背景:叶酸是中枢神经系统(CNS)正常生长和发育所必需的B9维生素家族。叶酸的运输由三种主要的运输蛋白介导:叶酸受体α(FRα),质子偶联叶酸转运蛋白(PCFT),和减少叶酸载体(RFC)。脑叶酸通过FRα和PCFT的协调作用发生在脉络丛(CP)上皮,或直接进入血管血脑屏障(BBB)的脑实质,由RFC介导。叶酸转运受损可能是由于FRα或PCFT功能突变的丧失,导致CSF叶酸水平次优。我们以前的报告已经证明,一旦被天然化合物吡咯并喹啉醌(PQQ)激活,核呼吸因子-1(NRF-1)的RFC上调。最近,我们已经确定了蛛网膜屏障(AB)的叶酸转运蛋白定位。本研究的目的是进一步表征叶酸转运体在AB细胞中的定位和功能。以及它们通过NRF-1/PGC-1α信号传导和叶酸缺乏的调节。
方法:在永生化小鼠AB细胞中,通过免疫细胞化学分析评估RFC和PCFT的极化定位,具有通过转运测定检查的RFC和PCFT功能。还研究了PQQ处理对RFC功能表达变化的影响。在叶酸缺乏条件下生长的小鼠AB细胞评估叶酸转运蛋白基因表达的变化,和其他关键转运蛋白和紧密连接蛋白。
结果:免疫细胞化学分析显示RFC在小鼠AB上皮的顶端定位,PCFT位于基底外侧和细胞内区室。PQQ导致RFC功能表达显着增加,由NRF-1/PGC-1α信号级联的激活介导。叶酸缺乏导致RFC表达显著增加,MRP3,P-gp,GLUT1和紧密连接蛋白claudin-5。
结论:这些结果揭示了RFC和PCFT在AB的极化表达,通过激活NRF-1/PGC-1α信号通路和叶酸缺乏诱导RFC功能表达。这些结果表明,AB可能有助于叶酸流入CSF,代表当CP处的叶酸转运受损时的额外途径。
BACKGROUND: Folates are a family of B9 vitamins essential for normal growth and development in the central nervous system (CNS). Transport of folates is mediated by three major transport proteins: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Brain folate uptake occurs at the choroid plexus (CP) epithelium through coordinated actions of FRα and PCFT, or directly into brain parenchyma at the vascular blood-brain barrier (BBB), mediated by RFC. Impaired folate transport can occur due to loss of function mutations in FRα or PCFT, resulting in suboptimal CSF folate levels. Our previous reports have demonstrated RFC upregulation by nuclear respiratory factor-1 (NRF-1) once activated by the natural compound pyrroloquinoline quinone (PQQ). More recently, we have identified folate transporter localization at the arachnoid barrier (AB). The purpose of the present study was to further characterize folate transporters localization and function in AB cells, as well as their regulation by NRF-1/PGC-1α signaling and folate deficiency.
METHODS: In immortalized mouse AB cells, polarized localization of RFC and PCFT was assessed by immunocytochemical analysis, with RFC and PCFT functionality examined with transport assays. The effects of PQQ treatment on changes in RFC functional expression were also investigated. Mouse AB cells grown in folate-deficient conditions were assessed for changes in gene expression of the folate transporters, and other key transporters and tight junction proteins.
RESULTS: Immunocytochemical analysis revealed apical localization of RFC at the mouse AB epithelium, with PCFT localized on the basolateral side and within intracellular compartments. PQQ led to significant increases in RFC functional expression, mediated by activation of the NRF-1/PGC-1α signalling cascade. Folate deficiency led to significant increases in expression of RFC, MRP3, P-gp, GLUT1 and the tight junction protein claudin-5.
CONCLUSIONS: These results uncover the polarized expression of RFC and PCFT at the AB, with induction of RFC functional expression by activation of the NRF-1/PGC-1α signalling pathway and folate deficiency. These results suggest that the AB may contribute to the flow of folates into the CSF, representing an additional pathway when folate transport at the CP is impaired.