Kir6.1/K-ATP Channel Modulates Microglial Phenotype:Implication In Parkinson’s Disease

Parkinson’s disease (PD) is the second most common age-related neurodegenerative diseases in the world, the clinical symptoms of which include tremor, bradykinesia, and rigidity. Considering the illness progress chronically, PD seriously affects the physical and mental health of the elderly, impairing their quality of life. Recent studies point out that the pathogenesis of PD may include:aging, environmental factors, mitochondrial dysfunction, oxidative stress and inflammation, etc. This complex factors become the restriction to breakthroughs of PD clinical therapeutic drugs. Levodopa (L-DOPA) replacement therapy, the golden standard for symptomatic therapy, does not retard the progressive of dopaminergic neurons loss. Besides, long-term administration leads to the serious complications like dyskinesia. Thus, searching key targets that improve the survival of dopaminergic neurons becomes one of the most important goal in PD study.The typical pathological characteristics of PD include progressive degeneration of DA neuron, Lewy bodies’ deposition and microglial excessive activation in substantia nigra pars compacta (SNpc). Microglia activation is mainly divided into classical activated M1 phenotype and alternative activated M2 phenotype. M1phenotype microglia produce amounts of cytokines causing inflammation, subsequently leading to the degeneration of the DA neurons. M2 phenotype microglia mainly secrete anti-inflammatory or nutritional factors promoting tissue repair, which is crucial to maintain the homeostasis of central nervous sysytem. Microglial phenotypes become various with the progress of PD. Therefore, it is a promising strategy for the treatment of PD via the key targets that can promote microglia polarization from neurotoxicity M1 phenotype to neuroprotective M2 phenotype.ATP-sensitive potassium channels (K-ATP), presented in many kinds of tissues with a variety of functions, provide a unique link between electrical excitability and cellular energetics. Our lab demonstrated that Kir6.1 and SUR2 constitute K-ATP channel in microglia, opening of which protects DA neurons in rotenone rat model by inhibiting microglia-mediated neuroinflammation. All these data indicate that Kir6.1/K-ATP channel in microglia maybe a new target of PD threapy for the modulation of microglial polarization.Therefore, in the first part of our work, we used several kinds of Kir6.1 mice to investigate the regulation of Kir6.1/K-ATP channel in MPTP mouse model of PD. Our results revealed that Kir6.1/K-ATP channel in microglia plays an important role in PD. In the second part, we focused on the role of Kir6.1/K-ATP channel in microglia polarization. The results showed that Kir6.1/K-ATP channel regulates microglia polarization by mediating p38 MAPK and NF-κB signaling. Finally, we explored the roles and the involved mechanisms of Kir6.1/K-ATP channel in LPS mouse model of PD. The conclusion is that Kir6.1/K-ATP channel is involved in PD through modulating microglial phenotype by mediating p38 MAPK signaling.Part I Role of Kir6.1/K-ATP channel in neural injury and glial activation in MPTP mouse model of PDAIM:To investigate the effect of Kir6.1/K-ATP channel on MPTP-induced neural injury using Kir6.1 heterozygote mice, astrocytic Kir6.1 knockout mice and microglial Kir6.1 knockout mice.METHODS:Kir6.1+/+and Kir6.1+/- mice were treated with MPTP intoxication protocol:20 mg/kg MPTP in saline was injected subcutaneously, and 250 mg/kg probenecid in DMSO was injected intraperitoneally every day for 5 consecutive days. Immunohistochemistry was taken for TH, GFAP and IBA-1 expression. Quantitative real-time PCR was used to analyze the mRNA level of cytokines such as TNF-α, IL-1β, iNOS, CCL3, Arg-1, CD206 