Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid
Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid
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DOI：
10.1021/acs.molpharmaceut.5b00580
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发表时间：
2015-12-01
影响因子：
4.9
通讯作者：
Nicolazzo, Joseph A.
中科院分区：
文献类型：
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作者：
Pan, Yijun;Scanlon, Martin J.;Nicolazzo, Joseph A.
大脑从omega-3脂肪酸前体合成必需多不饱和脂肪酸（PUFA）二十二碳六烯酸（DHA）的能力有限。因此，为了将PUPA的脑浓度维持在生理水平，血浆来源的DHA必须通过血脑屏障（BBB）运输。虽然DHA能够进入脑内皮细胞的管腔膜，但其低水溶性可能限制了其胞浆向管腔膜的转移，因此需要细胞内载体蛋白来促进该PUFA通过血脑屏障的运输。由于细胞内载体蛋白脂肪酸结合蛋白5 （FABPS）在人血脑屏障处表达，本研究在体外和体内分别评估了FABPS在脑内皮细胞摄取DHA和血脑屏障运输中的推测作用。在大肠杆菌C41（DE3）细胞中重组表达并纯化hfabb5，采用等温滴定量热法评估DHA与hFABPS的结合亲和力。评估FABPS siRNA对永生化人脑微血管内皮细胞（hCMEC/D3）摄取C-14-DHA的影响。优化C57BL/6小鼠原位经心灌注方法，比较fabps缺陷型（FABP5(-/-)）和野生型（FABP5(+/+)） C57BL/6小鼠血脑屏障内流率MO - C-14-DHA。DHA与hFABPS结合的平衡解离常数为155 +/- 8 nM（平均+/- SEM）。转染FABPS siRNA后，hCMEC/D3 mRNA和蛋白表达分别降低53.2 +/- 5.5%和44.8 +/- 13.7%，细胞摄取C-14-DHA减少14.1 +/- 2.7%。采用优化后的原位经心灌注条件（预灌注1 min (10 mL/min)，再灌注1 min）， C-14-DHA的Kin为0.04 +/- 0.01 mL/g/s。与FABP5（+/+）小鼠相比，FABP5（-/-）小鼠C-14-DHA K-in降低36.7 +/- 12.4%。本研究表明，FABPS与DHA结合并参与脑内皮细胞摄取和随后的DHA血脑屏障运输，证实了这种细胞质载体蛋白在中枢神经系统暴露这种对神经元功能至关重要的PUFA中的重要性。
The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUPA at physiological levels, plasma-derived DHA must be transported across the blood brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABPS) is expressed at the human BBB, the current study assessed the putative role of FABPS in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABPS assessed using isothermal titration calorimetry. The impact of FABPS siRNA on uptake of C-14-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate MO of C-14-DHA between FABPS-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABPS with an equilibrium dissociation constant of 155 +/- 8 nM (mean +/- SEM). FABPS siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABPS by 53.2 +/- 5.5% and 44.8 +/- 13.7%, respectively, which was associated with a 14.1 +/- 2.7% reduction in C-14-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of C-14-DHA (1 min)), the Kin of C-14-DHA was 0.04 +/- 0.01 mL/g/s. Relative to FABP5(+/+) mice, the K-in of C-14-DHA decreased 36.7 +/- 12.4% in FABP5(-/-)mice. This study demonstrates that FABPS binds to DHA and is involved in the brain endothelial cell uptake and subsequent BBB transport of DHA, confirming the importance of this cytoplasmic carrier protein in the CNS exposure of this PUFA essential for neuronal function.