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Glial activation and neuroinflammatory processes play an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and HIV dementia. Activated glia cells can secrete various proinflammatory cytokines and neurotoxic mediators, which may influence neuronal cell survival. Recent studies have demonstrated that glia cell-mediated neuroinflammation is also related to the pathophysiology of schizophrenia. In the present study, anti-inflammatory and neuroprotective effects of antipsychotics were investigated using cultured brain cells as a model. The results showed that spiperone significantly decreased the production of nitric oxide in lipopolysaccharide-stimulated BV-2 microglia cells, primary microglia and primary astrocyte cultures. Spiperone also significantly inhibited nitric oxide production in adenosine 5′-triphosphate (ATP)-stimulated primary microglia cultures. Spiperone markedly decreased the production of tumor necrosis factor-alpha in BV-2 microglia cells. Spiperone attenuated the expression of inducible nitric oxide synthase and proinflammatory cytokines such as interleukin-1β and tumor necrosis factor-alpha at mRNA levels in BV-2 microglia cells. Spiperone inhibited nuclear translocation and DNA binding of the p65 subunit of nuclear factor kappa B (NF-κB), inhibitor of kappa B (IκB) degradation, and phosphorylation of p38 mitogen-activated protein kinase in the lipopolysaccharide-stimulated BV-2 microglia cells. Moreover, spiperone was neuroprotective, as the drug reduced microglia-mediated neuroblastoma cell death in the microglia/neuron co-culture. These results imply that the antipsychotic spiperone has anti-inflammatory and neuroprotective effects in the central nervous system by modulating glial activation.
Neuroinflammation is actively involved in the pathogenesis of several neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and HIV-associated dementia (Block et al. 2007). Neuroglia including microglia and astrocytes are non-neuronal cells that play an important role in the homeostatic control of the neuronal extracellular environment (Chen and Swanson 2003). Microglia, which constitute about 10% of all glial cells, participate in the innate immunity in the brain, and they are also considered as the major cell type responsible for inflammation-mediated neurotoxicity (Liu and Hong 2003). Activation of microglia is observed after the exposure to lipopolysaccharide (LPS), interferon (IFN)-γ, β-amyloid or adenosine 5′-triphosphate (ATP). Activated microglia have the capability of secreting proinflammatory cytokines and neurotoxic mediators such as tumor necrosis factor-α (TNF-α), prostaglandin E2, interleukin (IL)-1β, IL-6, and free radicals such as nitric oxide (NO) and superoxide anion (Giulian et al. 1994; Zielasek and Hartung 1996). These proinflammatory cytokines and neurotoxic mediators are thought to contribute to neuronal injury and pathogenesis of the neuroinflammatory diseases (Minghetti and Levi 1998; Gonzalez-Scarano and Baltuch 1999). Over-activation of astrocytes also facilitates ongoing neurodegeneration by producing various neurotoxic factors such as NO and TNF-α (Suk 2005; Farina et al. 2007).
Although the pathological mechanisms of schizophrenia remain unclear, there is growing evidence that neuroinflammation is related to pathophysiology of schizophrenia (Steiner et al. 2006; Kato et al. 2007). Activation or increased number of microglia has been observed in postmortem brain of patients with schizophrenia (Bayer et al. 1999; Radewicz et al. 2000; Steiner et al. 2008). An increased microglia activation was also found in brain of patients with schizophrenia using technique of positron emission tomography with [11C] PK11195 radioligand, which specifically binds to the peripheral benzodiazepine-binding site (van Berckel et al. 2005,Cagnin et al. 2006). Recent studies have suggested that abnormal inflammatory and immune responses are also associated with pathogenesis of schizophrenia (Bernstein et al. 2005; Drzyzga et al. 2006). The beneficial effect of anti-inflammatory drugs celecoxib and minocycline, which are potent inhibitors of microglia activation, has been reported in patients with schizophrenia (Akhondzadeh et al. 2007; Miyaoka et al. 2007). Taken together, as inflammatory activation of microglia is actively involved in the pathogenesis of several neurodegenerative diseases and psychiatric disorders, inhibition of glial activation and subsequent neuroinflammation may be an effective therapeutic approach against these diseases.
