The Role Of LRRK2 In Manganese Neurotoxicity And Regulatory Mechanism

BackgroundManganese is one of the essential trace elements in the human body.It is involved in the synthesis of proteins and lipids and is a cofactor for many enzyme proteins.Under normal conditions,people can consume enough manganese from their diet.At the same time,manganese is an important industrial raw material.Over-aggregation of manganese in the human body can lead to manganese poisoning.Manganese neurotoxicity induces damage to dopaminergic neurons in the substantia nigra,causing clinical symptoms of Parkinson's disease.Activation of microglia plays an important role in the toxicity of manganese.Previous studies have shown that the neurotoxicity of manganese may not directly affect neuronal cells,but act on microglia to induce neurons through neuroinflammation;The inhibition of microglial activation partly relieves the neurotoxic effect of manganese,but the detailed mechanism is not very clear.Therefore,early detection of the key molecules regulating microglia function may open up new areas in the study of manganese toxicity.LRRK2 is a leucine kinase with multiple domain motifs and mainly exists in cytoplasm and mitochondrial membrane.LRRK2 mutation has been proved to play an important role in neurological diseases.Recent studies have found that LRRK2 is expressed in microglia and has higher expression levels than neurons,and inhibition of LRRK2 can inhibit the activation of microglia.Previous gene chip results showed that Mn could induce the alteration of LRRK2 in vivo,but the underlying mechanism is unknown.Our project tried to find the role of LRRK2 in Mn-induced neurotoxicity and neuroinflammation,which may supply new theoretical basis for Mn toxicity.Autophagy plays an important role in maintaining normal cell activity,and autophagy is involved in a variety of physiological processes.The phagocytosis of microglia is also regulated by autophagy.Autophagy plays an important role in maintaining normal cell activity,and autophagy is involved in many physiological processes.LRRK2 also plays a key role in regulating autophagy function.It was found that LRRK2 was present on autophagosome vesicles on the activated microglia.However,its role in manganese-induced microglial activation remains unclear,and whether manganese exposure affects microglial autophagy by regulating LRRK2 is unclear.So finding a key link in regulating autophagy may provide a new theoretical basis for the treatment and protection of manganese toxicity.AimsThis study intends to establish an in vitro and in vivo Mn exposure model to verify the effects of manganese exposure on the activation of microglia andneuroinflammation and the effect on LRRK2 expression.After determining the effect of Mn exposure on LRRK2 expression,the role of LRRK2 in Mn neurotox icity was confirmed by intervention of LRRK2 expression.By detecting the changes in autophagy and related signal transduction,our project tried to explore the mechanism of LRRK2 activation induced by manganese exposure,and provide new ideas and theoretical basis for prevention and treatment of manganese toxicity.Methods1.Model1)Animal ModelThe manganese exposure mouse models were divided into three groups:control group?0.9%NaCl?,manganese exposure group?100 mg/kg MnCl₂?and LPS positive control group?10 mg/ml LPS?.Mn and LPS were administrated 3 times by subcutaneous injection every other day.2)Cell model:In BV2 microglial cells,the concentration of manganese was 100?mol/L.LPS concentration is 1?g/ml,the stimulation time is 24 h.Atomic absorption spectrophotometry was used to detect the blood manganese and brain manganese content in mice.Behavioral experiments were to verify the effect of manganese exposure on exercise performance;Immunohistochemical staining and Western blot were used to detect the changes of TH protein after manganese exposure.Immunohistochemical staining,Western blot,qPCR,and ELISA were used to detect microglia morphology and expression of inflammatory factors after manganese exposure in vitro and in vivo.2.Immunofluorescence staining,Western blot and qPCR techniques were used to detect the expression of LRRK2 in vivo and in vitro exposure models.The inhibitory effects of LRRK2 on the expression of inflammatory cytokines in microglia induced by LRRK2 were observed using siRNA interference technology,inhibitors,and adenovirus assays.The knock-down LRRK2 virus was