The Protective Effect Of Rosiglitazone On Dopaminergic Neurons Through The Inhibition Of Microglia Activation

Parkinson's disease (PD) is a common neurodegenerative disorder among the old people, which affects approximately1-3%population. PD was originally described by James Parkinson in1817and its etiology is still unknown up to date. PD is characterized by clinical symptoms, namely bradykinesia, resting tremor, rigidity and postural instability. The primary pathological changes of the disease are the progressive loss of dopaminergic neurons within the substantia nigra pars compacta and the reduction of dopamine in the striatum. So far, researchers proposed that heredity, aging, environment, oxidative stress, mitochondrial dysfunction and apoptosis were probably of the main mechanisms. Recently, extensive evidences suggest the involvement of microglia in the degenerative process of PD.Microglia, the resident immune cells in the central nervous system (CNS), are considered to be the major cell type underlying the inflammatory process. Although microglia have beneficial effects in protective immunity and tissue repair, uncontrolled microglia activation has been implicated in contributing to host cell damage. In response to the pathological stimuli, microglia readily become activated and release various neurotoxic mediators, such as TNF-α, interleukin-1β (IL-1β), prostaglandin E2(PGE2), nitric oxide (NO) and reactive oxygen species (ROS), which work in concert to trigger neurodegeneration. The substantia nigra has an extremely high density of microglia which readily leads to intensive inflammation upon pathological stimuli. Since McGeer et al. described a large number of activated microglia in the substantia nigra of PD patient brains in1988, increasing studies have demonstrated that activated microglia may be associated with the development of PD and inhibition of microglia activation can protect dopaminergic neurons. On this basis, researchers have extensively searched for new agents to inhibit microglia activation for therapeutic intervention against neurodegeneration in PD.Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily which regulates transcription of distinct genes through heterodimerization with the retinoid X receptors (RXR). The receptor has been proven to play a critical role in glucose and lipid metabolism. In addition to adipocytes, cells of the monocyte/macrophage lineage also express PPARγ, suggesting the involvement of PPARγ in the function of these cells. Recently, PPARγ ligands have been reported to inhibit the expression of proinflammatory molecules from microglia in several in vitro and in vivo models of neurodegenerative diseases.Thiadiazolidinones (TZDs) are synthetic PPARγ ligands. They are small heterocyclic compounds with favorable pharmaceutic properties, such as oral bioavailability. Recently, it has been shown that TZDs have potent anti-inflammatory effects, such as inhibition of macrophage lineage activation, along with suppressing the expressions of TNF-α, IL-1β, NO and cyclooxygenase type2(COX-2). In the CNS, TZDs have similar anti-inflammatory effects on microglia. More recently, since several studies have shown that pioglitazone, a member of TZDs, exhibits a neuroprotective effect in various models of neuroinflammation, it is hypothesized that the TZDs could be used as a novel treatment approach to reduce the neuroinflammation in PD. Therefore, we sought to investigate the effect of rosiglitazone on microglia activated by lipopolysaccharide (LPS) and the protective effect on dopaminergic neurons. Along with this, we also examined its molecular mechanism regulating microglial production of inflammtory mediators such as TNF-α and NO. All the experiments were divided into3parts as following:Part1:Rosiglitazone protects dopaminergic neurons in primary cell cultureObjective: To study the protective effect of rosiglitazone on dopaminergic neurons through the inhibition of microglia activation in primary ventral mesencephalic cultures.Methods:1. Microglia were prepared from whole brains of1～2day-old Wistar rat pups. The enriched microglia were>98%in purity, as determined by immunostaining for microglia specific marker (OX-42). To establish an in vitro PD model, microglia were co-cultured with primary mesencephalic neuron-enriched cultures and then treated with LPS (1μg/ml) for72h. Dopaminergic neurons were detected with the anti-TH antibody and counted to assess the viability upon the insult of LPS.2. The above in vitro PD model was treated with rosiglitazone at indicated concentrations and with the same amount of vehicle as controls. To assess the protective effect of rosiglitazone on dopaminergic neurons through the inhibition of microglia activation, the dopaminergic neurons and activated microglia were stained with their respective antibodies and counted. Microglia activation was determined by the obvious morphological changes with the staining of the OX-42, including an irregular shape with an expanded transparent extension from the soma and intensified OX-42staining.3. To explore the effect of rosiglitazone on the release of inflammatory mediators in microglia, the levels of TNF-α, NO and Superoxide in the supernatants of microglia culture were measured with different detection kits according to the manufacture's instructions.Results:1. After48h of LPS exposure,70%～80%of the microglia were activated. The generation of TNF-α, NO and Superoxide are significantly increased in microglia-enriched cultures after LPS treatment for24h.2. In neuron-enriched cultures treated with LPS, no dopaminergic neuronal loss was found. In contrast, in neuron-microglia co-culture, LPS (1μg/ml) induced～45%loss of dopaminergic neurons after a72h treatment. Pretreatment with rosiglitazone (50μM)1h prior to LPS treatment significantly increased the viability of dopaminergic neuron compared with that LPS alone. These indicated that the loss of dopaminergic neuron was associated with the microglia activation.3. Rosiglitazone significantly decreased the number of activated microglia and the subsequent release of