Antonietta Bernardo

Role of the peroxisome proliferator-activated receptor-γ (PPAR-γ) and its natural ligand 15-deoxy-Δ12,14-prostaglandin J2 in the regulation of microglial functions

The peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is a member of a large group of nuclear receptors controlling the proliferation of peroxisomes that is involved in the downregulation of macrophage functions. Here, we report that PPAR‐γ was constitutively expressed in rat primary microglial cultures and that such expression was downregulated during microglial activation by endotoxin (LPS). The presence of the PPAR‐γ natural ligand 15‐deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) counteracted the repression of PPAR‐γ expression caused by LPS. In microglial cultures stimulated by LPS, interferon‐γ (IFN‐γ) or by their combination, 15d‐PGJ2 reduced the production of nitric oxide (NO) and the expression of inducible NO synthase (iNOS). The inhibitory effect was dose‐dependent and did not involve an elevation of cyclic AMP, a second messenger known to inhibit NOS expression in microglia. In addition, 15d‐PGJ2 down‐regulated other microglial functions, such as tumour necrosis factor‐α (TNF‐α) synthesis and major histocompatibility complex class II (MHC class II) expression. The effects of 15d‐PGJ2 occurred, at least in part, through the repression of two important transcription factors, the signal transducer and activator of transcription 1 and the nuclear factor κB, known to mediate IFN‐γ and LPS cell signalling. Our observations suggest that 15d‐PGJ2, the synthesis of which is likely to occur within the brain, could play an important role in preventing brain damage associated with excessive microglial activation.

PPAR-γ Agonists as Regulators of Microglial Activation and Brain Inflammation

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) belongs to a large group of nuclear receptors controlling reproduction, metabolism, development and immune response. Upon activation by specific agonists, these receptors form dimers and translocate to the nucleus, where they act as agonist-dependent transcription factors and regulate gene expression by binding to specific promoter regions of target genes. The observation that PPAR-gamma is involved in the regulation of macrophage differentiation and activation in the peripheral organs has prompted the investigation of the functional role of PPAR-gamma in microglial cells, the main macrophage population of the CNS. The present review summarizes the several lines of evidence supporting that PPAR-gamma natural and synthetic agonists may control brain inflammation by inhibiting several functions associated to microglial activation, such as the expression of surface antigens and the synthesis of nitric oxide, prostaglandins, inflammatory cytokines and chemokines. Moreover, one of the major natural PPAR-gamma agonist, 15d-prostaglandin J(2) may contribute to the safe elimination of activated microglia by inducing apoptosis. Synthetic PPAR-gamma agonists do not entirely reproduce the range of 15d-prostaglandin J(2) effects, suggesting that PPAR-gamma independent mechanisms are also involved in the action of this prostaglandin. In addition to microglia, PPAR-gamma agonists affect functions and survival of other neural cells, including astrocytes, oligodendrocytes and neurons. Although most of the evidence comes from in vitro observations, an increasing number of studies in animal models further supports the potential therapeutic use of PPAR-gamma agonists in human brain diseases including multiple sclerosis, Parkinsons disease and Alzheimers disease.

Nuclear Factor kB-independent Cytoprotective Pathways Originating at Tumor Necrosis Factor Receptor-associated Factor 2

Most normal and neoplastic cell types are resistant to tumor necrosis factor (TNF) cytotoxicity unless cotreated with protein or RNA synthesis inhibitors, such as cycloheximide and actinomycin D. Cellular resistance to TNF requires TNF receptor-associated factor 2 (TRAF2), which has been hypothesized to act mainly by mediating activation of the transcription factors nuclear factor kB (NFkB) and activator protein 1 (AP1). NFkB was proposed to switch on transcription of yet unidentified anti-apoptotic genes. To test the possible existence of NFkB-independent cytoprotective pathways, we systematically compared selective trans-dominant inhibitors of the NFkB pathway with inhibitors of TRAF2 signaling for their effect on TNF cytotoxicity. Blockade of TRAF2 function(s) by signaling-deficient oligomerization partners or by molecules affecting TRAF2 recruitment to the TNF receptor 1 complex completely abrogated the cytoprotective response. Conversely, sensitization to TNF cytotoxicity induced by a selective NFkB blockade affected only a fraction of TNF-treated cells in an apparently stochastic manner. No cytoprotective role for c-Jun amino-terminal kinases/stress-activated protein kinases (JNKs/SAPKs), which are activated by TRAF2 and contribute to stimulation of activator protein 1 activity, could be demonstrated in the cellular systems tested. Although required for cytoprotection, TRAF2 is not sufficient to protect cells from TNF + cycloheximide cytotoxicity when overexpressed in transfected cells, thus indicating an essential role of additional TNF receptor 1 complex components in the cytoprotective response. Our results indicate that TNF-induced cytoprotection is a complex function requiring the integration of multiple signal transduction pathways.

