PDGF-BB alleviates pericyte damage by the activation of the PHF19-PRC2 complex via the miR-221/BRCA1 axis in Alzheimer's disease

PDGF-BB alleviates pericyte damage by the activation of the PHF19-PRC2 complex via the miR-221/BRCA1 axis in Alzheimer's disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PDGF-BB alleviates pericyte damage by the activation of the PHF19-PRC2 complex via the miR-221/BRCA1 axis in Alzheimer's disease heyun yang, Jueyan Li, Keying Zhao, Ling Shi, Hongying Zhou, Yulan Li, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6395732/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background PDGF-BB is one of the important factors to maintain the function of pericytes. Pericyte damage accelerates the progression of Alzheimer's disease (AD). The role of PDGF-BB in AD was verified in this study. Methods Pericytes were treated with Aβ1-42 or combined with PDGF-BB. CCK-8 assay, EdU assay and flow cytometry examined cell viability, proliferation and apoptosis. Co-Immunoprecipitation verified the relationship among BRCA1, PHF19, EZH2, EED, SUZ12 and RbAp46/48.Luciferase reporter assay verified the relationship among BRCA1, miR-221-3p and miR-222-3p. APP/PS1 mice were administrated with PDGF-BB. Morris water maze test examined animal behaviors.Immunofluorescence staining and Evans Blue assay examined the pericyte coverage and blood brain barrier (BBB) integrity. Results PDGF-BB enhanced cell viability and proliferation, while inhibited apoptosis of Aβ1-42-treated pericytes, which was abrogated by BRCA1 overexpression. BRCA1 was up-regulated in Aβ1-42-treated pericytes. Additionally, PDGF-BB treatment caused a down-regulation of BRCA1 and up-regulation of PHF19-PRC2 complex members, PHF19, EZH2, EED, SUZ12 and RbAp46/48. BRCA1 interacted with PHF19-PRC2 complex members. MiR-221 repressed BRCA1 expression by targeting BRCA1. MiR-222 interacted with BRCA1 and had no influence on BRCA1 expression. In vivo , PDGF-BB treatment ameliorated learning and memory ability and elevated pericyte coverage and BBB integrity in AD mice. Conclusion PDGF-BB activated PHF19-PRC2 complex by regulating miR-221/BRCA1 axis, thereby reducing the permeability of BBB and ameliorating the learning and memory ability of AD mice. Thus, PDGF-BB may play a therapeutic role in AD development. Alzheimer's disease PDGF-BB PHF19-PRC2 complex Pericytes Blood brain barrier Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Alzheimer's disease (AD) is a kind of neurodegenerative disease with hidden onset and gradual aggravation [ 1 ]. The characteristic pathological changes of AD mainly include plaque deposition formed by amyloid β-protein (Aβ) and neurofibrillary tangles in nerve cells formed by tau protein hyperphosphorylation [ 2 , 3 ]. It eventually causes progressive cognitive decline and behavioral impairments [ 4 , 5 ]. Blood brain barrier (BBB) is a highly selective semipermeable membrane between blood and nerve tissues, which maintains the stable environment of central nervous system and normal neuron function [ 6 ]. Previous study has confirmed that the decomposition of BBB causes a vicious cycle of AD progress [ 7 ]. When BBB is damaged, it activates secretase to cause Aβ excess and induces oxidative stress damage, eventually leading to neuron damage, memory loss and cognitive decline [ 8 ]. Thus, regulation of BBB may be an effective way to treat AD. Platelet derived growth factor-BB (PDGF-BB) plays a key role in normal embryonic development, cell growth and differentiation [ 9 ]. Abnormal activity of PDGF-BB is related to physical process of various diseases, including osteoarthritis, diabetic wound healing and intervertebral disk degeneration [ 10 – 12 ]. PDGF-BB plays a protective role in Aβ-induced SH-SY5Y neuronal injury [ 13 ]. PDGF-BB has ability to maintain the function of brain pericytes [ 14 , 15 ]. Pericyte damage or dysfunction accelerates the neurodegenerative cascade of impaired responses in AD. Pericyte injury-induced BBB destruction causes vascular permeability increase and the regression of brain microvessels, and then accelerates the impaired neurodegenerative cascade in AD [ 16 ]. Thus, PDGF-BB can be used as a potential drug for AD treatment, while the underlying mechanism still unclear. Polycomb repressive complex 2 (PRC2) mainly includes four subunits: EZH2, EED, SUZ12 and RbAp46/48 [ 17 ]. The expression of PRC2 decreases with age, and defective expression of PRC2 increases the incidence rate of delayed AD [ 18 ]. Compared with EED and SUZ12, PHF19 itself does not participate in the formation and stability of PRC2. PHF19 interacts with EZH2 and EED in PRC2 complex, and is an important regulatory factor for PRC2 complex to perform its function [ 19 ]. PHF19 regulates PRC2 complex to affect various disease progression, such as multiple myeloma and prostate cancer [ 20 , 21 ]. Whether PHF19 can affect the progression of AD still remains unclear. As a gene expression regulator, microRNAs (miRNAs) regulate cell proliferation, cycle, apoptosis and other processes by targeting 3'-untranslated region (UTR) of mRNA after transcription [ 22 ]. In PDGF-BB-treated human aortic smooth muscle cells, miR-221 and miR-222 are observed to up-regulate [ 23 ]. Among them, miR-221 is abnormally reduced in peripheral blood of AD patients, and miR-222 is confirmed to decrease in brain tissues of AD mice [ 24 , 25 ]. Further analysis of the target mRNA of miR-221/miR-222 shows that breath cancer susceptibility gene 1 (BRCA1) may interact with these two miRNAs. BRCA1 is known to be a negative regulator of PRC2 complex [ 26 ]. Therefore, we proposed a scientific hypothesis: PDGF-BB may inhibit BRCA1 by up-regulating miR-221/miR-222 and then activate PHF19-PRC2 complex, thereby reducing the permeability of BBB and playing a therapeutic role in AD mice. Materials and methods Animals C57BL/6 mice (1–3 days old, 8 months old) and APP/PS1 mice (8 months old) were obtained from Shanghai SLAC Laboratory Animal Company (China). Mice were housed under SPF conditions. Isolation and enrichment of pericytes Pericytes were isolated from neonatal C57BL/6 mice as the previous study described with slight modification [ 27 ]. In brief, mice were anesthetized with CO 2 . Brains were isolated from mice under aseptic and low-temperature conditions. The meninges were allowed to strip from the brains under a dissection microscope. After that, brains were sliced into debris, and then treated with 0.1% Collagenase II (Sigma-Aldrich, St. Louis, MO, USA) and 1000 U/mL DNase I (Sigma-Aldrich) at 37°C for 1 h. Cell homogenate was mixed with 17% Percoll reagent (Sigma-Aldrich). After several centrifugations, the cell precipitates were collected and cultured in low-glucose DMEM (Thermo Fisher Scientific, San Jose, CA, USA) and 20% foetal bovine serum at 37°C and CO 2 . The medium was changed every other day. Third generation of pericytes were used for subsequent experiments. Cell treatment and transfection Pericytes were treated with 5, 10, 20 µmol/L of Aβ1–42 (Sigma-Aldrich) for 24 h, or then treated with 10, 25, 50, 100 ng/mL of PDGF-BB (MedChemExpress, NJ, USA) for 72 h. The pcDNA3.1 vector carrying BRCA1 (pcDNA3.1-BRCA1), miR-221/miR-222 mimic were constructed for overexpression of BRCA1, miR-221 and miR-222. SiRNA specifically targeting BRCA1 (si-BRCA1), miR-221/miR-222 inhibitor were synthesized for knockdown of BRCA1, miR-221 or miR-222. Empty pcDNA3.1 vector, scrambled siRNA (si-NC), mimic NC and inhibitor NC were served as control. These plasmids and oligonucleotides were obtained from Genepharm (Guangzhou, Shanghai). Pericytes were transfected with plasmids and oligonucleotides applying Lipofectamin 2000 reagent (Invitrogen, Carlsbad, CA, USA). Cell viability, proliferation and apoptosis Applying BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488, Cell Counting Kit-8 (CCK-8) and Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China), viability, proliferation and apoptosis of pericytes were examined as the protocol of manufacturer. EdU-positive cells were observed under a laser scanning confocal microscope (Olympus, Tokyo, Japan). The absorbance value of CCK-8-treated cells at 450 nm was detected by a multiplate reader (BioTek, Winooski, VT,USA). Apoptotic cells were examined by a Flow Cytometer (Becton, Dickinson and Company, CA, USA). Co-Immunoprecipitation (Co-IP) Pericytes were allowed to reach 80% confluence, and then treated with RIPA Lysis Buffer. The supernate was collected for Input or immunoprecipitation assay. The supernate was incubated with primary antibodies, anti-PHF19 (Thermo Fisher Scientific), anti-EZH2 (Proteintech, Wuhan, China), anti-EED (Abcam, Cambridge, MA, USA), anti-SUZ12 (Abcam), anti-RbAp46/48 (Cell Signaling Technology; Danvers, MA, USA) or anti-IgG (Abcam), followed by incubation of Agarose A + G magnetic beads (Thermo Fisher Scientific). After that, the supernate was washed with PBS for several times and collected by centrifugation. Finally, the supernate was incubated with boiling water for 5 min, and collected for WB analysis. Luciferase reporter assay Luciferase reporter assay was conducted as the previous study reported [ 28 ]. The luciferase vector pmir-GLO carrying BRCA1 containing the wild type/mutant of miR-221-3p binding sites (5’-UGUAGC-3’ and 5’-GACA-UGUAGC-3’) and miR-22-3p binding sites (5’-UGUAGC-3’) (Genepharm). 