and YM-1. We constructed astrocytic Kir6.1 knockout mice and microglia Kir6.1 knockout mice using Cre-LoxP system. Quantitative PCR was applied for genes identification. Immunohistochemistry was used to evaluate the expression of TH, GFAP and IBA-1 in MPTP model of astrocytic Kir6.1 knockout mice. Double-labeled immunohistofluoresence was used to evaluate the expression of TH and IBA-1 in MPTP model of microglia Kir6.1 knockout mice.RESULTS:1) There was no significant difference of TH neurons in SNpc in saline group between Kir6.1+/+ and Kir6.1+/- mice. Compared with Kir6.1+/+ mice, Kir6.1+/- mice showed increased TH neurons loss in response to MPTP treatment.2) Kir6.1 knockdown aggravated SNpc glial activation and proliferation in response to MPTP treatment, including astrocytes and microglia.3) The increase of TNF-a, IL-1β, iNOS, CCL3 mRNA levels and the decrease of Arg-1, CD206, YM-1 mRNA levels alter more fiercely in Kir6.1+/- mice after MPTP treatment.4) There was no significant difference of TH neurons and glial activation in SNpc neither in saline group nor in MPTP group between Kir6.1loxp/loxP and Kir6.1loxp/loxpGFAPCre/+mice. 5) Microglia Kir6.1 deficiency exacerbated TH neurons loss and microglia activation in response to MPTP treatment.CONCLUSIONS:1) Kir6.1 knockdown aggravates microglial polarization from M2 to M1 induced by MPTP, leading to severer neural injury.2) Astrocytic Kir6.1 deficiency has no significant effect on the neural injury in MPTP mouse model.3) Kir6.1/K-ATP channel in microglia plays an important role in PD.Part Ⅱ Modulation of Kir6.1/K-ATP channel in microglia polarizationAIM:To observe the effects of Kir6.1/K-ATP channel on microglia polarization to Ml phenotype induced by LPS plus IFN-y or M2 phenotype induced by IL-4 on BV2 cells.METHODS:After transfecting BV2 with Kir6.1 siRNA or pcDNA3.1(-)-Kir6.1 plasmids, we used western blotting to detect Kir6.1 expression in microglia. After regulating Kir6.1 expression, we stimulated BV2 with LPS (100 ng/ml) plus IFN-y (20 ng/ml). RT-PCR and Elisa were used to measure the mRNA levels and secretion of cytokines such as TNF-a, IL-1β and IL-6. Immunocytofluoresence and flow cytometry were used to analyze microglia polarization to M1 phenotype. Western blotting was applied to detect expression of MAPK and NF-κB signaling-related proteins. After regulating Kir6.1 expression, we stimulated BV2 with IL-4 (20 ng/mL). RT-PCR was used to measure the mRNA levels of Arg-1, CD-206 and YM-1. ELISA was used to measure the secretion of TGF-β and IL-10. Immunocytofluoresence and flow cytometry were used to analyze microglia polarization to M2 phenotype. Western blot was applied to detect phosphorylation level of Jakl and Stat6 protein.RESULTS:1) Kir6.1 down-regulation exacerbated increased cytokines levels such as TNF-α, IL-1β and iNOS induced by LPS plus IFN-γ, which was consistence with Kir6.1 over-expression.2) Microglia polarization to Ml phenotype induced by LPS plus IFN-γ was promoted by Kir6.1 down-regulation but reversed by Kir6.1 over-expression.3) Kir6.1 down-regulation inhibited increased Arg-1, CD206 and YM-1 mRNA levels induced by IL-4, which was consistence with Kir6.1 over-expression.4) The phosphorylation level of p38 induced by LPS plus IFN-γ was exacerbated by Kir6.1 down-regulation but attenuated by Kir6.1 over-expression. Neither down-regulating nor up-regulating Kir6.1 affected phosphorylation level of JNK and ERK.5) NF-κB activation induced by LPS plus IFN-y was aggravated by Kir6.1 down-regulation and attenuated by over-expression.6) Kir6.1 down-regulation exacerbated increased phosphorylation level of Statl induced by LPS plus IFN-γ, but Kir6.1 over-expression inhibited.7) Kir6.1 down-regulation inhibited increased phosphorylation level of Jakl and Stat6 induced by IL-4, which was consistence with Kir6.1 over-expression.CONCLUSIONS:1) Kir6.1/K-ATP channel mediates microglia polarization. 