The Molecular Mechanism Of Thioredoxin-1 Regulating Mitochondria Fission/Fussion Dysfunction In Parkinson’s Disease

Parkinson’s disease（PD）is a neurodegenerative disease characterized by decreased dopamine（DA）secretion in the substantia nigra pars compacta（SNpc）and striatum.Its main pathological manifestations are the loss of dopaminergic neurons in SNpc and the formation of Lewy body（LB）.Its clinical symptoms are movement symptoms such as resting tremor,myotonia and delayed action,bradykinesia,postural balance disorder,and often accompanied by non-motor symptoms such as hypoolusia,depression and memory impairment.Mitochondrial fission and fusion dysfunction plays an important role in the pathogenesis of PD,but the underlying molecular mechanism still remains unclear.Mitochondria are dynamic organelles that constantly undergo fission and fusion.This dynamic balance is essential for maintaining the function of mitochondria.Mitochondrial dynamics-related proteins 1（Drp1）,Optic atrophy protein 1（OPA1）,Mitochondrial fusion protein 1（Mfn1）and mitochondrial fusion protein 2（Mfn2）are the major proteins involved in mitochondrial dynamics.When mitochondria divide,Drp 1 and mitochondrial fission protein 1（Fis1）gather in the outer membrane of mitochondria,form helical structure and cause mitochondria to divide into two.After splitting,Mfn1,Mfn2 and OPA1mediate the fusion of outer mitochondrial membrane and inner membrane,respectively.In the process of mitochondrial fusion,Mfn1 and Mfn2 promote the connection of two adjacent outer mitochondrial membranes,and then OPA1 promotes the fusion of inner mitochondrial membranes.Mitochondrial gene mutations that regulate mitotic fusion are associated with the occurrence and development of neurodegenerative diseases.Mitochondrial fission and fusion dysfunction lead to increased production of reactive oxygen species（ROS）,oxidative stress damage and decreased synthesis of adenosine triphosphate（ATP）and changes in Ca²⁺homeostasis,which damage dopaminergic neurons.Phosphatase and tensin homolog deleted on chromosome ten（PTEN）-mediated protein kinase 1（PINK1）and E3 ubiquitinated ligase Parkin not only protect dopaminergic neurons,but also have important effects on mitochondrial dynamics,but the exact molecular mechanism is still unclear.Thioredoxin-1（Trx-1）is an important redox regulatory protein in prokaryotic and eukaryotic cells with a molecular weight of 12 kDa.Trx-1 has the biological characteristics of antioxidation,maintains the reductive environment of cells,regulates transcription factors,inhibits apoptosis signal regulated kinase 1（ASK1）regulation pathway and protects cells.Overexpression of Trx-1 resists the neurotoxicity of environmental toxicant1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine（MPTP）and its metabolite 1-methyl-4-phenylpyridine（MPP⁺）,and inhibits endoplasmic reticulum stress induced by MPTP/MPP⁺in PD model.It protects dopaminergic neurons from damage.However,the molecular mechanism of Trx-1 regulating mitochondrial fission and fusion dysfunction in PD remains unclear.The aim of this study is to further demonstrate the effects of Trx-1 on motor movement disorder in PD model and to explore the molecular mechanism of Trx-1regulating the dysfunction of mitochondrial fission and fusion in PD model.The results are as follows:（1）Overexpression of Trx-1 improved the motor movement disorder of PD in mice.Wild type,Transgenic mice（TG）mice were used to construct the mice model of PD.The exercise ability of the mice was tested by open-field test,scratch and rotation test.The results showed that the ability of the mice injected with MPTP in open-field was weakened,the distance of movement was shortened,the time of movement above rotation was shortened and the ability of scratch was reduced,which indicates that MPTP induces the motor movement disorder of the mice.Overexpression of Trx-1 significantly improved the decrease of MPTP-induced autonomic motor ability in mice,suggesting that overexpression of Trx-1 improves the decrease of MPTP-induced motor movement.（2）Downregulation of Trx-1 in the substantia nigra of the midbrain on the motor behavior of mice.Adeno-associated virus Trx-1（mTrx-1）was injected into the SNpc of mice by stereotaxis sugery to reduce the expression of Trx-1.A mouse model of PD was constructed with MPTP.Behavioral tests showed that after Trx-1 knockdown,the motor ability of mice was decreased significantly after MPTP treatment,and the motor ability of mice after Trx-1 knockdown was aggravated,indicating that knockdown of Trx-1aggravated the motor ability injury of mice induced by MPTP.（3）The effects of overexpression of Trx-1 on mitochondrial fission and fusion dysfunction and its molecular mechanism.Construction of mouse model with overexpression of Trx-1 under electron microscopy,the mitochondrial fragments increased and mitochondrial fission abnormalities were observed in the SNpc of mice injected with MPTP.The recovery of mitochondrial fission and fusion dysfunction in overexpression of Trx-1 transgenic mice was observed.These results suggest that Trx-1 regulates the balance of mitochondrial fission and fusion.Western Blot was used to detect the expressions of PINK1 and Parkin in the ventral tegmental area（VTA）.The results showed that the expressions of PINK1 and Parkin in the VTA of mice injected with MPTP were significantly increased,while the overexpression of Trx-1 significantly inhibited the increases of PINK1 and Parkin induced by MPTP.The expressions of Drp1 and Mfn1 in the VTA of mice treated with MPTP were increased significantly,while the overexpression of Trx-1 significantly inhibited the increases of Drp1 and Mfn1 induced by MPTP.（4）The effect of Trx-1 knockdown on mitochondrial fission and fusion dynamics molecules.The expressions of PINK1,Parkin,Drp1 and Mfn1 were detected by Wesrern Blot.The results showed that the expressions of PINK1,Parkin,Drp1 and Mfn1 were decreased after Trx-1 knockdown.After downregulation of Trx-1 and MPTP treatment,the expressions of PINK1,Parkin,Drp 1 and Mfn1were increased more significantly than those in negative group.In conclusion:Overexpression of Trx-1 improves the motor movement disorder of PD mice,while knockdown of Trx-1 aggravates motor movement disorder of PD mice.Trx-1 improves the dynamic balance between mitochondrial fission and fusion induced by MPTP through regulating the expressions of PINK1/Parkin and mitochondrial dynamics related molecules,restoring dynamic balance between mitochondrial fission and fusion,then improves the functions of mitochondria and protects dopaminergic neurons from the toxic damage of MPTP.The dynamic balance between mitochondrial fission and fusion regulated by Trx-1 may be an important strategy for the treatment of PD.