293T cells were transfected with BRCA1 WT/MUT and miR-221-3p/miR-22-3p mimic or mimic NC. Applying Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA), the luciferase activity was examined. Animal protocols APP/PS1 mice were intraperitoneally injected with 30 µL of 0.01% (w/v) PDGF-BB for 1 month, or injected with 200 µL of lentivirus-mediated pcDNA3.1-BRCA1 or lentivirus-mediated pcDNA3.1-NC into tail vein. APP/PS1 mice served as AD mouse model. Wild type C57BL/6 mice served as control. The pcDNA3.1-BRCA1 and pcDNA3.1-NC vectors were packaged into lentiviral particles (Genepharm). After modeling, the hippocampus and cortical tissues were separated from euthanized mice for further analysis. Behavioural testing Behaviours of mice after 25 days PDGF-BB and lentivirus of administration was tested by performing morris water maze (MWM) test following the previous study reported [ 29 ]. In brief, a circular pool with 120 cm of diameter was divided into four equal quadrants. The target quadrant was equipped with the escape platform. The circular pool contained opaque water and flooded the escape platform. Mice were allowed to train for 5 consecutive days, 4 times a day. The time of mice to find the platform was recorded. Mice that could not find the platform within 60 s were guided to the platform and held for 10 s. After that, the platform was removed. The mice were placed from the farthest end of the platform, the swimming trajectory of mice was recorded for 60 s. All images and data were analyzed using SAMRT 2.0 (Pan Lab, Barcelona, Spain). RNA extraction and qRT-PCR Total RNA and miRNA were extracted from pericytes, hippocampus and cortical tissues utilizing RNA Rapin Extraction Solution or PureLink™ miRNA Kit (Invitrogen). Total RNA and miRNA were served as template to synthesize cDNA applying High Capacity cDNA Kit or TaqMan MicroRNA (Applied Biosystem Foster, City, CA, USA). PCR reaction was carried out utilizing TB Green® Premix Ex Taq™ II or Mir-X miRNA qRT-PCR TB Green Kit (Takara, Beijing, China). The relative expression of mRNA and miRNA was normalized to GAPDH or U6, and analyzed by 2 −ΔΔCt method. Western blotting Pericytes, hippocampus and cortical tissues were treated with RIPA Lysis Buffer (Thermo Fisher Scientific) to extract total proteins. Protein samples were separated by 10% SDS-PAGE gel electrophoresis, and then transfered onto PVDF membranes. The membranes were incubated with primary and secondary antibodies. The antibodies, anti-PDGF-BB, anti-BRCA1, mouse anti-EZH2, anti-EED, anti-SUZ12, anti-α-SMA, anti-Desmin, anti-GAPDH and anti-IgG-HRP were obtained from Abcam, except for anti-RbAp46/48 (Cell Signaling Technology), anti-PHF19 (Proteintech) and anti-PDGFRβ (Proteintech). After developing with ECL reagent, the bands were analyzed by Image J software. Enzyme-linked immunosorbent assay (ELISA) The levels of Aβ40, Aβ42, PDGF-BB and PDGFRβ in hippocampus were examined applying ELISA kits. Mouse Aβ40, Aβ42, PDGF-BB and PDGFRβ ELISA Kit were obtained from EK-Bioscience (Shanghai, China). The absorbance value of each samples was detected by a multiplate reader. Evans Blue (EB) staining Mice were injected with 2% of EB dye (4 mL/kg; Sigma-Aldrich) into the left femoral vein, and allowed to circulate for 60 min. After that, the anesthetized mice were subjected to intracardial perfusion with PBS. The hippocampus was separated from brain of mice, and then fixed with 4% paraformaldehyde and embedded with paraffin. Paraffin sections of hippocampus were observed under a fluorescent microscopy (Nikon, Tokyo, Japan). Immunofluorescence (IF) staining Paraffin sections of hippocampus and cortical tissues were subjected to antigen retrieval with sodium citrate and blocking with bovine serum albumin. Sections were incubated with rabbit anti-PDGFRβ (Biolab, Beijing, China), mouse anti-CD31 (Thermo Fisher Scientific), and then stained with donkey anti-rabbit IgG-Alexa Fluor® 647 (Abcam) or goat anti-mouse IgG-Alexa Fluor® 488 antibodies (Abcam). The sections were stained with Hoechst dye. The sections were observed under a fluorescent microscopy. Statistical analysis Each assay was conducted for 3 times. Data were expressed as mean ± standard deviation. SPSS 22.0 statistical software (IBM, Armonk, NY, USA) was used for statistical analysis. Two-tailed Student’s t test and one-way ANOVA were used to analyze the statistical difference. P less than 0.05 was considered as a significant difference. Results PDGF-BB treatment promoted cell viability and proliferation, and repressed apoptosis of Aβ1-42-treated pericytes. To explore the functional role of PDGF-BB in AD, pericytes were treated with Aβ1–42 to mimic AD conditions in vitro . To test if PDGF-BB could affect cell viability, proliferation of Aβ1-42-treated pericytes, we conducted CCK-8 and EdU assays. As shown in Fig. 1A-C, Aβ1–42 reduced cell viability and proliferation of pericytes. PDGF-BB (25, 50, 100 ng/mL) enhanced cell viability and proliferation of Aβ1-42-treated pericytes, although at different extent. Results of flow cytomrtey revealed that PDGF-BB treatment reversed Aβ1-42-induced apoptosis of pericytes, especially 50 ng/mL of PDGF-BB (Fig. 1 D, E). Thus, PDGF-BB treatment promoted cell viability and proliferation, and repressed apoptosis of Aβ1-42-treated pericytes. PDGF-BB repressed BRCA1 expression in pericytes. GSE46564 data revealed the differentially expressed genes between Control and PDGF-BB-treated pericytes (Fig. 2 A). Protein-protein interaction (PPI) analyzed the proteins that bind to PHF19 in the up-regulated genes of Aβ1-42-treated pericytes. It showed that PHF19 interacted with the PHF19-PRC2 complex components, EZH2, EED, SUZ12 and RbAp46/48 (Fig. 2 B). Moreover, the expression of BRCA1 in pericytes was detected by qRT-PCR and western blotting. BRCA1 was down-regulated in pericytes in the presence of PDGF-BB (25, 50, 100 ng/mL) (Fig. 2 C-E). Whereas, Aβ1–42 treatment caused an up-regulation of BRCA1 in pericytes in a concentration-dependent manner (Fig. 2 F-H). PDGF-BB treatment affected cell viability, proliferation and apoptosis of Aβ1-42-treated pericytes by regulating BRCA1. We conducted BRCA1 overexpression or knockdown in Aβ1-42-treated pericytes. QRT-PCR and western blotting results uncovered that BRCA1 was up-regulated in Aβ1-42-treated pericytes. BRCA1 overexpression enhanced BRCA1 expression, while BRCA1 silencing inhibited BRCA1 expression in Aβ1-42-treated pericytes (Fig. 3 A-C). Using EdU assay, CCK-8 assay and flow cytometry, we observed that cell proliferation and viability was decreased, apoptosis was increased in pericytes following Aβ1–42 treatment. BRCA1 overexpression further reduced cell proliferation and viability, and promoted apoptosis of Aβ1-42-treated pericytes. The influence of Aβ1–42 treatment on cell proliferation, viability and apoptosis was abrogated by BRCA1 deficiency (Fig. 3 D-H). To reveal the mechanism of action of PDGF-BB in AD, the expression of BRCA1 in pericytes following Aβ1–42 and PDGF-BB treatment was examined. Figure 4 A-C showed that BRCA1 expression was increased in Aβ1-42-treated pericytes. Aβ1–42 treatment mediated up-regulation of BRCA1 in pericytes was repressed by PDGF-BB treatment, especially 50 ng/mL of PDGF-BB. Thus, PDGF-BB at 50 ng/mL was used to treat pericytes in further assays. Applying EdU assay, CCK-8 assay and flow cytometry, we found that cell proliferation and viability were decreased, apoptosis was increased in Aβ1-42-treated pericytes. PDGF-BB treatment led to a boost in cell proliferation and viability, and caused a decrease in apoptosis of Aβ1-42-treated pericytes. The influence conferred by PDGF-BB treatment was abolished by BRCA1 overexpression (Fig. 4 D-H). Therefore, PDGF-BB treatment affected cell viability, proliferation and apoptosis of Aβ1-42-treated pericytes by regulating BRCA1. PDGF-BB regulated BRCA1-mediated PHF19-PRC2 complex. The impact of PDGF-BB on PHF19-PRC2 complex components was detected by western blotting. Figure 5 A, B showed that the expression of PHF19, EZH2, EED, SUZ12 and RbAp46/48 was decreased in pericytes following Aβ1–42 treatment. These proteins were up-regulated in pericytes in the presence of Aβ1–42 combined with PDGF-BB treatment. Furthermore, the relationship between BRCA1 and PHF19-PRC2 complex components was verified by Co-IP. We found that BRCA1 interacted with PHF19, EZH2, EED, SUZ12 and RbAp46/48, respectively (Fig. 5 C-G). Thus, PDGF-BB inhibited BRCA1 to activate PHF19-PRC2 complex. PDGF-BB treatment repressed BRCA1 expression by regulating miR-221. Boinformatic analysis (TargetScan database) revealed that BRCA1 may be a target of miR-221 and miR-222. QRT-PCR results uncovered that miR-221 and miR-222 were down-regulated in Aβ1-42-treated pericytes. PDGF-BB treatment enhanced miR-221 and miR-222 expression in Aβ1-42-treated pericytes (Fig. 6 A, B). Luciferase reporter assay verified the relationship among BRCA1, miR-221-3p and miR-222-3p. Following transfection of miR-221-3p mimic and BRCA1 WT (5’-UGUAGC-3’), the luciferase activity of cells had no changes. Whereas, the luciferase activity was severely decreased in cells following transfection of miR-221-3p mimic and BRCA1 WT (5’-GACA-UGUAGC-3’). It implied that BRCA1 interacted with miR-221-3p at 5’-GACA-UGUAGC-3’. The luciferase activity was reduced in 293T cells in the presence of BRCA1 WT (5’-UGUAGC-3’) and miR-222-3p mimic, suggesting that BRCA1 was a target of miR-222-3p (Fig. 6 C-E). Furthermore, miR-221 or miR-221 overexpression reduced BRCA1 expression in pericytes. On the contrary, both miR-221 silencing and miR-222 knockdown caused an up-regulation of BRCA1 in pericytes (Fig. 7 A-F). Additionally, miR-221 inhibitor reversed PDGF-BB treatment-mediated down-regulation of BRCA1 in Aβ1-42-treated pericytes. MiR-222 deficiency had no influence on BRCA1 expression in pericytes following Aβ1–42 and PDGF-BB treatment. Therefore, PDGF-BB treatment repressed BRCA1 expression in pericytes by regulating miR-221 in Aβ1-42-treated pericytes. PDGF-BB treatment ameliorated learning and memory ability of AD mice by regulating BRCA1. Here, we constructed a AD mouse model, and verified the functional role of PDGF-BB in vivo . MWM test examined the behaviors of mice. Figure 8 A showed the trajectory of mice. Compared with WT mice, AD mice displayed higher escape latency and swimming distance, fewer number of platform crossings and less time spent in target quadrant. PDGF-BB treatment reduced escape latency and swimming distance, and elevated platform crossing number and time spent in target quadrant of AD mice. These influence conferred by PDGF-BB treatment was reversed by BRCA1 overexpression (Fig. 8 B-E). Utilizing ELISA kits, we found that the levels of PDGFRβ were decreased, the levels of PDGF-BB, Aβ40 and Aβ42 were increased in the hippocampus of mice. PDGF-BB treatment enhanced the levels of PDGFRβ and PDGF-BB, and reduced the levels of Aβ40 and Aβ42 in the hippocampus of mice, which was reversed by BRCA1 overexpression (Fig. 8 F-I). Additionally, IF staining examined the pericyte coverage in mice. The pericyte coverage was decreased in the hippocampus and cortical tissues of AD mice. BRCA1 overexpression reversed PDGF-BB treatment-caused promotion of pericyte coverage in the hippocampus and cortical tissues of AD mice (Fig. 9 A-D). These data suggested that PDGF-BB treatment ameliorated learning and memory ability of AD mice by regulating BRCA1. PDGF-BB treatment ameliorated BBB integrity in AD mice by regulating miR-221/BRCA1 axis-mediated