Identification of Blood–Brain Barrier-Associated Proteins in the Human Brain

The blood–brain barrier (BBB) is essential for cerebral homeostasis and controls the selective passage of molecules traveling in and out of the brain. Despite the crucial role of the BBB in a variety of brain diseases and its relevance for the development of drugs, there is little known about its molecular architecture. In particular, the composition of the basal lamina between the astrocytic end-feet and the endothelial cells is only partly known. Here, we present a proteomic analysis of the basal lamina of the human BBB. We combined laser capture microdissection with shotgun proteomics for selective enrichment and identification of specific proteins present in the cerebral microvasculature and arachnoidal vessels collected from normal human brain tissue specimens. Proteins found to be associated with the blood–brain barrier were validated by immunohistochemistry. Expression of membrane protein MLC1 was found in all brain barriers. Phosphoglucomutase-like protein 5 appeared to be variably present along the outer part of intracerebral vessels, and multidrug resistance protein 1 was identified in both intracerebral, as well as arachnoidal blood vessels. The results demonstrate the presence of so far unidentified proteins in the human BBB and illustrate topic differences in their expression. In conclusion, we showed that sample purification by microdissection followed by shotgun proteomics provides a list of proteins identified in the BBB. Subsequent immunohistochemistry detailed the respective expression sites of membrane protein MLC1 and phosphoglucomutase-related protein 5. The role of the identified proteins in the functioning of the BBB needs further investigations.

血脑屏障（blood–brain barrier, BBB）对脑内稳态至关重要，可调控分子选择性进出大脑。尽管血脑屏障在多种脑部疾病中发挥关键作用，且与药物研发息息相关，但学界对其分子结构的认知仍较为有限。其中，星形胶质细胞终足与内皮细胞之间的基膜组成，目前仅部分为人所知。本研究针对人类血脑屏障的基膜开展蛋白质组学分析，将激光捕获显微切割技术与鸟枪蛋白质组学相结合，对从正常人类脑组织标本中获取的脑微血管和蛛网膜血管内的特定蛋白进行选择性富集与鉴定。与血脑屏障相关的蛋白通过免疫组织化学方法完成验证：研究发现膜蛋白MLC1在所有脑屏障中均有表达，磷酸葡萄糖变位酶样蛋白5在脑内血管外层的表达存在异质性，而多药耐药蛋白1则在脑内血管及蛛网膜血管中均被检出。本研究结果证实人类血脑屏障中存在此前未被识别的蛋白，并揭示了其表达的部位差异。综上，我们证明通过显微切割进行样本纯化后结合鸟枪蛋白质组学，可获得血脑屏障中鉴定得到的蛋白列表；后续免疫组织化学实验明确了膜蛋白MLC1及磷酸葡萄糖变位酶相关蛋白5的具体表达位点。上述已鉴定蛋白在血脑屏障功能中所发挥的作用，仍有待进一步研究。