Research On Mechanisms That CXCL1 Regulate Proliferation And Differentiation Of Neural Stem Cells In APP/PS1 Mice

Background and Objective Alzheimer’s disease is a degenerative disease and the most common cause of dementia.In the Alzheimer’s disease,the damage of the neurons performing cognitive function leads to the dementia;the neurons that perform basic body functions are also damaged in the late stage,which causes the patient to be bedridden and eventually died of various complications.In the cerebrospinal fluid of patients with early and very early Alzheimer’s disease,the level of CXCL1 is elevated and is considered to be one of the indicators to the diagnosis of Alzheimer’s disease at early stage.The APP/PS1 mice carry mutant human presenilin gene and human/murine amyloid proprotein fusion gene,which is a classical animal model of Alzheimer’s disease.Does the level of CXCL1 in the cerebrospinal fluid have abnormal change in the APP/PS1 mice as in patients?The issue has not been clearly answered.In the peripheral tissues,CXCL1 is mainly secreted by vascular endothelial cells,activated macrophages,and fibroblasts.CXCL1 can be secreted by neurons,microglia,oligodendrocytes and activated astrocytes in the brain.We previously found that the monocytes with high expression of CXCL1 are involved in the disruption of the blood-brain barrier in APP/PS1 mice,suggesting that CXCL1 may be involved in the pathogenesis of Alzheimer’s disease,so it is necessary to elucidate the source of the CXCL1 in the brain.In this study,we analyzed the secretion of CXCL1 in Alzheimer’s disease by immunofluorescence co-localization analysis in vivo and inflammatory cell activation assay in vitro. Neural stem cells have two basic properties:the one is the self-renewal,neural stem cells can produce daughter neural stem cells through symmetric division and asymmetric division;the other is the differentiation,the neural stem cells can differentiate into astrocytes,neurons and oligodendrocytes.In the central nervous system of adult mammalian,the subventricular zone in the lateral ventricle and the subgranular layer in the hippocampus are the primary location where neural stem cells reside and neurogenesis occurs.The neural stem cells in the subventricular zone can directly contact the cerebrospinal fluid via cell processes.The cerebrospinal fluid contains many growth factors such as epidermal growth factor and fibroblast growth factor-2,which play an important role in maintaining self-renewal and neurogenesis of neural stem cells.Does the CXCL1 in the cerebrospinal fluid play a role in the biological behavior of neural stem cells located in the subependymal layer?To elucidate this problem,we used CXCL1 to stimulate neural stem cells in vivo and in vitro,and then analyzed its effects on the proliferation and differentiation of neural stem cells. Reactive oxygen species（ROS）is an important second messenger.The sources of endogenous ROS mainly include the mitochondrial pathway and the NADPH oxidase pathway.The NADPH oxidase family consists of seven members,NOX1-5,Doux1 and Doux2.The endogenous production of ROS is closely related to the proliferation of neural stem/progenitor cells.In the mechanism section,we analyzed the effect of CXCL1 on the production of reactive oxygen species in neural stem cells in vitro and confirmed it by in vivo rescue experiments.We further analyzed the downstream signaling pathways of reactive oxygen species.Method 1.The detection of CXCL1 protein level in the cerebrospinal fluid of APP/PS1 mice The genotype of APP/PS1 mice was identified by PCR.The cerebrospinal fluid of the4,8,and 12 month-old APP/PS1 mice and their wild-type littermate was obtained by puncture of medullary pool,and the protein level of CXCL1 was detected by ELISA. 2.The analysis of the source of CXCL1 in middle-aged APP/PS1 mice The co-localization of CXCL1 with neuronal marker MAP2,astrocytic marker GFAP,macrophage markers F4/80 was detected by immunofluorescence. 3.The determination of the secretion and synthesis of CXCL1 after activation of macrophages and microglia LPS and ATP were used alone or in combination to stimulate the macrophage cell line RAW264.7 and the microglial cell line BV2 cell line.The supernatant was collected,and the protein level of CXCL1 was detected by ELISA.The total RNA was extracted and the mRNA expression of CXCL1 was detected by Real-Time PCR.4.The effects of CXCL1 on the proliferation of neural stem cells in vitro The effect of CXCL1 on the viability of neural stem cells was examined by WST-8assay.The experiment was designed to include the control group,the low-dose CXCL1group and the high-dose CXCL1 group.The cell viability assay was lasted for 4 days.The effect of CXCL1 on neural stem cell proliferation in vitro was further analyzed by BrdU incorporation assay.The experiment was designed to include the control group,the low-dose CXCL1 group and the high-dose CXCL1 group.The BrdU⁺Nestin⁺cells was determined by immunofluorescence assay,and its percentage was calculated after 24 hours of cell culture. 5.The effect of CXCL1 on the proliferation of neural stem cells in vivoThe experiment was designed to include the control group,the low-dose CXCL1group and the high-dose CXCL1 group.The CXCL1 was delivered into the lateral ventricle of the wild-type mice by stereotactic injection,and the BrdU were intraperitoneally injected to label the proliferating neural stem cells.After the 7 days,the brain was taken,fixed,and oscillated.Immunofluorescence assay was used to detect the BrdU⁺Nestin⁺cells in the subventricular zone of the lateral ventricle,and its number was calculated. 