Ajaib S. Paintlia

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‐α, interleukin‐1β, and inducible nitric oxide synthase in fetal rat brains. There were significantly reduced numbers of TUNEL+ nuclei coimmunostained for platelet‐derived growth factor‐αR+ [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.

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. Paintlia, A. S., Paintlia, M. K., Khan, M., Vollmer, T., Singh, A. K., Singh, I. HMG‐CoA reductase inhibitor augments survival and differentiation of oligodendrocyte progenitors in animal model of multiple sclerosis. FASEB J. 19, 1407–1421 (2005)

Correlation of very long chain fatty acid accumulation and inflammatory disease progression in childhood X-ALD:: implications for potential therapies

This study was designed to understand the role of inflammatory mediators involved in the neurobiology of childhood adrenoleukodystrophy (cALD) by comparing the differential expression of the inflammatory mediators with metabolite very long chain fatty acids that accumulate in this disease. Histopathological examinations indicated extensive demyelination and accumulation of infiltrates in perivascular cuffs in plaque area (PA) and inflammatory area (IA) compared to normal looking area (NLA) of the cALD brain and controls. The PA had excessive accumulation of cholesterol ester (25-30-fold), VLC fatty acids (8-12-fold), and exhaustive depletion of cholesterol (60-70%) and sphingomyelin (50-55%) in comparison to controls. The mRNA expression of cytokines (IL-1alpha, IL-2, IL-3, IL-6, TNF-alpha, and GM-CSF), chemokines (CCL2, -4, -7, -11, -16, -21, -22, CXCL1, CX3CL1, and SDF-2) and iNOS in IA was significantly increased compared to NLA of the cALD and controls determined by gene array, semiquantitative RT-PCR, and immunohistochemistry. These results indicate that accumulation of VLC fatty acid contents in membrane domains associated with signal transduction pathways may trigger the inflammatory process through activation of resident glial cells (microglia and astrocytes) resulting in loss of myelin and oligodendrocytes.

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.

Inhibition of Rho Family Functions by Lovastatin Promotes Myelin Repair in Ameliorating Experimental Autoimmune Encephalomyelitis

Impaired remyelination is critical to neuroinflammation in multiple sclerosis (MS), which causes chronic and relapsing neurological impairments. Recent studies revealed that immunomodulatory activity of statins in an experimental autoimmune encephalomyelitis (EAE) model of MS are via depletion of isoprenoids (farnesyl-pyrophosphate and geranylgeranyl-pyrophosphate) rather than cholesterol in immune cells. In addition, we previously documented that lovastatin impedes demyelination and promotes myelin repair in treated EAE animals. To this end, we revealed the underlying mechanism of lovastatin-induced myelin repair in EAE using in vitro and in vivo approaches. Survival, proliferation (chondroitin sulfate proteoglycan-NG2+ and late oligodendrocyte progenitor marker+), and terminal-differentiation (myelin basic protein+) of OPs was significantly increased in association with induction of a promyelinating milieu by lovastatin in mixed glial cultures stimulated with proinflammatory cytokines. Lovastatin-induced effects were reversed by cotreatment with mevalonolactone or geranylgeranyl-pyrophosphate, but not by farnesyl-pyrophosphate or cholesterol, suggesting that depletion of geranygeranyl-pyrophosphate is more critical than farnesyl-pyrophosphate in glial cells. These effects of lovastatin were mimicked by inhibitors of geranylgeranyl-transferase (geranylgeranyl transferase inhibitor-298) and downstream effectors {i.e., Rho-family functions (C3-exoenzyme) and Rho kinase [Y27632 (N-(4-pyridyl)-4-(1-aminoethyl)cyclohexanecarboxamide dihydrochloride)]} but not by an inhibitor of farnesyl-transferase (farnesyl transferase inhibitor-277). Moreover, activities of Rho/Ras family GTPases were reduced by lovastatin in glial cells. Corresponding with these findings, EAE animals exhibiting demyelination (on peak clinical day; clinical scores ≥3.0) when treated with lovastatin and aforementioned agents validated these in vitro findings. Together, these data provide unprecedented evidence that—like immune cells—geranylgeranyl-pyrophosphate depletion and thus inhibition of Rho family functions in glial cells by lovastatin promotes myelin repair in ameliorating EAE.

IL-4-Induced Peroxisome Proliferator-Activated Receptor γ Activation Inhibits NF-κB 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 γ (PPAR-γ) antagonist revealed that inhibition of Cyt-Mix-induced iNOS expression and survival of differentiating OPs by IL-4 is via PPAR-γ activation. There was a coordinate increase in the expression of both PPAR-γ 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-κB-Luc and pTK-PPREx3-Luc) and 12/15-LOX small interfering RNA revealed that IL-4-induced PPAR-γ activation antagonizes NF-κB 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-κB transactivation in CNS glial cells by IL-4 via PPAR-γ activation, hence its implication for the protection of differentiating OPs during MS and other CNS demyelinating 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.

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

J. Neurochem. (2011) 116, 508–521.

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 ≥ 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 ≥ 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.

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.