The Effects And Therapeutics Of NLRP3 Inflammasome In Neuroinflammation Of Parkinson’s Disease

Parkinson’s disease (PD), the second most common neurodegenerative disease worldwide with a prevalence of 2% among the elderly over 65 years of age, is characterized by the selective loss of dopaminergic (DA) neurons in the substantia nigra and the deposition of Lowy body in the neurons. The cardinal motor symptoms of PD includ resting tremor, bradykinesia, rigidity, and postural instability, which has become a global health concern and brought about a huge burden on economy and society.. Clinically, the primary drug for the treatment of PD remains L-DOPA, which ameliorates the symptoms but fails to reverse the process of DA neuronal degeneration. And long-term use of L-DOPA can also produce a variety of motor complications, which seriously affect the quality of life of PD patients. Although aging, oxidative damage, neuroinflammation, and genetic factors have been recognized to play crucial roles in the pathogenesis of PD in recent decades, the precise etiology of these diseases is still unclear. So far researchers have focused on the chronic activation of neuroinflammation including those mediated by microglia and the resident CNS macrophages which are traditionally recognized to increase the risk of PD.The I kappa B kinase (IKK) β/nuclear factor kappa B (NF-κB) pathway is an important systemic inflammatory mediator, which regulates various inflammatory mediators and acts an important role in inflammatory responses. The most inflammatory reactions are mediated by pro-inflammatory cytokines, which are regulated in part by NF-κB. NF-κB is free and enters the nucleus and binds to the promoter region of genes such as interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-a), IL-6, and IL-18, which are associated with the development of PD. Increased serum peripheral cytokine levels due to systemic inflammation have been associated with AD and PD, suggesting a link between systemic inflammation and neurodegeneration. However, the detailed mechanism underlying the association of peripheral inflammation and PD remains unknown.The upregulation of mature caspase-1 is the premise of the activation of inflammasome. Activated caspase-1 is required for IL-1β and IL-18 release, and plays a key role in inflammation. Caspase-1 is an endogenous cysteine protease synthesized as inactive pro-caspase-1 and activated by dimerization and autoproteolysis within multiprotein complexes including ASC and NLRP3 inflammasome. NLRP3, as the highest expression of inflammasome in macrophages and microglia, which is able to be recognized as a component of the inflammatory process, is one of the best studied inflammasome, so that we focus on the NLRP3 inflammasome in our study. Among these responses, inflammasome has been shown to have important functions in neuroinflammation, yet the precise mechanism of NLRP3 inflammasome action in is not clear.Therefore, In the first part of our study we explored that the effect of GM1 on the MPTP-induced PD rat and determined whether GM1 reduces DA neuronal apoptosis via NLRP3 signaling pathway.In the second part of our study we prepared peripheral inflammation model with lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages to explore its activation on BV2 microglia. It was found that LPS induced the production of IL-1β, IL-6 and TNF-a in the culture medium of RAW264.7 macrophages. The LPS and ATP two-signal model has been involved in the inflammasome activation. After a first signal induced by LPS triggers accumulation of pro-inflammatory cytokines, exposure to high concentrations of ATP acts as a powerful second signal to elicit the processing of the inflammatory response. We further showed that LPS plus ATP activated inflammasome evidenced by the upregulation of caspase-1 and IL-1βp, which could be suppressed by z-YVAD, a caspase-1 inhibitor. Furthermore, the conditioned medium obtained from LPS-treated RAW264.7 macrophages activated BV2 microglia, stimulating the release of IL-1β, IL-6 and TNF-a from BV2 cells. z-YVAD pretreatment markedly suppressed BV2 microglia activation induced by RAW264.7 macrophage conditioned medium. Taken together, our study indicates that macrophage-mediated peripheral inflammation can evoke neuroinflammation and subsequently aggravate neural damage. NLRP3 inflammasome and caspase-1 may be potential targets for modulating systemic inflammatory responses in neurodegenerative diseases.Part I Caspase-1 inhibition attenuates the activation of BV2 microglia induced by LPS-treated RAW264.7 macrophagesAIM:To investigate whether z-YVAD, an caspase-1 inhibitor, protects against the activation of BV2 microglia induced by LPS-treated RAW264.7 macrophages, and explore its underlying mechanisms.METHODS:Murine RAW264.7 macrophages were treated with LPS (200ng/mL) for 1h followed by ATP (5mmol/L) incubation for 0.5h, then we