration of CD8(+) T cells around the perivascular niche and deep brain parenchyma of hemiparkinsonian monkeys as compared to control. The treatment of hemiparkinsonian monkeys with NBD peptides decreased the microglial expression of RANTES and attenuated the infiltration of CD8(+) T cells in nigra. These results indicate the possible involvement of chemokine-dependent adaptive immune response in Parkinsonism.

Role of cytokine p40 family in multiple sclerosis.

Over the last couple of decades of neuro-immunological research, the p40 family of cytokines has emerged out as one of the most intriguing areas of interest because of multi-faceted roles of these cytokine in immune-modulation and inflammation. The IL-12, the most widely studied cytokine of this family, is well-characterized for its Th1-favoring activity, and therefore plays a key role in the pathophysiology of Th1-mediated autoimmune diseases like multiple sclerosis (MS). On the other hand, the IL-23, another member of the p40 family with shared p40 subunit, drives polarization of Th17, a subset of T cell suspected to have a key role in the pathophysiology of MS and experimental allergic encephalomyelitis (EAE), poses a challenge to our current understanding of Th1/Th2 hypotheses in autoimmune diseases. However, the more puzzling issues, the researchers are currently confronted with, are the biological roles of other two members of this family, the p40 monomer and the p40(2), the homodimer. Predominance of the mRNA level of p40 over p35 in the central nervous system of EAE and MS suggests a possible involvement of p40 in the pathogenesis of MS. However, the distinctive biological role of monomeric and dimeric form of p40 is not clearly understood yet. Initially, it was thought that p402 does not have any biological activity and only involved in antagonizing bioactive IL-12 but according to recent evidences, both p402 and p40 appear to have a proinflammatory role, therefore might be a crucial molecule in the pathogenesis of MS. The current review focuses on biological function of p40 family of cytokines with particular emphasis on MS.

Lipid-lowering drugs.

Although a change in life-style is often the method of first choice for lipid lowering, lipid-lowering drugs, in general, help to control elevated levels of different forms of lipids in patients with hyperlipidemia. While one group of drugs, statins, lowers cholesterol, the other group, fibrates, is known to take care of fatty acids and triglycerides. In addition, other drugs, such as ezetimibe, colesevelam, torcetrapib, avasimibe, implitapide, and niacin are also being considered to manage hyperlipidemia. As lipids are very critical for cardiovascular diseases, these drugs reduce fatal and nonfatal cardiovascular abnormalities in the general population. However, a number of recent studies indicate that apart from their lipid-lowering activities, statins and fibrates exhibit multiple functions to modulate intracellular signaling pathways, inhibit inflammation, suppress the production of reactive oxygen species, and modulate T cell activity. Therefore, nowadays, these drugs are being considered as possible therapeutics for several forms of human disorders including cancer, autoimmunity, inflammation, and neurodegeneration. Here I discuss these applications in the light of newly discovered modes of action of these drugs.

HATs and HDACs in neurodegeneration: a tale of disconcerted acetylation homeostasis.

Gradual disclosure of the molecular basis of selective neuronal apoptosis during neurodegenerative diseases reveals active participation of acetylating and deacetylating agents during the process. Several studies have now successfully manipulated neuronal vulnerability by influencing the dose and enzymatic activity of histone acetyltransferases (HATs) and histone deacetylases (HDACs), enzymes regulating acetylation homeostasis within the nucleus, thus focusing on the importance of balanced acetylation status in neuronal vitality. It is now increasingly becoming clear that acetylation balance is greatly impaired during neurodegenerative conditions. Herein, we attempt to illuminate molecular means by which such impairment is manifested and how the compromised acetylation homeostasis is intimately coupled to neurodegeneration. Finally, we discuss the therapeutic potential of reinstating the HAT-HDAC balance to ameliorate neurodegenerative diseases.

Regulation of TNF-alpha-induced IL-6 production in MG-63 human osteoblast-like cells.

