The Role And Mechanism Of FUNDC1 In Regulating Parkinson’s Disease Under Hypoxia By Promoting Mitochondrial Autophagy

Objective: Parkinson’s disease(PD)is the second most common neurodegenerative disease after Alzheimer’s disease.The main clinical manifestations are quiescence tremor,myotonia,motor retardation and postural gait disorders,etc.Pathological changes were mainly manifested as progressive loss of dopaminergic neurons in the densification part of the substantia nigra,deposition of α-Synuclein(α-syn),and formation of intracellular Lewy body(LB).A large number of dopaminergic neurons had been involved when clinical symptoms of Parkinson’s disease appeared.As a result,early diagnosis is difficult,and current treatments can only relieve clinical symptoms,but cannot reverse neuronal death.Therefore,in-depth study of the pathogenesis of Parkinson’s disease and search for potential therapeutic targets are of great significance for early diagnosis and prevention of the disease process.Heredity,environment,oxidative stress and mitochondrial autophagy may be one of the causes of Parkinson’s disease.Mitochondrial autophagy homeostasis plays an important role in maintaining the integrity of neuronal cells and providing energy metabolism.Mitochondrial autophagy is in a state of disorder in dopaminergic neurons in the substania nigra of patients with Parkinson’s disease.Overactivated mitochondrial autophagy leads to serious damage to cell integrity and breakdown of energy metabolism,inducing the release of more inflammatory factors.Various etiologies will eventually cause cell hypoxia,and hypoxia seems to be the common or final pathway of various etiologies.It has been reported that hypoxia will disrupt mitochondrial autophagy of cells.Oxygen intake disorders and oxygen utilization disorders were closely related to these causes.Hypoxia could promotes alphasynaptonucleoprotein modification,aggregation,and propagation.Hypoxia regulation may be a key therapeutic target for Parkinson’s disease.It has been reported that a complete mitochondrial outer membrane protein FUNDC1(FUN14 domain containing 1,FUNDC1),which is the receptor of mitochondria autophagy induced by hypoxia,has been found.FUNDC1 can interact with autophagy protein LC3 through its binding domain Y(18)xx L(21)to regulate mitochondrial autophagy,but the role of FUNDC1 in Parkinson’s disease remains unclear.Based on this,this study explored whether hypoxia regulates Parkinson’s disease through mitochondrial autophagy mediated by FUNDC1,hoping to use FUNDC1 as a therapeutic target to find a new breakthrough and direction for the defense and treatment of Parkinson’s disease.Methods: In the first part,the mouse model of Parkinson’s disease was constructed by MPTP.The effects of hypoxia on motor capacity,dopaminergic neurons,mitochondrial autophagy,inflammatory factors,FUNDC1 and HIF-1α expression in MPTP-induced Parkinson’s disease mice were investigated by behavioral tests,immunohistochemical staining,Nis-type staining,transmission electron microscopy,western blot and q RT-PCR.In the second part,the MPP + concentration suitable for SH-SY5 Y was explored by cell viability assay(CCK8 method),and the SH-SY5 Y cell line was induced by MPP + to construct the cell model of Parkinson’s disease.The effects of hypoxia on mitochondrial autophagy,apoptosis,inflammatory,FUNDC1 and HIF-1α expression in Parkinson’s disease cell models were investigated by western blotting,transmission electron microscopy,flow cytometry and immunofluorescence.Then,the mechanism of the effect of hypoxia on mitochondrial autophagy in Parkinson’s disease cell models was studied by means of si RNA interference on FUNDC1 gene expression,immunocoprecipitation and transmission electron microscopy.In the third part,the rescue effects of oxygen therapy and FUNDC1 inhibitor cell penetrating peptide P on MPTP-induced Parkinson’s disease mouse model and Parkinson’s disease cell model under hypoxia were explored through the experimental methods of western blotting,immunohistochemical staining and immunocoprecipitation,and the mechanism of action were preliminatively clarified.Results: 1.The results of behavioral experiments showed that hypoxia could increase the exercise time of MPTP mice in the rod-climbing experiment and reduce the exercise time of MPTP mice in the rod-rotating experiment.2.The results of immunohistochemical staining and Nith staining showed that hypoxia could decrease TH expression,increaseα-syn expression,and reduce the survival number of neurons in the substantia nigra of MPTP mice.3.Western blots showed that hypoxia increased the expressions of PINK1,LC3 II and p62 and decreased the expression of TIM23 in the substantia nigra of MPTP mice.4.The results of transmission electron microscopy showed that the number of autophagic vesicles increased and the number of mitochondria decreased in the substantia