removed the medium and washed 2-3 times with sterile PBS solution. Subsequently, the RAW264.7 cells were treated with the fresh cell medium for 24h. We collected the RAW264.7 cell culture supernatants to induce the activation of BV2 cells for another 24h. RAW264.7 cells and BV2 cells were pretreated with z-YVAD (10μmol/L) for 1h before LPS or condition medium incubation respectively. The contents of IL-6, IL-1β, TNF-a in RAW264.7 and BV2 cells were evaluated with their respective commercially available ELISA kits according to the manufacturer’s instructions. Western blotting was used to analysis the expression of NF-κB pathway and NLRP3 inflammasome. The immunofluorescent staining showed the activation of NF-κB pathway.RESULTS:(1) these results showed that z-YVAD inhibited the synthesis and release of pro-inflammatory cytokines IL-1β, IL-6 and TNF-a in concentrate on-dependent manners in RAW264.7 cells stimulated by LPS and ATP; dual stimulation with LPS and ATP had a synergistic effect on the expression of the NF-κB signaling pathway by western blot analysis and immunofluorescent staining, which was inhibited by z-YVAD. (*p<0.05,**p<0.01 and***p<0.001)(2) these results showed that z-YVAD inhibited the synthesis and release of pro-inflammatory cytokines IL-1β, IL-6 and TNF-a in concentrate on-dependent manners in BV2 cells induced by LPS-treated RAW264.7 macrophages had a synergistic effect on the expression of the NF-κB signaling pathway by western blot analysis and immunofluorescent staining, which was inhibited by z-YVAD. (*p<0.05, **p<0.01 and***p<0.001)CONCLUSION:Our study indicates that z-YVAD protects against BV2 microglia induced by LPS-treated RAW264.7 macrophages and provides an insight into the potential involvement of the NLRP3 inflammasome pathway in the neuroprotection.Part II Neuroprotection of GM1 in MPTP-induced PD rat model and its mechanismAIM:To investigate whether GM1 protects against MPTP-induced dopaminergic (DA) neuronal apoptosis in PD rat model and explore the underlying mechanisms.METHODS:Experimental rats (5 weeks old) were randomly divided into three groups:vehicle, MPTP (Rot) and MPTP+GM1 (Rot+GM1). Day 1-3:Rot+GM1 rats received 25mg/kg/day GM1 under the skin of the back to established rat PD model, the other two grope were treated by the normal saline. Day 4-8:Rot and Rot+ GM1 rats were orally administered 25mg/kg MPTP under the skin of the back, while Rot+GM1 rats still treating 25mg/kg GM1. Four weeks later, the behavioral profile of animals was accessed by catalepsy tests. Immunohistochemistry was performed to detect the staining of tyrosine hydroxylase (TH) and the expression of microglia cells/astrocyte cells in substantia nigra (SN). Western blotting confirmed the expression of NF-κB pathway and NLRP3 inflammasome in SN. ELISA showed expression of IL-1β、IL-6、TNF-aboth in the blood seurum and in SN.RESULTS:(1) Chronic MPTP treatment resulted in a significantly prolonged descent latency compared with the vehicle group (p<0.01), and catalepsy induced by MPTP was significantly antagonized by treatment with GM1 (p<0.05).(2) In MPTP-treated rats, the number of TH-positive neurons and Nissl stained cells in the SNc was reduced significantly (p<0.01). However, the death rate of TH-positive cells and Nissl stained cells in SN of the Rot+GM1 group was reduced compared with the MPTP group (p<0.01). The activated microglia cells and astrocyte cell were also reduced in Rot+GM1 group.(3) Dietary treatment with GM1 abrogated the effect of MPTP on NF-κB pathway levels in SN determined by western blotting. And our data showed that expression levels of NLRP3 inflammasome in MPTP rats were increased significantly compared with vehicle rats, but decreased significantly after subsequent GM1 treatment (*p<0.05,**p<0.01).(4) The expression of IL-iβ、 IL-6、TNF-aboth in the blood seurum and in SN was significantly increased in MPTP-treated rats, when PD rats treated with GM1 displayed a considerable decrease. (*p<0.05,**p<0.01).CONCLUSION:In this study, we clarified that GM1 triggered neuroinflammation and induced DA neuron death in the PD rat model. And GM1 protected dopminergic neuron by reducing neuroinflammation. Additionally, the effect of GM1 may be via inhibiting activation of the NLRP3 signaling pathway.In summary, our present study has the following new concerns:1. GM1 protects against MPTP-induced DA neurons in the rat model of Parkinsonism involving ameliorating neuroinflammation, possibly via inhibiting activation of the NLRP3 signaling pathway, which could provide new insight into clinical therapeutics for PD.2. Caspase-1 inhibition protects against BV2 microglia induced by LPS-treated RAW264.7 macrophages. Although the effect of NLRP3 inflammasome activation on neuronal mechanism is complex, our study provides an insight into the potential involvement of NLRP3 inflammasome pathway in the neuroprotection of dopaminergic cell death.