Elsevier

Brain Research

Volume 941, Issues 1–2, 21 June 2002, Pages 1-10
Brain Research

Research report
Effects of peroxisome proliferator-activated receptor agonists on LPS-induced neuronal death in mixed cortical neurons: associated with iNOS and COX-2

https://doi.org/10.1016/S0006-8993(02)02480-0Get rights and content

Abstract

In neurodegenerative disease, the use of non-steroidal anti-inflammatory drugs (NSAIDs) has been regarded as beneficial. The NSAID, an inhibitor of cyclooxygenase (COX), has been also suggested as a ligand of the peroxisome proliferator-activated receptor (PPAR). In cortical neuron–glial co-cultures, we examined the effect of PPAR agonists on lipopolysaccharide(LPS)-induced neuronal death, which has been known to be NO-dependent. LPS induced iNOS expression and the release of nitric oxide in microglia, and COX-2 expression in neurons. PPAR-γ agonists such as 15d-PGJ2, ciglitazone and troglitazone prevented LPS-induced neuronal death and abolished LPS-induced NO and PGE2 release, however PPAR-α agonists such as clofibrate and WY14,643 did not produce the same results. PPAR-γ agonists also reduced LPS-induced iNOS and COX-2 expression, which suggested by interfering with the NF-κB signal pathway.

Introduction

Peroxisomal proliferator-activated receptors (PPARs) consist of a family of three nuclear receptors, (α, β (also referred to as NUC 1), and γ), which heterodimerize with retinoid X receptors and function as a transcriptional regulator of genes linked to lipid metabolism [19]. PPAR-α is found predominantly in the liver, heart and kidney, and PPAR-γ is found primarily in adipose tissue. PPAR-β is almost ubiquitously expressed, but its function is relatively unknown [18], [28]. Recently, it has been suggested that PPAR-α and -γ are important immunomodulatory mediators. Activation of PPAR-γ in monocyte/macrophage and activated microglia inhibits inflammatory mediator and cytokine production [16], [34]. PPAR can also be activated by non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin [21], which might be useful in the prevention or treatment of Alzheimer’s disease (AD).

Nitric oxide (NO) and prostanoid have physiological and pathological roles in mediating the interaction between neurons and glial cells within the central nervous system, as they are known to regulate inflammation, neurotransmission and neural cell survival [4], [29]. LPS-induced neuronal death is closely associated with iNOS/NO-dependent toxicity. Although recent report showed that neurons in degenerative conditions express iNOS [12], [13], microglial cells are the main source of LPS-induced iNOS/NO in neuron–glial culture and in vivo. It has been suggested that microglia-produced NO and reactive nitrogen oxides mediate neuronal cell death in ischemic and neurodegenerative disorders [2]. LPS also induced cyclooxygenase (COX), the key enzyme for prostanoid synthesis, has two isoforms, COX-1 and COX-2. While COX-1 is constitutively expressed in most cell types, COX-2 is rapidly induced by mitogens, cytokines and endotoxins. COX-2 increases in pathological conditions such as seizure, ischemia and inflammation [1]. In addition, altered mRNA expression of COX-2 in the brain with Alzheimer’s disease has been reported [6], [25], [32].

Recently, it has been shown that the intermediate of prostaglandin in the cell, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) acts as a PPAR-γ agonist and results in down-regulation of iNOS and proinflammatory cytokine in macrophage and microglial cells [17], [22], [33]. Heneka et al. [15] reported that 15d-PGJ2 protected NO-mediated cerebellar granule cell death through down-regulation of iNOS expression. In comparison with iNOS, the study of the regulation of COX-2 expression by 15d-PGJ2 is still controversial. 15d-PGJ2 induced COX-2 expression in immortalized epithelial cells [3], but suppressed COX-2 expression in fetal hepatocyte cells [5]. Recently, Koppal et al. [20] reported that 15d-PGJ2 decreased the production of COX-2 as well as iNOS in LPS-stimulated BV-2 microglial cells, and Subbaramaiah et al. [38] reported PPAR-γ ligands such as ciglitazone, troglitazone and 15d-PGJ2 suppressed the transcriptional activation of COX-2 in human epithelial cells. Thus, expression of iNOS and COX-2 through PPAR agonists might to be differently regulated in different cell types.

In the present study, we investigated the effects of PPAR-α or PPAR-γ agonists on lipopolysaccharide (LPS)-induced neuronal cell death from cortical neuron–glial culture, and then on the regulation of iNOS and COX-2 expression associated with LPS-induced neuronal death.

Section snippets

Materials

Lipopolysaccharide (Escherichia coli, 0127:B8), cytosine–β-arabinofuranoside (ARA-C), d-glucose, Triton X-100, trypsin, Nonidet P40 and phenylmethylsufonyl fluoride were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS), equine serum and glutamine were from Gibco-BRL (Gaithersburg, MD, USA). 15-Deoxy-Δ12,14-prostaglandin J2, Ciglitazone, NS398 and PGA1 were from Biomol (Plymouth Meeting, PA, USA). WY14,643 was obtained from Cayman (Ann Arbor, MI, USA). iNOS and COX-2

LPS-induced neuronal death associated with iNOS and COX-2

In mixed cortical neuron–glial coculture, LPS induced NO and PGE2 release, and also COX-2 and iNOS gene expression. To determine the source of COX-2 and iNOS, we used immunohistochemistry with antibodies of iNOS and COX-2. As a result, we ascertained that the iNOS was expressed in the microglia, and that COX-2 was mainly in the neurons. We also identified the neuron and microglia using immunocytochemisty with CD11b, microglia specific antibody and NeuN, neuronal specific antibody (Fig. 1). LPS

Discussion

In the present study, we examined the effects of PPAR isoform agonists on lipopolysaccharide (LPS)-induced cortical neuronal death. We found that PPAR-γ agonist prevented LPS-induced neuronal death and abolished the expression of iNOS and COX-2; however, PPAR-α agonists did not produce the same results. We also found that PPAR-γ agonists could inhibit iNOS and COX-2 expression, which may be accomplished by interfering with NF-κB activation. These results suggest that PPAR-γ activation can

Acknowledgments

This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the Brain Disease Research Center at Ajou University.

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