The Role Of Nrf2 In Cadmium-induced Microglial Activation, Its Contribution To Synaptic Damages And The Underlying Mechanism

Objective:Cadmium（Cd）,a widespread environmental pollutant,is one of the priority metals that are of great public health concerns.In daily life,people are exposed to Cd mainly by eating Cd-contaminated food and smoking.Besides,mining and smelting are common occupational Cd exposures routes for workers in the non-ferrous metals industry.As a neurotoxicant,Cd has been shown to pass through the blood–brain barrier and seriously affect the nervous system function.Epidemiological evidence shows that higher cumulative Cd exposure is associated with diminished neurocognitive performance in adults,and blood Cd concentration is inverse associated with cognitive function in old people.Synapses,the contact points of structures and functions between neurons,are the neurobiological basis of learning and memory.Microglia cells,the only immune cells of the central nervous system,play a pivotal role in the regulation of synaptic function.In addition to maintaining synaptic homeostasis,microglia cells can also engulf functional synapses and lead to learning and memory impairment.Activated microglia can lead to synaptic dysfunction and synaptic loss by secreting various proinflammatory mediators and enhancing phagocytosis.The hippocampus plays crucial roles in the consolidation of short-term to long-term memories,and spatial memory.Therefore,study of the changes of microglia in the hippocampus under Cd exposure has important implications for understanding the damage of neurons and learning and memory.Currently,it is considered that chronic or excessive inflammatory responses are the major mechanism by which Cd causes neurotoxicity.Several studies have shown that Cd can induce microglial activation.However,whether activated microglia play a role in the process of Cd induced nervous system damage,as well as the molecular mechanisms underlying nerve injury have yet to be elucidated.The nuclear factor-erythroid 2-related factor 2（Nrf2）is one of the core transcription factors regulates cellular redox homeostasis.Increasing evidence has reported that Nrf2exert a critical role in pathological and physiological processes,such as redox homeostasis regulation,heavy metal stress,the inflammatory response and the metabolism and detoxification.Nrf2 exerts many protective effects in the mammalian CNS.However,the detailed mechanism and the primary cell type that Nrf2 functions are still unclear.Therefore,in the present study,we first used NRF2 knockout（KO）mice and wild type littermates to explore sub-chronic Cd exposure induced neurotoxicity and neuroinflammation.Secondly,we created a NRF2 knockdown（KD）BV2 cell line to investigate the role of microglia-derived NRF2 in neuroinflammation and the activation of microglia.Finally,we developed either NRF2-KD or Scramble BV2 microglia in co-culture with primary hippocampal neurons from WT mice or HT22 mouse hippocampal neuronal cell line to assess the effects of NRF2 regulated microglial activation on synaptic damage.Methods:1.Study the role of Nrf2 in Cd exposure on learning and memory impairment and aberrant microglial activation in vivo.Groups of male Nrf2-KO or Nrf2-WT mice were provided either drinking water containing 100 ppm cadmium chloride or deionized water for 24 weeks,and changes in body weights were monitored on a weekly basis.Morris water maze test,open-field test,and T-maze continuous alternation test were tested after24 weeks Cd exposure to evaluate the learning and memory ability.After the behavioral experiment,mice were sacrificed under CO₂ anesthesia then beheaded for hippocampal tissues or perfused through the left ventricle using standard methods for pathological section.The Cd concentration in hippocampus tissue was analyzed by inductively coupled plasma-mass spectrometry（ICP-MS）;Golgi staining was used to detect the synaptic dendrite complexity,spine density and the morphology of neurons in hippocampus;Gene expression of oxidative stress and inflammation responses were determined by realtime PCR;Synapse-related proteins,the NFκB signaling pathway-related proteins,and microglial activation related proteins were determined by Western Blot;The neuronal dendrites and the microglia density and morphology were determined by immunofluorescence experiments.2.Investigating the type of cells（microglia or neuron）primarily affected by Nrf2 and the role of microglia-derived NRF2 in Cd induced neuroinflammation