Peroxisome proliferator-activated receptors (PPARs) and peroxisomes in rat cortical and cerebellar astrocytes

Astrocytes are the most versatile cells of the neural tissue. Numerous astrocytic functions—such as protection from oxidative damage, catabolism of neuroactive D-amino acids acting as neuromodulators, synthesis and catabolism of some lipid molecules, and, possibly, gluconeogenesis—reside in peroxisomes. The expression of several peroxisomal enzymes, particularly those of the acyl-CoA β-oxidation pathway, is regulated by a class of ligand-activated transcription factors, known as peroxisome proliferator-activated receptors (PPARs), acting on their target genes as heterodimers with the retinoid X receptors (RXRs). In this work, primary and secondary cultures of astrocytes from the cerebral cortices and cerebella of neonatal rats (2 and 7 days of postnatal age) were utilized to investigate the expression of peroxisomal enzymes, PPAR and RXR isotypes (α, β and γ), by both biochemical and immunological methods. The results obtained demonstrate that astrocytes in vitro express peroxisomal enzymes, PPARs, and RXRs and that differences dependent on brain area, animal age, and culture time are reminiscent of the in vivo situation. Therefore, primary cultures of astrocytes and, particularly, high purified subcultures may constitute a useful model for further studies aimed to gain further insights into the roles of peroxisomes and PPARs related to lipid and glucose metabolism in these cells.

Regulation of Glial Cell Functions by PPAR-gamma Natural and Synthetic Agonists.

In the recent years, the peroxisome proliferator-activated receptor-γ (PPAR-γ), a well known target for type II diabetes treatment, has received an increasing attention for its therapeutic potential in inflammatory and degenerative brain disorders. PPAR-γ agonists, which include naturally occurring compounds (such as long chain fatty acids and the cyclopentenone prostaglandin 15-deoxy Δ12,14 prostaglandin J2), and synthetic agonists (among which the thiazolidinediones and few nonsteroidal anti-inflammatory drugs) have shown anti-inflammatory and protective effects in several experimental models of Alzheimers and Parkinsons diseases, amyotrophic lateral sclerosis, multiple sclerosis and stroke, as well as in few clinical studies. The pleiotropic effects of PPAR-γ agonists are likely to be mediated by several mechanisms involving anti-inflammatory activities on peripheral immune cells (macrophages and lymphocytes), as well as direct effects on neural cells including cerebral vascular endothelial cells, neurons, and glia. In the present article, we will review the recent findings supporting a major role for PPAR-γ agonists in controlling neuroinflammation and neurodegeneration through their activities on glial cells, with a particular emphasis on microglial cells as major macrophage population of the brain parenchyma and main actors in brain inflammation.

Astrocytes contribute to neuronal impairment in βA toxicity increasing apoptosis in rat hippocampal neurons

Astrocytosis is a common feature of amyloid plaques, the hallmark of Alzheimers disease (AD), along with activated microglia, neurofibrillary tangles, and β‐amyloid (βA) deposition. However, the relationship between astrocytosis and neurodegeneration remains unclear. To assess whether βA‐stimulated astrocytes can damage neurons and contribute to βA neurotoxicity, we studied the effects of βA treatment in astrocytic/neuronal co‐cultures, obtained from rat embryonic brain tissue. We found that in neuronal cultures conditioned by βA‐treated astrocytes, but not directly in contact with βA, the number of apoptotic cells increased, doubling the values of controls. In astrocytes, βA did not cause astrocytic cell death, nor did produce changes in nitric oxide or prostaglandin E2 levels. In contrast, S‐100β expression was remarkably increased. Our data show for the first time that βA–astrocytic interaction produces a detrimental effect on neurons, which may contribute to neurodegeneration in AD. GLIA 34:68–72, 2001.

TNFα downregulates PPARδ expression in oligodendrocyte progenitor cells: Implications for demyelinating diseases

TNFα has been implicated in several demyelinating disorders, including multiple sclerosis (MS) and X‐adrenoleukodystrophy (X‐ALD). TNFα abundance is greatly increased in the areas surrounding damaged regions of the central nervous system of patients with MS and X‐ALD, but its role in the observed demyelination remains to be elucidated. A class of nuclear receptors, the peroxisome proliferator‐activated receptors (PPARs), has been implicated in several physiological and pathological processes. In particular, PPARδ has been shown to promote oligodendrocyte (OL) survival and differentiation and PPARγ has been implicated in inflammation. In the present study, we investigate on the effects of TNFα on OLs during differentiation in vitro. The results obtained show that TNFα treatment impairs PPARδ expression with concomitant decrease of lignocerolyl‐CoA synthase and very‐long‐chain fatty acid β‐oxidation as well as plasmalogen biosynthesis. We propose a hypothetical model possibly explaining the perturbation effects of proinflammatory cytokines on myelin synthesis, maturation, and turnover. GLIA 41:3–14, 2003.

Nuclear receptor peroxisome proliferator-activated receptor-gamma is activated in rat microglial cells by the anti-inflammatory drug HCT1026, a derivative of flurbiprofen.

The peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is constitutively expressed in primary cultures of rat microglia, the main population of brain resident macrophages, and its ligand‐dependent activation leads to the repression of several microglial functions. A few non‐steroidal anti‐inflammatory drugs (NSAIDs), e.g. indomethacin and ibuprofen, show PPAR‐γ agonistic properties. It has been proposed that PPAR‐γ activation contributes to the potential benefits of the long‐term use of certain NSAIDs in delaying the progression of Alzheimers disease (AD). Previous data have shown that the NSAID HCT1026 [2‐fluoro‐α‐methyl(1,1′‐biphenyl)4‐acetic acid‐4‐(nitrooxy)butyl ester], a derivative of flurbiprofen which releases nitric oxide (NO), reduces the number of reactive microglial cells in a variety of models. This evidence together with the chemical analogy with ibuprofen led us to investigate whether flurbiprofen and HCT1026 interact with PPAR‐γ and interfere with microglial activation. We found that a low concentration (1 µm) of HCT1026, but not flurbiprofen, activated PPAR‐γ in primary cultures of rat microglia, with kinetics similar to those of the synthetic agonist ciglitazone. The PPAR‐γ antagonist GW9662 (2‐chloro‐5‐nitrobenzanilide) prevented the activation of PPAR‐γ by HCT1026. Interestingly, unlike other NSAIDs that activate PPAR‐γ at concentrations higher than those required for cyclooxygenase inhibition, HCT1026 activated PPAR‐γ and inhibited prostaglandin E2 synthesis at the same low concentration (1 µm). The results suggest that HCT1026 may exert additional anti‐inflammatory actions through PPAR‐γ activation, allowing a more effective control of microglial activation and brain inflammation.

Synergistic stimulation of MHC class I and IRF-1 gene expression by IFN-gamma and TNF-alpha in oligodendrocytes

In order to understand the molecular basis of the synergistic action of interferon γ (IFN‐γ) and tumour necrosis factor α (TNF‐α) on rat oligodendrocyte development, we studied some aspects of the signalling pathways involved in the regulation of the major histocompatibility complex (MHC) class I and the interferon regulatory factor 1 (IRF‐1) gene expression. Two well‐defined inducible enhancers of the MHC class I gene promoter, the MHC class I regulatory element (MHC‐CRE) and the interferon consensus sequence (ICS), were analysed. Neither IFN‐γ nor TNF‐α was capable of inducing MHC‐CRE binding activity when administrated alone. Following the exposure of oligodendrocytes to IFN‐γ, TNF‐R1 expression was transcriptionally induced by the binding of signal transducer and activator of transcription (STAT‐1) homodimers to the IFN‐γ activated site (GAS) present in the gene promoter. The upregulation of TNF‐R1 allowed TNF‐α to induce the binding of nuclear factor‐κB (NF‐κB) to the MHC‐CRE site. With respect to ICS element, IFN‐γ induced IRF‐1 binding, that was further enhanced upon co‐treatment with TNF‐α. The existence of a synergism between IFN‐γ and TNF‐α in stimulating IRF‐1 expression at the transcriptional level was supported by IRF‐1 promoter analysis: IFN‐γ directly induced the binding of STAT‐1 homodimers to the GAS element, while NF‐κB binding to the κB sequence was activated by TNF‐α only after IFN‐γ treatment. This transcriptional regulation of IRF‐1 gene by IFN‐γ and TNF‐α was confirmed at the mRNA level. The synergism demonstrated in the present study highlights the importance of cytokine interactions in magnifying their biological effects during brain injury and inflammation.

Peroxisome Proliferator-Activated Receptor-γ Agonists Promote Differentiation and Antioxidant Defenses of Oligodendrocyte Progenitor Cells

Several lines of evidence suggest that peroxisome proliferator-activated receptor-&ggr; (PPAR-&ggr;) agonists may control brain inflammation and, therefore, may be useful for the treatment of human CNS inflammatory conditions. The PPAR-&ggr; agonists delay the onset and ameliorate clinical manifestations in animal demyelinating disease models, in which the beneficial effects are thought to be mainly related to anti-inflammatory effects on peripheral and brain immune cells. Direct effects on neurons, oligodendrocytes, and other CNS resident cells cannot be excluded, however. To analyze potential direct actions of PPAR-&ggr; agonists on oligodendrocytes, we investigated the effects of both natural (15-deoxy &Dgr;12,14 prostaglandin J2) and synthetic (pioglitazone) PPAR-&ggr; agonists in primary cultures of rat oligodendrocyte progenitor cells. The PPAR-&ggr; agonists promoted oligodendrocyte progenitor cell differentiation and enhanced their antioxidant defenses by increasing levels of catalase and copper-zinc superoxide dismutase while maintaining the overall homeostasis of the glutathione system. Protective effects were abolished in the presence of the specific PPAR-&ggr; antagonist GW9662, indicating that they are specifically dependent on PPAR-&ggr;. These observations suggest that in addition to their known anti-inflammatory effects, PPAR-&ggr; agonists may protect oligodendrocyte progenitor cells by preserving their integrity and favoring their differentiation into myelin-forming cells. Thus, PPAR-&ggr; may promote recovery from demyelination by direct effects on oligodendrocytes.