Mechanism Of Cu Exposure-induced Cognitive Impairment And Neurotoxicity In Mice

Background and objectivesGlobally,neurological disorders are the mainly causes of disability-adjusted life-years（DALYs）and the second cause of death.The characteristics of heavy metal pollution in the environment have undergone important changes in recent years,and the health risks of Cu exposure are increasing.Substantial evidence shows that excess Cu plays a role in human neurodegenerative diseases（NDD）,such as Alzheimer’s disease（AD）and Parkinson’s disease（PD）.The main clinical symptom of NDD is cognitive impairment,but its pathogenesis is still unclear so far.Elucidating its pathogenesis is very important for the early prevention and treatment of the disease.In this study,transcriptome sequencing technology（RNA-seq）was used to analyze the transcriptome information of Cu-iexposed C.elegans on the base of the observation of Cu-induced motor behavior,neuronal cell and synaptic morphological changes in C.elegans.Then,differentially expressed transcripts were screened,relevant signaling pathways affecting Cu-induced nerve injury were identified and conserved targets in mammals were explored,finding that Cu may induce neurotoxicity through oxidative damage and neuroregulatory signaling.Therefore,subsequent mouse and cellular models were used to validate the clues suggested by RNA-seq.Next,the Cu-exposed mouse cognitive impairment model was further constructed to analyze the effect of Cu on the learning and memory ability of mice in this study,and ICP-MS technology was used to carry out the exposomics analysis of Cu exposure in mice organs,blood and urine to explore the internal exposure level.The mechanism of Cu induced cognitive impairment in mice was preliminarily verified through the detection of the pathological damage,neuronal activity,synaptic structurally related proteins,neurotransmitters,oxidative stress levels and related signal pathways of the mouse hippocampus.Finally,the hippocampal neuron HT22 cells was used as the target cell to investigat the role and mechanism of key signaling molecules CREB and Nrf2 in copper induced neurotoxicity and oxidative damage of HT22.This study will help to elucidate the molecular network and mechanism of transcription factor CREB and oxidative damage in response to Cu induced cognitive impairment by regulating transcription factors CREB and Nrf2,which is of great significance for the discovery of biomarkers of neurodegenerative diseases and the selection of intervention targets.Methods:1.According to the LC50 of the pre-experimental Cu-treated nematodes,Cu exposure concentrations（0,0.01,0.1,1,and 10 mg/L）in C.elegans were set.The development（body length and body width）and motor behavior（head thrashes,body bending,pharyngeal pump and defecation cycle）were observed in C.elegans after Cu treated at 24 h,48 h and 72 h,besides,chemotaxis index and Cu levels were detected in adult worms.The L1 stage strains of BZ555,DA1240,EG1285 and LX929 were treated with Cu until adults,the fluorescence intensity was observed and analyzed.The genes expression of neurotransmitter transporters and receptors were analyzed by q RT-PCR.2.The whole genome sequencing of Cu-exposed C.elegans was analyzed through high-throughput transcriptome（RNA-seq）,differential transcripts were screened,GO and KEGG analysis were used to identify the biological functions and nerve injury-related signals of C.elegans in response to Cu stress.Further,GSEA was used to analyzed differential expression genes of C.elegans.Finally,the oxidative stress level and antioxidant reductase system of Cu-exposed C.elegans were validated on the basis of transcriptome analysis.3.Based on the previous study,C57BL/6 mice were exposed to Cu（0,100 and 500 mg/L）through free drinking water,and the water intake and body weight were recorded.Morris water maze test was conducted to analyze the learning and memory ability of mice after Cu exposure for one month,two months and three months.Then,ICP-MS was performed to detect Cu level in urine,serum and organs including brain,heart,liver,lungs,kidneys,stomach,spleen and intestine,to explore the effects of Cu on exposure and organ accumulation in mice.Based on the clues of RNA-seq,HE staining,immunohistochemistry,immunofluorescence,TUNEL