and the activation of microglia.We established a Cd-exposed Nrf2 KD BV2 cell line and a Cd-exposed Nrf2 KD HT22 cell line.Moreover,we created primary neurons models treated with and without Nrf2 inhibitor.CCK8 assay was used to detect BV2 and HT22 cell viability;NRF2 downstream effectors were detected by realtime PCR;Primary neurons synapse associated protein expression were detected by western blot analysis;The expression of antioxidant associated gene,Cd metabolites enzymes associated gene and inflammatory response associated gene were detected by realtime PCR;Anti-oxidant proteins,the NFκB signaling pathway-related proteins,and microglial activation related proteins were determined by Western Blot;FM1-43 dye was used to detect the endocytosis function of BV2 cells;The scratch wound-healing assay was employed to examine BV2cell migration;The secretion of inflammatory factors was quantitated using a customized Luminex assay;NFκB nuclear translocation,and the expression of CD68 and Iba1 were determined by immunofluorescence experiments.Mino was used to detect the inhibition of Cd-induced microglial activation by immunofluorescence,Realtime PCR,Luminex and Western Blot assays.3.Investigating the role of microglia activation in Cd-induced neuronal synaptic damage.To define the role of microglia during Cd induced synapses injury,we applied the in vivo model in Method 1.In addition,mouse primary neurons or HT22 cells were cultured alone or co-cultured with BV2 microglial cells（with or without Nrf2 knockdown）.We established two different strategies of the co-culture:the neurons were cultured with BV2 microglial cells-conditioned medium（noncontact co-culture）,or co-cultured with BV2 microglial cells（contact co-culture）.Immunohistochemistry and Phalloidin staining were performed to analyze the dendritic spine density;The expression of synapse-related proteins was determined by Western Blot and immunofluorescence experiments.Immunofluorescence analysis of localization was carried out to analyze the phagocytosis of synapse by microglia;The expression of“eat me”signaling pathway-related proteins was detected by Western Blot analysis.Results:1.The effect of Nrf2 deficiency on Cd-induced learning and memory impairment1.1 Subchronic Cd exposure increases Cd content in mouse hippocampus.After Cd exposure,the Cd content in the hippocampus was significantly increased in both Nrf2-WT and Nrf2-KO mice（P<0.05）.1.2.Nrf2 deficiency exacerbates Cd-induced oxidative stress.After chronic Cd exposure,the Nrf2,Gclc and Hmox1 gene expression levels significantly increased in Nrf2-WT mice（P<0.05）.However,the level of Gclc,Gclm and Hmox1 were not significantly changed in Nrf2-KO mice.Cd exposure increased levels of nuclear staining of 8-OHd G in the hippocampus of Nrf2-WT and Nrf2-KO mice.Importantly,compared to Nrf2-WT mice,Nrf2-KO mice have more positive 8-OHd G immunofluorescence staining following Cd exposure.1.3.Nrf2 deficiency exacerbates Cd-induced learning and memory deficits.To investigate the effects of Nrf2 on the deficits of learning and memory under Cd exposure,Nrf2-KO and WT mice underwent the Morris Water Maze test,the open-field test,and the T-maze test at the end of Cd exposure.Morris Water Maze test shows that there were no significant differences in swimming speed among groups.During the acquisition phase,the mice with Cd treatment displayed a significant increased escape latency compared to WT-CON mice（P<0.05）.During the probe trial,it was revealed that the KO-Cd group and WT-Cd group increased escape latency to hidden platform（P<0.01 and P<0.05respectively）,spent less time in hidden platform and decreased the number of platform crossings（P<0.01 and P<0.05 respectively）than the WT-CON group.Compared with the WT-CON group,the KO-Cd group spent less time in target quadrant and increased escape latency to target quadrant（P<0.05）.Furthermore,compared with the WT-Cd group,the KO-Cd group increased escape latency to target quadrant and hidden platform（P<0.05）.The open-field test shows that there were no differences on movement velocity among groups.Moreover,the mice in KO-Cd group and WT-Cd group spent less time in the central area than the WT-CON group（P<0.05）.We assessed spontaneous alternation using the T-maze test.The result showed that KO-Cd group exhibited a significant reduction in spontaneous alternation compared to the WT-CON group（P<0.05）.2.The effect of Nrf2 deficiency on Cd-induced neuronal structural damage2.1 Cd-induced dendrite loss is exacerbated in the