2) Kir6.1/K-ATP channel is involved in signaling related with microglia polarization, such as p38 MAPK, NF-κB and Jak-Stat etc.Part III Effect of Kir6.1-mediated microglial polarization on neural injury in PDAIM:To clarify the mechanism that Kir6.1/K-ATP channel modulate microglia polarization in PD.METHODS:After down-regulating Kir6.1 with siRNA, we treated BV2 with SB203580 (10μM) for 1h, following treatment with LPS (100 ng/ml) plus IFN-γ (20 ng/ml). RT-PCR and ELISA were used to measure the mRNA levels and secretion of cytokines such as TNF-a, IL-1β and IL-6. Immunocytofluoresence was used to analyze microglia polarization to M1 phenotype. Supernatant were collected to stimulate SHSY5Y neurons. Western blot was applied to detect apoptosis-related proteins such as Bax, Bcl-2 and caspase-3. In vivo, after injecting LPS (5μg) into substantia nigra, we injected SB203580 (5 mg/kg) intraperitoneally every day for 7 consecutive days in Kir6.1+/+ and Kir6.1+/- mice. Western blot was used to measure the expression of p38 MAPK, NF-κB signaling-related proteins and IL-1β, CD206 protein levels. Immunohistochemistry was taken for detecting microglia activation and TH neurons loss.RESULTS:1) SB203580 inhibited LPS plus IFN-y-induced increase of p38 phosphorylation, NF-κB activation and cytokines such as TNF-a and IL-1β, causing decrease microglia polarization to M1 phenotype.2) It was attenuated by SB203580 that down-regulating Kir6.1 increased CM-induced neurons apoptosis.3) Kir6.1 knockdown aggravated LPS-induced increase of p38 phosphorylation, NF-κB activation, IL-1β level and decrease of CD206 level.4) Kir6.1 knockdown exacerbated microglia activation and TH neurons loss induced by LPS, which is alleviated by SB203580.CONCLUSIONS:1) SB203580 counteractes the effect of Kir6.1 down-regulation on M1 polarization, playing a neuroprotective role.2) Kir6.1 knockdown aggravates LPS-induced microglia polarization from M2 phenotype to M1 phenotype in midbrain.3) Kir6.1 knockdown exacerbates neural injury in LPS intoxication PD model, which is counteraed by SB203580.The major contributions of the present study lie in:1. Kir6.1/K-ATP channel in microglia played an important role in PD. Kir6.1 knockdown exacerbated microgliosis induced by MPTP or LPS, leading to severer neural injury in PD mouse model. Astrocytic Kir6.1 deficiency had no significant effect on the neural injury in MPTP mouse model. Microglia Kir6.1 deficiency aggravated neural injury induced by MPTP. These results indicate that Kir6.1/K-ATP channel in microglia is one of key targets in PD, which expands the study of Kir6.1/K-ATP channel in PD.2. Kir6.1/K-ATP channel mediated microglia polarization. Kir6.1 knockdown aggravated microglia polarization from M2 to M1 phenotype induced by MPTP or LPS, leading to severer neural injury in midbrain. Kir6.1 down-regulation exacerbated microglia polarization to M1 phenotype induced by LPS plus IFN-y and inhibited to M2 phenotype induced by IL-4, which was consistence with Kir6.1 over-expression. These data suggest that Kir6.1/K-ATP channel is one important target in regulating microglia polarization, providing us a novel target to develop neuroprotective agents modulating microglia functions.3. Kir6.1/K-ATP channel mediated microglia polarization via modulating p38 MAPK signaling. SB203580 counteractes the effect of Kir6.1 down-regulation on M1 polarization, playing a neuroprotective role. Kir6.1 knockdown exacerbates neural injury in LPS intoxication PD model, which is counteraed by SB203580. These results provide us with promising experimental evidence in the application of microglia modulating-drugs for PD therapy.