Anti-Inflammatory Effect And Mechanism Of Mitochondrial Glutamine Transporter SLC1A5＿var On Astrocytes In Parkinson’s Disease Model Mice

Parkinson’s disease(PD)is a common neurodegenerative disease in middle-aged and elderly people.With the increasing problem of population aging,the incidence rate is also increasing year by year,causing certain economic pressure to the society.Despite significant advances in the pathophysiology of Parkinson’s disease,there is still no effective cure.The pathological feature of Parkinson’s disease is the progressive loss of dopaminergic(DA)neurons in the nigrostriatal pathway,accompanied by the formation of Lewy bodies and the activation and proliferation of glial cells.Studies have shown that oxidative stress,lysosomal and mitochondrial dysfunction are involved in the pathological process of Parkinson’s disease.When mitochondrial function is damaged,respiratory chain complexes will be affected,and the disturbance of respiratory chain complexes is closely related to the production of reactive oxygen species,suggesting that oxidative stress may be the pathogenic mechanism of mitochondrial dysfunction-related Parkinson’s disease.In addition,neuroinflammation is also involved in the glial cell-activated immune response that is closely related to Parkinson’s disease.The stability of the central nervous system requires many nutrients to maintain,such as amino acids and growth factors,among which amino acids can not only provide energy for cells,but also function as neurotransmitters to transmit nerve signals.As an excitatory neurotransmitter,glutamate is involved in all brain cell activities and is one of the most abundant amino acids in the brain;glutamine can be converted into glutamate to provide ATP for the brain and is an important source of energy for brain cells.The ability of astrocytes to take up glutamate is much higher than that of neurons,so there is a very critical metabolic coupling pathway between the two,that is,the glutamate-glutamine cycle,which is taken up by astrocytes.Glutamate is converted into glutamine,which is then taken up by neurons,where the glutamine transporter can transport glutamine synthesized in astrocytes into neurons and provide energy for neurons.Changes in the expression of glutamine transporter protein levels and amino acid residue modifications can affect glutamate and glutamine cycling between astrocytes and neurons.Previous studies in our laboratory have shown that the glutamine transporter SLC1A5 can promote the activation of the NLRP3 inflammasome and aggravate neuroinflammation by binding to NLRP3,thereby affecting the pathological changes of Parkinson’s disease.In 2020,the journal Cell Metabolism reported that a variant of SLC1A5(SLC1A5_var)exists on the mitochondrial membrane of cells,which has a mitochondrial-localized targeting signal,and knockdown of mitochondrial SLC1A5_var can inhibit cancer cell growth.On this basis,the previous study of this experiment found that astrocyte mitochondrial SLC1A5_var was opposite to the glutamine transporter SLC1A5 on the cell membrane,and the mitochondrial glutamine transporter SLC1A5_var could significantly inhibit the levels of various inflammatory factors in astrocytes.Object:To explore the anti-inflammatory effect and mechanism of mitochondrial glutamine transporter(SLC1A5_var)on astrocytes of Parkinson’s disease model mice.Methods:The MPTP subacute PD model was established in 3-month-old C57BL/6 mice,and the dopaminergic neuron marker TH and astrocyte marker GFAP were detected by immunohistochemistry in the substantia nigra pars compacta,and detected by multiple fluorescence staining.Co-labeling of SLC1A5_var,GFAP and Tom20,Western blot was used to detect midbrain TH,and Q-PCR was used to detect the levels of IL-1β,TNF-α and IL-6 inflammatory factors in midbrain tissue and the expression of A1 and A2 phenotype-specific markers.mRNA levels.Primary astrocytes were cultured,stimulated with LPS,and given mitochondrial glutamine transporter overexpression virus and small interference.The level of mitochondrial oxidative phosphorylation in primary astrocytes was detected by Seahorse cell energy metabolism analyzer.The expression of mitochondrial protein in primary astrocytes and mitochondrial protein SLC1A5_var in mouse midbrain was detected by WB.The levels of IL-1β,TNF-α,IL-6 and IL18 in primary astrocytes stimulated with LPS after