Small Extracellular Vesicles From MPP~+-stimulated Astrocytes Induce Neuronal Apoptosis Through MiR-543-3p/Sirt1 Mediated Accumulation Of Fatty Acids

Objective:Parkinson’s disease(PD),the second most popular neurodegenerative disease only after Alzheimer disease,is most common in people around the age of 60.The onset of PD is mostly insidious and the progression is slow.When the motor function damage such as slow movement,stiffness and tremor occurs,it is already in the advanced stage of pathology.The major pathological change is the death of dopaminergic neuronal cells in the substantia nigra,which leads to the notable reduction of dopamine content in the striatum,but the cause and specific mechanism remain unclear.Recent reports show that the excess fatty acid(FA)may cause lipid peroxidation,oxidative stress,mitochondrial uncoupling and dysfunction of neuronal cells,and participate in the pathological changes of PD.Therefore,fine control and timely elimination of free FA in neuronal cells are essential to prevent PD-related neurodegeneration.Astrocytes are the most common cell type in the central nervous system and are major immune and response cells.Reactive astrocytes release a variety of molecules that increase inflammation and affect neuronal cell function.Small extracellular vesicles(s EVs)are small vesicles with membrane structure produced and released by cells,which can mediate communication between cells,and micro RNAs(miRNAs)carried by them have been shown to be associated with PD.Recent studies have found that miR-543-3p is highly expressed in MPP~+-treated astrocytes,regulating glutamate transporters and causing motor dysfunction,suggesting that miR-543-3p may be involved in the occurrence and development of PD.Our previous study found that miR-543-3p was highly expressed in s EVs released by PD-reactive astrocytes.Do astrocytes influence neuronal cell death through miR-543-3p in s EVs.By bioinformatics analysis,we found that Silent information regulator 1(Sirt1)is the target gene of miR-543-3p.Studies have shown that Sirt1 can regulate lipid metabolism and autophagy,and regulate neuronal cell apoptosis to reduce the damage caused by neurodegenerative diseases.Based on the above studies,we hypothesized that s EVs released by PD-reactive astrocytes lead to neuronal apoptosis through miR-543-3p/Sirt1-mediated fatty acid retention.Methods:1.First of all,reactive astrocytes were obtained by MPP~+treatment,and the expression of reactive astrocyte marker proteins and miR-543-3p was verified by Western blot and Quantitative Real-time polymerase chain reaction,respectively.2.Ultrafiltration and ultracentrifugation were used to separate the small extracellular vesicles of the supernatant of astrocytes culture.The small extracellular vesicles were identified by Flow Nano Analyzer,transmission electron microscopy and Western blot,and the expression of miR-543-3p in the small extracellular vesicles was detected by Quantitative Real-time polymerase chain reaction.3.A co-culture system of astrocytes and neuronal cells was established,and immunofluorescence and Quantitative Real-time polymerase chain reaction were used to verify that miR-543-3p of astrocytes could be transferred to neuronal cells through small extracellular vesicles.4.Then,the neuronal cell activity treated with astrocytes-derived small extracellular vesicles was detected,and the apoptosis of neuronal cells was detected by Western blot.The apoptosis of neuronal cells was verified by co-culture and miR-543-3p mimics.5.Bioinformatics analysis was used to predict the target genes of miR-543-3p,and their functional enrichment was analyzed.6.Then fatty acid content in neurons treated with astrocytes-derived small extracellular vesicles was detected,and lipid peroxidation in neuronal cells was detected.Lipid droplets were observed by immunofluorescence,and oxidative stress and mitochondrial function were detected by Western blot.7.miR-543-3p mimics and antioxidant N-Acetyl-L-cysteine(NAC)were used to explore the specific mechanism of miR-543-3p induced fatty acid retention in neuronal cells.8.The si Sirt1 knockdown neuronal cells was used to verify the function of Sirt1 by immunofluorescence and Western blot.9.Finally,the target genes of miR-543-3p were verified by Quantitative Real-time polymerase chain reaction,Western blot and Dual-luciferase reporter gene assay.Results:1.The expression of miR-543-3p increased in reactive astrocytes activated by MPP~+,and the difference was statistically significant(P=0.0004).2.The size of small extracellular vesicles ranged from 50 nm to 200 nm.Under transmission electron microscope,the small extracellular vesicles were double-concave disks.Three positive protein markers ALIX,TSG101 and CD9 of small extracellular vesicles were detected by Western blot,while the negative protein marker GM130 was negative.3.Quantitative Real-time polymerase chain reaction and immunofluorescence observed miR-543-3p transfer from astrocytes to neuronal cells via small extracellular vesicles.4.Cell activity and Western blot results showed that MPP~+-stimulated astrocytes-derived small extracellular vesicles induced neuronal apoptosis through miR-543-3p.5.The target gene of miR-543-3p is enriched in regulating fatty acid transport,and its fatty acid toxicity may lead to neuronal apoptosis.6.MPP~+-stimulated astrocytes-derived small extracellular vesicles mediated fatty acid retention through miR-543-3p,leading to lipid peroxidation,oxidative stress and mitochondrial damage in neuronal cells.The specific mechanism is that miR-543-3p is highly expressed in neuronal cells through small extracellular vesicles,which affects the extracellular transport of fatty acids and leads to fatty acid toxicity.7.miR-543-3p regulated fatty acid transport by targeting Sirt1 through small extracellular vesicles,leading to its retention in neuron cells.Conclusions:1.miR-543-3p is highly expressed in MPP~+-stimulated astrocytes and their secreted small extracellular vesicles.2.miR-543-3p induce neuronal apoptosis by MPP~+-stimulated astrocytes-derived small extracellular vesicles.3.miR-543-3p mediated neuronal fatty acid retention through MPP~+-stimulated astrocytes-derived small extracellular vesicles,leading to lipid peroxidation,oxidative stress and mitochondrial damage.4.miR-543-3p mediated fatty acid transport dysfunction in neuronal cells by targeting Sirt1through small extracellular vesicles,leading to fatty acid toxicity.