Paraquat Combined With Maneb Co-exposure Induces Noradrenergic Locus Coeruleus Neurodegeneration Though Microglial Activation And Intervention Of Taurine

Objective:Parkinson’s disease(PD)is the second most common neurodegenerative disease after Alzheimer’s disease,and its prevalence increases year by year.Previous studies have shown that long-term exposure to environmental toxicants such as pesticides increase the risk of PD.Paraquat(PQ)and maneb(Mb)are widely used pesticides in agriculture.Epidemiological studies have revealed that combined exposure of PQ and Mb significantly increases the risk of PD.PQ can pass blood-brain barrier,resulting in damage dopaminergic(DA)neurons in the substantia nigra.Similar to PQ,Mb exposure also leads to DA neurodegeneration,and synergistic neurotoxicity is observed with combined with PQ.The combined application of PQ and Mb has become a common method to prepare PD models.Recent studies have demonstrated that the pathological damage of PD is not only limited in DA neuron,but also noradrenergic(NE)neuron in the locus coeruleus(LC)of brainstem.The LC/NE neurodegeneration precedes damage of DA neuron and is even more severe than DA neurodegeneration in PD.However,it is not clear whether the combined exposure of PQ and Mb could damage LC/NE neuron.Neuroinflammation mediated by microglia is closely related to the pathogenesis of multiple neurodegenerative diseases,including PD.Activated microglia can be divided into M1 and M2 polarization phenotypes.M1 belongs to the classical activation and can produce a large number of proinflammatory factors and reactive oxygen species(ROS)as well as other neurotoxic substances to promote damage of nervous system.While,M2 microglia secrete anti-inflammatory factors.We previously found that PQ and Mb combined exposure stimulates activation of microglia in primary cell cultures.However,the phenotypes of microglial activation induced by PQ and Mb and whether microglial activation might be related to PQ and Mb-induced LC/NE neurodegeneration as well as whether blocking microglial activation could alleviate PQ and Mb-induced damage of LC/NE nerons remain unclear.In this study,the toxic effects and underlying mechanisms of combined exposure of PQ and Mb on LC/NE neurons were investigated in mice.The protective effects of Taurine(Tau)against PQ and Mb-induced LC/NE neuronal damage were further explored.The results of this study will provide experimental evidence for the mechanistic research and development of treatment strategy of LC/NE neurodegeneration injured by PQ and Mb.Materials and Methods:(1)PQ and Mb model:Forty adult SPF male C57BL/6J mice were fed adaptively for 7days.They were randomly divided into control(Con)and model(PQ+Mb)groups(n=20 mice in each group).Mice in model group were injected intraperitoneally with PQ(10 mg/kg·bw)and Mb(30 mg/kg·bw)for 6 weeks(twice per week).The control mice were given the same dose of saline.After intoxication,mice(n=5 mice/group)were anesthetized and were perfused with 4%paraformaldehyde for 10 minutes.Brain tissues were dissected for pathological observation.The rest of mice(n=15 mice/group)were perfused with normal saline only.The brainstem samples were separated quickly on ice and then were frozen in liquid nitrogen,followed by stored at-80 C.(2)Tau model:60 adult SPF male C57BL/6J mice were randomly divided into control(Con),model(PQ+Mb)and Tau intervention group(Tau+PQ+Mb)after 7 days of adaptive feeding.The treatment strategy of PQ and Mb was the same with(1).Tau(150mg/kg)was injected intraperitoneally into mice 30 minutes prior to PQ and Mb exposure for 6 weeks(twice per week).After intoxication,mice(n=5 mice/group)were anesthetized and were perfused with 4%paraformaldehyde for 10 minutes.Brain tissues were dissected for pathological observation.The rest of mice(n=15 mice/group)were perfused with normal saline only.The brainstem samples were separated quickly on ice and then were frozen in liquid nitrogen,followed by stored at-80 C.(3)Immunohistochemical analysis:The frozen sections of the brainstem were prepared and the LC/NE neurons was observed by immunohistochemical staining with anti-TH(LC/NE neuron marker)antibody.The anti-Iba-1 and anti-CD11b(microglia marker)antibodies were used for immunohistochemical staining to observe the activation of microglia.(4)Quantitative RT-PCR assay:The mRNA levels of M1(iNOS,TNF-α,IL-1β)and M2(Arg-1,Ym-1)markers in the brainstem of mice in each group were analysized by quantitative RT-PCR assay.(5)Western blot detection:The expression and aggregation levels of a-syn as well as the phosphorylation levels of STAT and NF-κB signaling pathways in the brainstem of mice were analysized by Western blot technology.Results:Part I The role of microglial polarization in damage of LC/NE neuron induced by PQ and Mb(1)The effect of combined exposure of PQ and Mb on body weight of mice:The weight gain of mice exposed to PQ+Mb was slightly lower than that of Con group,but there was no significant difference between groups(P>0.05).