Effects Of Long-term Low Dose Copper Exposure On Substantia Nigra Protein Profile And Mitochondrial Function In Mice Model Of Parkinson’s Disease

Objective:To investigate the effects of low-dose copper exposure on nigrostriatal protein profiles and mitochondrial function in SNCA*A53T transgenic PD model mice,as well as the effects of low copper on intracellular mitochondrial function in PD model neurons,and to further explore the pathological mechanisms of α-syn deposition.Methods:Twelve-month-old SNCA*A53T transgenic PD model mice and WT mice were randomly divided into four groups:WT mice group,copper-treated WT mice group,A53T mice group,and copper-treated A53T mice group,with 8 mice in each group.The mice in the coppertreated group were given drinking water containing 0.13 ppm copper chloride,and the rest were given normal drinking water treatment for a 4-month experimental period.In addition,a CCK8 assay was performed on human A53T mutant α-syn overexpression PD model SHSY5Y cells,and the 1/2 IC50 value of CuCl2 was used as the concentration of poison.The experiment was divided into 2 groups,the control group and CuCl2 treatment group,and the medium containing Cu2+was added to the treatment group and incubated for 24h before the experiment.The mice were tested for locomotor function by the open-field experiment,and the protein profile in the substantia nigra was examined by proteomics technology.Western blot assay was used to detect the mitochondrial and neuron-related proteins in the nigrostriatal nigra,and then immunohistochemical staining and Nissl staining were used to detect the changes in the number of neurons and Iba1 in the nigrostriatal nigra,and finally,the changes of inflammatory factors in the substantia nigra and the contents of LDH,ROS and ATP were detected by ELISA.Results:(1)The results of the open-field experiments showed that the total and central zone locomotor distances were significantly reduced in copper-treated WT and A53T mice(both P<0.05).(2)Proteomics identified a total of 2828 highly expressed proteins,40 differentially expressed proteins(DEPs)in WT compared to Cu-WT mice,136 DEPs in WT compared to A53T mice,and 180 DEPs in A53T compared to Cu-A53T mice.The above DEPs are involved in the process of mitochondrial functional impairment,neurodevelopmental impairment,and immune response activation.(3)Cluster analysis of WT,A53T,and Cu-A53T mice yielded six categories of DEPs,with a decreasing trend for cluster 1 and an increasing trend for cluster 3 KEGG analysis revealed that both up-and down-regulated DEPs were involved in the PD pathway and identified biological processes involved in mitochondrial energy metabolism and transport,mitochondrial respiratory/electron transport chain,and neuronal axonal transport in the PD pathway.(4)Western blot showed that compared with WT mice,NDUFA10,ATP5A,Mfn1,TH,and DAT protein expression were weakened in Cu-WT mice(P<0.05),A53T mice’s NDUFA10,ATP5A,Mfn1,TH,and DAT protein expression were diminished(all P<0.05)and Drpl protein expression was enhanced(P<0.05);compared with A53T mice,UQCRFS1,OPA1,Mfn1,LC3BⅡ/Ⅰ,Pink1,and TH protein expression were diminished(all P<0.05)and P62,Drp1,and α-syn protein expression were increased(all P<0.05),in Cu-A53T mice the results suggest that copper treatment impaired mitochondrial function and neuronal cell development in mice,which is consistent with the histological results.(5)The results of the staining experiments showed that the number of TH+and Nissl+were reduced(P<0.05)for both Cu-WT and A53T mice compared with WT mice and the number of IBal was increased(P<0.05);the number of Nissl+was reduced in Cu-A53T mice compared with A53T mice(P<0.01)and The number of IBal increased(P<0.01),and the results suggest that copper treatment reduced nerve cells in mice and induced neuroinflammation,which is consistent with the histological results.(6)The results of the enzyme-linked immunosorbent assay showed that compared with WT mice,the levels of inflammatory cytokines IL-6,IL-1β,and TNF-α in the substantia nigra of Cu-WT mice were increased(P<0.05),while the levels of TGF-β1 were decreased(P<0.05).In A53T mice,the levels of inflammatory cytokines IL-6,IL-1β,and TNF-α were increased(P<0.05),while the levels of IL-4 were decreased(P<0.05).Compared with A53T mice,the levels of inflammatory cytokines IL-6 and IL-1β in the substantia nigra of Cu-A53T mice were increased(P<0.05),and the results suggest that copper treatment induced neuroinflammation in mice,which is consistent with the histological results.In addition,ATP(P<0.05)content decreased,LDH(P<0.05)and ROS(P<0.05)production increased in CuCl2 treatment group compared with control group,suggesting that copper enhanced cytotoxicity.Conclusion:Low-dose copper exposure altered the protein profile of mitochondrial function,neurodevelopmental and immune response processes in the substantia nigra of mice,induced further elevation of α-syn levels in the substantia nigra of PD model mice,and ultimately affected the motor function of mice,and low copper also induced neuro cytotoxicity and reduced production capacity.The above results suggest that copper exposure may have disrupted the structure of neural cells,disturbed the brain function of WT mice,and accelerated the process of neurodegeneration in PD mouse models.