TNF-a, NO and Superoxide.Conclusion:Activated microglia secrete several neurotoxic mediators which have detrimental effects on dopaminergic neurons. Rosiglitazone protects dopaminergic neurons against LPS-induced neurotoxicity through the inhibition of microglia activation and subsequent release of inflammatory mediators.Part2:Neurotoxic mechanisms of inflammatory mediators in dopaminergic neurons and rosiglitazone-mediated reduction of the neurotoxicityObjective:1. To study the mechanisms that inflammatory mediators are toxic to dopaminergic neurons.2. To explore whether rosiglitazone can protect dopaminergic neurons through reducing the release of inflammatory mediators.Methods:1. The supernatant of BV-2cells treated with LPS was used as inflammation-conditioned media and the level of TNF-a, NO and Superoxide was measured with ELISA, Griess and Superoxide assay kits.2. The viability of MN9D cell cultured in the inflammation-conditioned media was measured by MTT and Trypan Blue staining. To clarify the toxicity of inflammatory mediators secreted by activated BV-2cells, the amount of MDA (an indicator of oxidative stress) in MN9D cells was measured by colorimetry and the mitochondrial membrane potential were measured with JC-1mitochondrial membrane potential assay kit. The apoptosis of BV-2cell was detected with TUNEL and Hoechst33258staining.3. After the administration of rosiglitazone in BV-2cells, viability, level of MDA, mitochondrial membrane potential and apoptosis of MN9D cells were measured with same methods.Results:1. Stimulation of BV-2cells with LPS led to a significant increase in the TNF-a, NO and Superoxide levels although the morphology of BV-2cells had no obvious changes upon LPS exposure.2. Mitochondrial membrane potential was greatly decreased and the amount of MDA was obviously increased when MN9D cells were cultured in inflammation-conditioned media. These indicated that MN9D cells underwent oxidative stress and mitochondrial dysfunction. Consistent with the above results, survival of MN9D cells cultured in inflammation-conditioned media was greatly decreased, and the apoptosis of MN9D cells was significantly increased.3. After the administration of rosiglitazone in BV-2cells, the viability of MN9D cells was obviously increased, mitochondrial membrane potential was partly restored, the amount of MDA was greatly decreased and the apoptosis was significantly decreased, when compared with that in MN9D cells cultured in inflammation-conditioned media, indicating the protective effects of rosiglitazone.Conclusion:MN9D cells cultured in inflammatory conditioned-media exhibit oxidative stress and mitochondrial dysfunction which lead to apoptosis. Rosiglitazone reduces apoptosis in MN9D cells through the attenuation of oxidative stress and restoration of mitochondrial membrane potential. In summary, rosiglitazone protects MN9D cells through the attenuation of oxidative stress and mitochondrial dysfunction which were derived from the insults of inflammatory mediators.Part3:The molecular mechanisms of rosiglitazone-mediated inhibitory effect on microglia activationObjective:To elucidate the mechanism responsible for the inhibitory effect on microglia of rosiglitazone.Methods: 1. To elucidate the mechanism responsible for the inhibitory effect of rosiglitazone on TNF-a and NO production, we examined TNF-a and iNOS mRNA expression levels with RT-PCR.2. To study the influence of rosiglitazone on the inflammatory marker protein, iNOS expression was measured with Western blot.3. To assess whether the inhibitive effect of rosiglitazone on gene expression occurred via blockade of NF-κB activity in BV-2cells, the expression of IKBa and the phosphorylation levels of NF-κB p65was detected by Western blot. The translocation of NF-κB p65from cytoplasm to the nucleus was determined with immunofluorescence analysis.4. To determine whether the repressive effect of rosiglitazone on synthesis and release of inflammatory mediators occurred via MAPK signaling pathway, the expression of p38MAPK, JNK and ERK were measured with Western blot.Results:1. Consistent with the results obtained from the cytokine production assays, LPS-upregulated mRNA levels of TNF-a and iNOS were inhibited by rosiglitazone, suggesting that rosiglitazone negatively regulated the production of TNF-a and NO at the transcriptional level in the LPS-stimulated BV-2cells.2. Rosiglitazone significantly inhibited LPS-induced phosphorylation of NF-κB p65subunit and cytosol-unclear translocation of NF-κB p65in BV-2cells. The results indicated the NF-κB might potentially involved in suppressing TNF-a and NO production by rosiglitazone.3. The signal pathways of p38MAPK, JNK and ERK were activated in BV-2cells upon LPS treatment. Administration of rosiglitazone exhibited inhibitory effects on p38MAPK and JNK activation and no effect on ERK activation. These findings indicated that rosiglitazone inhibited the generation of TNF-a and NO involving the suppression of p38MAPK and JNK signal pathway.Conclusion:Rosiglitazone inhibits LPS-induced gene expression of inflammatory mediators in microglia. In addition, the anti-inflammatory property of rosiglitazone is associated with the inhibition of NF-κB, p38MAPK and JNK activation in LPS-stimulated BV-2cells.SummaryUsing in vitro inflammatory PD model, we systematically studied the inhibitive effect of PPARy agonist rosiglitazone on microglia activation which is involved in the degenerative process of PD. For the first time we demonstrated that rosiglitazone inhibited the activation of microglia through the suppression of NF-κB, p38MAPK and JNK pathway. Additionally, our study simulated the inflammatory insult on MN9D cells with conditioned media from activated BV-2cells and visually showed the inhibitive effect of rosiglitazone on oxidative stress, mitochondrial dysfunction and apoptosis via reducing the release of inflammatory mediators. Our study provided more data to understand the mechanism underlying the inhibition of rosiglitazone on microglia activation and to discuss the potential of TZDs compounds for the treatment of PD.