overexpression of mitochondrial glutamine transporter were detected by Q-PCR,and their effect on inflammatory factors was detected.effect;flow cytometry was used to detect the changes of mitochondrial ROS and membrane potential in primary astrocytes overexpressing mitochondrial glutamine transporter and administered LPS.Immunofluorescence was used to detect mitochondrial ROS,membrane potential changes and p65 nuclear entry after overexpression of mitochondrial glutamine transporter and LPS stimulation.WB detected the phosphorylation levels of p65,p38,IKK and other proteins in the downstream pathways of TLR4 stimulated by LPS after overexpression of mitochondrial glutamine transporter.Results:1.Mitochondrial SLC1A5_var ameliorates the neuronal damage and increased inflammation in MPTP-induced PD model mice.C57BL/6 mice were used for stereotaxic injection of LPS and subcutaneous injection of MPTP for PD modeling,and the mitochondrial SLC1A5_var protein and RNA in the midbrain of mice were detected.The results showed that the expression of glutamine transporter in the midbrain of LPS and MPTP model mice was significantly reduced;suggesting that SLC1A5_var was significantly down-regulated in PD animal models.Compared with the control group,the MPTP group significantly lost TH-positive neurons and activated GFAP proliferation.while overexpression of SLC1A5_var attenuated MPTP-induced loss of TH-positive neurons and inhibited the proliferation and activation of GFAP in mice.In addition,the levels of inflammatory factors IL-1β,IL-6,and TNF-α in the midbrain of the MPTP model group were increased,and the reactive astrocyte A1 was activated,while overexpression of SLC1A5_var could significantly reduce IL-1β,IL-6,TNF-α levels,and reduced astrocyte A1 transformation.2.The expression of SLC1A5_var in primary astrocytes stimulated by LPS and MPP+was significantly decreased.The cultured primary astrocytes were treated with PBS,LPS and MPP+,respectively.The mitochondrial proteins were extracted and found that compared with the PBS control group.LPS and The protein expression of SLC1A5_var in the MPP+group was significantly decreased,and the mRNA level was also significantly down-regulated.3.SLC1A5_var improves LPS-induced mitochondrial dysfunction in astrocytes.After overexpressing SLC1A5_var,LPS was administered to primary astrocytes,and it was found that compared with the control group,the maximum respiratory potential and basal respiratory value decreased after LPS stimulation,that is,the level of oxidative phosphorylation decreased,while overexpression of SLC1A5_var ameliorated the decreased level of mitochondrial oxidative phosphorylation in astrocytes induced by LPS;in addition,compared with the control group,the mitochondrial membrane potential JC-1 decreased and the level of mitochondrial ROS increased in the LPS group high,while overexpression of SLC1A5_var inhibited LPS-induced decrease in mitochondrial JC-1 and increase in ROS level.4.Mitochondrial SLC1A5_var of astrocytes reduces LPS-mediated increase of inflammatory factors.LPS-stimulated astrocytes can observe the entry of p65 into the nucleus,while overexpression of SLC1A5_var can abolish the entry of p65;LPS stimulation leads to IKK,p3 8,AKT,JNK phosphorylation levels and inflammatory factors IL-1β,IL-6,IL-18,TNF-α levels increased,while overexpression of SLC1A5_var reduces IKK,p38,AKT,JNK phosphorylation levels and inflammation factors IL-1β,IL-6,IL-18,TNF-α levels.Conclusions:1.Overexpression of mitochondrial SLC1A5_var can alleviate the inflammatory response of astrocytes in Parkinson’s disease model mice and has neuroprotective effects on MPTP model mice.2.SLC1A5_var promotes mitochondrial oxidative phosphorylation levels in astrocytes,reduces mitochondrial reactive oxygen species levels and maintains membrane potential stability.3.SLC1A5_var can improve the inflammatory process of astrocytes stimulated by LPS and MPP+,and reduce the level of inflammatory factors.The major contributions of the present study lie in:1.This study found that there are different glutamine transporters on the mitochondrial membrane of astrocytes and the plasma membrane,and they play an important role in mitochondrial function.2.In this study,it was found that the mitochondrial glutamine transporter SLC1A5_var can improve astrocyte inflammation caused by LPS,which provides a new idea for the study of neuroinflammation.