The Role And Mechanism Of Netrin-1 In Striatal Dopamine Depletion And Motor Dysfunction Induced By Manganese In Mice

Objective:Manganese（Mn）is one of the essential trace elements in humans.It can accumulate in the striatum through the blood-brain barrier（BBB）,and excessive accumulation of Mn can cause neurotoxicity in the central nervous system.This is mainly due to decreased dopamine（DA）levels in the striatum and impairment of nigrostriatal DAergic neuronal projections leading to extrapyramidal dysfunction.Although there are many studies on the mechanisms of manganese neurotoxicity,the specific mechanisms of manganese-induced DA function and impairment of nigrostriatal dopaminergic neuronal projections leading to dyskinesia remain to be further investigated.Netrin-1,as an axon guidance factor,regulates normal DA transmission and plays a vital role in axonal traction in nigrostriatal dopaminergic neuronal projections.Netrin-1 is also a secreted laminin family trophic factor with anti-inflammatory,anti-apoptotic,and neuronal survival effects and plays an essential role in supporting neuronal morphogenesis and signaling.However,the role of netrin-1 in manganese-induced neurotoxicity has been little explored.This study aimed to verify whether manganese overexposure inhibits the axon guidance factor Netrin-1,which impairs the function of striatal dopaminergic neurons in mice and the motor impairment caused by projection damage.It provides new ideas and targets for preventing and treating manganese overexposure and in-depth studies on the neurotoxic mechanisms of manganese impairment of motor function.Accordingly,this study will use an integrated molecular biology approach,neurobehavior,morphology,and constructing a netrin-1recombinant protein mouse model through animal experiments.We will explore the key role of manganese-dependent axon guidance molecule netrin-1 downregulation in manganese-induced transport function and projection damage of DA neurons,and further elucidate the mechanism of manganese-induced neurotoxicity.Methods:The common manganese dyeing model used male and female SPF adult wild-type C57BL/6 mice weighing 20±2 g.The mice were divided into 4 groups at random,based on body weight.16 mice in each group,64 mice in total,as the control group,low Mn group,medium Mn group,and high Mn group,respectively.In order to build a model of manganese poisoning in mice,Mn Cl₂was administered intraperitoneally with 0.9%Na Cl,12.5 mg/kg,25 mg/kg and 50 mg/kg Mn Cl₂at a volume of 5 ml/kg for 14 days.64 mice with the same conditions as above were selected for the Netrin-1 recombinant intervention model and randomly divided into 4groups according to body weight,16 mice in each group.The netrin-1 recombinant protein negative control group（Con-NC）,netrin-1 recombinant protein treated group（Con-Treated）,50 mg/kg Mn Cl₂group（Mn-NC）,and netrin-1 recombinant protein+50 mg/kg Mn Cl₂group（Mn-Treated）.Mice in the control group were injected intraperitoneally with 0.9%Na Cl,and mice in the Mn-dyed group were injected intraperitoneally with 12.5,25,and 50 mg/kg Mn Cl₂in a volume of 5 m L/kg for 14days,once daily.For netrin-1 recombinant protein injection:mice were injected with0.2μL PBS or netrin-1 recombinant protein into the striatal region via brain stereotaxic localization.After the model was successfully established,behavioral tests were performed to measure the movement of each group of mice.The pathological changes of striatum morphology were observed by HE staining.The pathological changes of neuronal Nissl body in the striatum were observed by Nissl staining.silver-plated staining was used to observe the damage of nerve fibers in the striatum of each group of mice.Immunofluorescence double-staining experiment,fluorescent gold retrograde tracer labeling experiment,LS18 tiling light film The nigrostriatal dopaminergic neuronal projection damage was observed in each group of mice by 3D fluorescence imaging.ELISA kits were used to detect the serum netrin-1 level and striatal DA level in each group of mice.The expressions of netrin-1,TH,DAT1,DRD3 were determined by Western blotting.The level of m RNA expression of netrin-1,TH,DAT1,and DRD3 was determined by RT-q PCR.Results:The manganese content in the striatum of mice increased significantly with the increase in the dose of poisoning.The results of behavioral experiments showed that the mice in the 50 mg/kg Mn group had limited voluntary activity,reached fatigue with significantly reduced running distance,stick time,moving distance,and moving speed in the open field,and Netrin-1 recombinant protein injection could reverse the motor impairment caused by manganese exposure.Netrin-1 recombinant protein injection improved the damage of striatal tissue induced by Mn exposure.The results of silver plating staining showed that the nerve fiber damage in the striatal tissue of mice in the 50 mg/kg Mn group was severe,and netrin-1recombinant protein injection improved the nerve fiber damage in the striatal tissue induced by manganese exposure.The results of immunofluorescence double-staining,fluorescence gold retrograde tracer labeling,and 3D fluorescence imaging by LS18 tiling microscope showed that the number of nigrostriatal DAergic projection neurons was significantly reduced,and the projection pathway was significantly damaged in the 50 mg/kg Mn group.Compared with the control group,DA levels in the striatum and netrin-1levels in the serum of mice were significantly reduced in the 50 mg/kg Mn group.Compared with the control group,the m RNA and protein expression of netrin-1,TH,DAT1,and DRD3 were decreased in the striatum of mice at50 mg/kg Mn.m RNA and protein expression of netrin-1,TH,DAT1,and DRD3 decreased after Netrin-1 recombinant protein injection.Conclusion:1.Mn causes impairment of the nigrostriatal dopaminergic neuronal projection pathway and motor dysfunction in mice by inhibiting netrin-1 expression.2.Mn causes downregulation of TH,DAT1,and DRD3expression through downregulation of netrin-1 expression,affecting dopamine function.