injected through brain stereotaxic and the changes of exercise ability and related proteins in mice exposed to manganese were observed.3.Immunofluorescence staining,transmission electron microscopy,Western blot and qPCR were used to detect the expression of autophagy-related molecules in vivo and in vitro exposed to manganese.Intervention of LRRK2 expression by various means to observe the expression of autophagy-related molecules.Western blot was used to detect the interaction between LRRK2 and AMPK and mTOR signaling pathways.Using the specific inhibitors of the corresponding proteins,the effect of signaling pathway-associated proteins on LRRK2 expression was observed.Results1.Manganese exposure can induce microglial activation and cause neuroinflammationThe results of atomic absorption in vivo showed that compared with the control group,blood manganese and brain manganese were significantly increased in the Mn-treated group?P<0.05?;The results of open-field tests and climbing rod tests showed that the ability of mice in the Mn-exposed group was significantly decreased.The results of immunohistochemical staining showed that exposure to manganese could reduce the expression of TH protein in the substantia nigra and striatum,and increase the proportion of Iba-1 and CD68 positive cells?P<0.05?.Western blot and qPCR results showed that both in vivo and exogenous manganese exposures could increase the expression of inflammation-related molecules?P<0.05?.ELISA results suggested that manganese exposure could induce the release of inflammatory factors?P<0.05?.2.Role of LRRK2 in manganese-induced microglial activation and neuroinflammationWestern blot and immunofluorescence staining showed that Mn exposure significantly increased the expression of LRRK2 in the substantia nigra and striatum?P<0.05?.The results of immunofluorescence staining showed that manganese exposure could increase the proportion of LRRK2 in BV2 cells?P<0.05?.Western blot and qPCR results demonstrated that manganese stimulation significantly increased the expression of LRRK2 in BV2 cells?P<0.05?.Through LRRK2 inhibitor,AV-LRRK2 adenovirus and siRNA-LRRK2 technology,the results showed that inhibition of LRRK2 expression can significantly inhibit the expression of inflammatory factors in BV2 cells?P<0.05?;After stereotaxic injection of AAV-LRRK2 virus,the results suggested that reducing the expression of LRRK2 in vivo can significantly inhibit the effect of manganese exposure on the exercise ability of mice?P<0.05?;Western blot results suggested that reducing LRRK2 expression in vivo reduced the effects of manganese exposure on TH protein and significantly inhibited the activation of microglia induced by manganese exposure.The results suggest that LRRK2 plays an important role in manganese exposure-induced microglial activation.3.Role of autophagy and signaling pathways in the induction of LRRK2 changes by manganese exposureWestern blot and qPCR results suggested that the expression of autophagy-related molecules in vivo and in vitro was increased after manganese exposure?P<0.05?.Transmission electron microscopy showed that the number of autophagic vacuoles increased significantly after manganese exposure?P<0.05?;Inhibition of LRRK2expression by LRRK2 inhibitor,AV-LRRK2 adenovirus and siRNA-LRRK2technology significantly inhibited autophagy-related protein and mRNA expression in BV2 cells?P<0.05?.Western blot results showed that manganese exposure significantly reduced the level of mTOR phosphorylation in the substantia nigra and striatum of mice?P<0.05?.At the same time increase the level of phosphorylation of AMPK.Inhibition of LRRK2 could significantly inhibit the effect of manganese on the phosphorylation of mTOR?P<0.05?.Inhibition of LRRK2 did not alter the effect of manganese exposure on the phosphorylation level of AMPK,whereas the addition of AMPK inhibitor significantly inhibited the toxic effects of manganese and the alteration of LRRK2.ConclusionIn summary,our study first discovered that LRRK2 is an important cause of manganese-induced microglial activation;Decreasing the expression of LRRK2 may inhibit the manganese-induced microglial activation and reduce the toxic effects of manganese.LRRK2 is responsible for the dysfunction of microglia by regulating the autophagic function of microglia;AMPK signaling pathway and mTOR signaling pathway may be key signaling pathways for manganese-regulated autophagy.