| Objective:Pericytes are mural cells covering brain capillaries and small veins and play an important role in neurodegenerative diseases such as Alzheimer’s disease,Parkinson’s disease,and amyotrophic lateral sclerosis.The main manifestation is the loss of pericytes leading to damage of the blood-brain barrier,which activates inflammatory signals and affects adjacent neurovascular units,ultimately leading to neurodegeneration.Elucidating the molecular mechanisms affecting pericyte function in adult and aged brain,improving pericyte coverage and maintaining the integrity of the blood-brain barrier may provide new targets for the treatment of related neurodegenerative diseases.YAP(Yes-associated protein)is a transcriptional co-activator expressed in a variety of neuronal cells and can participate in the development of neurodegenerative diseases by regulating vascular function.The present study confirms the pathological mechanism by which YAP deletion on pericytes mediates the abnormal mechanical stress that alters vascular structure and thus leads to blood-brain barrier damage,and provides a preliminary explanation for the age-dependent cognitive deficits triggered by specific knockdown of YAP on pericytes.Part Ⅰ.Pericyte YAP knockout causes learning memory impairment in adult miceMethod:(1)To construct a pathological model of adult chronic social isolation and to detect the expression level of YAP gene in the hippocampal region of adult chronic social isolation model mice by immunofluorescence staining assay.(2)To detect the expression of YAP gene in adult mouse hippocampal pericytes and on primary pericytes and pericyte lines by immunofluorescence staining assay.(3)Adult mouse models of pericyte-specific knockdown of the YAP gene were obtained using YAPfl/fl mice crossed with PDGFRβ-Cre ERT2 tool mice,and changes in YAP knockdown efficiency on pericytes were detected by immunostaining experiments.(4)The behavioral phenotypes of mice at different stages(3 months of age,10months of age)after specific knockdown of YAP gene on pericytes were examined using learning memory-related behavioral(water maze,new object,new location,three-box socialization,etc.)and motor ability-related behavioral(stick spinning,pole climbing,mine field,etc.)and emotion-related behavioral(elevated cross maze,etc.).Results:(1)YAP expression is downregulated in the hippocampal region of adult social isolation model mice.A pathological model of social memory disorder was constructed by performing chronic social isolation for 2 weeks in 2-month-old C57BL/6J mice.Immunofluorescence staining results showed that YAP gene expression was significantly down-regulated in the hippocampus of adult social isolation model mice.(2)YAP was widely expressed in pericytes in the hippocampal region of adult mice.Immunofluorescence co-staining of YAP gene and pericytes in the hippocampal region of adult C57BL/6J mice showed that YAP gene was co-localized with the pericyte marker PDGFRβin the hippocampal region of adult mice.The co-localization of YAP gene with pericyte markers NG2,PDGFRβandα-SMA was also verified in vitro using pericyte cell lines.We also extracted adult 2-month-old primary pericytes and determined the purification of pericytes by immunostaining of endothelial cells,pericytes and astrocytes,and then co-stained with the pericyte marker PDGFRβand YAP,and verified the co-localization of YAP and adult primary pericytes.(3)Construction of an adult stage pericyte YAP knockout mouse model.A mouse model of YAP knockdown in adult primary pericytes was constructed,and the mice were injected with tamoxifen intraperitoneally at the age of 2 months and induced stably for one month,and the enzymatic efficiency of YAP gene expression on the pericyte marker PDGFRβwas analyzed by immunofluorescence staining.The results showed a decrease in fluorescence intensity and fluorescence density of the YAP gene,suggesting the successful construction of adult pericyte YAP knockout mice.