Oxygen and steroids affect the regulatory role of natriuretic peptide receptor-C on surfactant secretion by type II cells.

Atrial natriuretic peptide (ANP) and its receptors natriuretic peptide receptor (NPR)-A and NPR-C are all highly expressed in alveolar epithelial type II cells (AEC2s) in the late-gestation ovine fetal lung and are dramatically decreased postnatally. However, of all the components, NPR-C stimulation inhibits ANP-mediated surfactant secretion. Since alveolar oxygen increases dramatically after birth, and steroids are administered to mothers antenatally to enhance surfactant lung maturity, we investigated the effects of O2 concentration and steroids on NPR-C-mediated surfactant secretion in AEC2s. NPR-C expression was highest at 5% O2 while being suppressed by 21% O2, in cultured mouse lung epithelial cells (MLE-15s) and/or human primary AEC2s. Surfactant protein-B (SP-B) was significantly elevated in media from both in vitro and ex vivo culture at 13% O2 versus 21% O2 in the presence of ANP or terbutaline (TER). Both ANP and C-ANP (an NPR-C agonist) attenuated TER-induced SP-B secretion; this effect was reversed by dexamethasone (DEX) pretreatment in AEC2s and by transfection with NPR-C siRNA in MLE-15 cells. DEX markedly reduced AEC2 NPR-C expression, and pregnant ewes treated with betamethasone showed reduced ANP in fetal sheep lung fluid. These data suggest that elevated O2 downregulates AEC2 NPR-C and that steroid-mediated NPR-C downregulation in neonatal lungs may provide a novel mechanism for their effect on perinatal surfactant production.

Effect of vitamin D3 intake on the onset of disease in a murine model of human Krabbe disease.

Low vitamin D level is a risk factor for various late-onset CNS demyelinating disorders. We investigated whether vitamin D deficiency influences disease in twitcher mice (GALC(twi/twi) ; twi), a murine model of Krabbe disease (KD), an inherited disorder caused by galactocerebrosidase (GALC) deficiency that leads to psychosine accumulation, oligodendrocyte (OL) loss, and CNS demyelination. We found that the in situ 1,25-dihydroxyvitamin D3 level was reduced, with a parallel increase in the expression of inflammatory cytokines and vitamin D-catabolizing enzymes in the brains of KD and twi mice compared with age-matched controls. Pups maintained on milk from lactating heterozygous (GALC(twi/+) ) mothers that were fed a vitamin D3-supplemented diet until weaning and then fed a vitamin D3-supplemented diet demonstrated delayed body weight loss and development of disease in twi mice. This delayed the onset of tremors and locomotor disabilities that eventually impacted the life span of twi mice (50 ± 2 days). Accordingly, the expression of antioxidant enzymes was increased with delayed psychosine accumulation, lipid peroxidation, and inflammatory response that eventually protected CNS myelin and axonal integrity in twi mice. In vitro studies revealed that 1,25-dihydroxyvitamin D3 enhances antioxidant defenses in OLs deficient for GALC or incubated with psychosine. Together these data provide the first evidence that vitamin D deficiency affects disease development in twi mice and that vitamin D3 supplementation has the potential to improve the efficacy of KD therapeutics.

S-nitrosoglutathione induces ciliary neurotrophic factor expression in astrocytes, which has implications to protect the central nervous system under pathological conditions.

Accumulating evidence suggests that reactive astrogliosis has beneficial and detrimental outcomes in various CNS disorders, but the mechanism behind this dichotomy is unclear. Recent advances in this direction suggested that NO signaling is critical to regulate the outcomes of reactive astrogliosis in vivo. Using biochemical and genetic approaches, we here investigated the effect of S-nitrosoglutathione (GSNO; a physiological NO donor) in astrocytes in vitro settings. GSNO enhanced the expressions of glial fibrillary acidic protein and neurotrophic factors including ciliary neurotrophic factor (CNTF) in astrocytes in a dose-dependent manner. The enhanced CNTF expression in GSNO-treated astrocytes was ascribed to NO-mediated sGC/cGMP/PKG signaling. It was associated with p38 MAPK-dependent increased peroxisome proliferator-activated receptor-γ transactivation. In addition, the chromatin accessibility of peroxisome proliferator-activated receptor-γ accompanied with ATF2 and CREB (cAMP-response element-binding protein) was enhanced across the CNTF gene promoter in GSNO treated astrocytes. Interestingly, secreted CNTF was responsible for increased expression of glial fibrillary acidic protein in GSNO-treated astrocytes in an autocrine manner via a JAK2- and STAT3-dependent mechanism. In addition, CNTF secreted by GSNO-treated astrocytes enhanced the differentiation of immature oligodendrocytes in vitro. These effects of GSNO were consistent with an endogenously produced NO in astrocytes stimulated with proinflammatory cytokines in vitro. We conclude that NO signaling induces CNTF expression in astrocytes that favors the beneficial outcomes of reactive astrogliosis in vivo. Our data suggest that the endogenously produced NO or its exogenous source has potential to modulate the outcomes of reactive astrogliosis to protect CNS under pathological conditions.