Tumor necrosis factor-alpha (TNF-alpha) stimulates osteoblast production of interleukin-6 (IL-6), an inflammatory cytokine implicated in osteoclastic bone resorption. Therefore, we tested the hypothesis that TNF-alpha-induced IL-6 production in MG-63 osteosarcoma cells occurs via the p38 mitogen-activated protein kinase (MAPK) pathway. TNF-alpha activated p38 MAPK and stimulated IL-6 secretion by MG-63 cells, and pre-incubation of cells with the p38 MAPK inhibitor abrogated TNF-alpha-dependent IL-6 secretion. Transfection of IL-6 full-length and 5-deletion gene promoter reporter constructs indicated that p38 MAPK activation by TNF-alpha enhanced IL-6 gene expression, and that the p38 MAPK-responsive region resided in the proximal 260-bp segment. Transfection of NFkappaB and C/EBPbeta-sensitive reporter promoter constructs demonstrated that NFkappaB activity was enhanced and that constitutive C/EBPbeta was inhibited by TNF-alpha, with both effects being p38 MAPK-dependent. In conclusion, although p38 MAPK activation by TNF-alpha stimulates IL-6 secretion by MG-63 cells, it has opposing effects on c/EBPbeta and NFkappaB activity.

Ligation of CD40 stimulates the induction of nitric-oxide synthase in microglial cells.

The present study was undertaken to investigate the role of CD40 ligation in the expression of inducible nitric-oxide synthase (iNOS) in mouse BV-2 microglial cells and primary microglia. Ligation of CD40 alone by either cross-linking antibodies against CD40 or a recombinant CD40 ligand (CD154) was unable to induce the production of NO in BV-2 microglial cells. The absence of induction of NO production by CD40 ligation alone even in CD40-overexpressed BV-2 microglial cells suggests that a signal transduced by the ligation of CD40 alone is not sufficient to induce NO production. However, CD40 ligation markedly stimulated interferon-gamma (IFN-gamma)-mediated NO production. Ligation of CD40 in CD40-overexpressed cells further stimulated IFN-gamma-induced production of NO. This stimulation of NO production was accompanied by stimulation of the iNOS protein and mRNA. In addition to BV-2 glial cells, CD40 ligation also stimulated IFN-gamma-mediated NO production in mouse primary microglia and peritoneal macrophages. To understand the mechanism of induction/stimulation of iNOS, we investigated the roles of nuclear factor kappaB (NF-kappaB) and CCAAT/enhancer-binding protein beta (C/EBPbeta), transcription factors responsible for the induction of iNOS. IFN-gamma alone was able to induce the activation of NF-kappaB as well as C/EBPbeta. However, CD40 ligation alone induced the activation of only NF-kappaB but not of C/EBPbeta, suggesting that the activation of NF-kappaB alone by CD40 ligation is not sufficient to induce the expression of iNOS and that the activation of C/EBPbeta is also necessary for the expression of iNOS. Consistently, dominant-negative mutants of p65 (Deltap65) and C/EBPbeta (DeltaC/EBPbeta) inhibited the expression of iNOS in BV-2 microglial cells that were stimulated with the combination of IFN-gamma and CD40 ligand. Stimulation of IFN-gamma-mediated activation of NF-kappaB but not of C/EBPbeta by CD40 ligation suggests that CD40 ligation stimulates the expression of iNOS in IFN-gamma-treated BV-2 microglial cells through the stimulation of NF-kappaB activation. This study illustrates a novel role for CD40 ligation in stimulating the expression of iNOS in microglial cells, which may participate in the pathogenesis of neuroinflammatory diseases.

Induction of nitric-oxide synthase and activation of NF-kappaB by interleukin-12 p40 in microglial cells.