PHF19-PRC2 complex. We further detected the expression of pericyte markers in AD mice by western blotting. The expression of α-SMA, Desmin and PDGFRβ was decreased in hippocampus and cortical tissues of AD mice, which was up-regulated by PDGF-BB treatment. BRCA1 overexpression reversed PDGF-BB-mediated up-regulation of α-SMA, Desmin and PDGFRβ in hippocampus and cortical tissues of AD mice (Fig. 10 A, B and E, F). Moreover, the expression of PHF19-PRC2 complex components, PHF19, EZH2, EED, SUZ12, RbAp46/48 was decreased, and BRCA1 was up-regulated in hippocampus and cortical tissues of AD mice. PDGF-BB caused a down-regulation of BRCA1 and an up-regulation of PHF19, EZH2, EED, SUZ12, RbAp46/48 in hippocampus and cortical tissues of AD mice, which was abolished by BRCA1 up-regulation (Fig. 10 C, D and G, H). QRT-PCR results showed that miR-221 was down-regulated, BRCA1 was up-regulated in hippocampus and cortical tissues of AD mice. PDGF-BB treatment enhanced miR-221 expression and reduced BRCA1 expression in hippocampus and cortical tissues of AD mice. The influence conferred by PDGF-BB treatment was abrogated by BRCA1 overexpression (Fig. 11 A, B). Additionally, we carried out EB assay to detect the BBB integrity in mice. As shown in Fig. 11 C, D revealed that the BBB integrity was decreased in AD mice, which was rescued by PDGF-BB treatment. BRCA1 overexpression destroyed the protection of PDGF-BB on BBB integrity in AD mice. All these findings suggested that PDGF-BB treatment ameliorated BBB integrity in AD mice by regulating miR-221/BRCA1 axis-mediated PHF19-PRC2 complex. Discussion AD seriously threatens the health of the human, especially elderly. In this work, we found that PDGF-BB gave boost to cell viability and proliferation, while inhibited apoptosis of Aβ1-42-treated pericytes. PDGF-BB treatment caused a down-regulation of BRCA1 in Aβ1-42-treated pericytes. The influence conferred by PDGF-BB was abrogated by BRCA1 overexpression. Mechanism studies have shown that BRCA1 interacted with PHF19-PRC2 complex, and was a target of miR-221 and miR-222. Thus, PDGF-BB activated PHF19-PRC2 complex by regulating miR-221/BRCA1 axis in Aβ1-42-treated pericytes. In vivo , PDGF-BB treatment ameliorated learning and memory ability and elevated BBB integrity in AD mice. All these data suggested that PDGF-BB regulated miR-221/BRCA1 axis to activate PHF19-PRC2 complex, thereby alleviating AD development in mice. PDGF-BB is a growth factor secreted and released by platelets, endothelial cells, macrophages, epithelial cells and smooth muscle cells. It specifically interacts with PDGFR to promote the proliferation and differentiation of connective tissue cells, endothelial cells and smooth muscle cells [ 30 ]. PDGF-BB participates in the progression of various diseases. Gianni-Barrera et al. have revealed the functional role of PDGF-BB in aberrant angiogenesis. PDGF-BB regulates VEGF-R2 signaling pathway to prevent VEGF-induced aberrant angiogenesis [ 31 ]. Inhibition of PDGF-BB secretion promotes healing bisphosphonate-related osteonecrosis of the jaw, which attributes to elevate angiogenesis and osteogenesis [ 32 ]. Additionally, previous study has found that PDGF-BB treatment exerts protective effects in Aβ-induced neuroblastoma cell damage [ 13 ]. PDGF-BB treatment reduces pericyte loss and BBB impairment by regulating ERK/Akt pathways, thereby alleviating AD development [ 14 ]. Consistently, we also confirmed the therapeutical effects of PDGF-BB in AD. Moreover, we found that PDGF-BB treatment promoted cell viability, proliferation and repressed apoptosis of Aβ1-42-treated pericytes. In vivo , PDGF-BB treatment ameliorated learning and memory ability and alleviated BBB impairment in AD mice. The role of BRCA1 in AD is well known. Mano et al. have conducted pyrosequencing to examine BRCA1 methylation levels in peripheral blood of AD patients, suggesting that BRCA1 methylation is a risk factor for AD [ 33 ]. Hypomethylation of BRCA1-mediated up-regulation of BRCA1 in brain of AD patients is highly insoluble and mislocated in the cytoplasm, which leads to DNA damage in AD mice [ 34 ]. BRCA1 is intense and specific localizated at neurofibrillary tangles of brain tissues in AD patients [ 35 ]. In the present work, we determined the role of BRCA1 in AD. BRCA1 was up-regulated in Aβ1-42-treated pericytes and repressed by PDGF-BB treatment. BRCA1 overexpression reversed PDGF-BB-mediated increase of cell viability and proliferation and inhibition of apoptosis in Aβ1-42-treated pericytes. BRCA1 was a potential target of miR-221 and miR-222. MiR-221 repressed BRCA1 expression by targeting BRCA1, while miR-222 inhibitor had no influence on BRCA1 expression. Thus, PDGF-BB treatment alleviated AD development by regulating miR-221/BRCA1 axis, instead of miR-222/BRCA1 axis. PHF19-PRC2 complex takes part in the development of many solid tumors [ 36 ]. The abnormally expressed of PRC2 complex members, especially EZH2, is closely correlated with the malignancy of the tumors [ 37 ]. PRC2 complex inhibitors have been used in clinical tumor therapy [ 38 ]. In the present work, we first revealed the functional role of PHF19-PRC2 complex in the progression of AD. BRCA1 interacted with PHF19-PRC2 complex members, PHF19, EZH2, EED, SUZ12, RbAp46/48, which was regulated by PDGF-BB treatment. Thus, PDGF-BB treatment ameliorated AD development by activating BRCA1-mediated PHF19-PRC2 complex in vitro and in vivo . Conclusion This work demonstrated that PDGF-BB activated PHF19-PRC2 complex by regulating miR-221/BRCA1 axis, thereby reducing the permeability of BBB and ameliorating the learning and memory ability of AD mice. Thus, PDGF-BB may play a therapeutic role in AD development. Abbreviations AD Alzheimer's disease Aβ Amyloid β-protein BBB Blood brain barrier BRCA1 Breath cancer susceptibility gene 1 CCK-8 Cell Counting Kit-8 Co-IP Co-Immunoprecipitation EB Evans Blue ELISA Enzyme-linked immuno sorbent assay IF Immunofluorescence miRNAs MicroRNAs MWM Morris water maze PDGF-BB Platelet derived growth factor-BB PRC2 Polycomb repressive complex 2 UTR Untranslated region Declarations Acknowledgements 1. National Natural Science Foundation of China: A novel mechanism of blood-brain barrier dysfunction promoting Alzheimer's disease: PDGF-BB/PDGFRβ/lncRNA Pat1-mediated pericellular deletion. Project number: 82060215. 2. Yunnan Provincial Science and Technology Department - Kunming Medical joint special project: Study on the treatment and mechanism of human umbilical cord mesenchymal stem cells combined with sulforaphane in transgenic mice with Alzheimer's disease. Project number: 202001AY070001-267. 3. Education Department of Yunnan Province - Teacher project: Preliminary study on the effect of chronic cerebral hypoperfusion on blood-brain barrier permeability in transgenic mice with Alzheimer's disease. Project number: 2020J0221. 4. Science and Technology Talents and Platform Program of Yunnan Provincial Department of Science and Technology -- Reserve talents for provincial young and middle-aged academic and technical leaders. Project number: 202305AC160081. 5. "Spring City Plan" high-level talents - famous doctors in Spring City. Certificate number: C202012002. Author contributions Heyun Yang conceived, designed and supervised the study. Heyun Yang, Keying Zhao, Yulan Li, JinRong Ya, Chongmin Wu interpreted the results and drafted the manuscript. Heyun Yang, Jueyan Li, Keying Zhao, Ling Shi, Hongying Zhou, Yulan Li, Qiong Zhao, JinRong Ya,Chongmin Wu, Jinxia Yan performed all experiments. Heyun Yang, Yulan Li, Qiong Zhao, JinRong Ya,Chongmin Wu, performed statistical analysis. All authors have approved the fnal version of the manuscript. Funding This work was financially supported by the National Natural Science Foundation of China (No. 82060215), Yunnan Provincial Science and Technology Department - Kunming Medical joint special project (No. 202001AY070001-267), Education Department of Yunnan Province - Teacher project (No. 2020J0221), Science and Technology Talents and Platform Program of Yunnan Provincial Department of Science and Technology (No. 202305AC160081), and 'Spring City Plan' high-level talents (No. C202012002). Ethics approval and consent to participate Informed consent was obtained from all participants, and approval was granted by the Ethics Committee of Kunming First People's Hospital (YLS2020-20). Consent for publication Not applicable Competing interests The authors declare that they have no competing interests. Author details 1 Department of Neurology, Kunming First People's Hospital, Yunnan, China. 2 Calmette Hospital Affiliated to Kunming Medical University, Yunnan, China. 3 Kunming Medical University, Yunnan, China. References Weller J, Budson A. Current understanding of Alzheimer's disease diagnosis and treatment. F1000Res, 2018. 7. Khan S, Barve KH, Kumar MS. Recent Advancements in Pathogenesis, Diagnostics and Treatment of Alzheimer's Disease. Curr Neuropharmacol, 2020. 18(11): p. 1106-1125. Lyketsos CG, Carrillo MC, Ryan JM, Khachaturian AS, Trzepacz P, Amatniek J, et al. Neuropsychiatric symptoms in Alzheimer's disease. Alzheimers Dement, 2011. 7(5): p. 532-9. Agrawal Y, Smith PF, Rosenberg PB. Vestibular impairment, cognitive decline and Alzheimer's disease: balancing the evidence. Aging Ment Health, 2020. 24(5): p. 705-708. Deardorff WJ, Grossberg GT. Behavioral and psychological symptoms in Alzheimer's dementia and vascular dementia. Handb Clin Neurol, 2019. 165: p. 5-32. 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Yu JR, Lee CH, Oksuz O, Stafford JM, Reinberg D. PRC2 is high maintenance. Genes Dev, 2019. 33(15-16): p. 903-935. Cai L, Rothbart SB, Lu R, Xu B, Chen WY, Tripathy A, et al. An H3K36 methylation-engaging Tudor motif of polycomb-like proteins mediates PRC2 complex targeting . Mol Cell, 2013. 49(3): p. 571-82. Ren Z, Ahn JH, Liu H, Tsai YH, Bhanu NV, Koss B, et al. PHF19 promotes multiple myeloma tumorigenicity through PRC2 activation and broad H3K27me3 domain formation. Blood, 2019. 134(14): p. 1176-1189. Schinke CD, Bird JT, Qu P, Yaccoby S, Lyzogubov VV, Shelton R, et al. PHF19 inhibition as a therapeutic target in multiple myeloma. Curr Res Transl Med, 2021. 69(3): p. 103290. Jain P, Ballare C, Blanco E, Vizan P, Croce LD. PHF19 mediated regulation of proliferation and invasiveness in prostate cancer cells. eLife, 2020. 9. Saliminejad K, Khorshid HRK, Fard SS, Ghaffari SH. An overview of microRNAs: Biology, functions, therapeutics, and analysis methods. J Cell Physiol, 2019. 