(4)Adult stage pericyte YAP knockout mice showed cognitive memory impairment.The behavioral results showed that in the novel object recognition experiments of 3-month-old and 10-month-old pericyte YAP knockout mice(Conditional knock out,cKO),the preference indices of cKO mice and control mice were similar in the adaptation stage,indicating that the mice had no specific bias;in the detection stage cKO mice showed a decrease in the recognition index,suggesting that cKO mice had working memory impairment.Similarly,in the new position recognition,the preference indices of cKO mice and control mice were similar in the adaptation phase;however,cKO mice showed a decrease in recognition index in the detection phase,suggesting that cKO mice have spatial memory impairment.In the water maze experiment,during the training phase,the locomotor speed of cKO mice and control mice were the same,indicating that the locomotor ability of the two groups of mice was similar,but the escape latency of cKO mice was longer than that of the control group;meanwhile,the number of crossing and dwell time of cKO mice for the escape platform and the number of crossing and dwell time of the quadrant where the escape platform was located during the testing phase showed a decrease compared with the control group.These results suggest that cKO mice have long-term spatial memory impairment.In addition,in the three-box socialization experiment,the two groups of mice showed preference for unfamiliar mice during the adaptation phase and maintained normal socialization ability;however,in the detection phase,cKO mice had less contact with unfamiliar mice than the control group,indicating that cKO mice also had social memory impairment.Otherwise,there was no difference in motor ability and anxiety between my cKO and control mice.These results suggest that the YAP gene on pericytes has an important role in cognitive memory.Part Ⅱ.Effect of pericyte YAP knockout on the structure and permeability of the blood-brain barrier in the hippocampal region of miceMethods:(1)Neurons in the hippocampal region were activated by three-box socialization experiments in cKO mice and control mice,and neuronal activation of c Fos in the hippocampal region was detected using immunofluorescence assays.(2)To detect the pericyte coverage in the hippocampus of pericyte YAP knockout mice at different age stages(3 months old,10 months old,22 months old),and to detect the coverage area of the pericyte marker PDGFRβon the endothelial cell marker CD31using immunofluorescence staining.(3)To analyze the cerebral vascular structures in the hippocampus of pericyte YAP knockout mice at adult and aged stages,and to detect the diameter abundance,vascular folding and curvature,vascular breakage,vascular length and vascular branching of the microvascular structures of pericyte marker PDGFRβand endothelial cell marker CD31using immunofluorescence staining.(4)To detect blood-brain barrier permeability in the hippocampus of pericyte YAP knockout mice at different age stages(3 months old,10 months old,22 months old),and to detect extravasation of different molecular weights around endothelial cells by immunofluorescence staining using exogenous dextran sign tracing and endogenous fibronectin.(5)Pericyte YAP knockout mice at different ages(3 months,10 months,22 months)were tested for endothelial barrier function and paracrine transport in the hippocampus,and the expression of paracrine transport proteins and tight junction proteins on the vascular endothelium was observed using AQP4,ZO-1,Cluadin-5 and endothelial cell marker CD31/Glut1 co-staining.Results:(1)Pericyte YAP knockout reduces neural activity in the hippocampal region of mice.The c Fos in the hippocampal region of adult 10-month-old pericyte YAP knockout mice were examined after a three-box social experiment,and the immunofluorescence staining results showed that the number of c Fos in the hippocampal region was significantly reduced in cKO mice compared with the control group.We also specifically analyzed CA1(short-term memory),CA2(social memory),CA3(spatial memory),and DG(memory formation)in different regions of the hippocampus,and the results showed that cKO mice exhibited a decrease in c Fos-activated cells in different regions of the hippocampus.This suggests that the reduction in c Fos exhibited by our cKO is consistent with different learning memory-related behavioral phenotypes.(2)Pericyte YAP knockdown leads to reduced pericyte coverage in the hippocampus of mice.By immunofluorescence staining of endothelial and pericytes in cKO mice at different ages of 3 months,10 months and 22 months,the results showed that pericyte YAP knockout mice caused approximately 60%decrease in pericyte coverage and decreased area of pericytes at different ages compared to control mice,but at 3 months stage and 22 months stage cKO mice did not showed changes in endothelial cell area only in mid-adult 10-month-old cKO mice,suggesting that pericyte YAP gene knockdown caused a decrease in pericyte coverage and thus secondary effects on pericyte area and endothelial cell area.