AMP-activated protein kinase signaling protects oligodendrocytes that restore central nervous system functions in an experimental autoimmune encephalomyelitis model.

AMP-activated protein kinase (AMPK) signaling is reported to protect neurons under pathologic conditions; however, its effect on oligodendrocytes (OLs) remains to be elucidated. We investigated whether AMPK signaling protects OLs to restore central nervous system (CNS) functions in an experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Increased inflammation and demyelination in the CNS and peripheral immune responses were consistent with the observed clinical impairments in EAE animals, which were attenuated by treatment with metformin compared with vehicle. In addition, expressions of neurotrophic factors and of signatory genes of OL lineages were increased in the CNS of metformin-treated EAE animals. Likewise, metformin attenuated inflammatory response and enhanced expressions of neurotrophic factors, thereby protecting OLs via AMPK activation in mixed glial cultures stimulated with lipopolysaccharide/interferon γ in vitro, as evidenced by analysis of the expression of signatory genes of O1(+)/MBP(+) OLs and their cellular populations. Metformin also attenuated oxidative stress and malondialdehyde-containing protein levels, with corresponding induction of antioxidative defenses in OLs exposed to cytokines via AMPK activation. These effects of metformin were evident in the CNS of EAE animals. These data provide evidence that AMPK signaling is crucial to protect OLs and, thus, CNS functions in EAE animals. We conclude that AMPK activators, including metformin, have the potential to limit neurologic deficits in multiple sclerosis and related neurodegenerative disorders.

Modulation of Rho-Rock signaling pathway protects oligodendrocytes against cytokine toxicity via PPAR-α-dependent mechanism.

We earlier documented that lovastatin (LOV)-mediated inhibition of small Rho GTPases activity protects vulnerable oligodendrocytes (OLs) in mixed glial cell cultures stimulated with Th1 cytokines and in a murine model of multiple sclerosis (MS). However, the precise mechanism of OL protection remains unclear. We here employed genetic and biochemical approaches to elucidate the underlying mechanism that protects LOV treated OLs from Th1 (tumor necrosis factor-α) and Th17 (interleukin-17) cytokines toxicity in in vitro. Cytokines enhanced the reactive oxygen species (ROS) generation and mitochondrial membrane depolarization with corresponding lowering of glutathione (reduced) level in OLs and that were reverted by LOV. In addition, the expression of ROS detoxifying enzymes (catalase and superoxide-dismutase 2) and the transactivation of peroxisome proliferators-activated receptor (PPAR)-α/-ß/-γ including PPAR-γ coactivator-1α were enhanced by LOV in similarly treated OLs. Interestingly, LOV-mediated inhibition of small Rho GTPases, i.e., RhoA and cdc42, and Rho-associated kinase (ROCK) activity enhanced the levels of PPAR ligands in OLs via extracellular signal regulated kinase (1/2)/p38 mitogen-activated protein kinase/cytoplasmic phospholipase 2/cyclooxygenase-2 signaling cascade activation. Small hairpin RNA transfection-based studies established that LOV mainly enhances PPAR-α and less so of PPAR-ß and PPAR-γ transactivation that enhances ROS detoxifying defense in OLs. In support of this, the observed LOV-mediated protection was lacking in PPAR-α-deficient OLs exposed to cytokines. Collectively, these data provide unprecedented evidence that LOV-mediated inhibition of the Rho-ROCK signaling pathway boosts ROS detoxifying defense in OLs via PPAR-α-dependent mechanism that has implication in neurodegenerative disorders including MS.

Interference with RhoA-ROCK signaling mechanism in autoreactive CD4+ T cells enhances the bioavailability of 1,25-dihydroxyvitamin D3 in experimental autoimmune encephalomyelitis.