234(5): p. 5451-5465. Higashi Y, Mummidi S, Sukhanov S, Yoshida T, Noda M, Delafontaine P, et al. Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression. Cell Signal, 2019. 57: p. 10-20. Wang X, Xu Y, Zhu H, Ma C, Dai X, Qin C. Downregulated microRNA-222 is correlated with increased p27Kip¹ expression in a double transgenic mouse model of Alzheimer's disease. Mol Med Rep, 2015. 12(5): p. 7687-92. Ding H, Huang Z, Chen M, Wang C, Chen X, Chen J, et al. Identification of a panel of five serum miRNAs as a biomarker for Parkinson's disease. Parkinsonism Relat Disord, 2016. 22: p. 68-73. Wang L, Zeng X, Chen S, Ding L, Zhong J, Zhao JC, et al. BRCA1 is a negative modulator of the PRC2 complex. Embo j, 2013. 32(11): p. 1584-97. Bernard-Patrzynski F, Lécuyer MA, Puscas I, Boukhatem I, Charabati M, Bourbonnière L, et al. Isolation of endothelial cells, pericytes and astrocytes from mouse brain. PloS one, 2019. 14(12): p. e0226302. Brzuzan P, Mazur-Marzec H, Florczyk M, Stefaniak F, Fidor A, Konkel R, et al. Luciferase reporter assay for small-molecule inhibitors of MIR92b-3p function: Screening cyanopeptolins produced by Nostoc from the Baltic Sea. Toxicol In Vitro, 2020. 68: p. 104951. Vorhees CV, Williams MT. Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc, 2006. 1(2): p. 848-58. Xu J, Xie L, Guo W. PDGF/PDGFR effects in osteosarcoma and the "add-on" strategy. Clin Sarcoma Res, 2018. 8: p. 15. Gianni-Barrera R, Butschkau A, Uccelli A, Certelli A, Valente P, Bartolomeo M, et al. PDGF-BB regulates splitting angiogenesis in skeletal muscle by limiting VEGF-induced endothelial proliferation. Angio, 2018. 21(4): p. 883-900. Gao SY, Lin RB, Huang SH, Liang YJ, Li X, Zhang SE, et al. PDGF-BB exhibited therapeutic effects on rat model of bisphosphonate-related osteonecrosis of the jaw by enhancing angiogenesis and osteogenesis. Bone, 2021. 144: p. 115117. Mano T, Sato K, Ikeuchi T, Toda T, Iwatsubo T, Iwata A, et al. Peripheral Blood BRCA1 Methylation Positively Correlates with Major Alzheimer's Disease Risk Factors. J Prev Alzheimers Dis, 2021. 8(4): p. 477-482. Mano T, Nagata K, Nonaka T, Tarutani A, Imamura T, Hashimoto T, et al. Neuron-specific methylome analysis reveals epigenetic regulation and tau-related dysfunction of BRCA1 in Alzheimer's disease. PNAS, 2017. 114(45): p. E9645-e9654. Evans TA, Raina AK, Delacourte A, Aprelikova O, Lee H, Zhu X, et al. BRCA1 may modulate neuronal cell cycle re-entry in Alzheimer disease. Int J Med Sci, 2007. 4(3): p. 140-5. Duan R, Du W, Guo W. EZH2: a novel target for cancer treatment. J Hematol Oncol, 2020. 13(1): p. 104. Wan L, Wan L, Xu K, Wei Y, Zhang J, Han T, et al. Phosphorylation of EZH2 by AMPK Suppresses PRC2 Methyltransferase Activity and Oncogenic Function. Mol Cell, 2018. 69(2): p. 279-291.e5. Miranda TB, Cortez CC, Yoo CB, Liang G, Abe M, Kelly TK, et al. DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation. Mol Cancer Ther, 2009. 8(6): p. 1579-88. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6395732","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452008683,"identity":"2e702d8b-fb13-4e7a-b969-9861ec72b1f2","order_by":0,"name":"heyun yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYJACZjDJ3gblHiBaC88xkrVIpBGpxeD42cOvCyoOJ26XfJa66WYbgxzfjQTGzwX4tJzJS7OeceZw4s7Zacdu57YxGEveSGCWnoFPy4EcM2PetsOJG26nt4G0JG64kcDGzINPy/k3QC3/gFpuHgdrqSes5UaO8WPeBqCWG2xghyUYENIieeONGTPPsXTjDWfS0m7nnJMwnHnmYbM0Pi1853OMP/PUWMtuOH7M7HZOmY083/Hkg5/xaVE4wMAmwcDQDOMD2QyMDXg0MDDINzAwf2BgqMOraBSMglEwCkY4AADwVVXFFSE3vQAAAABJRU5ErkJggg==","orcid":"","institution":"Kunming First People's Hospital: Affiliated Calmette Hospital of Kunming Medical University","correspondingAuthor":true,"prefix":"","firstName":"heyun","middleName":"","lastName":"yang","suffix":""},{"id":452008684,"identity":"efef3986-8dc9-4d5c-9063-abc53ebadd6c","order_by":1,"name":"Jueyan Li","email":"","orcid":"","institution":"Kunming First People's Hospital: Affiliated Calmette Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jueyan","middleName":"","lastName":"Li","suffix":""},{"id":452008685,"identity":"199d951d-51e0-4b19-be13-2f0db6ff5523","order_by":2,"name":"Keying Zhao","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Keying","middleName":"","lastName":"Zhao","suffix":""},{"id":452008686,"identity":"e823961c-24b0-462d-943d-664019f7126c","order_by":3,"name":"Ling Shi","email":"","orcid":"","institution":"Kunming First People's Hospital: Affiliated Calmette Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Shi","suffix":""},{"id":452008687,"identity":"49ac8043-8444-4d36-9275-1b55119bebaa","order_by":4,"name":"Hongying Zhou","email":"","orcid":"","institution":"Kunming First People's Hospital: Affiliated Calmette Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hongying","middleName":"","lastName":"Zhou","suffix":""},{"id":452008688,"identity":"60f7d374-7a33-4e21-aaf4-73a8f1b93ff3","order_by":5,"name":"Yulan Li","email":"","orcid":"","institution":"Kunming First People's Hospital: Affiliated Calmette Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yulan","middleName":"","lastName":"Li","suffix":""},{"id":452008689,"identity":"4105d46d-3072-4031-8c7b-afc661fa19b6","order_by":6,"name":"Qiong Zhao","email":"","orcid":"","institution":"Kunming First People's Hospital: Affiliated Calmette Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qiong","middleName":"","lastName":"Zhao","suffix":""},{"id":452008690,"identity":"041783d5-d391-4093-920b-3d88566a1a35","order_by":7,"name":"JinRong Ya","email":"","orcid":"","institution":"Kunming First People's Hospital: Affiliated Calmette Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"JinRong","middleName":"","lastName":"Ya","suffix":""},{"id":452008691,"identity":"495e1ced-ea1c-4507-a404-115a4fa6e01e","order_by":8,"name":"Chongmin Wu","email":"","orcid":"","institution":"Kunming First People's Hospital: Affiliated Calmette Hospital of Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chongmin","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-04-07 15:25:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6395732/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6395732/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82268234,"identity":"1d4d1b1e-2122-45e1-93a9-2e6a429661fb","added_by":"auto","created_at":"2025-05-08 13:41:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80715495,"visible":true,"origin":"","legend":"\u003cp\u003ePDGF-BB treatment promoted cell viability, proliferation and reduced apoptosis of Aβ1-42-treated pericytes. Pericytes were treated with Aβ1-42 or combined with PDGF-BB treatment (10, 25, 50, 100 ng/mL). CCK-8 assay (A), EdU assay( B, C) and \u0026nbsp;flow cytometry (D, E) examined viability, proliferation and apoptosis of pericytes. Data are expressed as mean±SD. (\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6395732/v1/9e352e82944e079e5b05fd93.png"},{"id":82267492,"identity":"fb6226be-6a5e-4bd6-8521-4f44811e2f0a","added_by":"auto","created_at":"2025-05-08 13:33:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":109888151,"visible":true,"origin":"","legend":"\u003cp\u003ePDGF-BB treatment repressed BRCA1 expression in Aβ1-42-treated pericytes.(A) Heatmap of differentially expressed genes between Control and PDGF-BB-treated pericytes from GSE46564 data. (B) PPI analyzed the target proteins binding to PHF19. QRT-PCR (C) and western blotting (D, E) detected BRCA1 expression in pericytes following PDGF-BB treatment (0, 10, 25, 50, 100 ng/mL). QRT-PCR (F) and western blotting (G, H) assessed BRCA1 expression in pericytes following Aβ1-42 treatment (0, 5, 10, 20 μM). Data are expressed as mean±SD. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6395732/v1/a4805f7e2dd35cfe9a8d4168.png"},{"id":82268230,"identity":"56caa228-b8d2-42b3-b153-c644b2d4bc88","added_by":"auto","created_at":"2025-05-08 13:41:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":46045322,"visible":true,"origin":"","legend":"\u003cp\u003eBRCA1 overexpression reduced cell viability, proliferation and enhanced apoptosis of Aβ1-42-treated pericytes. Pericytes were treated with Aβ1-42, and then transfected with pcDNA3.1-BRCA1, Vector, si-BRCA1 or si-NC. QRT-PCR (A) and werstern blotting (B, C) assessed the expression of BRCA1 in pericytes. EdU assay (D, E), CCK-8 assay (F) and flow cytometry (G, H) examined viability, proliferation and apoptosis of pericytes. Data are expressed as mean±SD.\u003csup\u003e \u003c/sup\u003e(*\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6395732/v1/c57ca27f1af5b2b431cae3bb.png"},{"id":82267485,"identity":"abb397ce-7f2b-4a40-a27b-a2c1dd4a2c0f","added_by":"auto","created_at":"2025-05-08 13:33:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68678871,"visible":true,"origin":"","legend":"\u003cp\u003ePDGF-BB treatment affected cell viability, proliferation and apoptosis of Aβ1-42-treated pericytes by regulating BRCA1. Pericytes were treated with Aβ1-42 or combined with PDGF-BB treatment (10, 25, 50, 100 ng/mL). QRT-PCR (A) and werstern blotting (B, C) assessed the expression of BRCA1 in pericytes. Pericytes were treated with Aβ1-42, and then treated PDGF-BB and transfected with pcDNA3.1-BRCA1, Vector. EdU assay (D, E), CCK-8 assay (F) and flow cytometry (G, H) examined viability, proliferation and apoptosis of pericytes. Data are expressed as mean±SD. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6395732/v1/64b95225c50169142e4798cb.png"},{"id":82267445,"identity":"445493e3-f74d-450e-bc09-57a71619d9a0","added_by":"auto","created_at":"2025-05-08 13:33:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":13198876,"visible":true,"origin":"","legend":"\u003cp\u003ePDGF-BB regulated BRCA1-mediated PHF19-PRC2 complex. (A) Western blotting detected the expression of PHF19, EZH2, EED, SUZ12 and RbAp46/48 in pericytes following Aβ1-42 or combined with PDGF-BB treatment. (C-G) Co-IP verified the relationship among BRCA1, PHF19, EZH2, EED, SUZ12 and RbAp46/48. Data are expressed as mean±SD. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6395732/v1/b321e412b7908ee5455fdabd.png"},{"id":82267446,"identity":"5a7f7c36-000f-479d-bd28-c57ad27a132a","added_by":"auto","created_at":"2025-05-08 13:33:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11181710,"visible":true,"origin":"","legend":"\u003cp\u003eBRCA1 interacted with miR-221 and miR-222. QRT-PCR detected the expression of miR-221 (A) and miR-222 (B) in pericytes following Aβ1-42 or combined with PDGF-BB treatment (0, 10, 25, 50, 100 ng/mL). (C-E) Luciferase reporter assay verified the relationship among BRCA1, miR-221-3p and miR-222-3p. Data are expressed as mean±SD. (\u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6395732/v1/f07c7139244a343a3ce6f1a7.png"},{"id":82267447,"identity":"c7eafc93-5ed5-468f-830c-380ce0864bb7","added_by":"auto","created_at":"2025-05-08 13:33:36","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":15627477,"visible":true,"origin":"","legend":"\u003cp\u003ePDGF-BB treatment repressed BRCA1 expression by regulating miR-221. QRT-PCR (A, D) and western blotting (B, C, E, F) examined the expression of BRCA1 in pericytes following transfection of miR-221 mimic, miR-222 mimic, mimic NC, miR-221 inhibitor, miR-222 inhibitor or inhibitor NC. QRT-PCR (G) and western blotting (H, I) examined the expression of BRCA1 in pericytes following Aβ1-42 treatment or combined with transfection of miR-221 inhibitor, miR-222 inhibitor, inhibitor NC. Data are expressed as mean±SD. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-6395732/v1/adfc4fdc20a4860ec476d1ee.png"},{"id":82267468,"identity":"2827d81e-8dc2-49cf-8311-5b89a1899150","added_by":"auto","created_at":"2025-05-08 13:33:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":33225757,"visible":true,"origin":"","legend":"\u003cp\u003ePDGF-BB alleviated AD development in mice by regulating BRCA1. APP/PS1 mice were administrated with PDGF-BB or combined with BRCA1, Vector. (A) Trajectory map of mice. Morris water maze test examined the escape latency (B), swimming distance (C), number of platform crossings (D) and time spent in target quadrant (E) of mice. ELISA detected the levels of PDGFRβ (F), PDGF-BB (G), Aβ40 (H) and Aβ42 (I) in the hippocampus of mice. Data are expressed as mean±SD. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-6395732/v1/e7cd40e83ed09866de7794e0.png"},{"id":82267494,"identity":"a038125f-e53f-4e65-b507-5f73535beb81","added_by":"auto","created_at":"2025-05-08 13:33:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":352165015,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;PDGF-BB enhanced the pericyte coverage in AD mice by regulating BRCA1.\u003c/p\u003e\n\u003cp\u003eAPP/PS1 mice were administrated with PDGF-BB or combined with BRCA1, Vector. IF staining examined the pericyte coverage in the hippocampus (A, C) and cortical tissues (B, D) of mice. Data are expressed as mean±SD. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-6395732/v1/42fb25f162f3002bb42a3ab7.png"},{"id":82268227,"identity":"1f95ff9e-9c93-428e-82d7-6059143d10a0","added_by":"auto","created_at":"2025-05-08 13:41:36","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":16974918,"visible":true,"origin":"","legend":"\u003cp\u003ePDGF-BB treatment regulated PHF19-PRC2 complex by regulating BRCA1. APP/PS1 mice were administrated with PDGF-BB or combined with BRCA1, Vector.\u003c/p\u003e\n\u003cp\u003eWestern blotting assessed the expression of α-SMA, Desmin, PDGFRβ, BRCA1, PHF19, EZH2, EED, SUZ12 and RbAp46/48 in the hippocampus (A-D) and cortical tissues (E-H) of mice. Data are expressed as mean±SD. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-6395732/v1/d730a95d398ce01eecce992a.png"},{"id":82267490,"identity":"fca5b63c-fec8-4b2f-9136-fa52b183b18b","added_by":"auto","created_at":"2025-05-08 13:33:38","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":67895506,"visible":true,"origin":"","legend":"\u003cp\u003ePDGF-BB treatment ameliorated BBB integrity in AD mice by regulating miR-221/BRCA1 axis. APP/PS1 mice were administrated with PDGF-BB or combined with BRCA1, Vector. QRT-PCR assessed the expression of miR-221 and BRCA1 in the hippocampus (A) and cortical tissues (B) of mice. (C-D) EB assay detected the BBB integrity in mice. Data are expressed as mean±SD. (\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Fig11.png","url":"https://assets-eu.researchsquare.com/files/rs-6395732/v1/e43f8fc8ea40d5f58f12c3cb.png"}],"financialInterests":"","formattedTitle":"\u003cp\u003ePDGF-BB alleviates pericyte damage by the activation of the PHF19-PRC2 complex via the miR-221/BRCA1 axis in Alzheimer's disease\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlzheimer's disease (AD) is a kind of neurodegenerative disease with hidden onset and gradual aggravation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The characteristic pathological changes of AD mainly include plaque deposition formed by amyloid β-protein (Aβ) and neurofibrillary tangles in nerve cells formed by tau protein hyperphosphorylation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It eventually causes progressive cognitive decline and behavioral impairments [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Blood brain barrier (BBB) is a highly selective semipermeable membrane between blood and nerve tissues, which maintains the stable environment of central nervous system and normal neuron function [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Previous study has confirmed that the decomposition of BBB causes a vicious cycle of AD progress [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. When BBB is damaged, it activates secretase to cause Aβ excess and induces oxidative stress damage, eventually leading to neuron damage, memory loss and cognitive decline [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, regulation of BBB may be an effective way to treat AD.\u003c/p\u003e \u003cp\u003ePlatelet derived growth factor-BB (PDGF-BB) plays a key role in normal embryonic development, cell growth and differentiation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Abnormal activity of PDGF-BB is related to physical process of various diseases, including osteoarthritis, diabetic wound healing and intervertebral disk degeneration [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. PDGF-BB plays a protective role in Aβ-induced SH-SY5Y neuronal injury [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. PDGF-BB has ability to maintain the function of brain pericytes [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Pericyte damage or dysfunction accelerates the neurodegenerative cascade of impaired responses in AD. Pericyte injury-induced BBB destruction causes vascular permeability increase and the regression of brain microvessels, and then accelerates the impaired neurodegenerative cascade in AD [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Thus, PDGF-BB can be used as a potential drug for AD treatment, while the underlying mechanism still unclear.\u003c/p\u003e \u003cp\u003ePolycomb repressive complex 2 (PRC2) mainly includes four subunits: EZH2, EED, SUZ12 and RbAp46/48 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The expression of PRC2 decreases with age, and defective expression of PRC2 increases the incidence rate of delayed AD [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Compared with EED and SUZ12, PHF19 itself does not participate in the formation and stability of PRC2. PHF19 interacts with EZH2 and EED in PRC2 complex, and is an important regulatory factor for PRC2 complex to perform its function [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. PHF19 regulates PRC2 complex to affect various disease progression, such as multiple myeloma and prostate cancer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Whether PHF19 can affect the progression of AD still remains unclear.\u003c/p\u003e \u003cp\u003eAs a gene expression regulator, microRNAs (miRNAs) regulate cell proliferation, cycle, apoptosis and other processes by targeting 3'-untranslated region (UTR) of mRNA after transcription [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In PDGF-BB-treated human aortic smooth muscle cells, miR-221 and miR-222 are observed to up-regulate [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Among them, miR-221 is abnormally reduced in peripheral blood of AD patients, and miR-222 is confirmed to decrease in brain tissues of AD mice [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Further analysis of the target mRNA of miR-221/miR-222 shows that breath cancer susceptibility gene 1 (BRCA1) may interact with these two miRNAs. BRCA1 is known to be a negative regulator of PRC2 complex [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, we proposed a scientific hypothesis: PDGF-BB may inhibit BRCA1 by up-regulating miR-221/miR-222 and then activate PHF19-PRC2 complex, thereby reducing the permeability of BBB and playing a therapeutic role in AD mice.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eC57BL/6 mice (1\u0026ndash;3 days old, 8 months old) and APP/PS1 mice (8 months old) were obtained from Shanghai SLAC Laboratory Animal Company (China). Mice were housed under SPF conditions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsolation and enrichment of pericytes\u003c/h3\u003e\n\u003cp\u003ePericytes were isolated from neonatal C57BL/6 mice as the previous study described with slight modification [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In brief, mice were anesthetized with CO\u003csub\u003e2\u003c/sub\u003e. Brains were isolated from mice under aseptic and low-temperature conditions. The meninges were allowed to strip from the brains under a dissection microscope. After that, brains were sliced into debris, and then treated with 0.1% Collagenase II (Sigma-Aldrich, St. Louis, MO, USA) and 1000 U/mL DNase I (Sigma-Aldrich) at 37\u0026deg;C for 1 h. Cell homogenate was mixed with 17% Percoll reagent (Sigma-Aldrich). After several centrifugations, the cell precipitates were collected and cultured in low-glucose DMEM (Thermo Fisher Scientific, San Jose, CA, USA) and 20% foetal bovine serum at 37\u0026deg;C and CO\u003csub\u003e2\u003c/sub\u003e. The medium was changed every other day. Third generation of pericytes were used for subsequent experiments.\u003c/p\u003e\n\u003ch3\u003eCell treatment and transfection\u003c/h3\u003e\n\u003cp\u003ePericytes were treated with 5, 10, 20 \u0026micro;mol/L of Aβ1\u0026ndash;42 (Sigma-Aldrich) for 24 h, or then treated with 10, 25, 50, 100 ng/mL of PDGF-BB (MedChemExpress, NJ, USA) for 72 h. The pcDNA3.1 vector carrying BRCA1 (pcDNA3.1-BRCA1), miR-221/miR-222 mimic were constructed for overexpression of BRCA1, miR-221 and miR-222. SiRNA specifically targeting BRCA1 (si-BRCA1), miR-221/miR-222 inhibitor were synthesized for knockdown of BRCA1, miR-221 or miR-222. Empty pcDNA3.1 vector, scrambled siRNA (si-NC), mimic NC and inhibitor NC were served as control. These plasmids and oligonucleotides were obtained from Genepharm (Guangzhou, Shanghai). Pericytes were transfected with plasmids and oligonucleotides applying Lipofectamin 2000 reagent (Invitrogen, Carlsbad, CA, USA).