detection,ELISA and WB were conducted to analyze the level of pathological injury and apoptosis of hippocampal tissue in mice;Oxidative stress level and the expression of Keap1/Nrf2 in hippocampus of mice;Expression of neurotransmitters,neurotrophic factors,key proteins of synaptic structure,PI3K/Akt and CREB/BDNF signaling pathways in mouse hippocampus.To investigate the effect of Cu exposure on neurological function and oxidative stress level in mouse hippocampus.4.On the basis of RNA-seq analysis and animal experiments,the mouse hippocampal neuron HT22cells were further used as target cells to analyze the effects of Cu on cell activity,apoptosis,mitochondrial damage,synaptic proteins,neurotrophic factors and oxidative stress levels.To investigate the role of Keap1/Nrf2 signaling pathway in Cu-induced oxidative stress in HT22 cells by scavenging ROS with reducing agent Nac.The effects of CREB on HT22 proliferation and apoptosis,and the regulation of CREB/BDNF signaling pathway and upstream and downstream proteins were observed after overexpression and knockdown of CREB by gene silencing and gene overexpression technology.Results1.The neurotoxic effect of Cu on C.elegans1.1 The effect of Cu on development and movement behavior of C.elegansThe LC50 of L1 stage N2 exposed to Cu were 713.814,202.441 and 101.457mg/L at 24 h,48 h and72 h.Cu exposure for 24 h had no effect on development（body length and body width）and defecation cycle,the head thrashes,body bends and pharyngeal pump were inhibited of C.elegans（P<0.05）.After C.elegans exposure to Cu for 48 h,the body length and width were shortened,the head thrashes,body bends,pharyngeal pump and defecation cycle were inhibited（P<0.05）.Cu exposure for 72 h,body length was shortened,body width was increased,the head thrashes,body bends,pharyngeal pump and defecation cycle were inhibited（P<0.05）.L1 stage N2 larvae exposure to Cu until adults,the chemotaxis index was decreased and the Cu content were gradually increased（P<0.05）.1.2 The effect of Cu on neurons in C.elegansL1 stage reporter gene strain（BZ555,DA1240,EG1285 and LX929）were treated with Cu until adult,the fluorescence intensity of dopaminergic neurons,glutamatergic neurons,γ-aminobutyric acid neurons and cholinergic neurons corresponding to GFP labeling showed a significant downward trend in a dose-dependent manner（P<0.05）.Morphological images showed significant loss of relevant dendrite of neuron,abnormal branching,spots and axonal discontinuities.The results of q RT-PCR showed that gene expression of the dopamine neuron transporter dat-1 and receptors dop-1,dop-3,serotonin receptors mod-1 and ser-4,glutamate receptor genes glr-1,glr-2,glr-6,mgl-1 and nmr-1 were decreased with the increase concentration of Cu exposure（P<0.05）,while the expression of transporter mod-5 and glutamate transporter eat-4,glt-1 and glt-3 were increased under 0.01mg/L Cu exposure then decreased with increasing Cu exposure concentration（P<0.05）.2.Mechanism of Cu induced nerve damage in in C.elegans2.1 Construction of a gene database for Cu-related nerve damage based on RNA-seqCu induced 2332 differential expression genes（567 up-and 1765 down-regulated genes）in the 1mg/L group and 2449 differential expression genes（724 up-and 1725 down-regulated genes）in the2 mg/L group.The top 20 DEGs were mainly related to the structure of the stratum corneum,immune response,metabolism,epigenetic and gene expression regulation.GO and KEGG analysis revealed that Cu stimulated multiple signaling pathways,such as steroid biosynthesis,longevity regulatory pathways,alanine,aspartate and glutamate metabolism and synaptic vesicle cycling signaling pathways,thereby affecting nematode cells processes,biological processes,metabolic processes and biological regulation.GSEA analysis of GO database showed that oxidoreductase activity（133 DEGs,NES=-1.728,P<0.001）and neuropeptide signaling pathway（132 DEGs,NES=-1.899,P<0.001）enriched the most DEGs.GSEA analysis of the KEGG database revealed oxidative phosphorylation（104 DEGs,NES=-1.400,P=0.001）and lysosomal signaling（169 DEGs,NES=-1.548,P<0.001）enriched the most DEGs.2.2 Mechanism of Cu induced oxidative damage in C.elegansCu stimulated the production of reactive oxygen species（ROS）,promoted the expression of oxidative