Vitamin D deficiency is a major risk factor for central nervous system (CNS) demyelinating diseases including multiple sclerosis (MS) and its animal model, that of experimental autoimmune encephalomyelitis (EAE). Both vitamin D(3) and 1, 25-dihydroxyviatmin-D(3) (calcitriol) had beneficial effects in EAE/MS. However, the exact cause of vitamin D deficiency in EAE/MS is not clear. Previously, we documented that lovastatin (LOV) provides protection in EAE animals via inhibition of RhoA-ROCK signaling. Herein, we demonstrate that LOV prevents the lowering of circulating 25-hydroxyvitamin-D(3) and 1,25-dihydroxyviatmin-D(3) levels including 1,25-dihydroxyviatmin-D(3) levels in the peripheral lymphoid organs and CNS of treated EAE animals. These effects of LOV were attributed to enhanced expression of vitamin D synthesizing enzyme (1α-hydroxylase) in kidney and the CNS, with corresponding reduction of vitamin D catabolizing enzyme (24-hydorxylase) expression in the CNS of EAE animals via inhibition of RhoA-ROCK signaling. Ex vivo and in vitro studies established that autoreactive Th1/Th17 cells had higher expression of 24-hydroxylase than Th2/T regulatory cells, that was reverted by LOV or ROCK inhibitor. Interestingly, LOV-mediated regulation of vitamin D metabolism had improved vitamin D(3) efficacy to confer protection in EAE animals and that was ascribed to the LOV- and calcitriol-induced immunomodulatory synergy. Together, these data provide evidence that interfering with RhoA-ROCK signaling in autoreactive Th1/Th17 cells can improve vitamin D(3) efficacy in clinical trials of MS and related neurodegenerative disorders.

Synergistic activity of interleukin-17 and tumor necrosis factor-α enhances oxidative stress-mediated oligodendrocyte apoptosis.

Th1 cytokine-induced loss of oligodendrocytes (OLs) is associated with axonal loss in CNS demyelinating diseases such as multiple sclerosis (MS)that contributes to neurological disabilities in affected individuals. Recent studies indicated that, in addition to Th1-phenotype cytokines including tumor necrosis factor (TNF)-α, Th17 phenotype cytokine, interleukin (IL)-17 also involved in the development of MS. In this study, we investigated the direct effect of IL-17 on the survival of OLs in the presence of TNF-α and individually in vitro settings. Our findings suggest that IL-17 alone, however, was not able to affect the survival of OLs, but it exacerbates the TNF-α-induced OL apoptosis as compared with individual TNF-α treatment. This effect of cytokines was ascribed to an inhibition of cell-survival mechanisms, co-localization of Bid/Bax proteins in the mitochondrial membrane and caspase 8 activation mediated release of apoptosis inducing factor from mitochondria in treated OLs. In addition, cytokine treatment disturbed the mitochondrial membrane potential in OLs with corresponding increase in the generation of reactive oxygen species, which were attenuated by N-acetyl cysteine treatment. In addition, combining of these cytokines induced cell-cycle arrest at G1/S phases in OL-like cells and inhibited the maturation of OL progenitor cells that was attenuated by peroxisome proliferator-activated receptor-γ/-ß agonists. Collectively, these data provide initial evidence that IL-17 exacerbates TNF-α-induced OL loss and inhibits the differentiation of OL progenitor cells suggesting that antioxidant- or peroxisome proliferator-activated receptor agonist-based therapies have potential to limit CNS demyelination in MS or other related demyelinating disorders.

Metformin attenuated the autoimmune disease of the central nervous system in animal models of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the CNS. Metformin is the most widely used drug for diabetes and mediates its action via activating AMP-activated protein kinase (AMPK). We provide evidence that metformin attenuates the induction of EAE by restricting the infiltration of mononuclear cells into the CNS, down-regulating the expression of proinflammatory cytokines (IFN-gamma, TNF-alpha, IL-6, IL-17, and inducible NO synthase (iNOS)), cell adhesion molecules, matrix metalloproteinase 9, and chemokine (RANTES). Furthermore, the AMPK activity and lipids alterations (total phospholipids and in free fatty acids) were restored by metformin treatment in the CNS of treated EAE animals, suggesting the possible involvement of AMPK. Metformin activated AMPK in macrophages and thereby inhibited biosynthesis of phospholipids as well as neutral lipids and also down-regulated the expression of endotoxin (LPS)-induced proinflammatory cytokines and their mediators (iNOS and cyclooxygenase 2). It also attenuated IFN-gamma and IL-17-induced iNOS and cyclooxygenase 2 expression in RAW267.4 cells, further supporting its anti-inflammatory property. Metformin inhibited T cell-mediated immune responses including Ag-specific recall responses and production of Th1 or Th17 cytokines, while it induced the generation of IL-10 in spleen cells of treated EAE animals. Altogether these findings reveal that metformin may have a possible therapeutic value for the treatment of multiple sclerosis and other inflammatory diseases.