\u003c/p\u003e\n\u003ch3\u003eCell viability, proliferation and apoptosis\u003c/h3\u003e\n\u003cp\u003eApplying BeyoClick\u0026trade; EdU Cell Proliferation Kit with Alexa Fluor 488, Cell Counting Kit-8 (CCK-8) and Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China), viability, proliferation and apoptosis of pericytes were examined as the protocol of manufacturer. EdU-positive cells were observed under a laser scanning\u003c/p\u003e \u003cp\u003econfocal microscope (Olympus, Tokyo, Japan). The absorbance value of CCK-8-treated cells at 450 nm was detected by a multiplate reader (BioTek, Winooski, VT,USA). Apoptotic cells were examined by a Flow Cytometer (Becton, Dickinson and Company, CA, USA).\u003c/p\u003e\n\u003ch3\u003eCo-Immunoprecipitation (Co-IP)\u003c/h3\u003e\n\u003cp\u003ePericytes were allowed to reach 80% confluence, and then treated with RIPA Lysis Buffer. The supernate was collected for Input or immunoprecipitation assay. The supernate was incubated with primary antibodies, anti-PHF19 (Thermo Fisher Scientific), anti-EZH2 (Proteintech, Wuhan, China), anti-EED (Abcam, Cambridge, MA, USA), anti-SUZ12 (Abcam), anti-RbAp46/48 (Cell Signaling Technology; Danvers, MA, USA) or anti-IgG (Abcam), followed by incubation of Agarose A\u0026thinsp;+\u0026thinsp;G magnetic beads (Thermo Fisher Scientific). After that, the supernate was washed with PBS for several times and collected by centrifugation. Finally, the supernate was incubated with boiling water for 5 min, and collected for WB analysis.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase reporter assay\u003c/h2\u003e \u003cp\u003eLuciferase reporter assay was conducted as the previous study reported [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The luciferase vector pmir-GLO carrying BRCA1 containing the wild type/mutant of miR-221-3p binding sites (5\u0026rsquo;-UGUAGC-3\u0026rsquo; and 5\u0026rsquo;-GACA-UGUAGC-3\u0026rsquo;) and miR-22-3p binding sites (5\u0026rsquo;-UGUAGC-3\u0026rsquo;) (Genepharm). 293T cells were transfected with BRCA1 WT/MUT and miR-221-3p/miR-22-3p mimic or mimic NC. Applying Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA), the luciferase activity was examined.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimal protocols\u003c/h3\u003e\n\u003cp\u003eAPP/PS1 mice were intraperitoneally injected with 30 \u0026micro;L of 0.01% (w/v) PDGF-BB for 1 month, or injected with 200 \u0026micro;L of lentivirus-mediated pcDNA3.1-BRCA1 or lentivirus-mediated pcDNA3.1-NC into tail vein. APP/PS1 mice served as AD mouse model. Wild type C57BL/6 mice served as control. The pcDNA3.1-BRCA1 and pcDNA3.1-NC vectors were packaged into lentiviral particles (Genepharm). After modeling, the hippocampus and cortical tissues were separated from euthanized mice for further analysis.\u003c/p\u003e\n\u003ch3\u003eBehavioural testing\u003c/h3\u003e\n\u003cp\u003eBehaviours of mice after 25 days PDGF-BB and lentivirus of administration was tested by performing morris water maze (MWM) test following the previous study reported [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In brief, a circular pool with 120 cm of diameter was divided into four equal quadrants. The target quadrant was equipped with the escape platform. The circular pool contained opaque water and flooded the escape platform. Mice were allowed to train for 5 consecutive days, 4 times a day. The time of mice to find the platform was recorded. Mice that could not find the platform within 60 s were guided to the platform and held for 10 s. After that, the platform was removed. The mice were placed from the farthest end of the platform, the swimming trajectory of mice was recorded for 60 s. All images and data were analyzed using SAMRT 2.0 (Pan Lab, Barcelona, Spain).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and qRT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA and miRNA were extracted from pericytes, hippocampus and cortical tissues utilizing RNA Rapin Extraction Solution or PureLink\u0026trade; miRNA Kit (Invitrogen). Total RNA and miRNA were served as template to synthesize cDNA applying High Capacity cDNA Kit or TaqMan MicroRNA (Applied Biosystem Foster, City, CA, USA). PCR reaction was carried out utilizing TB Green\u0026reg; Premix Ex Taq\u0026trade; II or Mir-X miRNA qRT-PCR TB Green Kit (Takara, Beijing, China). The relative expression of mRNA and miRNA was normalized to GAPDH or U6, and analyzed by 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003ePericytes, hippocampus and cortical tissues were treated with RIPA Lysis Buffer (Thermo Fisher Scientific) to extract total proteins. Protein samples were separated by 10% SDS-PAGE gel electrophoresis, and then transfered onto PVDF membranes. The membranes were incubated with primary and secondary antibodies. The antibodies, anti-PDGF-BB, anti-BRCA1, mouse anti-EZH2, anti-EED, anti-SUZ12, anti-α-SMA, anti-Desmin, anti-GAPDH and anti-IgG-HRP were obtained from Abcam, except for anti-RbAp46/48 (Cell Signaling Technology), anti-PHF19 (Proteintech) and anti-PDGFRβ (Proteintech). After developing with ECL reagent, the bands were analyzed by Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eThe levels of Aβ40, Aβ42, PDGF-BB and PDGFRβ in hippocampus were examined applying ELISA kits. Mouse Aβ40, Aβ42, PDGF-BB and PDGFRβ ELISA Kit were obtained from EK-Bioscience (Shanghai, China). The absorbance value of each samples was detected by a multiplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEvans Blue (EB) staining\u003c/h2\u003e \u003cp\u003eMice were injected with 2% of EB dye (4 mL/kg; Sigma-Aldrich) into the left femoral vein, and allowed to circulate for 60 min. After that, the anesthetized mice were subjected to intracardial perfusion with PBS. The hippocampus was separated from brain of mice, and then fixed with 4% paraformaldehyde and embedded with paraffin. Paraffin sections of hippocampus were observed under a fluorescent microscopy (Nikon, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence (IF) staining\u003c/h2\u003e \u003cp\u003eParaffin sections of hippocampus and cortical tissues were subjected to antigen retrieval with sodium citrate and blocking with bovine serum albumin. Sections were incubated with rabbit anti-PDGFRβ (Biolab, Beijing, China), mouse anti-CD31 (Thermo Fisher Scientific), and then stained with donkey anti-rabbit IgG-Alexa Fluor\u0026reg; 647 (Abcam) or goat anti-mouse IgG-Alexa Fluor\u0026reg; 488 antibodies (Abcam). The sections were stained with Hoechst dye. The sections were observed under a fluorescent microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eEach assay was conducted for 3 times. Data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. SPSS 22.0 statistical software (IBM, Armonk, NY, USA) was used for statistical analysis. Two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test and one-way ANOVA were used to analyze the statistical difference. \u003cem\u003eP\u003c/em\u003e less than 0.05 was considered as a significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePDGF-BB treatment promoted cell viability and proliferation, and repressed apoptosis of Aβ1-42-treated pericytes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo explore the functional role of PDGF-BB in AD, pericytes were treated with Aβ1\u0026ndash;42 to mimic AD conditions \u003cem\u003ein vitro\u003c/em\u003e. To test if PDGF-BB could affect cell viability, proliferation of Aβ1-42-treated pericytes, we conducted CCK-8 and EdU assays. As shown in Fig.\u0026nbsp;1A-C, Aβ1\u0026ndash;42 reduced cell viability and proliferation of pericytes. PDGF-BB (25, 50, 100 ng/mL) enhanced cell viability and proliferation of Aβ1-42-treated pericytes, although at different extent. Results of flow cytomrtey revealed that PDGF-BB treatment reversed Aβ1-42-induced apoptosis of pericytes, especially 50 ng/mL of PDGF-BB (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Thus, PDGF-BB treatment promoted cell viability and proliferation, and repressed apoptosis of Aβ1-42-treated pericytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePDGF-BB repressed BRCA1 expression in pericytes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGSE46564 data revealed the differentially expressed genes between Control and PDGF-BB-treated pericytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Protein-protein interaction (PPI) analyzed the proteins that bind to PHF19 in the up-regulated genes of Aβ1-42-treated pericytes. It showed that PHF19 interacted with the PHF19-PRC2 complex components, EZH2, EED, SUZ12 and RbAp46/48 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Moreover, the expression of BRCA1 in pericytes was detected by qRT-PCR and western blotting. BRCA1 was down-regulated in pericytes in the presence of PDGF-BB (25, 50, 100 ng/mL) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). Whereas, Aβ1\u0026ndash;42 treatment caused an up-regulation of BRCA1 in pericytes in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePDGF-BB treatment affected cell viability, proliferation and apoptosis of Aβ1-42-treated pericytes by regulating BRCA1.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe conducted BRCA1 overexpression or knockdown in Aβ1-42-treated pericytes. QRT-PCR and western blotting results uncovered that BRCA1 was up-regulated in Aβ1-42-treated pericytes. BRCA1 overexpression enhanced BRCA1 expression, while BRCA1 silencing inhibited BRCA1 expression in Aβ1-42-treated pericytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Using EdU assay, CCK-8 assay and flow cytometry, we observed that cell proliferation and viability was decreased, apoptosis was increased in pericytes following Aβ1\u0026ndash;42 treatment. BRCA1 overexpression further reduced cell proliferation and viability, and promoted apoptosis of Aβ1-42-treated pericytes. The influence of Aβ1\u0026ndash;42 treatment on cell proliferation, viability and apoptosis was abrogated by BRCA1 deficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-H). To reveal the mechanism of action of PDGF-BB in AD, the expression of BRCA1 in pericytes following Aβ1\u0026ndash;42 and PDGF-BB treatment was examined. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C showed that BRCA1 expression was increased in Aβ1-42-treated pericytes. Aβ1\u0026ndash;42 treatment mediated up-regulation of BRCA1 in pericytes was repressed by PDGF-BB treatment, especially 50 ng/mL of PDGF-BB. Thus, PDGF-BB at 50 ng/mL was used to treat pericytes in further assays. Applying EdU assay, CCK-8 assay and flow cytometry, we found that cell proliferation and viability were decreased, apoptosis was increased in Aβ1-42-treated pericytes. PDGF-BB treatment led to a boost in cell proliferation and viability, and caused a decrease in apoptosis of Aβ1-42-treated pericytes. The influence conferred by PDGF-BB treatment was abolished by BRCA1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-H). Therefore, PDGF-BB treatment affected cell viability, proliferation and apoptosis of Aβ1-42-treated pericytes by regulating BRCA1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePDGF-BB regulated BRCA1-mediated PHF19-PRC2 complex.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe impact of PDGF-BB on PHF19-PRC2 complex components was detected by western blotting. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B showed that the expression of PHF19, EZH2, EED, SUZ12 and RbAp46/48 was decreased in pericytes following Aβ1\u0026ndash;42 treatment. These proteins were up-regulated in pericytes in the presence of Aβ1\u0026ndash;42 combined with PDGF-BB treatment. Furthermore, the relationship between BRCA1 and PHF19-PRC2 complex components was verified by Co-IP. We found that BRCA1 interacted with PHF19, EZH2, EED, SUZ12 and RbAp46/48, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-G). Thus, PDGF-BB inhibited BRCA1 to activate PHF19-PRC2 complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePDGF-BB treatment repressed BRCA1 expression by regulating miR-221.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBoinformatic analysis (TargetScan database) revealed that BRCA1 may be a target of miR-221 and miR-222. QRT-PCR results uncovered that miR-221 and miR-222 were down-regulated in Aβ1-42-treated pericytes. PDGF-BB treatment enhanced miR-221 and miR-222 expression in Aβ1-42-treated pericytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Luciferase reporter assay verified the relationship among BRCA1, miR-221-3p and miR-222-3p. Following transfection of miR-221-3p mimic and BRCA1 WT (5\u0026rsquo;-UGUAGC-3\u0026rsquo;), the luciferase activity of cells had no changes. Whereas, the luciferase activity was severely decreased in cells following transfection of miR-221-3p mimic and BRCA1 WT (5\u0026rsquo;-GACA-UGUAGC-3\u0026rsquo;). It implied that BRCA1 interacted with miR-221-3p at 5\u0026rsquo;-GACA-UGUAGC-3\u0026rsquo;. The luciferase activity was reduced in 293T cells in the presence of BRCA1 WT (5\u0026rsquo;-UGUAGC-3\u0026rsquo;) and miR-222-3p mimic, suggesting that BRCA1 was a target of miR-222-3p (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-E). Furthermore, miR-221 or miR-221 overexpression reduced BRCA1 expression in pericytes. On the contrary, both miR-221 silencing and miR-222 knockdown caused an up-regulation of BRCA1 in pericytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-F). Additionally, miR-221 inhibitor reversed PDGF-BB treatment-mediated down-regulation of BRCA1 in Aβ1-42-treated pericytes. MiR-222 deficiency had no influence on BRCA1 expression in pericytes following Aβ1\u0026ndash;42 and PDGF-BB treatment. Therefore, PDGF-BB treatment repressed BRCA1 expression in pericytes by regulating miR-221 in Aβ1-42-treated pericytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePDGF-BB treatment ameliorated learning and memory ability of AD mice by regulating BRCA1.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHere, we constructed a AD mouse model, and verified the functional role of PDGF-BB \u003cem\u003ein vivo\u003c/em\u003e. MWM test examined the behaviors of mice. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA showed the trajectory of mice. Compared with WT mice, AD mice displayed higher escape latency and swimming distance, fewer number of platform crossings and less time spent in target quadrant. PDGF-BB treatment reduced escape latency and swimming distance, and elevated platform crossing number and time spent in target quadrant of AD mice. These influence conferred by PDGF-BB treatment was reversed by BRCA1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-E). Utilizing ELISA kits, we found that the levels of PDGFRβ were decreased, the levels of PDGF-BB, Aβ40 and Aβ42 were increased in the hippocampus of mice. PDGF-BB treatment enhanced the levels of PDGFRβ and PDGF-BB, and reduced the levels of Aβ40 and Aβ42 in the hippocampus of mice, which was reversed by BRCA1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF-I). Additionally, IF staining examined the pericyte coverage in mice. The pericyte coverage was decreased in the hippocampus and cortical tissues of AD mice. BRCA1 overexpression reversed PDGF-BB treatment-caused promotion of pericyte coverage in the hippocampus and cortical tissues of AD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-D). These data suggested that PDGF-BB treatment ameliorated learning and memory ability of AD mice by regulating BRCA1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePDGF-BB treatment ameliorated BBB integrity in AD mice by regulating miR-221/BRCA1 axis-mediated PHF19-PRC2 complex.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe further detected the expression of pericyte markers in AD mice by western blotting. The expression of α-SMA, Desmin and PDGFRβ was decreased in hippocampus and cortical tissues of AD mice, which was up-regulated by PDGF-BB treatment. BRCA1 overexpression reversed PDGF-BB-mediated up-regulation of α-SMA, Desmin and PDGFRβ in hippocampus and cortical tissues of AD mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA, B and E, F). Moreover, the expression of PHF19-PRC2 complex components, PHF19, EZH2, EED, SUZ12, RbAp46/48 was decreased, and BRCA1 was up-regulated in hippocampus and cortical tissues of AD mice. PDGF-BB caused a down-regulation of BRCA1 and an up-regulation of PHF19, EZH2, EED, SUZ12, RbAp46/48 in hippocampus and cortical tissues of AD mice, which was abolished by BRCA1 up-regulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC, D and G, H). QRT-PCR results showed that miR-221 was down-regulated, BRCA1 was up-regulated in hippocampus and cortical tissues of AD mice. PDGF-BB treatment enhanced miR-221 expression and reduced BRCA1 expression in hippocampus and cortical tissues of AD mice. The influence conferred by PDGF-BB treatment was abrogated by BRCA1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA, B). Additionally, we carried out EB assay to detect the BBB integrity in mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eC, D revealed that the BBB integrity was decreased in AD mice, which was rescued by PDGF-BB treatment. BRCA1 overexpression destroyed the protection of PDGF-BB on BBB integrity in AD mice. All these findings suggested that PDGF-BB treatment ameliorated BBB integrity in AD mice by regulating miR-221/BRCA1 axis-mediated PHF19-PRC2 complex.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAD seriously threatens the health of the human, especially elderly. In this work, we found that PDGF-BB gave boost to cell viability and proliferation, while inhibited apoptosis of Aβ1-42-treated pericytes. PDGF-BB treatment caused a down-regulation of BRCA1 in Aβ1-42-treated pericytes. The influence conferred by PDGF-BB was abrogated by BRCA1 overexpression. Mechanism studies have shown that BRCA1 interacted with PHF19-PRC2 complex, and was a target of miR-221 and miR-222. Thus, PDGF-BB activated PHF19-PRC2 complex by regulating miR-221/BRCA1 axis in Aβ1-42-treated pericytes. \u003cem\u003eIn vivo\u003c/em\u003e, PDGF-BB treatment ameliorated learning and memory ability and elevated BBB integrity in AD mice. All these data suggested that PDGF-BB regulated miR-221/BRCA1 axis to activate PHF19-PRC2 complex, thereby alleviating AD development in mice.\u003c/p\u003e \u003cp\u003ePDGF-BB is a growth factor secreted and released by platelets, endothelial cells, macrophages, epithelial cells and smooth muscle cells. It specifically interacts with PDGFR to promote the proliferation and differentiation of connective tissue cells, endothelial cells and smooth muscle cells [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. PDGF-BB participates in the progression of various diseases. Gianni-Barrera et al. have revealed the functional role of PDGF-BB in aberrant angiogenesis. PDGF-BB regulates VEGF-R2 signaling pathway to prevent VEGF-induced aberrant angiogenesis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Inhibition of PDGF-BB secretion promotes healing bisphosphonate-related osteonecrosis of the jaw, which attributes to elevate angiogenesis and osteogenesis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Additionally, previous study has found that PDGF-BB treatment exerts protective effects in Aβ-induced neuroblastoma cell damage [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. PDGF-BB treatment reduces pericyte loss and BBB impairment by regulating ERK/Akt pathways, thereby alleviating AD development [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Consistently, we also confirmed the therapeutical effects of PDGF-BB in AD. Moreover, we found that PDGF-BB treatment promoted cell viability, proliferation and repressed apoptosis of Aβ1-42-treated pericytes. \u003cem\u003eIn vivo\u003c/em\u003e, PDGF-BB treatment ameliorated learning and memory ability and alleviated BBB impairment in AD mice.\u003c/p\u003e \u003cp\u003eThe role of BRCA1 in AD is well known. Mano et al. have conducted pyrosequencing to examine BRCA1 methylation levels in peripheral blood of AD patients, suggesting that BRCA1 methylation is a risk factor for AD [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Hypomethylation of BRCA1-mediated up-regulation of BRCA1 in brain of AD patients is highly insoluble and mislocated in the cytoplasm, which leads to DNA damage in AD mice [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. BRCA1 is intense and specific localizated at neurofibrillary tangles of brain tissues in AD patients [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In the present work, we determined the role of BRCA1 in AD. BRCA1 was up-regulated in Aβ1-42-treated pericytes and repressed by PDGF-BB treatment. BRCA1 overexpression reversed PDGF-BB-mediated increase of cell viability and proliferation and inhibition of apoptosis in Aβ1-42-treated pericytes. BRCA1 was a potential target of miR-221 and miR-222. MiR-221 repressed BRCA1 expression by targeting BRCA1, while miR-222 inhibitor had no influence on BRCA1 expression. Thus, PDGF-BB treatment alleviated AD development by regulating miR-221/BRCA1 axis, instead of miR-222/BRCA1 axis.