Peripheral benzodiazepine receptor ligand PK11195 reduces microglial activation and neuronal death in quinolinic acid-injected rat striatum

Abstract

The effects of the peripheral benzodiazepine receptor (PBR) ligand, PK11195, were investigated in the rat striatum following the administration of quinolinic acid (QUIN). Intrastriatal QUIN injection caused an increase of PBR expression in the lesioned striatum as demonstrated by immunohistochemical analysis. Double immunofluorescent staining indicated PBR was primarily expressed in ED1-immunoreactive microglia but not in GFAP-immunoreactive astrocytes or NeuN-immunoreactive neurons. PK11195 treatment significantly reduced the level of microglial activation and the expression of pro-inflammatory cytokines and iNOS in QUIN-injected striatum. Oxidative-mediated striatal QUIN damage, characterized by increased expression of markers for lipid peroxidation (4-HNE) and oxidative DNA damage (8-OHdG), was significantly diminished by PK11195 administration. Furthermore, intrastriatal injection of PK11195 with QUIN significantly reduced striatal lesions induced by the excitatory amino acid and diminished QUIN-mediated caspase-3 activation in striatal neurons. These results suggest that inflammatory responses from activated microglia are damaging to striatal neurons and pharmacological targeting of PBR in microglia may be an effective strategy in protecting neurons in neurological disorders such as Huntington's disease.

Introduction

Excitotoxic processes contribute to the progressive cell death in Huntington's disease (HD), an inherited neurodegenerative disorder. Studies of brains from HD patients have revealed a relatively selective loss of neurons expressing the glutamate subtype N-methyl-d-aspartate (NMDA) receptor (Young et al., 1988). In HD animal models, intrastriatal injection of NMDA receptor agonists such as quinolinic acid (QUIN) leads to characteristic pathological responses resembling those found in HD brain (Beal et al., 1991, Schwarcz et al., 1983). Recent studies have documented the enhanced vulnerability of striatal neurons to excitotoxic injury in cells expressing mutant huntingtin protein (Li et al., 2003, Zeron et al., 2001) and increased level of quinolinate in neostriatum and cortex of early grade HD (Guidetti et al., 2004).

Microglia, the resident immune responsive cells of the brain, constitute an important source for the production of excitotoxins. Upon activation with inflammatory stimuli, microglia can release high amounts of QUIN (Espey et al., 1997, Heyes et al., 1996) and glutamate (Piani et al., 1992). Elevated levels of QUIN have been reported following brain damage in vivo with immunoreactivity of the excitotoxin localized to microglia (Lehrmann et al., 2001). Results from immunohistochemical analysis have also provided evidence for the progressive accumulation of reactive microglia in affected regions of HD brain (Sapp et al., 2001). At present, however, the specific roles of microglial responses in the pathology of HD are not well understood.

Activated microglia show high expressions of the peripheral benzodiazepine receptor (PBR) located on the outer membrane of mitochondria. PBR have been implicated in a host of cellular functions including regulation of mitochondrial lipid metabolism, apoptosis, cell proliferation, and immune system function (Casellas et al., 2002, Gavish et al., 1999). Importantly, autoradiographic data has established increased PBR density in an excitotoxic rat model of HD (Belloli et al., 2004, Levivier and Przedborski, 1998) and in affected striatum of HD patient brains (Messmer and Reynolds, 1998, Schoemaker et al., 1982). Ligands for the PBR include PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide) and this compound has been shown to possess anti-inflammatory actions in vitro and in vivo. PK11195 inhibited the secretion of pro-inflammatory cytokines (Klegeris et al., 2000) and proliferation of monocytes (Bessler et al., 1992) and reduced the production of NO and TNF-α with lipopolysaccharide (LPS) stimulation of cultured rodent microglia (Wilms et al., 2003). In this laboratory, we have documented PK11195 to block the expression and production of TNF-α and COX-2 induced with LPS treatment of human microglia (Choi et al., 2002). In vivo, PBR-specific ligands including PK11195 have reported therapeutic effects in animal models of inflammatory disorders such as rheumatoid arthritis (Waterfield et al., 1999), carrageenan-induced pleurisy (Torres et al., 2000) and pulmonary inflammation (Bribes et al., 2003). These clinical and experimental data raise the possibility that modulation of PBR may have utility to inhibit neuronal degeneration in HD.

In this study we have used an excitotoxin rat model of HD, with injection of QUIN into rat striatum, to investigate the effects of PK11195 to block microglial inflammatory responses and to confer neuroprotection. The results suggest a wide-spectrum of efficacy for PK11195 including inhibition of microglial-mediated inflammatory responses, resulting in reduction of pro-inflammatory cytokines and products of lipid peroxidation and oxidative DNA damage, and blockade of QUIN-induced caspase-3 activation and degeneration of striatal neurons.