6.The effect of CXCL1 on the differentiation of neural stem cells in vitro The experiment was designed to include the control group,the low-dose CXCL1group and the high-dose CXCL1 group.After the 6 days for astrocytic differentiation culture,the GFAP⁺cells,MAP2⁺cells,and the Nestin⁺cells was determined by immunofluorescence assay,and their percentages were calculated.The expression of GFAP and Tuji-1 in the neural stem cells were detected by immunoblotting.7.The effects of CXCL1 on the production of reactive oxygen species in neural stem cells in vitro The experiment was designed to include the control group,the low-dose CXCL1group and the high-dose CXCL1 group.The nuclei were stained with Hoechst 33342,H2DCFDA was used to label the reactive oxygen species generated in the cells,then the average fluorescence intensity of the cells was calculated. The expression of NOX2/gp91phox protein was detected by immunoblotting. 8.The study of the intracellular signaling pathway of the CXCL1 Immunoblotting was used to detect the phosphorylation levels of Akt and Stat3 in neural stem cells. 9.The effect of the inhibition of production of reactive oxygen species on the action of the CXCL1 in vivo The experiment includes the 10%DMSO group,the CXCL1 group and the Apocynin plus CXCL1 group.The CXCL1 and Apocynin were administrated into the lateral ventricle of the wild-type mice by the stereotactic injection,and the BrdU used to label proliferating neural stem cells were intraperitoneally injected to the mice.After the 7 days,the brain was taken,fixed,and oscillated.Immunofluorescence assay was used to detect the BrdU⁺Nestin⁺cells in the subventricular zone of the lateral ventricle. 10.The effect of CXCL1 on the mitochondrial membrane potential of the neural stem cells The experiment includes the control group,the low-dose CXCL1 group and the high-dose CXCL1 group.The mitochondrial membrane potential of the neural stem cells were detected by JC-1 staining.Result 1.In the cerebrospinal fluid of the middle-aged APP/PS1 mice,the levels of CXCL1were higher than the one in the wild-type littermate,and higher than the one in the adult APP/PS1 mice（4 month old）.2.The CXCL1 colocalizes with neurons and macrophages in the brain of APP/PS1mice. 3.The activated macrophages produce and secrete more CXCL1,while the synthesis and secretion of the CXCL1 did not change in the activated microglia. The mRNA expression and protein secretion of the CXCL1 increased significantly after RAW264.7 cells were activated by LPS.LPS combined with ATP stimulated RAW264.7 cells to produce and secret more CXCL1 than LPS alone.The stimulation of LPS and ATP had no significant effect on the synthesis and secretion of the CXCL1 in BV2cells.4.The CXCL1 promotes the proliferation of the neural stem cells in vitro The WST-8 experiments showed that the viability of the high-dose CXCL1-treated neural stem cells increased significantly on the 3^rd,the 4^th days,and the low-dose CXCL1-treated neural stem cells increased significantly on the 4^th days,compared with the control group. The BrdU incorporation experiments showed that the percentage of BrdU⁺Nestin⁺cells increased significantly after treatment with the high-dose CXCL1 for 24 hours.There were no significant difference between the low-dose group and the control group. 5.The CXCL1 promotes the proliferation of the neural stem cells in the subventricular zone. After the delivery of the high-dose CXCL1,the number of the BrdU⁺Nestin⁺cells in the subventricular zone increased significantly.There were no significant difference between the low-dose group and the control group in the number of the BrdU⁺Nestin⁺cells.6.The CXCL1 inhibits the differentiation of neural stem cells to astrocytes in vitro On the 6^th day,compared with the control group,the percentage of the GFAP⁺cells in the high-dose CXCL1-treated group decreased significantly,and the percentage of MAP2⁺cells and Nestin⁺cells did not change significantly.There was no significant difference between the low-dose group and the control group in the differentiation of the neural stem cells.The levels of GFAP in the high-dose CXCL1-treated neural stem cells were also decreased in immmunoblotting expriment. 7.The CXCL1 promotes the expression of NOX2/gp91phox in vitro and increases the production of reactive oxygen species in the neural stem cells The high-dose of CXCL1 increased reactive oxygen species in the neural stem cells,but the low-dose CXCL1 had no significant effect. The high-dose of CXCL1 increased the NOX2/gp91 phox,and the low-dose of CXCL1 had no significant effect.The results suggest that CXCL1 promotes reactive oxygen species generation by the NADPH oxidase pathway. 8.The CXCL1 increased Akt phosphorylation,but has no effect on Stat3phosphorylation in the neural stem cells The high-dose of CXCL1made the ratio of pAkt to Akt increased in the 12^th and 24^thh hours in the neural stem cells,but the low-dose of CXCL1 had no significant effect. The CXCL1 had no significant effect on the ratio between pStat3 and Stat3 in the 12^thh and 24^th hours in the neural stem cells.9.The action of the CXCL1 on the neural stem cells can be blocked by the inhibition of the production of reactive oxygen species. The number of the BrdU⁺Nestin⁺cells in the subventricular zone was significantly reduced by the delivery of the Apocynin in the mice which had been given the high-dose CXCL1.There was no significant difference between the mice given Apocynin and CXCL1 and the mice given only 10%DMSO in the number of the BrdU⁺Nestin⁺cells in the subventricular zone.10.The CXCL1 has not effect on the mitochondrial membrane potential of neural stem cells.The CXCL1（100ng/mL,10ng/mL）had no significant effect on the mitochondrial membrane potential of neural stem cells in the 24^th hours.Conclusion In Alzheimer’s disease,the neuron-and activated macrophage-derived CXCL1 can act on the neural stem cells located in the subependymal layer through cerebrospinal fluid,promotes the proliferation of the neural stem cells by the NOX2-ROS-PI3K/Akt pathway,and inhibits the differentiation of the neural stem cells to astrocyte,thereby playing a protective role.