MT1 Regulates The Phagocytosis Of α-synuclein By Microglia And The Underlying Mechanisms

Aim:To explore the role and underlying mechanism of Melatonin Receptor 1(MTI)in microglial phagocytosis and its effect on the pathological aggregation of α-synuclein(αsyn),with the goal of clarifying the function of MT1 in microglia and its role in the pathogenesis of Parkinson’s disease(PD),which may provide a novel insight into the pathology and treatment of PD.Methods:Mouse microglial cell line BV2,primary microglia and primary cortical neurons were utilized as experimental subjects.The study was conducted in the following stages:1.Small interference RNA si-Mtnrla was employed to knock down the gene and protein expression of MT1 in BV2 cells,and their phagocytic function of latex beads and zymosan was assessed via immunofluorescence(IF).2.Wild-type(WT)and Mtnr1a knockout(MT1-KO)mice were generated,from which primary microglia were isolated.Phagocytosis of latex beads and zymosan by these cells was evaluated using IF.3.RT-qPCR was used to determine the expression of LC3-associated phagocytosis(LAP)-related genes(such as Rubicon,Nox2,and Uvrag)in si-Mtnr1a treated BV2 cells.Western Blotting(WB)was employed to analyze the expression of LAP-related molecules(e.g.,RUBICON,PI3KCIII,UVRAG,Beclin-1,NOX2,ATG5,and LC3).Additionally,WB was utilized to assess LAP-related protein expression in WT and MT1-KO mouse primary microglia.4.MT1 overexpression plasmid and MT1 agonist Ramelteon were used to treat BV2 cells and subsequent changes in LAP-related proteins were analyzed.5.The ability to phagocytose αsyn-GFP monomer of WT and MT1-KO mouse primary microglia were observed via IF.Moreover,BV2 cells,WT,and MT1-KO mouse primary microglia were treated with preformed fibril(PFF)α-syn to examine MT1 expression and α-syn aggregation.6.A neuron-microglia co-culture model was established by inducing the accumulation of α-syn in primary neurons using PFF to observe the clearance ability of WT and MT1-KO primary microglia on pathological α-syn in neurons.The distribution and expression of phosphorylated α-syn at Ser129(pS129-α-syn)were detected by IF and the levels of soluble and insoluble α-syn was evaluated using WB,clarifying the role of microglial MT1 in α-syn progression of PD.Results:1.MT1 suppression diminished the capacity of BV2 cells to phagocytose latex beads and zymosan.RT-qPCR results revealed a reduction in the expression of LAPassociated genes(e.g.,Rubicon,Nox2,and Uvrag)following Mtnr1a knockdown.WB indicated a decrease in LAP-related protein expression,including RUBICON,PI3KC-Ⅲ,UVRAG,Beclin-1,NOX2,ATG5,and LC3.2.Compared to the WT group,MT1-KO microglia demonstrated reduced the phagocytic capacity for latex beads and zymosan.WB results displayed a decrease in the expression of LAP-related proteins,such as RUBICON,PI3KC-Ⅲ,Beclin-1 and LC3.3.Overexpression or activation of MT1 in BV2 cells led to increased LAP-related protein expression,consistent with the results of the small interfering RNA knockdown test.IF results revealed that RUBICON expression in primary microglia was enhanced after MT1 stimulation.4.The result of IF demonstrated that the phagocytosis of α-syn-GFP monomer in primary microglia from MT1-KO mice was decreased compared to the WT group.PFF treatment reduced MT1 expression in BV2 cells,and after PFF treatment of primary microglia,the MT1-KO group accumulated more α-syn in primary microglia compared to the WT group.5.PFF was able to induce the accumulation of α-syn in primary neurons.Upon co-culture with microglia,the phagocytic and scavenging abilities of microglia for neuropathic α-syn were impaired,and the levels of pS129-α-syn and insoluble α-syn aggregates were higher in the MT1-KO group compared to the WT group.Conclusion:MT1 plays a regulatory role in microglial phagocytosis,which is related to LC3-associated phagocytosis.Deletion of MT1 exacerbates α-syn aggregation in microglia and impairs phagocytosis and clearance of pathological α-syn in neurons.