hippocampus of Nrf2-KO mice.We determined the alteration in dendrite,which is critically involved in learning and memory deficits,in both DG and CA1 subregions of the hippocampus.Immunofluorescent staining against MAP2 showed that the dendrites were greatly reduced in KO-Cd group,and slightly reduced in WT-Cd group.We performed Golgi-Cox staining and analyzed dendritic arborization and we found that Cd reduced the dendritic complexity and dendritic length of the hippocampal neurons.Besides,Nrf2 deficiency presented significantly greater dendrite impairment than WT mice after Cd treatment.2.2 Cd-induced dendritic spine loss is exacerbated in the hippocampus of Nrf2-KO mice.We conducted Golgi-Cox staining to examine spine density and morphology in the hippocampus.A significant reduction in overall dendritic spine density was found in WT-Cd group and KO-Cd group,whereas KO-CON group had no significant effect compared with WT-CON group.Further assessment of spine morphology showed that treatment with Cd significantly increased the number of immature spines and decreased the number of mature spines in hippocampal subdomains,and the decrease of mature spines was more obvious in Nrf2 KO mice than WT mice（P<0.05）.2.3 Cd-induced synapse-related markers loss is exacerbated in the hippocampus of Nrf2-KO mice.Exposure to Cd significantly reduced the levels of PSD-95 and SYN.Importantly,the expression of PSD-95 in Nrf2-KO mice was significantly lower than those in Nrf2-WT mice.3.The effect of Nrf2 deficiency on Cd-induced microglia activation and inflammatory response3.1 The effect of Cd on hippocampal microgliosis is increased in Nrf2-KO mice.We observed the changes in basal or Cd-induced levels of Iba1 in both genetic backgrounds.By contrast,after Cd treatment,Nrf2-KO mice have a higher Iba1 immunoreactivity than Nrf2-WT mice.In the hippocampus of WT-CON and KO-CON mice,the majority of microglia were ramified microglia.After Cd treatment,the percentage of microglia with ramified morphology was markedly decreased,whereas the percentage of amoeboid and round microglia was increased.Importantly,the proportion of ramified microglia was significantly decreased,while the proportion of round microglia was a significant increase in the hippocampus of KO-Cd mice compared to WT-Cd mice（P<0.05）.The expression of inflammatory factor-related genes in the hippocampus was detected by RT-q PCR.The results showed that the m RNA levels of classical inflammatory factors were significantly increased in KO-Cd and WT-Cd group,however,the m RNA levels in KO-Cd group were higher than those in WT-Cd group（P<0.05）.Western Blot was used to detect the expression of NFκB/IκB-αsignaling pathway-related proteins in the hippocampus.The results showed that p-IκB-αand p-NFκB protein expressions were significantly increased after subchronic cadmium exposure,but the level of protein increase in KO-Cd group was higher than that in WT-Cd group（P<0.05）.In addition,the IκB-αprotein level of KO-Cd group significantly decreased compared with WT-CON group（P<0.05）.Both immunofluorescence staining and Western Blot experiments showed that the expression of CD68 in the hippocampus was increased after subchronic Cd exposure,but the increase in Nrf2-KO mice was greater than that in Nrf2-WT mice（P<0.05）.4.Effects of Nrf2 knockdown or inhibition on the oxidative stress level and cell viability of cultured microglia and neurons in vitro4.1 Nrf2 deficient microglia cells are more vulnerable to Cd stimulation compared to neuronal cells.We observed that in BV2 microglia cells,Nrf2 knockdown dramatically reduced the m RNA levels of Nrf2 and its downstream genes Gclc,Gclm,Hmox1 and Nqo1.In contrast,although the m RNA levels of Nrf2 were dramatically decreased in Nrf2-KD HT22hippocampal neuronal cells,Nrf2 knockdown did not affect its downstream genes levels in neurons.4.2 The effects of Cd on the viability of BV2 and HT22 cells.We found that exposure to Cd greater than 1μM resulted in an obvious cell death in BV2cells.However,treatment with 1–3μM of Cd did not affect cellular viability of HT22 cells.We also found the cell viability was no difference between the Scramble cells and the Nrf2-KD cells in HT22 at each Cd concentration.However,when treated by 1–5μM Cd,the BV2 cell viability significantly differs between the Scramble cells and the Nrf2-KD cells.4.3 Effects of Nrf2 inhibition on primary neurons after Cd exposure.Nrf2 was blocked in primary neurons using the small-molecule inhibitor of NRF2（ML385）.Similar to the