Activation of PPAR-γ and PTEN cascade participates in lovastatin-mediated accelerated differentiation of oligodendrocyte progenitor cells.

Previously, we and others documented that statins including-lovastatin (LOV) promote the differentiation of oligodendrocyte progenitor cells (OPCs) and remyelination in experimental autoimmune encephalomyelitis (EAE), an multiple sclerosis (MS) model. Conversely, some recent studies demonstrated that statins negatively influence oligodendrocyte (OL) differentiation in vitro and remyelination in a cuprizone-CNS demyelinating model. Therefore, herein, we first investigated the cause of impaired differentiation of OLs by statins in vitro settings. Our observations indicated that the depletion of cholesterol was detrimental to LOV treated OPCs under cholesterol/serum-deprived culture conditions similar to that were used in conflicting studies. However, the depletion of geranylgeranyl-pp under normal cholesterol homeostasis conditions enhanced the phenotypic commitment and differentiation of LOV-treated OPCs ascribed to inhibition of RhoA-Rho kinase. Interestingly, this effect of LOV was associated with increased activation and expression of both PPAR-γ and PTEN in OPCs as confirmed by various pharmacological and molecular based approaches. Furthermore, PTEN was involved in an inhibition of OPCs proliferation via PI3K-Akt inhibition and induction of cell cycle arrest at G1 phase, but without affecting their cell survival. These effects of LOV on OPCs in vitro were absent in the CNS of normal rats chronically treated with LOV concentrations used in EAE indicating that PPAR-γ induction in normal brain may be tightly regulated-providing evidences that statins are therapeutically safe for humans. Collectively, these data provide initial evidence that statin-mediated activation of the PPAR-γ-PTEN cascade participates in OL differentiation, thus suggesting new therapeutic-interventions for MS or related CNS-demyelinating diseases.

Combination therapy of lovastatin and rolipram provides neuroprotection and promotes neurorepair in inflammatory demyelination model of multiple sclerosis.

Drug combination therapies for central nervous system (CNS) demyelinating diseases including multiple sclerosis (MS) are gaining momentum over monotherapy. Over the past decade, both in vitro and in vivo studies established that statins (HMG-CoA reductase inhibitors) and rolipram (phosphodiesterase-4 inhibitor; blocks the degradation of intracellular cyclic AMP) can prevent the progression of MS in affected individuals via different mechanisms of action. In this study, we evaluated the effectiveness of lovastatin (LOV) and rolipram (RLP) in combination therapy to promote neurorepair in an inflammatory CNS demyelination model of MS, experimental autoimmune encephalomyelitis (EAE). Combination treatment with suboptimal doses of these drugs in an established case of EAE (clinical disease score > or = 2.0) significantly attenuated the infiltration of inflammatory cells and protected myelin sheath and axonal integrity in the CNS. It was accompanied with elevated level of cyclic AMP and activation of its associated protein kinase A. Interestingly, combination treatment with these drugs impeded neurodegeneration and promoted neurorepair in established EAE animals (clinical disease score > or = 3.5) as verified by quantitative real-time polymerase chain reaction, immunohistochemistry and electron microscopic analyses. These effects of combination therapy were minimal and/or absent with either drug alone in these settings. Together, these data suggest that combination therapy with LOV and RLP has the potential to provide neuroprotection and promote neurorepair in MS, and may have uses in other related CNS demyelinating diseases.

Modulation of peroxisome proliferator-activated receptor-alpha activity by N-acetyl cysteine attenuates inhibition of oligodendrocyte development in lipopolysaccharide stimulated mixed glial cultures.