Interleukin-12 (IL-12) is composed of two different subunits, p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the central nervous system of patients with multiple sclerosis (MS) suggests that IL-12 p40 may have a role in the pathogenesis of the disease. However, the mode of action of p40 is completely unknown. Because nitric oxide produced from the induction of nitric-oxide synthase (iNOS) also plays a vital role in the pathophysiology of MS, the present study was undertaken to explore the role of p40 in the induction of NO production and the expression of iNOS in microglia. Both IL-12 and p40(2), the p40 homodimer, dose-dependently induced the production of NO in BV-2 microglial cells. This induction of NO production was accompanied by an induction of iNOS protein and mRNA. Induction of NO production by the expression of mouse p40 cDNA but not that of the mouse p35 cDNA suggests that the p40 but not the p35 subunit of IL-12 is involved in the expression of iNOS. In addition to BV-2 glial cells, p40(2) also induced the production of NO in mouse primary microglia and peritoneal macrophages. However, both IL-12 and p40(2) were unable to induce the production of NO in mouse primary astrocytes. Because activation of NF-kappaB is important for the expression of iNOS, we investigated the effect of p40(2) on the activation of NF-kappaB. Induction of the DNA binding as well as the transcriptional activity of NF-kappaB by p40(2) and inhibition of p40(2)-induced expression of iNOS by SN50, a cell-permeable peptide carrying the nuclear localization sequence of p50 NF-kappaB, but not by SN50M, a nonfunctional peptide mutant, suggests that p40(2) induces the expression of iNOS through the activation of NF-kappaB. This study delineates a novel role of IL-12 p40 in inducing the expression of iNOS in microglial cells, which may participate in the pathogenesis of neuroinflammatory diseases.

Interleukin-10 and interleukin-13 inhibit proinflammatory cytokine-induced ceramide production through the activation of phosphatidylinositol 3-kinase.

Ceramide produced by hydrolysis of plasma membrane sphingomyelin (SM) in different cells including brain cells in response to proinflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta)] plays an important role in coordinating cellular responses to stress, growth suppression, and apoptosis. The present study underlines the importance of IL-10 and IL-13, cytokines with potent antiinflammatory properties, in inhibiting the proinflammatory cytokine (TNF-alpha and IL-1beta)-mediated degradation of SM to ceramide in rat primary astrocytes. Treatment of rat primary astrocytes with TNF-alpha or IL-1beta led to rapid degradation of SM to ceramide, whereas IL-10 and IL-13 by themselves were unable to induce the degradation of SM to ceramide. Interestingly, both IL-10 and IL-13 prevented proinflammatory cytokine-induced degradation of SM to ceramide. Both IL-10 and IL-13 caused rapid activation of phosphatidylinositol (PI) 3-kinase, and inhibition of that kinase activity by wortmannin and LY294002 potently blocked the inhibitory effect of IL-10 and IL-13 on proinflammatory cytokine-mediated induction of ceramide production. This study suggests that the inhibition of proinflammatory cytokine-mediated degradation of SM to ceramide by IL-10 and IL-13 is mediated through the activation of PI 3-kinase. As ceramide induces apoptosis and IL-10 and IL-13 inhibit the induction of ceramide production, we examined the effect of IL-10 and IL-13 on proinflammatory cytokine-mediated apoptosis. Inhibition of TNF-alpha-induced apoptosis by IL-10 and IL-13 suggests that the antiapoptotic nature of IL-10 and IL-13 is probably due to the inhibition of ceramide production.

Activation of nuclear factor-kB in the spinal cord of experimental allergic encephalomyelitis.

The DNA-binding activity of nuclear factor (NF-kB) was found to be induced in the spinal cord of rats with experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), from the onset of the disease. This activation of NF-kB persisted throughout the disease period and decreased thereafter in the recovery phase. Supershift analysis of NF-kB DNA-binding activity in nuclear extracts of spinal cords showed that RelA/p65 and p50 subunits but not c-Rel/p75, RelB/p68 and p52 subunits were involved in DNA binding. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kB activation, markedly inhibited the in vivo activation of NF-kB in the spinal cord of EAE rats and attenuated the clinical symptoms of EAE. These studies suggest that activation of NF-kB plays an important role in the pathogenesis of EAE and inhibitors of NF-kB activation may have therapeutic importance in MS.