results with HT22,the cell viability was no difference between the vehicle group and the ML385 group at each Cd concentration.Besides,the cell viability was not significantly changed until the concentration reached 10μM.Western Blot experiments showed that 5μM Cd Cl2 significantly reduced the expression of PSD95 and SYN proteins in primary neurons（P<0.05）,while Nrf2 inhibition did not affect the expression levels of PSD95 and SYN proteins in neurons.5.Effects of Nrf2 knockdown on Cd-induced microglia activation and inflammatory in vitro5.1 Nrf2 deficient microglia cells are more sensitive to oxidative stress and inflammatory responses induced by Cd exposure.The expressions of antioxidant-related genes and Cd metabolism-related enzymes in Scramble BV2 microglia were significantly increased after Cd treatment（P<0.05）.The expression of antioxidant-related genes and Cd metabolism-related enzymes in Nrf2-KD BV2 microglia cells was significantly lower than that in Scramble BV2 microglia cells after Cd treatment（P<0.05）.After being treated with Cd or LPS,the migration ability and phagocytic ability of BV2 microglia were significantly increased,and the increase was more obvious in Nrf2-KD BV2 microglia than Scramble BV2 microglia（P<0.05）.After Cd or LPS exposure,the NFκB/IκB-αsignaling pathway was activated,and the translocation of NFκB proteins to the nucleus was increased,the expression of inflammatory factor-related genes was increased,the secretion of inflammatory factor proteins in the supernatant was increased in BV2 microglia.Moreover,this situation was more evident in Nrf2-KD BV2 microglia.5.2 Minocycline inhibits the activation of Scramble BV2 microglia.Mino intervention reduced the expression of CD68,the expression of inflammatory factor-related genes,and the secretion of inflammatory factors in Scramble BV2 cells after Cd treatment（P<0.05）.Moreover,Mino intervention significantly reduced the activation of the NFκB/IκB-αsignal pathway.However,Mino intervention did not significantly moderate the intervention effect after Cd treatment in Nrf2-KD BV2 cells.6.The role of Cd-induced microglial activation and its contribution to synaptic damage6.1 The phagocytosis of synapses by microglia in vivo after subchronic Cd exposure.The expression of SYN co-localized with microglial and the expression of CX3CR1protein was increased（P<0.05）in Nrf2-WT mice after Cd exposure.The expression of SYN co-localized with microglial and the expression of CX3CL1,CX3CR1,C3 and CR3proteins were increased（P<0.05）in Nrf2-KO mice after Cd exposure.6.2 Effects of Cd exposure on synapses in cultured neurons in vitro.5μM and 10μM Cd treatment significantly reduced the density of dendritic spines and the expression of synapse-related protein SYN in primary cultured neurons（P<0.05）.5μM Cd treatment significantly decreased the expression levels of synapse-related proteins PSD95 and SYN and dendritic microtubule-related protein MAP2 in HT22 hippocampal neurons（P<0.05）.6.3 The aberrantly activated microglia in co-culture system exacerbated Cd induced neuronal synaptic damage.In direct contact coculture system of primary neurons and BV2 microglia,we found that 2μM Cd significantly increased the synaptophysin localization in the cytoplasm of microglia.2μM Cd significantly decreased the level of synaptic associated protein in HT22cocultured with BV2 scramble.In addition,“eat-me”signals associated protein were dramatically increased in both HT22-BV2 scramble mixed culture after Cd treatment.In conditioned medium coculture conditions,the 2μM Cd BV2 conditioned medium group showed fewer dendritic spines and fewer synapse-associated proteins compared with the control group.Deficiency of Nrf2 in microglia aggravates microglial activation after Cd treatment,thereby aggravating Cd-induced synaptic damage in a co-culture model.Mino attenuates Cd-induced synaptic damage in cultured neurons by inhibiting microglia activation.Conclusion:1.Nrf2 deficiency exacerbates Cd-induced learning and memory deficits,dendrite and dendritic spine loss,and the aberrant activation of microglia.2.Nrf2 deficiency aggravates microglial activation caused by Cd,which may be a contributing cause of more serious nerve damage3.Nrf2 deficiency enhanced Cd-induced abnormal activation of microglia which subsequently damages the synapse by enhancing the phagocytosis and the secretion of inflammatory cytokines.Complement receptor pathway and chemokine pathway are involved in direct phagocytosis of synapses by microglia after Cd exposure.