The Effects Of TMEM16F-mediated Microglia Polarization On The Neuroinflammation Of Alzheimer’s Disease And Its Regulatory Mechanism

Objective:Alzheimer’s disease（AD）is a common progressive neurodegenerative disease,which is clinically characterized as cognitive dysfunction and memory loss,accompanied by personality changes and behavioral abnormalities.The main pathological manifestations are senile plaques formed by continuous deposition of Aβoligomer and neurofibrillary tangles formed by phosphorylated tau protein.The pathogenesis of AD is complex and unclear,and there is no effective strategy for prevention and treatment.The existing therapeutic drugs can only delay the progression of the disease.In recent years,microglia-mediated neuroinflammation has been shown to play a crucial role in the pathogenesis of AD.The dynamic process that activated microglia show the different functional phenotypes according to the different of microenvironment and the stimulus is called microglia polarization,which can be divided into the proinflammatory M1 phenotype and anti-inflammatory M2phenotype.In the pathogenesis of AD,activated microglia gather around Aβplaques,and the accumulating Aβoligomer can induce microglia polarization to M1-type with releasing pro-inflammatory factors,aggravating AD neuron loss,accelerating tau protein phosphorylation and causing synaptic dysfunction.M2 microglia play its anti-inflammatory effect while enhancing phagocytosis activity and secrete Aβ-related degradation enzyme to remove Aβplaques.Therefore,how to regulate activated microglia to convert them into M2 phenotype has become a hot topic in AD treatment.As a member of Anoctamins（ANO）family,transmembrane protein 16F（TMEM16F）has the dual functions of calcium-activated chloride channel and scramblase.In the central nervous system,TMEM16F can participate in the phenotypic transformation of microglia and regulate the migration and phagocytic activity of microglia.Studies have shown that TMEM16F can adjust the excitability of anterior horn motor neurons of spinal cord in amyotrophic lateral sclerosis.Moreover,neuronal TMEM16F can make phosphatidylserine expose to the surface of cells,which induces phagocytosis of apoptotic neurons in the ischemic penumbra by microglia and aggravates the ischemia-reperfusion injury of ischemic stroke.These results suggest that TMEM16F may be one of the participants in the pathogenesis of AD.NLRP3 inflammasome is composed of NLRP3,ASC and Caspase1 precursor proteins,which are involved in the occurrence and development of AD and are closely related to the neuroinflammation mediated by AD.During the pathogenesis of AD,NLRP3inflammasome is overactivated.It can increase the production of pro-inflammatory factor IL-1β,weaken the phagocytosis ability of microglia to Aβ,promote Aβaggregation and tau protein formation.In this study,the regulation of TMEM16F on microglia polarization in AD model and its relationship with NLRP3 inflammatory body were observed from two aspects:in vivo animal experiment and in vitro cell experiment.Methods:1.Expression and distribution of TMEM16F in Alzheimer’s animal and cell models:（1）C57BL/6 mice and APP/PS1 transgenic mice aged 3,6,9 and 12 months were divided into AD model group and Control group with 15 mice in each group.The expression level of TMEM16F in each group was detected by western blot.The distribution and expression of TMEM16F in hippocampal CA1,CA3 and DG regions were detected by immunohistochemistry.The co-expression of iba1 and TMEM16F in the hippocampus was shown as the immunofluorescence figures.（2）Human microglia HMC3 was stimulated by Aβ_25-35to construct AD microglia model.The optimal concentration of Aβ_25-35was selected by CCK8 assay.Weternblot was used to detect the effect of different concentrations of Aβ25-35 on TMEM16F expression in microglia.2.Effects of TMEM16F-mediated microglia polarization on cognitive behavior and pathological damage in APP/PS1 mice:Nine-month-old APP/PS1transgenic mice and their litter wild-type mice were selected.siRNA-TMEM16F in vivo was injected into bilateral hippocampi of the mice by stereotactic technique,and AD animal model with TMEM16F knockdown was constructed.The experiment was divided into four groups:Wild type group（WT group）,APP/PS1 group（AD model animal group）,APP/PS1-NC（APP/PS1 mice were stereoscopically injected with siRNA negative control）,and APP/PS1 siRNA-TMEM16F group（APP/PS1 mice were stereoscopically injected with siRNA-TMEM16F in vivo）.Morris water maze was used to evaluate the spatial learning and memory ability of mice in each group,and their escape latency,retention time in the target quadrant and times of crossing the platform in the exploration experiment were recorded.TMEM16F,M1 marker i NOS,M2 marker Arg1,NLRP3 inflammasome-related protein（NLRP3,pro-Caspase 1,ASC）and apoptosis-related protein（Bcl-2,Bax and cl-Caspase 3）were detected by western blot.The co-expression of iba1 with i NOS and NLRP3 in the cortex and hippocampus were detected by immunofluorescence assay.The secretion of IL-4,IL-6,IL-18 and IL-1βin brain tissues were detected by ELISA.Futhermore,the expression and distribution of Aβand Caspase 