Expression of a dominant-negative mutant of p21(ras) inhibits induction of nitric oxide synthase and activation of nuclear factor-kappaB in primary astrocytes.

The present study underlines the importance of p21(ras) in regulating the inducible nitric oxide synthase (iNOS) in primary astrocytes. Bacterial lipopolysaccharides induced the GTP loading of p21(ras), and the expression of a dominant-negative mutant of p21(ras) (Deltap21(ras)) inhibited lipopolysaccharide-induced GTP loading in rat primary astrocytes. To delineate the role of p21(ras) in the induction of iNOS, we examined the effect of Deltap21(ras) on the expression of iNOS and the production of nitric oxide. It is interesting that expression of Deltap21(ras) markedly inhibited the production of nitric oxide and the expression of iNOS in lipopolysaccharide- and proinflammatory cytokine (tumor necrosis factor-alpha, interleukin-1beta; interferon-gamma)-stimulated rat and human primary astrocytes. Inhibition of iNOS promoter-derived chloramphenicol acetyltransferase activity by Deltap21(ras) suggests that p21(ras) is involved in the transcription of iNOS. As activation of nuclear factor-kappaB (NF-kappaB) is necessary for the transcription of iNOS, we examined the effect of Deltap21(ras) on the activation of NF-kappaB. Expression of Deltap21(ras) inhibited the DNA binding as well as the transcriptional activity of NF-kappaB in activated astrocytes, suggesting that Deltap21(ras) inhibits the expression of iNOS by inhibiting the activation of NF-kappaB. These studies also suggest that inhibitors of p21(ras) may be used as therapeutics in nitric oxide- and cytokine-mediated neuroinflammatory diseases.

Expression of a constitutively active form of phosphatidylinositol 3-kinase inhibits the induction of nitric oxide synthase in human astrocytes.

The present study underlines the importance of phosphatidylinositol 3-kinase (PI 3-kinase) in attenuating the induction of nitric oxide synthase (iNOS) in human astrocytes. Proinflammatory cytokines induced the production of nitric oxide (NO) and the expression of iNOS in human U373MG astrocytoma cells and primary astrocytes. Expression of a catalytically active p110 subunit (p110*) of PI 3-kinase but not that of a kinase-deficient mutant of p110 (p110-kd) induced an increase in PI 3-kinase activity and inhibited cytokine-induced production of NO and expression of iNOS. However, expression of p110* had no effect on the activation of NF-kB, suggesting that p110* inhibits the expression of iNOS without inhibiting the activation of NF-kB.

Amelioration of experimental allergic encephalomyelitis in Lewis rats by lovastatin.

Proinflammatory cytokines and inducible nitric oxide synthase (iNOS) are involved in the pathogenesis of experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We have previously reported that lovastatin (Pahan, K., Sheikh., F.G., Namboodiri, A. and Singh, I., Lovastatin and Phenylacetate inhibit the induction of nitric oxide synthase and cytokines in rat primary astrocytes, microglia and macrophages. J. Clin. Invest., 100 (1997) 2671-2679.), an inhibitor of the mevalonate pathway, inhibits the expression of iNOS and proinflammatory cytokines in rat primary glial cells (astroglia and microglia) and macrophages. The present study underlines the therapeutic importance of lovastatin in ameliorating the neuroinflammatory disease process in the central nervous system of EAE rats. Immunohistochemical results show a higher degree of expression of iNOS, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in brains of rats with acute monophasic EAE relative to the control animals. Administration of lovastatin inhibited the expression of iNOS, TNF-alpha and IFN-gamma in the CNS of EAE rats and improved the clinical signs of EAE suggesting that this compound may have therapeutic potential in the treatment of neuroinflammatory diseases like MS.