(3)Pericyte YAP knockdown leads to abnormal vascular structure in the hippocampus of mice.Immunofluorescence staining of endothelial cells and pericytes was performed by immunofluorescence staining of adult 3-month-old and aged 22-month-old cKO mice.The results showed that early adult 3-month-old and aged 22-month-old cKO mice exhibited widening of pericyte and endothelial cell diameters,and showed vascular zigzag lamination and significant breakage of pericytes.It is suggested that pericyte YAP knockdown leads to abnormal vascular structure.(4)Pericyte YAP knockdown resulted in increased permeability of the hippocampal blood-brain barrier in mice.By performing BBB functional assays in3-month-old,10-month-old,and 22-month-old cKO mice,different molecular weight extravasations around the endothelial vasculature were observed by exogenous and endogenous tracing.The results showed that for the large molecular weight of about150 KDa of murine Ig G,significant deposition of lg G immunoprotein appeared at 10months of age and 22 months of age.For endogenous toxic fibrin and albumin of medium molecular weight around 70 KDa,significant deposits of Albumin and Fibrinogen appeared around the vascular endothelium of cKO mice at 3 months of age,10 months of age and 22 months of age.In contrast,for exogenous dextran injections of small molecular weight around 4 KDa,significant FITC signal deposition was detected around the vascular endothelium of 3-month-old cKO mice.The results suggest that cellular YAP knockdown leads to blood-brain barrier dysfunction and causes severe extravasation in an age-dependent manner.(5)Pericyte YAP knockdown leads to reduced vascular endothelial barrier and paracrine transport function in the hippocampus of mice.Immunofluorescence staining was performed to detect the tight junction proteins ZO-1 and Claudin-5 and the paratransit protein AQP4 between the endothelium and astrocytes through the endothelial barrier in 3-,10-,and 22-month-old cKO mice.The results showed that pericyte YAP knockdown led to decreased levels of proteins related to vascular endothelial barrier and paratransit function in the hippocampus of mice at different stages of 3,10 and 22 months of age.Part Ⅲ.Effect of pericyte YAP knockout on vascular inflammation and neuroinflammation in the hippocampal region of miceMethods:The expression of endothelial cell markers CD31,Glut1 homogeneous microglia Iba1 and astrocyte GFAP in 3-month-old,10-month-old and 22-month-old pericyte YAP knockout mice was detected using immunofluorescence staining to observe the level of vascular inflammation and neuroinflammation caused by abnormal blood-brain barrier function.Results:(1)Pericyte YAP knockout leads to vascular inflammation in the hippocampus of mice.Detection of inflammatory signaling markers GFAP and Iba1overlying the hippocampal vascular endothelium by immunofluorescence showed a significant increase in the density of GFAP~+and Iba1~+cells in the hippocampus of cKO mice at 3 months of age,10 months of age,and 22 months of age stages in terms of endothelial cell area.It is suggested that pericyte YAP knockdown leads to disruption of the microvascular endothelial barrier and endothelial cell tight junctions,erythrocyte exudation,and leukocyte infiltration into brain parenchyma,triggering vasculitic inflammation.(2)Pericyte YAP knockdown in the aged stage led to neuroinflammation in the hippocampus of mice in an age-dependent manner,and verified that neuroinflammation was delayed from vascular inflammation.The cell density of the hippocampal inflammatory cell markers GFAP and Iba1 was detected by immunofluorescence assay,and the results showed that the hippocampal Iba1~+cell density of cKO mice at the adult stage of 3 months and 10 months of age showed an increase,but the GFAP~+cell density did not change.In contrast,both Iba1~+and GFAP~+cell densities appeared to increase in 22-month-old cKO mice at the older stage.It is suggested that pericyte YAP knockdown in adulthood leads to mild neuroinflammation and that neuroinflammation is delayed over vascular inflammation.With the aging process,pericyte YAP knockout exhibited severe neuroinflammation in old age.Part Ⅳ.Effect of Pericyte YAP Knockout on Cellular and Myelin Proteins in Oligodendrocyte Spectrum of Mouse Hippocampal RegionMethods:Immunofluorescence co-staining was used to detect Olig2~+PDGFRα~+cells in hippocampal tissues of 3-month-old,10-month-old and 22-month-old pericyte YAP knockout mice to observe the proliferation ability of oligodendrocyte progenitors;CC1~+and ASPA~+immunofluorescence staining was used to detect