Glial cells secrete proinflammatory mediators in the brain in response to exogenous stimuli such as infection and injury. Previously, we documented that systemic maternal lipopolysaccharide (LPS)-exposure at embryonic gestation day 18 causes oligodendrocyte (OL)-injury/hypomyelination in the developing brain which can be attenuated by N-acetyl cysteine (NAC; precursor of glutathione). The present study delineates the underlying mechanism of NAC-mediated attenuation of inhibition of OL development in LPS-stimulated mixed glial cultures. Factors released by LPS-stimulated mixed glial cultures inhibited OL development as shown by decrease in both proliferation 3bromo-deoxyuridine+/chondroitin sulfate proteoglycan-NG2+, hereafter BrdU+/NG+ and differentiation (O4+ and myelin basic protein+) of OL-progenitors. Correspondingly, an impairment of peroxisomal proliferation was shown by a decrease in the level of peroxisomal proteins in the developing OLs following exposure to LPS-conditioned media (LCM). Both NAC and WY14643, a peroxisome proliferator-activated receptor (PPAR)-alpha agonist attenuated these LCM-induced effects in OL-progenitors. Similar to WY14643, NAC attenuated LCM-induced inhibition of PPAR-alpha activity in developing OLs. Studies conducted with cytokines and diamide (a thiol-depleting agent) confirmed that cytokines are active agents in LCM which may be responsible for inhibition of OL development via peroxisomal dysfunction and induction of oxidative stress. These findings were further corroborated by similar treatment of developing OLs generated from PPAR-alpha(-/-) and wild-type mice or B12 oligodendroglial cells co-transfected with PPAR-alpha small interfering RNAs/pTK-PPREx3-Luc plasmids. Collectively, these data provide evidence that the modulation of PPAR-alpha activity, thus peroxisomal function by NAC attenuates LPS-induced glial factors-mediated inhibition of OL development suggesting new therapeutic interventions to prevent the devastating effects of maternal infections.

Attenuation of lipopolysaccharide-induced inflammatory response and phospholipids metabolism at the feto-maternal interface by N-acetyl cysteine.

Maternal microbial infections cause adverse fetal developmental outcomes including embryonic resorption, intrauterine fetal death, and preterm labor. Recent studies demonstrated that oxidative-stress plays an important role in chorioamniotitis pathogenesis. Herein we investigated the effect of N-acetyl cysteine (NAC) on lipopolysaccharide (LPS)-induced preterm labor and fetal demise in murine model. Lipopolysaccharide exposure at embryonic day 18 demonstrated an increase in the abortion rate and fetal demise in pregnant rats. This was associated with increase in an inflammatory response (cytokines, chemokines, and iNOS expression) and infiltration of leukocytes (monocytes and polymorphonuclear cells) in the placenta. There was increased expression of cytosolic and secretary phospholipase A2 with increased secretion of prostaglandin-2 and leukotriene B4 in the placenta, suggestive of increased metabolism of phospholipids. In addition, expression of cycloxygenase-2 and malondialdehyde production (oxidative-stress marker) was increased in the placenta. Conversely, NAC pretreatment abolished these effects of LPS in the placenta. Collectively, these data provide evidence that LPS-induced increased inflammation and metabolism of phospholipids at the feto-maternal interface (placenta) is critical for preterm labor and fetal demise during maternal microbial infections which could be blocked by antioxidant-based therapies.

GSNO promotes functional recovery in experimental TBI by stabilizing HIF-1α.

Traumatic brain injury (TBI) causes sustained disability due to compromised neurorepair mechanisms. Crucial to neurorepair and functional recovery following both TBI and stroke is hypoxia-inducible factor-1 alpha (HIF-1α). Based on reports that HIF-1α could be stabilized via S-nitrosylation, we tested the hypothesis that the S-nitrosylating agent S-nitrosoglutathione (GSNO) would stabilize HIF-1α, thereby stimulating neurorepair mechanisms and aiding in functional recovery. TBI was induced by controlled cortical impact (CCI) in adult rats. GSNO (0.05mg/kg) was administered at two hours after CCI. The treatment was repeated daily until the 14th day after CCI. Functional recovery was assessed by motor and cognitive functions, and the recovery was compared with the expression of HIF-1α. The mechanisms of GSNO-mediated S-nitrosylation of HIF-1α were determined using brain endothelial cells. While non-treated TBI animals showed sustained neurobehavioral deficits, GSNO treatment of TBI improved neurobehavioral functions. GSNO also increased the expression of HIF-1α and VEGF. The beneficial effects of GSNO on neurobehavioral functions in TBI animals were blocked by treatment with the HIF-1α inhibitor 2-methoxyestradiol (2-ME). The stimulatory effect of GSNO on VEGF was reversed not only by 2-ME but also by the denitrosylating agent dithiothreitol, confirming our hypothesis that GSNO's benefits are mediated by the stabilization of HIF-1α via S-nitrosylation. GSNO's S-nitrosylation of HIF-1α was further confirmed using a biotin switch assay in endothelial cells. The data provide evidence that GSNO treatment of TBI aids functional recovery through stabilizing HIF-1α via S-nitrosylation. GSNO is a natural component of the human brain/body, and its exogenous administration has not shown adverse effects in humans. Therefore, the translational potential of GSNO therapy in TBI is high.