Induction of the manganese superoxide dismutase gene by sphingomyelinase and ceramide.

The present study reports the effect of ceramide generated by hydrolysis of membrane sphingomyelin with bacterial sphingomyelinase (SMase) and of cell-permeable ceramide analogues on the expression of manganese superoxide dismutase (MnSOD). Incubation of the rat primary astrocytes with SMase led to a time- and dose-dependent increase in MnSOD activity. The increase in MnSOD activity was accompanied by an increase in MnSOD protein and mRNA. A similar effect on the expression of MnSOD was observed with the addition of cell-permeable ceramide analogues (C2 and C6). On the other hand, C2-dihydroceramide (N-acetylsphinganine), which lacks the functional critical double bond, was ineffective in inducing the expression of MnSOD. Nuclear run-on analysis showed that SMase and ceramide increased the rate of transcription of the MnSOD gene. Besides astrocytes, SMase was also found to induce the expression of MnSOD in rat mesangial cells, C6 glial cells, PC12 cells, and human skin fibroblasts. Markedly higher expression of mRNA, protein, and activity of MnSOD in skin fibroblasts from patients with Farber disease, a human disorder with pathognomonic accumulation of ceramide due to a deficiency of ceramidase, than in normal skin fibroblasts indicate that ceramide may act as a physiological inducer of MnSOD gene expression. However, stimulation of ceramide-mediated DNA fragmentation by antisense knockdown of MnSOD suggests that induction of MnSOD by ceramide is a protective response of the cell.

Inhibition of phosphatidylinositol 3-kinase induces nitric-oxide synthase in lipopolysaccharide- or cytokine-stimulated C6 glial cells.

Nitric oxide (NO) produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of demyelinating and neurodegenerative diseases. The present study underlines the importance of phosphatidylinositol 3-kinase (PI 3-kinase) in the expression of iNOS in C6 glial cells and rat primary astrocytes. Bacterial lipopolysaccharide (LPS) or interleukin-1beta (IL-1beta) was unable to induce the expression of iNOS and the production of NO in rat C6 glial cells. Similarly, wortmannin and LY294002, compounds that inhibit PI 3-kinase, were also unable to induce the expression of iNOS and the production of NO. However, a combination of wortmannin or LY294002 with LPS or IL-1beta induced the expression of iNOS and the production of NO in C6 glial cells. Consistent with the induction of iNOS, wortmannin also induced iNOS promoter-derived chloramphenicol acetyltransferase activity in LPS- or IL-1beta-treated C6 glial cells. The expression of iNOS by LPS in C6 glial cells expressing a dominant-negative mutant of p85alpha, the regulatory subunit of PI 3-kinase, further supports the conclusion that inhibition of PI 3-kinase provides a necessary signal for the induction of iNOS. Next we examined the effect of wortmannin on the activation of mitogen-activated protein (MAP) kinase and nuclear factor NF-kappaB in LPS- or IL-1beta-stimulated C6 glial cells. In contrast to the inability of LPS and IL-1beta alone to induce the expression of iNOS, both LPS and IL-1beta individually stimulated MAP kinase activity and induced DNA binding and transcriptional activity of NF-kappaB. Wortmannin alone was unable to activate MAP kinase and NF-kappaB. Moreover, wortmannin had no effect on LPS- or IL-1beta-mediated activation of MAP kinase and NF-kappaB, suggesting that wortmannin induced the expression of iNOS in LPS- or IL-1beta-stimulated C6 glial cells without modulating the activation of MAP kinase and NF-kappaB. Similar to C6 glial cells, wortmannin also stimulated LPS-mediated expression of iNOS and production of NO in astrocytes without affecting the LPS-mediated activation of NF-kappaB. Taken together, the results from specific chemical inhibitors and dominant-negative mutant expression studies demonstrate that apart from the activation of NF-kappaB, inhibition of PI 3-kinase is also necessary for the expression of iNOS and production of NO.

