| Objectives:Patients with Alzheimer’s disease(AD)have a higher incidence of osteoporosis than healthy adults,but the underlying mechanism is not clear.The aim of this study was to investigate the effect of brain derived extracellular vesicles(B-EVs)from APPswe/PS1 dE9(AD)mice on the differentiation fate of bone marrow mesenchymal stem cells(BMSCs)and their role in AD-induced bone loss.Methods:(1)To determine whether B-EVs can be transported to bone and whether there is a difference in the amount of AD-B-EVs transported to bone compared with WT-B-EVs,mice were intracerebroventricularly or intravenously injected with fluorescent labeled WT-B-EVs,AD-B-EVs or equal volume of the vehicle.(2)The effects of AD-B-EVs or WT-B-EVs on the differentiation of BMSCs were observed during the induction of osteogenic and adipogenic differentiation;the positive areas of calcium nodules in osteogenic differentiation were detected by Alizarin Red S(ARS)staining,and the lipid droplets in adipogenic differentiation were detected by Oil Red O(ORO)staining.The expression levels of osteogenic-related genes(Ocn and Alpl)and adipogenic-related genes(Cebpa and Pparg)were also detected using qRT-PCR analysis.The effects of AD-B-EVs or WT-B-EVs on osteoclastic differentiation of RAW264.7 cells were observed using Tartrate-resistant acid phosphatase(TRAP)staining to detect the number of osteoclasts.(3)Four-month-old C57BL/6 male mice were used in this study.For B-EV treatment,WT-B-EVs,AD-B-EVs or an equal volume of the vehicle was injected into the mice intravenously once a week for 2months.Bone mineral density(BMD)and bone microstructure changes were assessed by microcomputed tomography(μCT);the double calcein labeling was performed to detect the mineral apposition rate(MAR)and bone formation rate per bone surface(BFR/BS)of mouse femur samples;OCN-positive osteoblasts or TRAP-positive osteoclasts were detected by immunohistochemistry staining;the levels of serum OCN and CTX-Ⅰ were measured by ELISA,and the levels of perilipin A-positive adipocytes were detected by immunofluorescence staining.(4)qRT-PCR analysis was used to validate the expression of several miRNAs(miR-483-5p,miR-34c-5p and miR-141-3p)which were previously reported to be related to bone metabolism in AD-B-EVs,brain tissue from AD mice and plasma-derivedEVs from AD patients(AD-P-EVs).(5)BMSCs were treated with AD-P-EVs or sex-and age-matched normal control plasma EVs(CN-P-EVs)during the induction of osteogenic and adipogenic differentiation,and ARS and ORO staining were performed.(6)Specific antagomiR was used to silence the significantly increased miRNA in AD-B-EVs,the effects of antagomiR pretreated AD-B-EVs on the differentiation of BMSCs were observed during the induction of osteogenic and adipogenic differentiation.(7)Mice were intravenously injected with antagomiR pretreated AD-B-EVs once a week,μCT,immunohistochemistry,immunofluorescence,andELISA were detected after 2 months of injection.(8)Target genes of miRNAs in BMSCs were inhibited using si RNA,and the role of target genes in osteogenic and adipogenic differentiation of BMSCs was verified using ARS or ORO staining and qRT-PCR analysis.Results:(1)Fluorescence labeled WT-B-EVs and AD-B-EVs can be detected in the bone and other tissues,with stronger signals in the liver,bone,and the kidney,followed by the spleen,lung,and the heart.No significant difference of fluorescence intensity was found between the WT-B-EVs and AD-B-EVs.We also detected the distribution of fluorescence labeled WT-B-EVs or AD-B-EVs in different tissues after intravenous injection for 24 h.Fluorescent signals were detected in all organs,with stronger signals in the liver,spleen,and the lung,followed by the bone,kidney,heart,and the brain.No significant difference of fluorescence intensity was found between the WT-B-EVs and AD-B-EVs.