\u003c/p\u003e \u003cp\u003ePHF19-PRC2 complex takes part in the development of many solid tumors [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The abnormally expressed of PRC2 complex members, especially EZH2, is closely correlated with the malignancy of the tumors [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. PRC2 complex inhibitors have been used in clinical tumor therapy [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In the present work, we first revealed the functional role of PHF19-PRC2 complex in the progression of AD. BRCA1 interacted with PHF19-PRC2 complex members, PHF19, EZH2, EED, SUZ12, RbAp46/48, which was regulated by PDGF-BB treatment. Thus, PDGF-BB treatment ameliorated AD development by activating BRCA1-mediated PHF19-PRC2 complex \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis work demonstrated that PDGF-BB activated PHF19-PRC2 complex by regulating miR-221/BRCA1 axis, thereby reducing the permeability of BBB and ameliorating the learning and memory ability of AD mice. Thus, PDGF-BB may play a therapeutic role in AD development.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eAD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eAlzheimer\u0026apos;s disease\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eA\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eAmyloid \u0026beta;-protein\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eBBB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eBlood brain barrier\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eBRCA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eBreath cancer susceptibility gene 1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eCCK-8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eCell Counting Kit-8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eCo-IP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eCo-Immunoprecipitation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eEB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eEvans Blue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eELISA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eEnzyme-linked immuno sorbent assay\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eIF\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eImmunofluorescence\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003emiRNAs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eMicroRNAs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eMWM\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eMorris water maze\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003ePDGF-BB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003ePlatelet derived growth factor-BB\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003ePRC2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003ePolycomb repressive complex 2\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 94px;\"\u003e\n \u003cp\u003eUTR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 459px;\"\u003e\n \u003cp\u003eUntranslated region\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. National Natural Science Foundation of China: A novel mechanism of blood-brain barrier dysfunction promoting Alzheimer's disease: PDGF-BB/PDGFRβ/lncRNA Pat1-mediated pericellular deletion. Project number: 82060215.\u003c/p\u003e\n\u003cp\u003e2. Yunnan Provincial Science and Technology Department - Kunming Medical joint special project: Study on the treatment and mechanism of human umbilical cord mesenchymal stem cells combined with sulforaphane in transgenic mice with Alzheimer's disease. Project number: 202001AY070001-267.\u003c/p\u003e\n\u003cp\u003e3. Education Department of Yunnan Province - Teacher project: Preliminary study on the effect of chronic cerebral hypoperfusion on blood-brain barrier permeability in transgenic mice with Alzheimer's disease. Project number: 2020J0221.\u003c/p\u003e\n\u003cp\u003e4. Science and Technology Talents and Platform Program of Yunnan Provincial Department of Science and Technology -- Reserve talents for provincial young and middle-aged academic and technical leaders. Project number: 202305AC160081.\u003c/p\u003e\n\u003cp\u003e5. \"Spring City Plan\" high-level talents - famous doctors in Spring City. Certificate number: C202012002.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHeyun Yang conceived, designed and supervised the study. Heyun Yang, Keying Zhao, Yulan Li, JinRong Ya, Chongmin Wu interpreted the results and drafted the manuscript. Heyun Yang, Jueyan Li, Keying Zhao, Ling Shi, Hongying Zhou, Yulan Li, Qiong Zhao, JinRong Ya,Chongmin Wu, Jinxia Yan performed all experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHeyun Yang, Yulan Li, Qiong Zhao, JinRong Ya,Chongmin Wu, performed statistical analysis. All authors have approved the fnal version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the National Natural Science Foundation of China (No. 82060215), Yunnan Provincial Science and Technology Department - Kunming Medical joint special project (No. 202001AY070001-267), Education Department of Yunnan Province - Teacher project (No. 2020J0221), Science and Technology Talents and Platform Program of Yunnan Provincial Department of Science and Technology (No. 202305AC160081), and 'Spring City Plan' high-level talents \u0026nbsp;(No. C202012002).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all participants, and approval was granted by the Ethics Committee of Kunming First People's Hospital (YLS2020-20).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eDepartment of Neurology, Kunming First People's Hospital, Yunnan, China.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eCalmette Hospital Affiliated to Kunming Medical University, Yunnan, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u0026nbsp;\u003c/sup\u003eKunming Medical University, Yunnan, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eWeller J, Budson A. 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J Prev Alzheimers Dis, 2021. 8(4): p. 477-482.\u003c/li\u003e\n \u003cli\u003eMano T, Nagata K, Nonaka T, Tarutani A, Imamura T, Hashimoto T, et al. Neuron-specific methylome analysis reveals epigenetic regulation and tau-related dysfunction of BRCA1 in Alzheimer\u0026apos;s disease. PNAS, 2017. 114(45): p. E9645-e9654.\u003c/li\u003e\n \u003cli\u003eEvans TA, Raina AK, Delacourte A, Aprelikova O, Lee H, Zhu X, et al. BRCA1 may modulate neuronal cell cycle re-entry in Alzheimer disease. Int J Med Sci, 2007. 4(3): p. 140-5.\u003c/li\u003e\n \u003cli\u003eDuan R, Du W, Guo W. EZH2: a novel target for cancer treatment. J Hematol Oncol, 2020. 13(1): p. 104.\u003c/li\u003e\n \u003cli\u003eWan L, Wan L, Xu K, Wei Y, Zhang J, Han T, et al. Phosphorylation of EZH2 by AMPK Suppresses PRC2 Methyltransferase Activity and Oncogenic Function. Mol Cell, 2018. 69(2): p. 279-291.e5.\u003c/li\u003e\n \u003cli\u003eMiranda TB, Cortez CC, Yoo CB, Liang G, Abe M, Kelly TK, et al. DZNep is a global histone methylation inhibitor that reactivates developmental genes not silenced by DNA methylation. Mol Cancer Ther, 2009. 8(6): p. 1579-88.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alzheimer's disease, PDGF-BB, PHF19-PRC2 complex, Pericytes, Blood brain barrier ","lastPublishedDoi":"10.21203/rs.3.rs-6395732/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6395732/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003ePDGF-BB is one of the important factors to maintain the function of pericytes. Pericyte damage accelerates the progression of Alzheimer's disease (AD). The role of PDGF-BB in AD was verified in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003ePericytes were treated with Aβ1-42 or combined with PDGF-BB. CCK-8 assay, EdU assay and flow cytometry examined cell viability, proliferation and apoptosis. Co-Immunoprecipitation verified the relationship among BRCA1, PHF19, EZH2, EED, SUZ12 and RbAp46/48.Luciferase reporter assay verified the relationship among BRCA1, miR-221-3p and miR-222-3p. APP/PS1 mice were administrated with PDGF-BB. Morris water maze test examined animal behaviors.Immunofluorescence staining and Evans Blue assay examined the pericyte coverage and blood brain barrier (BBB) integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003ePDGF-BB enhanced cell viability and proliferation, while inhibited apoptosis of Aβ1-42-treated pericytes, which was abrogated by BRCA1 overexpression. BRCA1 was up-regulated in Aβ1-42-treated pericytes. Additionally, PDGF-BB treatment caused a down-regulation of BRCA1 and up-regulation of PHF19-PRC2 complex members, PHF19, EZH2, EED, SUZ12 and RbAp46/48. BRCA1 interacted with PHF19-PRC2 complex members. MiR-221 repressed BRCA1 expression by targeting BRCA1. MiR-222 interacted with BRCA1 and had no influence on BRCA1 expression. \u003cem\u003eIn vivo\u003c/em\u003e, PDGF-BB treatment ameliorated learning and memory ability and elevated pericyte coverage and BBB integrity in AD mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003ePDGF-BB activated PHF19-PRC2 complex by regulating miR-221/BRCA1 axis, thereby reducing the permeability of BBB and ameliorating the learning and memory ability of AD mice. Thus, PDGF-BB may play a therapeutic role in AD development.\u003c/p\u003e","manuscriptTitle":"PDGF-BB alleviates pericyte damage by the activation of the PHF19-PRC2 complex via the miR-221/BRCA1 axis in Alzheimer's disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-08 13:33:31","doi":"10.21203/rs.3.rs-6395732/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ff2f10ef-e7dc-4ae8-8922-dfd53ab37143","owner":[],"postedDate":"May 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-24T00:57:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-08 13:33:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6395732","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6395732","identity":"rs-6395732","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}