HMG-CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis.

Impaired remyelination due to degeneration of both postmitotic oligodendrocytes and oligodendrocyte progenitors (OPs) is the major hallmark of inflammatory demyelination in multiple sclerosis (MS) lesions and experimental autoimmune encephalomyelitis (EAE). Here, we have demonstrated the potential of lovastatin, a HMG-CoA reductase inhibitor, for the restoration of impaired remyelination mediated through enhanced survival and differentiation of OPs in the spinal cord of treated EAE animals. Lovastatin treatment significantly increased the level of myelin lipids in the spinal cord of treated EAE animals, coinciding with the attenuation of disease severity and inflammatory demyelination as compared with untreated EAE animals. The increased expression of myelin proteins and transcription factors associated with differentiating oligodendrocytes along with the increase in number of NG2+/BrdU- and NG2+/BrdU+ cells, and the expression of proliferating OP-specific proteins, demonstrated the restoration of remyelination in the spinal cord of lovastatin-treated EAE animals. Corresponding to this, in vitro studies further corroborated the increased survival and differentiation of OPs in lovastatin-treated activated mixed glial cells suggesting that lovastatin protects against the degeneration of OPs and enhances their differentiation through induction of a pro-remyelinating environment in the spinal cord of treated EAE animals. Together, these data demonstrate that lovastatin has the potential to augment remyelination in MS lesions and other neuroinflammatory diseases.

Impaired peroxisomal function in the central nervous system with inflammatory disease of experimental autoimmune encephalomyelitis animals and protection by lovastatin treatment.

Peroxisomes are ubiquitous subcellular organelles and abnormality in their biogenesis and specific gene defects leads to fatal demyelinating disorders. We report that neuroinflammatory disease in brain of experimental autoimmune encephalomyelitis (EAE) rats decreased the peroxisomal functions. Degradation of very long chain fatty acids decreased by 47% and resulted in its accumulation (C26:0, 40%). Decreased activity (66% of control) of dihydroxyacetonephosphate acyltransferase (DHAP-AT), first enzyme in plasmalogens biosynthesis, resulted in decreased levels of plasmalogens (16-30%). Catalase activity, a peroxisomal enzyme, was also reduced (37%). Gene microarray analysis of EAE spinal cord showed significant decrease in transcripts encoding peroxisomal proteins including catalase (folds 3.2; p<0.001) and DHAP-AT (folds 2.6; p<0.001). These changes were confirmed by quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis, suggesting that decrease of peroxisomal functions in the central nervous system will have negative consequences for myelin integrity and repair because these lipids are major constituents of myelin. However, lovastatin (a cholesterol lowering and anti-inflammatory drug) administered during EAE induction provided protection against loss/down-regulation of peroxisomal functions. Attenuation of induction of neuroinflammatory mediators by statins in cultured brain cells [J. Clin. Invest. 100 (1997) 2671-2679], and in central nervous system of EAE animals and thus the EAE disease [J. Neurosci. Res. 66 (2001) 155-162] and the studies described here indicate that inflammatory mediators have a marked negative effect on peroxisomal functions and thus on myelin assembly and that these effects can be prevented by treatment with statins. These observations are of importance because statins are presently being tested as therapeutic agents against a number of neuroinflammatory demyelinating diseases.

Combined medication of lovastatin with rolipram suppresses severity of experimental autoimmune encephalomyelitis.