3 were detected by immunohistochemistry,and the pathological injury of brain tissues was detected by HE staining.The ultrastructure of hippocampal tissue was detected by transmission electron microscope.3.Study on the mechanism of NLRP3 inflammasome participating in the microglia polarization mediated by TMEM16F in AD:HMC3cells were transiently transfected and AD microglia with TMEM16F knockdown were constructed by Aβ_25-35stimulation.The experiment was divided into four groups:Blank Control group（Control group）,AD model group(Aβ_25-35treated group),siRNA-NC group(Aβ_25-35treated after transfection of siRNA-NC)and siRNA-TMEM16F group(Aβ_25-35treated after transfection of siRNA-NC).TMEM16F,M1 microglia markers（i NOS,Cox2）,M2 microglia markers（Arg1,Socs3）,NLRP3 inflammasome associated proteins（NLRP3,pro-Caspase 1,ASC）were analyzed by western blot.Real-time quantitative PCR was used to detect the expressions of pro-inflammatory cytokines IL-6,IL-1,TNF-αand anti-inflammatory cytokines IL-4,IL-10,TGF-βin microglia of each group,and the secretion levels of IL-1βand IL-18 in microglia of each group were detected by ELISA.The effect of microglia conditioned medium on the apoptosis ratio of SH-SY5Y cell were evaluated by flow cytometry.Results:1.（1）Compared with the litter wild-type control group,the expression of TMEM16F in APP/PS1 mice at the age of 6 months,9 months and 12 months was increased and the trend of TMEM16F in APP/PS1 mice at the age of 9 months was the most significant.The distribution of TMEM16F was observed in the hippocampus DG,CA1 and CA3 regions,and the distribution of TMEM16F in the CA3 region was more obvious.Compared with the Control group,the positive proportion of TMEM16F in the hippocampus of APP/PS1 mice at the age of 6,9 and 12 months increased.The proportion of TMEM16F positive cells was the highest at 9 months of age.Moreover,the co-expression level of iba1 and TMEM16F was upregulated in APP/PS1 mice.（2）TMEM16F was highly expressed in AD cell model,and 20μM Aβ_25-35was the optimal concentration for modeling.Morris water maze test revealed compared with the APP/PS1-NC group,the escape latency of APP/PS1siRNA-TMEM16F group was significantly shortened.In the probe test,APP/PS1siRNA-TMEM16F group had more retention time in target quadrant and times of crossing platform than APP/PS1-NC group.The western blot results showed that compared with APP/PS1-NC group,the expression levels of TMEM16F,i NOS,NLRP3,pro-Caspase 1,ASC in APP/PS1 siRNA-TMEM16F group decreased,while the expression level of Arg1 increased.The expression of micorglial i NOS and NLRP3 was observed by immunofluorescence double staining.After TMEM16F knockdown,the co-expression level of iba1 with inos and NLRP3 respectively were downregulated.After TMEM16F knockdown,Caspase 1 activity of hippocampus and cortex decreased in APP/PS1 mice.Compared with APP/PS1-NC group,the secretion levels of pro-inflammatory factors IL-6,IL-18 and IL-1βin APP/PS1siRNA-TMEM16F group were decreased,while the anti-inflammatory factor IL-4level was increased.Moreover,the ultrastructure of APP/PS1 mice hippocampus was improved by knocking down TMEM16F.Compared with APP/PS1-NC group,APP/PS1 siRNA-TMEM16F group increased the level of apoptosis-related protein Bcl-2,and decreased the expression levels of Bax and cl-Caspase 3.The proportion of Caspase 3 positive cells in cortex and hippocampus decreased,the damage of brain tissue was improved,and the deposition of Aβin brain was decreased.3.Knocking down TMEM16F can reduce the levels of i NOS and Cox2 in AD microglia and up-regulate the expression levels of M2 microglia markers（Arg1 and Socs3）.Further,real-time quantitative PCR was used to detect the expression levels of inflammatory factors.The results showed that TMEM16F knockdown decreased the levels of pro-inflammatory factors（IL-6,IL-1,TNF-α）and up-regulated the expressions of IL-4,IL-10,TGF-βin AD microglia.Compared with siRNA-NC group,the expression levels of NLRP3,pro-caspase 1 and ASC in siRNA-TMEM16F group were down-regulated and the secretion levels of IL-1βand IL-18 were decreased.After the intervention of NLPR3 agonist,Nigericin,the expression of M1 microglia markers（i NOS,Cox2）increased in the siRNA-TMEM16F+Nigericin group compared to the siRNA-TME16F group.Meanwhile,the expression of Arg1,a marker of M2 microglia,decreased,and the secretion levels of IL-1βand IL-18increased.Compared with the Control group,addition of conditioned medium from Aβ_25-35treated group could increase the level of apoptosis,while conditioned medium from siRNA-TMEM16F group could down-regulate the level of apoptosis and exert inhibitory effect on the activity of Caspase 3.Conclusions:1.TMEM16F is highly expressed in AD model;2.Knocking down TMEM16F can improve the spatial memory ability of APP/PS1 mice,promote the transformation of microglia into anti-inflammatory M2 type,inhibit the activation of NLRP3 inflammation,reduce brain cell apoptosis and Aβdeposition,and improve brain tissue injury.3.TMEM16F knockdown can convert microglia into M2phenotype through inhibiting the activity of NLRP3 inflammasome in AD;4.TMEM16F is expected to be a new therapeutic target for AD.