Cytokine-mediated induction of ceramide production is redox-sensitive. Implications to proinflammatory cytokine-mediated apoptosis in demyelinating diseases.

The present study underlines the importance of reactive oxygen species in cytokine-mediated degradation of sphingomyelin (SM) to ceramide. Treatment of rat primary astrocytes with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta led to marked alteration in cellular redox (decrease in intracellular GSH) and rapid degradation of SM to ceramide. Interestingly, pretreatment of astrocytes with N-acetylcysteine (NAC), an antioxidant and efficient thiol source for glutathione, prevented cytokine-induced decrease in GSH and degradation of sphingomyelin to ceramide, whereas treatment of astrocytes with diamide, a thiol-depleting agent, alone caused degradation of SM to ceramide. Moreover, potent activation of SM hydrolysis and ceramide generation were observed by direct addition of an oxidant like hydrogen peroxide or a prooxidant like aminotriazole. Similar to NAC, pyrrolidinedithiocarbamate, another antioxidant, was also found to be a potent inhibitor of cytokine-induced degradation of SM to ceramide indicating that cytokine-induced hydrolysis of sphingomyelin is redox-sensitive. Besides astrocytes, NAC also blocked cytokine-mediated ceramide production in rat primary oligodendrocytes, microglia, and C6 glial cells. Inhibition of TNF-alpha- and diamide-mediated depletion of GSH, elevation of ceramide level, and DNA fragmentation (apoptosis) in primary oligodendrocytes by NAC, and observed depletion of GSH, elevation of ceramide level, and apoptosis in banked human brains from patients with neuroinflammatory diseases (e.g. X-adrenoleukodystrophy and multiple sclerosis) suggest that the intracellular level of GSH may play a critical role in the regulation of cytokine-induced generation of ceramide leading to apoptosis of brain cells in these diseases.

Cytokine-induced accumulation of very long-chain fatty acids in rat C6 glial cells: implication for X-adrenoleukodystrophy.

X-Adrenoleukodystrophy (X-ALD) is an inherited metabolic disorder of very long-chain fatty acids (VLCFA) with subsequent manifestation of neuroinflammatory disease. To investigate the possible role of proinflammatory cytokines in the X-ALD disease process, we examined the effect of cytokines on the metabolism of VLCFA in C6 glial cells expressing oligodendrocyte-like properties. C6 glial cells under serum-free conditions were treated with different combinations of cytokines (tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma) or cytokine with bacterial lipopolysaccharide (LPS). Cytokine-treated C6 cells had higher concentrations of VLCFA, measured as percent weight and also as C26:0/C22:0 ratio, which were 300-400% as compared with the controls. We also found increased levels of C26:1 in cytokine-treated cells. The accumulation of VLCFA paralleled the decrease (35-55%) in peroxisomal beta-oxidation activity and a 12- to 14-fold increase in the production of nitric oxide (NO). Individual cytokines were unable either to produce NO or to increase the levels of VLCFA in C6 cells. Inhibition of cytokine-induced NO production by L-N-methylarginine, an inhibitor of NO synthase (NOS), and N-acetylcysteine, an inhibitor of cytokine-mediated induction of inducible NOS, normalized the peroxisomal beta-oxidation activity and the levels of VLCFA, suggesting a role for the proinflammatory cytokines and NO toxicity in the neuropathological changes associated with abnormal VLCFA metabolism (e.g., X-ALD). X-ALD is a peroxisomal disease having deficient oxidation of VLCFA, resulting in the excessive accumulation of VLCFA in all tissues but especially in brain. We observed greater increase in levels of VLCFA in the inflammatory region of ALD brain (in the demyelinating plaque and the area around the plaque) than in the normal-looking area away from the plaque; this also indicates that cytokines in the proinflammatory region may augment the VLCFA defect caused by the inherited abnormality in X-ALD brain. Although C6 glial cultured cells do not reflect the X-ALD model precisely, the observed relationship between the cytokine-induced inhibition of the oxidation of VLCFA, excessive accumulation of VLCFA, and excessive production of NO and their normalization by inhibitors of NOS in C6 glial cells suggests that NO-mediated toxicity may play a role in VLCFA-associated neuroinflammatory diseases (e.g., X-ALD).