stress biomarkers H₂O₂,lipid peroxide MDA,the expression of antioxidant enzyme SOD decreased first then increased,and GPX significantly decreased in C.elegans.In addition,the expressions of SOD-encoded genes were deregulated,the expressions of GPX and metal detoxification protein-related genes were decreased,and the expression of heavy metal response factors HMT and NUMR were increased.These results suggested that Cu promoted the production of ROS,simultaneously led to the imbalance of antioxidant defense system and inhibits the expression of metal detoxification enzymes,resulting in the dysfunction of antioxidant defense and detoxification in nematodes.3.Effects of Cu on cognitive function and mechanism of neurotoxic effect in mice3.1 Effects of Cu on learning and memory ability in miceIn this study,a mice model of cognitive impairment caused by Cu exposure was established.After one month exposure,the average daily Cu intake of each mouse were 0.43 mg/mouse in 100 mg/L group and 1.32 mg/mouse in 500 mg/L group.Cu intake per unit body weight of mice were 0.02mg/g in 100 mg/L group and 0.06 mg/g in 500 mg/L group.After two months exposure,the average daily Cu intake of each mouse were 0.37 mg/mouse in 100 mg/L group and 1.15 mg/mouse in 500 mg/L group.Cu intake per unit body weight of mice were 0.02 mg/g in 100 mg/L group and0.06 mg/g in 500 mg/L group.After three months exposure,the average daily Cu intake of each mouse were 0.41mg/mouse in 100 mg/L group and 1.41 mg/mouse in 500 mg/L group.The average daily Cu intake of each mouse were 0.02 mg/g in 100 mg/L group and 0.06 mg/g in 500mg/L group.Cu exposure for one month,the target quadrant latency in probe trial of the 500 mg/L group was significantly increased（P<0.05）.Cu exposure for two months,the results of training test showed that the platform latency of 500 mg/L group increased significantly（P<0.05）,the results of probe trial showed that the platform latency and target quadrant latency were significantly increased in the 500 mg/L group（P<0.05）.Cu exposure for three months,The training test results showed the target quadrant latency and platform latency were significantly increased in the 500 mg/L group（P<0.05）,the target quadrant latency and platform latency of probe trial were significantly increased in the 500 mg/L group（P<0.05）.The urinary Cu levels of mice of one month,two months and three months were promoted with the increasing exposure of Cu,and the differences were statistically significant with or without creatinine correction（P<0.05）.There was no statistical difference in the serum Cu levels of mice among the groups.The results of organ Cu levels showed that Cu exposure for one month,the level of in the spleen and intestine were significantly increased（P<0.05）,and the Cu levels in the brain,liver,lungs and stomach were slightly increased with no statistical difference.Cu exposure for two months,Cu levels in the brain,spleen,liver,lung,kidney and intestine were promoted（P<0.05）.Cu exposure for three months,Cu levels in brain,spleen,liver,lung,kidney,stomach and intestine were all increased significantly（P<0.05）.In addition,the cumulative level of Cu in organs were time-dependent with Cu exposure.3.2 Effects of Cu on neurological function of mice hippocampusThe pathological results showed that Cu exposure caused the neuronal cells shrink and number decrease in the DG and CA3 regions of the mice hippocampus.With prolonged Cu exposure,pyknotic and hyperchromatic,pyramidal neuron cell necrosis,as well as more extensive karyopyknosis and karyolysi were occurred.In addition,increased expression of GFAP in the mice hippocampus indicated the production of inflammation,enhanced TNNEL fluorescence and increased Neu N antibody expression suggested that Cu promoted apoptosis of mice hippocampus.3.3 Effects of Cu exposure on oxidative damage in mice hippocampusCu induced oxidative damage in mice hippocampus.With Cu exposure,the production of MDA was significantly increased,the activity of SOD and GPX were gradually inhibited（P<0.05）.The Keap1/Nrf2 antioxidant signaling pathway was activated.The expression of NRF2,HO-1 and NQO-1 were promoted,while the expression of Keap1 was inhibited（P<0.05）.In addition,the expressions of antioxidant enzymes SOD-1 and SOD-2 were decreased（P<0.05）,which