Combinations of new medications or existing therapies are gaining momentum over monotherapy to treat central nervous system (CNS) demyelinating diseases including multiple sclerosis (MS). Recent studies established that statins (HMG-CoA reductase inhibitors) are effective in experimental autoimmune encephalomyelitis (EAE), an MS model and are promising candidates for future MS medication. Another drug, rolipram (phosphodiesterase-4 inhibitor) ameliorates the clinical severity of EAE via induction of various anti-inflammatory and neuroprotective activities. In this study, we tested whether combining the suboptimal doses of these drugs can suppress the severity of EAE. Prophylactic studies revealed that combined treatment with suboptimal doses of statins perform better than their individually administered optimal doses in EAE as evidenced by delayed clinical scores, reduced disease severity, and rapid recovery. Importantly, combination therapy suppressed the progression of disease in an established EAE case via attenuation of inflammation, axonal loss and demyelination. Combination treatment attenuated inflammatory T(H)1 and T(H)17 immune responses and induced T(H)2-biased immunity in the peripheral and CNS as revealed by serological, quantitative, and immunosorbant assay-based analyses. Moreover, the expansion of T regulatory (CD25(+)/Foxp3(+)) cells and self-immune tolerance was apparent in the CNS. These effects of combined drugs were reduced or minimal with either drug alone in this setting. In conclusion, our findings demonstrate that the combination of these drugs suppresses EAE severity and provides neuroprotection thereby suggesting that this pharmacological approach could be a better future therapeutic strategy to treat MS patients.

Lipopolysaccharide-induced peroxisomal dysfunction exacerbates cerebral white matter injury: attenuation by N-acetyl cysteine.

Cerebral white matter injury during prenatal maternal infection characterized as periventricular leukomalacia is the main substrate for cerebral palsy (CP) in premature infants. Previously, we reported that maternal LPS exposure causes oligodendrocyte (OL)-injury/hypomyelination in the developing brain which can be attenuated by an antioxidant agent, N-acetyl cysteine (NAC). Herein, we elucidated the role of peroxisomes in LPS-induced neuroinflammation and cerebral white matter injury. Peroxisomes are important for detoxification of reactive oxidative species (ROS) and metabolism of myelin-lipids in OLs. Maternal LPS exposure induced selective depletion of developing OLs in the fetal brain which was associated with ROS generation, glutathione depletion and peroxisomal dysfunction. Likewise, hypomyelination in the postnatal brain was associated with decrease in peroxisomes and OLs after maternal LPS exposure. Conversely, NAC abolished these LPS-induced effects in the developing brain. CP brains imitated these observed changes in peroxisomal/myelin proteins in the postnatal brain after maternal LPS exposure. In vitro studies revealed that pro-inflammatory cytokines cause OL-injury via peroxisomal dysfunction and ROS generation. NAC or WY14643 (peroxisome proliferators activated receptor (PPAR)-alpha agonist) reverses these effects of pro-inflammatory cytokines in the wild-type OLs, but not in PPAR-alpha(-/-) OLs. Similarly treated B12 oligodenroglial cells co-transfected with PPAR-alpha siRNAs/pTK-PPREx3-Luc, and LPS exposed PPAR-alpha(-/-) pregnant mice treated with NAC or WY14643 further suggested that PPAR-alpha activity mediates NAC-induced protective effects. Collectively, these data provide unprecedented evidence that LPS-induced peroxisomal dysfunction exacerbates cerebral white matter injury and its attenuation by NAC via a PPAR-alpha dependent mechanism expands therapeutic avenues for CP and related demyelinating diseases.

Immunomodulatory effect of combination therapy with lovastatin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside alleviates neurodegeneration in experimental autoimmune encephalomyelitis.

Combination therapy with multiple sclerosis (MS) therapeutics is gaining momentum over monotherapy for improving MS. Lovastatin, an HMG-CoA reductase inhibitor (statin), was immunomodulatory in an experimental autoimmune encephalomyelitis (EAE) model of MS. Lovastatin biases the immune response from Th1 to a protective Th2 response in EAE by a different mechanism than 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, an immunomodulating agent that activates AMP-activated protein kinase. Here we tested these agents in combination in an EAE model of MS. Suboptimal doses of these drugs in combination were additive in efficacy against the induction of EAE; clinical symptoms were delayed and severity and duration of disease was reduced. In the central nervous system, the cellular infiltration and proinflammatory immune response was decreased while the anti-inflammatory immune response was increased. Combination treatment biased the class of elicited myelin basic protein antibodies from IgG2a to IgG1 and IgG2b, suggesting a shift from Th1 to Th2 response. In addition, combination therapy lessened inflammation-associated neurodegeneration in the central nervous system of EAE animals. These effects were absent in EAE animals treated with either drug alone at the same dose. Thus, our data suggest that agents with different mechanisms of action such as lovastatin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, when used in combination, could improve therapy for central nervous system demyelinating diseases and provide a rationale for testing them in MS patients.

