Development of a method for isolating brain capillaries from a single neonatal mouse brain and comparison of proteomic profiles between neonatal and adult brain capillaries

Fluids and Barriers of the CNS

Research

Seiryo Ogata¹  Yudai Hamada²  Shingo Ito³  Takeshi Masuda³  Sumio Ohtsuki³ 

[1] Department of Environmental Medicine and Molecular Toxicology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-Machi, Aoba-Ku, 980-8575, Sendai, Japan;Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Chuo-Ku, 862-0973, Kumamoto, Japan;Department of Pharmaceutical Microbiology, School of Pharmacy, Kumamoto University, 5-1 Oe-Honmachi, Chuo-Ku, 862-0973, Kumamoto, Japan;Department of Pharmaceutical Microbiology, School of Pharmacy, Kumamoto University, 5-1 Oe-Honmachi, Chuo-Ku, 862-0973, Kumamoto, Japan;Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Chuo-Ku, 862-0973, Kumamoto, Japan;Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, 5-1 Oe-Honmachi, Chuo-Ku, 862-0973, Kumamoto, Japan;

关键词: Brain capillaries;    Isolation;    Neonate;    Proteomics;    Single brain;    Age;    Transporter;    Extracellular matrix;   

DOI  :  10.1186/s12987-023-00449-w

 received in 2023-01-27, accepted in 2023-06-05,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundThe functions and protein expressions of the blood–brain barrier are changed throughout brain development following birth. This study aimed to develop a method to isolate brain capillaries from a single frozen neonatal mouse brain and elucidate the enrichment of brain capillaries by quantitative proteomic analysis. We further compared the expression profile of proteins between neonatal and adult brain capillary fractions.MethodsThe brain capillary fraction was prepared by the optimized method from a single frozen mouse neonatal brain on postnatal day 7. The brain capillary fractions and brain lysates were digested by trypsin and analyzed by liquid chromatography-mass spectrometry for quantitative proteomics.ResultsBy optimizing the isolation method, we observed brain capillaries in the fraction prepared from a single neonatal mouse brain (nBC fraction). A protein amount of 31.5 μg, which is enough for proteomic analysis, was recovered from the nBC fraction. By proteomics analysis, the brain capillary selective proteins, including Abcb1a/Mdr1, Slc2a1/Glut1, Claudin-5, and Pecam-1, were found to be concentrated > 13.4-fold more in nBC fractions than in whole brain lysates. The marker proteins for neurons and astrocytes were not concentrated in nBC fractions, while those of pericytes and microglia were concentrated. Compared to adult mouse brain capillary fractions (aBC fractions), the expressions of Abcb1a/Mdr1a, Abcc4/Mrp4, and Slc2a1/Glut1 were significantly lower in nBC fractions than in aBC fractions, whereas those of Slc1a4/Asct1, Slc1a5/Asct2, Slc7a1/Cat1, and Slc16a1/Mct1 were significantly higher. Amino acid transporters, Slc38a5/Snat5, showed the greatest nBC-to-aBC ratio among transporters (9.83-fold). Network analysis of proteins expressed differentially between nBC and aBC fractions revealed that the proteins with terms related to the extracellular matrix were enriched.ConclusionsWe succeeded in isolating brain capillaries from a single frozen brain of a neonatal mouse at postnatal day 7. Proteomic analysis revealed the differential expression in brain capillaries between neonatal and adult mice. Specifically, amino acid transporters, including Slc1a5/Asct2 and Slc38a5/Snat5, were found to be induced in neonatal brain capillaries. The present isolation method will promote the study of the function and expression of the neonatal blood–brain barrier.

【 授权许可】

CC BY   
© The Author(s) 2023

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