Therapy for X-adrenoleukodystrophy: normalization of very long chain fatty acids and inhibition of induction of cytokines by cAMP.

X-adrenoleukodystrophy (X-ALD) is an inherited fatty acid metabolic disorder with secondary manifestation of neuroinflammatory disease process. We report that compounds (forskolin, 8-bromo cAMP, and rolipram) that increase cAMP and activate protein kinase A (PKA) were found to stimulate the peroxisomal beta-oxidation of lignoceric acid (C24:0) whereas compounds (H-89 and myristoylated PKI) that decrease cAMP and PKA activity inhibited the peroxisomal beta-oxidation of lignoceric acid in cultured skin fibroblasts from X-ALD patients. Consistent with the stimulation of beta-oxidation of lignoceric acid, activators of PKA normalized the level of very long chain fatty acids (VLCFA) in X-ALD cultured skin fibroblasts. This normalization of VLCFA in X-ALD cells with forskolin, 8-Br cAMP or with rolipram, an inhibitor of cAMP phosphodiesterase, was realized independent of expression of mRNA or protein of the ALD gene, suggesting that cAMP derivatives can correct the metabolic defect in X-ALD fibroblasts without involving the candidate gene for the disease. Because astrocytes and microglia in demyelinating lesions of X-ALD brain express proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), we examined the effect of cAMP derivatives or rolipram on lipopolysaccharide-stimulated rat primary astrocytes and microglia and found that cAMP derivatives and rolipram inhibited the induction of TNF-alpha and IL-1beta in both astrocytes and microglia. The ability of cAMP derivatives and rolipram to block the induction of TNF-alpha and IL-1beta in astrocytes and microglia and to normalize the fatty acid pathogen in skin fibroblasts of x-adrenoleukodystrophy (X-ALD) clearly identify cAMP analogs or rolipram as candidates for potential therapy for X-ALD patients.

Inhibitors of protein phosphatase 1 and 2A differentially regulate the expression of inducible nitric-oxide synthase in rat astrocytes and macrophages.

Nitric oxide produced by inducible nitric-oxide synthase (iNOS) in different cells including brain cells in response to proinflammatory cytokines plays an important role in the pathophysiology of stroke and other neurodegenerative diseases. The present study underlines the importance of protein phosphatase (PP) 1 and 2A in the regulation of the differential expression of iNOS in rat primary astrocytes and macrophages. Compounds (calyculin A, microcystin, okadaic acid, and cantharidin) that inhibit PP 1 and 2A were found to stimulate the lipopolysaccharide (LPS)- and cytokine-mediated expression of iNOS and production of NO in rat primary astrocytes and C6 glial cells. However, these inhibitors inhibited the LPS- and cytokine-mediated expression of iNOS and production of NO in rat resident macrophages and RAW 264.7 cells. Similarly, okadaic acid, an inhibitor of PP 1/2A, stimulated the iNOS promoter-derived chloramphenicol acetyltransferase activity in astrocytes and inhibited the iNOS promoter-derived chloramphenicol acetyltransferase activity in macrophages, indicating that okadaic acid also differentially regulates the transcription of the iNOS gene in astrocytes and macrophages. The observed stimulation of the expression of iNOS in astrocytes and the inhibition of the expression of iNOS in macrophages with the inhibition of PP 1/2A activity clearly delineate a novel role of PP 1/2A in the differential regulation of iNOS in rat astrocytes and macrophages. Because the activation of NF-kappaB is necessary for the induction of iNOS and the expression of tumor necrosis factor (TNF)-alpha also depends on the activation of NF-kappaB, we examined the effect of okadaic acid on the LPS-mediated activation of NF-kappaB and production of TNF-alpha in rat primary astrocytes and macrophages. Interestingly, in both cell types, okadaic acid stimulated the LPS-mediated DNA binding as well as transcriptional activity of NF-kappaB and production of TNF-alpha. This study suggests that the stimulation of iNOS expression in astrocytes by inhibitors of PP 1/2A is possibly due to the stimulation of NF-kappaB activation; however, activation of NF-kappaB is not sufficient for the induction of iNOS in macrophages and that apart from NF-kappaB some other signaling pathway(s) sensitive to PP 1 and/or PP 2A is/are possibly involved in the regulation of iNOS in macrophages. This differential induction of iNOS as compared with similar activation of NF-kappaB by inhibitors of PP 1/2A indicates the involvement of different intracellular signaling events for the induction of iNOS in two cell types of the same animal species.