IL-4-induced peroxisome proliferator-activated receptor gamma activation inhibits NF-kappaB trans activation in central nervous system (CNS) glial cells and protects oligodendrocyte progenitors under neuroinflammatory disease conditions: implication for CNS-demyelinating diseases.

Th2 phenotype cytokine, IL-4, plays an important role in the regulation of Th1 cell responses and spontaneous remission of inflammatory CNS demyelinating diseases such as multiple sclerosis (MS). In this study we demonstrate IL-4-induced down-regulation of inducible NO synthase (iNOS) expression and survival of differentiating oligodendrocyte progenitors (OPs) in proinflammatory cytokine (Cyt-Mix)-treated CNS glial cells, which is a condition similar to that observed in the brain of a patient with MS. IL-4 treatment of Cyt-Mix-treated CNS glial cells significantly decreased iNOS expression/NO release with a parallel increase in survival of differentiating OPs. IL-4 effects were concentration-dependent and could be reversed by anti-IL-4R Abs. The use of inhibitors for Akt, p38 MAPK, and peroxisome proliferator-activated receptor gamma (PPAR-gamma) antagonist revealed that inhibition of Cyt-Mix-induced iNOS expression and survival of differentiating OPs by IL-4 is via PPAR-gamma activation. There was a coordinate increase in the expression of both PPAR-gamma and its natural ligand-producing enzyme 12/15-lipoxygenase (12/15-LOX) in IL-4-treated cells. Next, EMSA, immunoblots, and transient cotransfection studies with reporter plasmids (pNF-kappaB-Luc and pTK-PPREx3-Luc) and 12/15-LOX small interfering RNA revealed that IL-4-induced PPAR-gamma activation antagonizes NF-kappaB transactivation in Cyt-Mix-treated astrocytes. In support of this finding, similarly treated 12/15-LOX(-/-) CNS glial cells further corroborated the result. Furthermore, there was reversal of IL-4 inductive effects in the brain of LPS-challenged 12/15-LOX(-/-) mice when compared with LPS-challenged wild-type mice. Together, these data for the first time demonstrate the inhibition of Cyt-Mix-induced NF-kappaB transactivation in CNS glial cells by IL-4 via PPAR-gamma activation, hence its implication for the protection of differentiating OPs during MS and other CNS demyelinating diseases.

N-acetylcysteine prevents endotoxin-induced degeneration of oligodendrocyte progenitors and hypomyelination in developing rat brain.

Periventricular leukomalacia (PVL), the dominant form of brain injury in premature infants, is characterized by diffuse white matter injury and is associated with cerebral palsy (CP). Maternal and placental infections are major causes of prematurity and identifiable etiology of PVL and CP. Here we have evaluated the therapeutic efficacy of N-acetylcysteine (NAC), a potent antioxidant and precursor of glutathione, to attenuate lipopolysaccharide (LPS)-induced white matter injury and hypomyelination in the developing rat brain, an animal model of PVL. Intraperitoneal pretreatment of pregnant female rats with NAC (50 mg/kg), 2 hr prior to administration of LPS at embryonic day 18 (E18), attenuated the LPS-induced expression of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1beta, and inducible nitric oxide synthase in fetal rat brains. There were significantly reduced numbers of TUNEL(+) nuclei coimmunostained for platelet-derived growth factor-alphaR(+) [a surface marker for oligodendrocyte progenitor cells (OPCs)] at E20 in the subventricular zone of fetal rat brain in the NAC + LPS group compared with the untreated LPS group. Interestingly, immunostaining for O4 and O1 as markers for late OPCs and immature oligodendrocytes demonstrated fewer O4(+) and O1(+) cells in the LPS group compared with the NAC + LPS and control groups. Consistent with O4(+)/O1(+) cell counts, the expression of myelin proteins such as myelin basic protein, proteolipid protein, and 2'3'-cyclic nucleotide phosphodiesterase, including transcription factors such as MyT1 and Gtx, was less in the LPS group at late postnatal days, indicating severe hypomyelination in the developing rat brain when compared with NAC + LPS and control groups. Collectively, these data support the hypothesis that NAC may provide neuroprotection and attenuate the degeneration of OPCs against LPS evoked inflammatory response and white matter injury in developing rat brain. Moreover, these data suggest the possible use of NAC as a treatment for pregnant women with maternal or placental infection as a means of minimizing the risk of PVL and CP.