oligodendrocyte maturation and differentiation ability;MBP~+NF200~+immuno-co-staining was used to detect myelin protein renewal and regeneration.The expression of MBP and MAG myelin-related proteins in the hippocampal region was also investigated by Western Blot.Results:(1)Pericyte YAP knockdown resulted in decreased proliferation of oligodendrocyte progenitor cells in the hippocampal region of mice.The immunofluorescence detection of Olig2~+PDGFRα~+double positive cells,a marker of oligodendrocyte progenitor cells in the hippocampal region,showed that oligodendrocyte progenitor cell density was reduced in the hippocampal region of cKO mice at 3 months,10 months and 22 months of age,suggesting that pericyte YAP knockdown affects the proliferative capacity of oligodendrocyte progenitor cells.(2)Pericyte YAP knockdown causes delayed maturation of mouse hippocampal oligodendrocytes.Immunofluorescence detection of oligodendrocyte markers CC1~+and ASPA~+positive cells in the hippocampal region showed that oligodendrocyte density was reduced in the hippocampal region of cKO mice at 3months of age,10 months of age and 22 months of age,suggesting that pericyte YAP knockdown affects the ability of oligodendrocyte progenitor cells to mature and differentiate.(3)Pericyte YAP knockdown resulted in decreased myelin renewal and regeneration in the hippocampal region of mice.The results showed that myelin protein expression was decreased in the hippocampal region of cKO mice at 3 months of age,10 months of age and 22 months of age by immunofluorescence detection of the myelin protein marker MBP~+NF200~+,and the results further demonstrated that the hippocampal region of cKO mice showed myelin The results further demonstrated that cKO mice showed decreased myelin protein expression in the hippocampal region.It is suggested that the knockdown of YAP gene in pericytes affects the ability of oligodendrocyte progenitor cells to mature and differentiate.Part Ⅴ.Effects of pericyte YAP knockout on neurons and axons in the hippocampal region of miceMethods:Axonal degeneration was detected by NF200~+immunofluorescence staining in 3-month-old,10-month-old and 22-month-old pericyte YAP knockout mice;neuronal changes were detected by Neu N~+immunofluorescence staining.Results:(1)Pericyte YAP knockout caused neurodegeneration in the hippocampal region of mice.Detection of neuronal marker Neu N~+positive cells in the hippocampal region by immunofluorescence showed no change in neuronal density in the hippocampal region of cKO mice and controls at 3 months of age.However,a significant decrease in neuronal density was observed in the hippocampal region of cKO mice at the 10-month-old and 22-month-old stages compared to controls,suggesting that pericyte YAP knockdown affects neuronal density with the aging process,leading to neurodegeneration.(2)Pericyte YAP knockout resulted in axonal degeneration in the hippocampal region of mice.The neurofilament marker NF200~+in the hippocampal region was detected by immunofluorescence,and the results showed that the hippocampal region of cKO mice did not show differential changes at 3 months of age and 10 months of age,but the hippocampal region of cKO mice showed down-regulation of NF200~+fluorescence compared with the control group at 22 months of age.It is suggested that pericyte YAP knockout causes mice to affect axonal degeneration in the hippocampal region in an age-dependent manner.Conclusions:(1)The YAP gene is widely expressed in pericytes in the hippocampal region of adult mice.Pericyte YAP knockout causes cognitive memory dysfunction in adult mice.(2)Pericyte YAP knockdown resulted in an age-dependent decrease in pericyte coverage,vasoconstriction,and blood-brain barrier permeability in the mouse hippocampus.(3)Pericyte YAP knockout caused blood-brain barrier leakage and led to vascular inflammation and neuroinflammation.(4)Pericyte YAP knockdown-induced neurovascular inflammation leads to delayed cell renewal and impaired myelin regeneration in oligodendrocyte lineages.(5)Pericyte YAP knockout affects myelin changes,causing neuronal damage and axonal degeneration,leading to neurodegeneration.Accordingly,in this study,we analyzed the synergistic protective effects and mechanisms of YAP-mediated pericytes against vascular diseases by using a pericyte-specific mouse model with multiple assays to analyze the structural function of blood vessels in the brain and changes in the response of neighboring neurovascular units,respectively. |
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