Lovastatin and sodium phenylacetate normalize the levels of very long chain fatty acids in skin fibroblasts of X- adrenoleukodystrophy.

The present study underlines the importance of lovastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, and the sodium salt of phenylacetic acid (NaPA), an inhibitor of mevalonate pyrophosphate decarboxylase, in normalizing the pathognomonic accumulation of saturated very long chain fatty acids (VLCFA) in cultured skin fibroblasts of X-adrenoleukodystrophy (X-ALD) in which the ALD gene is either mutated or deleted. Lovastatin or NaPA alone or in combination stimulated the beta-oxidation of lignoceric acid (C24:0) and normalized the elevated levels of VLCFA in skin fibroblasts of X-ALD. Ability of lovastatin and NaPA to normalize the pathognomonic accumulation of VLCFA in skin fibroblasts of X-ALD may identify these drugs as possible therapeutics for X-ALD.

Abnormality in catalase import into peroxisomes leads to severe neurological disorder.

Peroxisomal disorders are lethal inherited diseases caused by either defects in peroxisome assembly or dysfunction of single or multiple enzymatic function(s). The peroxisomal matrix proteins are targeted to peroxisomes via the interaction of peroxisomal targeting signal sequences 1 and 2 (PTS1 or PTS2) with their respective cytosolic receptors. We have studied human skin fibroblast cell lines that have multiple peroxisomal dysfunctions with normal packaging of PTS1 and PTS2 signal-containing proteins but lack catalase in peroxisomes. To understand the defect in targeting of catalase to peroxisomes and the loss of multiple enzyme activities, we transfected the mutant cells with normal catalase modified to contain either PTS1 or PTS2 signal sequence. We demonstrate the integrity of these pathways by targeting catalase into peroxisomes via PTS1 or PTS2 pathways. Furthermore, restoration of peroxisomal functions by targeting catalase-SKL protein (a catalase fused to the PTS1 sequence) to peroxisomes indicates that loss of multiple functions may be due to their inactivation by H2O2 or other oxygen species in these catalase-negative peroxisomes. In addition to enzyme activities, targeting of catalase-SKL chimera to peroxisomes also corrected the in situ levels of fatty acids and plasmalogens in these mutant cell lines. In normal fibroblasts treated with aminotriazole to inhibit catalase, we found that peroxisomal functions were inhibited to the level found in mutant cells, an observation that supports the conclusion that multiple peroxisomal enzyme defects in these patients are caused by H2O2 toxicity in catalase-negative peroxisomes. Moreover, targeting of catalase to peroxisomes via PTS1 and PTS2 pathways in these mutant cell lines suggests that there is another pathway for catalase import into peroxisomes and that an abnormality in this pathway manifests as a peroxisomal disease.