Antipsychotics are broadly divided into two groups, the typical or first-generation antipsychotics and the atypical or second-generation antipsychotics. Haloperidol, chlorpromazine, and spiperone belong to the typical antipsychotics, while clozapine, rimcazole, and olanzapine belong to the atypical antipsychotics. Almost all antipsychotics have a property that antagonizes dopamine D2 receptor and subsequently blocks dopamine pathway in brain (Ereshefsky 1999). Recently, several studies have demonstrated that antipsychotic drugs have an effect that is related to the modulation of inflammatory responses (Drzyzga et al. 2006). Olanzapine reduced NO production in LPS-stimulated N9 microglia cells (Hou et al. 2006). Atypical antipsychotics such as risperidone, perospirone, ziprasidone, quetiapine, and aripprazole significantly suppressed IFN-γ or LPS-induced NO or TNF-α production in murine 6-3 microglia cells (Kato et al. 2007, 2008; Bian et al. 2008). Flupentixol and trifluperidol reduced the IL-1β, IL-2, TNF-α and NO release in LPS-stimulated mixed glia and microglia cultures (Kowalski et al. 2003, 2004). It has been also reported that chlorpromazine and loxapine reduced LPS-induced IL-1β and IL-2 secretion in rat mixed glia and microglia cultures (Labuzek et al. 2005).
Although previous studies have demonstrated anti-inflammatory effects of antipsychotics in glia, the detailed molecular mechanisms underlying the anti-inflammatory effects are not completely understood. Furthermore, effects of antipsychotics on glial neurotoxicity have not been investigated. Here, the effect of several antipsychotics on the inflammatory activation and neurotoxicity of microglia, as well as astrocytes, has been determined. This study showed that spiperone and olanzapine inhibited LPS-induced production of NO, TNF-α, and their gene expression in microglia. Particularly, a butyrophemone derivative spiperone suppressed NF-κB and p38 MAPK activation in LPS-stimulated microglia cells. The anti-inflammatory effect of spiperone was also found in astrocytes and macrophage. Lastly, spiperone showed neuroprotective effect by attenuating microglia neurotoxicity in a microglia-neuroblastoma co-culture model.
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In the present study, it has been demonstrated that antipsychotics such as spiperone and olanzapine inhibit inflammatory activation of glia cells. Particularly, spiperone significantly reduced NO or TNF-α production in LPS-stimulated microglia cell lines, primary microglia and astrocyte cultures. The RT-PCR analysis showed that spiperone markedly suppressed the iNOS, TNF-α, and IL-1β gene expression at the transcriptional level in BV-2 microglia cells. NF-κB and p38 MAPK pathways were at least partly involved in the anti-inflammatory mechanisms of spiperone in BV-2 cells. In addition, spiperone showed neuroprotective effects by attenuating microglial neurotoxicity in a microglia-neuron co-culture.
Activation of microglia is related to disease progression and pathology in several neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and HIV dementia (Block et al. 2007). Recent studies have reported that microglia activation is also closely related to pathogenesis of schizophrenia (Muller and Ackenheil 1998; Bayer et al. 1999; Radewicz et al. 2000; Steiner et al. 2008). Under the neurodegenerative conditions, microglia are activated and release various inflammatory mediators including proinflammatory cytokines and free radicals such as NO and superoxide anion (Aloisi 2001). Because NO is one of the main proinflammatory mediators and plays an important role in neuroinflammatory diseases, the effects of the antipsychotics on the NO production in LPS-stimulated microglia cells were examined. Spiperone and olanzapine markedly reduced NO production in LPS-simulated microglia cells without a significant cytotoxicity (Fig. 1). TNF-α is another major proinflammatory mediator, which plays an important role in the process of neuroinflammatory diseases (Jongeneel 1995). Our results showed that the spiperone also inhibited TNF-α production in LPS-stimulated BV-2 microglia cells. These results are consistent with previous reports where olanzapine decreased NO and TNF-α production in rat primary microglia or N9 microglia cells (Wilms et al. 2003; Hou et al. 2006). In the subsequent studies that focused on spiperone, a strong inhibitory effect of the drug on the inflammatory gene expression and inflammatory signaling pathways was found. Spiperone, an antagonist of dopamine D2 receptor and serotonin 5-HT1A/5-HT2A receptor, is widely used as a pharmacological tool. Especially, the [3H]spiperone was long used as a radioligand to label the receptors such as dopamine D2 and serotonin 5-HT1A/5-HT2A receptors (Metwally et al. 1998). Now, the present studies indicate a novel role of spiperone as a potent inhibitor of neuroinflammation: spiperone not only inhibits the production and expression of inflammatory mediators in BV-2 microglia cells or primary microglia cells, but it also suppresses glial inflammatory signal transduction pathways. Moreover, spiperone also inhibited NO production in ATP-stimulated primary microglia cells. Recently, it has been reported that spiperone blocks dopamine-induced microglial chemotaxis (Mastroeni et al. 2008). In that report, spiperone has been used to antagonize the microglia-chemotactic action of dopamine in an attempt to explain the selective vulnerability of dopamine neurons in Parkinson’s disease. Although microglia cells expressed dopamine D receptors as determined by RT-PCR (Kato et al. 2008; Mastroeni et al. 2008), anti-inflammatory effects of the high-affinity dopamine D2 receptor partial agonist aripiprazole were apparently not mediated through dopamine D2 receptor in microglia cells (Kato et al. 2008). In that study, a full agonist of dopamine D2 receptor quinpirole did not show anti-inflammatory effects, while aripiprazole was anti-inflammatory by inhibiting IFN-γ induced elevation of [Ca2+] in microglia. Moreover, in the current study, not all D2 receptor antagonists showed anti-inflammatory effects. For example, haloperidol and chlorpromazine did not suppress NO production in LPS-stimulated microglia at non-toxic concentration (Fig. 1). Thus, it is suggested that spiperone exerts its anti-inflammatory effects on glia in a dopamine receptor-independent manner.