nigra neurons of MPTP mice under hypoxia condition.5.Western blots showed that hypoxia decreased the expression of apoptotic protein Bcl-2,but increased the expression of apoptotic protein Bax and Cleaved caspase3.6.The results of immunohistochemical staining and western blot showed that hypoxia increased the expression of i NOS and COX2 in substantia nigra of MPTP mice.7.Western blotting and real-time quantitative PCR results showed that hypoxia increased the m RNA transcription and protein expression level of HIF-1α in the substantia nigra of MPTP mice,and decreased the m RNA transcription and protein expression level of FUNDC1.8.The results of CCK-8 experiment showed that the cytotoxicity of MPP+ on SH-SY5 Y cells and the optimal concentration of administration were 3m M.9.Western blots showed that hypoxia increased the expression of PINK1,LC3 II,p62,Bax and Cleaved caspase3,decreased the expression of TIM23 and Bcl-2,increased the expression of i NOS and COX2,and increased the expression of HIF-1α in MPP+ treated SH-SY5 Y cells.The expression of FUNDC1 was decreased,and the results of transmission electron microscopy showed that hypoxia could increase the formation of autophagy vacuolar and reduce the number of mitochondria in MPP+ treated SH-SY5 Y cells.The results of apoptosis flow cytometry showed that hypoxia could increase the apoptosis of MPP+treated SH-SY5 Y cells,and the addition of 3-MA could partially reverse the above changes.10.Cellular immune fluorescence results showed that anoxia could increase the fluorescence intensity of COX2 and i NOS in MPP+ treated SH-SY5 Y cells,and the addition of 3-MA could reduce the fluorescence intensity of COX2 and i NOS.11.Western blot results showed that silencing FUNDC1 could decrease the expressions of PINK1 and LC3 II,increase the expression of TIM23,and decrease the expression of COX2 and i NOS in MPP+ treated SH-SY5 Y cells under hypoxia condition.12.Transmission electron microscopy results showed that silencing FUNDC1 could reduce the formation of autophagic vesicles and increase the number of mitochondria in MPP+cells under hypoxia.13.The results of co-immunoprecipitation showed that hypoxia could increase the interaction between LC3 II and FUNDC1 in MPP+-treated SH-SY5 Y cells,and HIF-1α inhibitor could decrease the interaction between LC3 II and FUNDC1.14.Transmission electron microscopy(TEM)showed that HIF-1α inhibitors could reduce the formation of autophagic vesiculae and increase the number of mitochondria in MPP+ treated SH-SY5 Y cells under hypoxia caused by FUNDC1 overexpression.15.Western blots showed that cell-penetrating peptide P could decrease the expression of PINK1,i NOS and COX2 and increase the expression of TIM23 in MPP+ treated SHSY5 Y cells under hypoxia condition.16.Co-immunoprecipitation results showed that cell penetration of peptide P could reduce the interaction between LC3 II and FUNDC1 in MPP+ treated SH-SY5 Y cells.17.Immunohistochemical and Nysch staining results showed that both oxygen administration and cell penetrating peptide P increased TH expression in the substantia nigra of MPTP mice under hypoxia condition,decreased α-syn expression and increased the survival number of neurons,and oxygen administration combined with FUNDC1 inhibitor cell penetrating peptide P could increase the above effects more significantly.18.The results of immunohistochemical staining showed that both oxygen administration and cell penetrating peptide P could reduce the expression of i NOS and COX2 in the substantia nigrum of MPTP mice under hypoxia condition,and oxygen administration combined with cell penetrating peptide P could significantly reduce the expression of i NOS and COX2.Conclusion: 1.Hypoxia can aggravate the movement disorder of MPTP-induced Parkinson’s disease mice,increase the death of dopaminergic neurons in the substantia nigra,promote mitochondrial autophagy,hinder the process of autophagy flow,promote inflammatory response and apoptosis,and decrease the expression of FUNDC1 and increase the expression of HIF-1α under hypoxia.2.Hypoxia can increase mitochondrial autophagy and promote inflammation and apoptosis in SH-SY5 Y cells treated with MPP+,and 3-MA and silting FUNDC1 can partially save the above changes.Mitochondrial autophagy in MPP+-treated SH-SY5 Y cells mediated by Fund C1-mediated hypoxia is regulated by HIF-1α/FUNDC1/LC3 pathway.3.FUNDC1 inhibitor can reduce mitochondrial autophagy and inflammatory response in MPP+ treated SHSY5 Y cells under hypoxia condition.Oxygen administration and FUNDC1 inhibitor can both reduce MPTP-induced dopaminergic neuron death in the substancia nigra of Parkinson’s disease mice and inhibit neuroinflammatory response.Oxygen administration combined with FUNDC1 inhibitors significantly inhibited neuroinflammatory responses and played a neuroprotective role in MPTP-induced Parkinson’s disease mice.