(2)ARS staining revealed that AD-B-EVs could induce a significant suppression of osteogenesis of BMSCs(P < 0.05).qRT-PCR also showed decreased expression of osteogenesis-related genes(Ocn and Alpl)of BMSCs treated with AD-B-EVs(P < 0.05).ORO staining showed that compared with WT-B-EVs and vehicle groups,AD-B-EVs could significantly promote adipogenesis of BMSCs(P < 0.01)and upregulate the expression of adipogenesis-related genes(Cebpa and Pparg)(P <0.05).In addition,AD-B-EVs had no significant effect on osteoclastic differentiation of RAW264.7 cells compared with WT-B-EVs group.(3)μCT analysis revealed that AD-B-EVs induced significant decreases in BMD and impaired bone microstructures(P < 0.01).Double calcein labeling analysis showed marked decreases in mineral apposition rate(MAR)and bone formation rate per bone surface(BFR/BS)in AD-B-EVs-treated mice compared with those treated with vehicle or WT-B-EVs(P < 0.05).Immunohistochemical staining for OCN showed remarkably reduced numbers of OCN-positive osteoblasts on the surface of trabecular bones from AD-B-EVs-treated mice than those from vehicle-or WT-B-EVs-treated mice(P < 0.0001).ELISA for serum OCN revealed the level of serum OCN was markedly decreased after AD-B-EVs treatment(P < 0.05).Immunofluorescence staining revealed significantly increased numbers of perilipin A-positive adipocytes within the bone marrow in AD-B-EVs group compared with other two groups(P< 0.05).TRAP staining revealed that there was no significant difference in the number of osteoclasts among AD-B-EVs,WT-B-EVs and vehicle treatment groups.ELISA for serum bone resorption marker C-terminal telopeptides of type I collagen(CTX-Ⅰ)showed no significant difference among these three groups.(4)qRT-PCR analysis revealed that miR-483-5p was significantly elevated in B-EVs and brain tissue of AD mice(P < 0.01);miR-483-5p levels were significantly increased in plasma EVs of AD patients(P <0.05),and miR-483-5p levels were negatively correlated with BMD(T-Score)of femoral neck in AD patients(r =-0.688,P < 0.001).(5)ARS and ORO staining showed that AD-P-EVs also exhibited notable anti-osteogenic and pro-adipogenic effects compared with CN-P-EVs(P < 0.001).(6)We found the expression of miR-483-5p in BMSCs treated with AD-B-EVs was significant increased(P < 0.01).ARS staining showed that inhibition of miR-483-5p by antagomiR-483-5p significantly rescue the anti-osteogenic effects of AD-B-EVs on mouse BMSCs(P < 0.01).The expression of Ocn and Alpl were also upregulated(P < 0.05).ORO staining and qRT-PCR analysis revealed that inhibition of miR-483-5p significantly suppressed the lipid droplet formation(P < 0.05)and the expression of Cebpa and Pparg stimulated by AD-B-EVs(P < 0.05).(7)μCT analysis showed that the after inhibition of miR-483-5p by antagomiR-483-5p,the reduction of BMD and impaired bone microstructures caused by AD-B-EVs were improved compared with antagomiR-NC-pre-treated AD-B-EVs group(P < 0.01).Double calcein labeling analysis also showed an increase of MAR and BFR/BS(P <0.05).The numbers of OCN-positive osteoblasts and the OCN levels in serum were also increased while the numbers of perilipin A-positive adipocytes were reduced(P < 0.05).Similarly,no significant changes were observed in the number of TRAP-positive osteoclasts as well as the level of serum CTX-Ⅰ between the groups.(8)The expression of IGF2 was remarkably decreased in AD-B-EVs treated BMSCs from both western blot and qRT-PCR analysis(P < 0.01),while inhibition of miR-483-5p in AD-B-EVs can partially reverse the effect of AD-B-EVs induced decrease of IGF2(P < 0.05).Furthermore,inhibition of IGF2 in BMSCs can also have anti-osteogenesis and pro-adipogenesis effects(P < 0.0001 and P < 0.01)and qRT-PCR analysis showed that osteogenesis-associated genes were down-regulated(P < 0.05)and adipogenesis-associated genes were up-regulated(P <0.05),respectively.Conclusion:AD-B-EVs can be transported across the blood brain barrier to the bone,and could significantly decrease osteogenesis and increase adipogenesis of BMSCs and induce bone-fat imbalance in adult mice by carrying miR-483-5p.Furthermore,miR-483-5p mediated the anti-osteogenic,pro-adipogenic,and pro-osteoporotic effects of AD-B-EVs associated with the inhibition of IGF2. |
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