NF-κB is one of the major transcription factors that are activated by inflammatory signal transduction pathways. NF-κB plays a critical role in the expression of proinflammatory cytokines and enzymes including iNOS, IL-1β, and TNF-α, which are responsible for the innate and adaptive immune responses (Baeuerle and Henkel 1994). The molecular mechanisms of NF-κB activation have been well studied, and they involve a cascade activation of cytoplasmic proteins and the ultimate nuclear translocation and DNA binding of p65 subunit of NF-κB (Delhase et al. 2000; Karin and Ben-Neriah 2000). In the cytoplasm, NF-κB is bound and controlled by its inhibitory subunit, IκB. Inflammatory signals such as LPS stimulate the nuclear translocation and DNA binding of p65 subunit of NF-κB through IκB degradation. In the present study, it was found that spiperone inhibited IκB degradation and the subsequent nuclear translocation and DNA binding of p65 in BV-2 microglia cells (Fig. 5). Additionally, it was found that spiperone inhibited LPS-induced activation of p38 MAPK in BV-2 cells (Fig. 5d), which has been previously implicated in the signal transduction pathways responsible for the induction of iNOS and TNF-α gene expression in glia cells or macrophages (Bhat et al. 1998; Koistinaho and Koistinaho 2002). Taken together, our results indicated that NF-κB and p38 MAPK pathways might be involved in the suppressive effects of spiperone on the gene expression of iNOS, TNF-α, and IL-1β in BV-2 microglia cells.
Glial activation has both destructive and beneficial effects on neuronal survival (Block et al. 2007). Over-activation of glia, however, may contribute to neurodegenerative processes through the production of various neurotoxic factors including free radicals and proinflammatory cytokines (Klegeris et al. 2007). In fact, a number of anti-inflammatory agents, which inhibited glial activation or production of proinflammatory mediators, attenuated neuronal degeneration. Thus, it is suggested that a search for the efficient anti-inflammatory compounds that prevent glial activation may lead to an effective therapeutic approach against many inflammation-mediated neurodegenerative conditions. The current study showed that spiperone protected neuroblastoma cells against microglial toxicity in a microglia/neuron co-culture (Fig. 6). The neuroprotective effect of spiperone is likely because of the inhibition of microglia activation, but not a protective action on the neuroblastoma cells, because the spiperone-pretreated microglia cultures were washed before the addition of the neuroblastoma cells for the co-culture: the B35 neuroblastoma cells had not been exposed to spiperone. Although the co-culture of the LPS-stimulated microglia with neuroblastoma cell line may not be the same as the in vivo conditions, it partially reflects the pathological condition where activated microglia influence the survival of neuronal cells in neurodegenerative diseases. Further studies are, however, required to evaluate a neuroprotective property of the spiperone in the animal models of neurodegenerative diseases and to understand the precise molecular mechanisms of anti-inflammatory actions of the antipsychotics in vitro as well as in vivo. Nevertheless, the present study suggests the protective effects of the antipsychotics, spiperone in particular, against inflammation-mediated neurodegeneration. Future works along this line will clarify the detailed molecular mechanisms underlying the anti-inflammatory effects of spiperone on glia cells.