目的通过单细胞转录组数据,探究牙本质基质蛋白1(dentin matrix protein 1,DMP1)突变小鼠血脑屏障通透性改变的分子机制。方法通过同源重组的方法,在C57BL/6J背景的小鼠体内敲入一段突变序列,将DMP1糖基化位点的丝氨酸错义突变为分子量...目的通过单细胞转录组数据,探究牙本质基质蛋白1(dentin matrix protein 1,DMP1)突变小鼠血脑屏障通透性改变的分子机制。方法通过同源重组的方法,在C57BL/6J背景的小鼠体内敲入一段突变序列,将DMP1糖基化位点的丝氨酸错义突变为分子量更小的甘氨酸,构建S89G-DMP1小鼠模型。前序研究发现DMP1糖基化位点无义突变的S89G-DMP1小鼠会导致血脑屏障(blood brain barrier,BBB)功能障碍。分离小鼠脑组织后通过酶消化制备单细胞悬液,上机测序得到24167个细胞的转录组数据。在R语言中利用Seurat包进行细胞分群,寻找各亚群的差异基因后进行细胞类型定义。结合血脑屏障损伤对神经炎症和神经功能的影响,分析对照组(野生组,简称WT组)与S89G-DMP1组的神经元、小胶质细胞、少突胶质细胞、星形胶质细胞等细胞群体的变化和细胞类型特异性基因表达差异,找到影响血脑屏障完整性的关键细胞和因子。结果在S89G-DMP1小鼠大脑中,少突细胞比例明显上升,神经细胞比例下降。S89G-DMP1组与WT组神经细胞的基因表达较为接近,S89G-DMP1组与WT组星形胶质细胞的基因表达差异较大,共筛选出10个上调基因,85个下调基因,通过GO分析,发现下调基因显著富集在细胞黏附、离子跨膜转运等生理功能上。除此之外,在小胶质细胞、少突胶质细胞中也发现与细胞黏附生理功能的基因在S89G-DMP1小鼠大脑中显著下调。结论S89G-DMP1小鼠大脑神经细胞的比例降低,可能与BBB功能障碍互为因果,但基因表达谱无明显改变,主要通过下调星形胶质细胞、少突胶质细胞和小胶质细胞中与细胞黏附功能相关的基因表达来增加小鼠BBB的通透性,导致BBB功能障碍。展开更多
The blood-brain barrier （BBB） is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed mo...The blood-brain barrier （BBB） is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein I （DMP1）-proteoglycan （PG）, which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMPI-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 （S89） in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine （S89G）, which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that reg- ulated BBB formation, but also assigned a new function to DMP1-PG.展开更多
基金We thank Dr. Chunlin Qin （Bayler colleage of dentistry, Texas A＆M University） for providing us DMP1 antibody. This study was supported by China National Key Research and Development Program （2016YFA0100801 YS）, and the National Natural Science Foundation of China （Grant Nos. 8133030 YS and 31620103904 YS）, and grants： 2016YFC102705 YS 2014BAI04B07 WZL+1 种基金 81470715 YS TJ1504219036 WZL： 2017BR009 YS.
文摘The blood-brain barrier （BBB） is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein I （DMP1）-proteoglycan （PG）, which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMPI-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 （S89） in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine （S89G）, which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that reg- ulated BBB formation, but also assigned a new function to DMP1-PG.