led to anti-oxidative imbalance in mice brain tissue.3.4 Effects of Cu on neurotransmitter and related signals in mice hippocampusCu inhibited the secretion of neurotransmitters,reduced expression of postsynaptic membrane compact protein PSD-95 and synaptophysin SYP,resulting in decreased synaptic plasticity.Cu inhibits the phosphorylation of CREB,reduced the expression of neurotrophic factor BDNF and its receptor Trk B,and led to the dysfunction of CREB/BDNF signaling.In addition,Cu may inhibit PI3K/Akt and CREB/BDNF signaling,promote the downstream apoptotic protein Bax and inhibit the expression of anti-apoptotic protein Bcl-2,to induce apoptosis of hippocampal cells.4.Mechanism of Cu-induced HT22 cell damage4.1 Effects of Cu exposure on proliferation,apoptosis and mitochondrial function of HT22cellsIn vitro studies showed that Cu exposure inhibited the proliferation activity and promoted apoptosis of HT22 cells in a dose-dependent manner at 48 h,and the intracellular Cu²⁺content was promoted with the increasing of Cu treatment concentration.In addition,Cu destructed mitochondria of HT22cells,including mitochondrial swelling,rupture and reduced cristae,decreased mitochondrial membrane potential and increased autophagosome,and increased expression of autophagy-related proteins LC3B and P62.4.2 The role of transcription factor Nrf2 in Cu-induced oxidative stress in HT22 cellsCu induced oxidative stress,promoted the production of lipid peroxidation product MDA,and decreased the antioxidant enzyme activity of GPX in HT22 cells.The oxidative stress induced by Cu accelerated Keap1/Nrf2 complex dissociation,promoted the nuclear translocation of Nrf2,thereby activating the expression of antioxidant molecules HO-1 and NQO1 locating at the downstream.Besides,Cu-induced oxidative stress also promoted the expression of Bax and inhibited the expression of Bcl-2.Therefore,Cu-induced ROS not only activated the antioxidant defense signaling pathway Nrf2/HO-1/NQO1,but also promoted mitochondrial apoptosis signaling.4.3 The role of transcription factor CREB in Cu-induced cytotoxicity in HT22 cellsIn vitro studies showed have shown that Cu treatment reduced the expression levels of PSD-95,SYP,BDNF and Trk B proteins in HT22 cells,suggesting that Cu inhibited the expression of neurotrophic factors and membrane receptor proteinss,and inhibited the expression of key proteins before and after synapses,leading to synaptic plasticity disorders.In addition,Cu inhibited the phosphorylation of CREB,and the phosphorylation levels of upstream proteins ERK,PI3K and Akt in CREB/BDNF signaling pathway were decreased,at the same time downstream proteins BDNF,Trk B and Bcl-2 were inhibited.The results of overexpression and knockdown indicated that Cu exposure may reduce the transcription of downstream genes BDNF and Bcl-2 by inhibiting CREB phosphorylation,inhibited the activation of Trk B by BDNF,further inhibited the drive of PSD-95by PI3K/Akt signaling and the activation of ERK,ultimately leading to cytotoxicity of HT22.Preliminary conclusions:1.Cu inhibits the development,locomotion and learning behavior of C.elegans,damages dopaminergic,glutamatergic,cholinergic andγ-aminobutyric acid neurons of C.elegans,and inhibits the expression of corresponding neurotransmitter transporters and receptors.2.RNA-seq results suggested that Cu may induce neurotoxicity in C.elegans by inhibiting neuroregulatory signaling pathways and oxidative stress,resulting in abnormal motor behavior.3.Cu induces cognitive impairment in mice,produces progressive decline in learning and memory with prolonged exposure.Cu induced cognitive dysfunction may be related to the accumulation of Cu in mouse brain tissue.4.Cu activates the Nrf2/HO-1/NQO1 signaling pathway by stimulating oxidative damage in the hippocampus and inhibits the CREB/BDNF and PI3K/AKT signaling pathways,resulting in decreased synaptic plasticity and neuronal apoptosis,to induce cognitive impairment in mice.5.Cu exposure leads to decreased viability,apoptosis,mitochondrial dysfunction,synaptic plasticity disorder and oxidative stress in HT22 cells.The Nrf2/HO-1/NQO1 and CREB/BDNF signaling pathways may play important roles in Cu-mediated oxidative stress,neuronal fine damage and synaptic plasticity impairment.