(2)The effects of combined exposure of PQ and Mb on LC/NE neuron in mice:Immunohistochemistery showed that compared with Con group,a decreased trend of the number of LC/NE neurons(TH~+cells)in PQ+Mb-treated mice was observed after two weeks of exposure,but no statistical difference was detected(P>0.05).After 4weeks of intoxication,the number of LC/NE neurons in the PQ+Mb group was significantly lower than that of Con mice(P<0.05),and decreased to 41.5%in Con group after 6 weeks of exposure(P<0.01).Consistent with degeneration of LC/NE neuron,Western blot analysis showed that the levels ofα-syn oligomer in the brainstem of PQ+Mb-treated mice were significantly increased after 6 weeks of exposure,although the monomer ofα-syn remained unchanged.The effects of combined exposure of PQ and MB on the activation and M1polarization of microglia in the brainstem of mice:Immunohistochemistery showed that compared with Con group,after 2,4 and 6 week of intoxication,microglial activation in the brainstem of mice in PQ+MB group was observed by showing enlarged body size and intensified Iba-1 and CD11b immunostaining(P<0.05).Compared with Con group,PQ and Mb exposure significantly increased the mRNA levels of M1 markers,including iNOS,TNF-αand IL-1βin the brainstem of mice(P<0.05),these results suggest that PQ and MB combined exposure induced microglial activation and M1 polarization,which preceded LC/NE neurodegeneration.(4)The effects of combined exposure of PQ and Mb on JAK-STATs and NF-κB signaling pathway in the brainstem of mice:Compared with Con group,the increased levels of p-STAT1,p-STAT3,p-p65 and p-IκBαas well as decreased levels of total IκBαin the brainstem of mice treated with PQ+Mb were observed after 6 weeks of exposure.Part II The protective effects of Tau against PQ and Mb-induced LC/NE neurodegeneration through inhibition of microglial M1 polarization(1)The effects of Tau on the body weight of mice exposed to PQ and Mb:No significant difference of the body weight of mice in each group was observed(P>0.05).(2)The effects of Tau on the activation and polarization of microglia in mice exposed to PQ and Mb:Compared with Con group,microglial activation in the brainstem of mice in PQ+Mb group was observed by showing enlarged body size and intensified Iba-1 and CD11b immunostaining.Tau treatment significantly reduced the activation of microglia induced by PQ+Mb,and the difference was statistically significant(P<0.01).Compared with Con group,PQ and Mb exposure significantly increased the mRNA levels of M1 markers,including iNOS,TNF-αand IL-1βin the brainstem of mice(P<0.05),which were significantly reduced by Tau(P<0.01).The transcript levels of M2 markers Arg-1 and Ym-1 in the brainstem of PQ+Mb group were also significantly increased compared with Con mice(P<0.05);however,Tau treatment failed to interfere with the increase of M2 markers in PQ+Mb-treated mice(P>0.05).(3)The effects of Tau on the activation of JAK-STATs and NF-κB pathways in the brainstem of mice exposed to PQ and Mb:Compared with Con group,the phosphorylation levels of STAT1,STAT3 and NF-κB in the brainstem of mice in PQ+Mb group were significantly increased(P<0.05),indicating activation of JAK-STATs and NF-κB signalings.Tau treatment significantly reduced PQ+Mb-induced activation of NF-κB,but failed to interefere with STAT1 and STAT3activation in mice.(4)The effects of Tau on LC/NE neurodegeneration and a-syn expression in mice exposed to PQ and Mb:The number of LC/NE neurons(TH~+cells)in the PQ+Mb group was significantly lower than that of Con mice(P<0.01);Tau treatment significantly reduced LC/NE neurodegeneration in PQ and Mb-intoxicated mice,and the difference was statistically significant(P<0.01).Compared with Con group,the levels ofα-syn oligomer in the brainstem of mice in PQ+Mb group were significantly increased(P<0.01),which were significantly reduced by Tau treatment(P<0.01).Conclusion:1.PQ and Mb co-exposure induced damage of LC/NE neuron in mice.2.PQ and Mb combined exposure induced microglial activation and M1 polarization,which preceded LC/NE neurodegeneration.3.PQ and Mb-induced microglial M1 polarization might be related to the activation of JAK-STATs and NF-κB signaling pathways.4.Tau treatment attenuated LC/NE neurodegeneration in PQ and Mb-intoxicated mice.5.The protective effects of Tau against PQ and Mb-induced LC/NE neurons through inhibition of microglial M1 polarization.6.The neuroprotective effects of Tau might be attributed to the inhibition of microglal M1 polarization via NF-κB signaling pathway.