Regulatory Mechanisms of Folate Transporters by Transcription Factors in the Brain

叶酸（Vit B9）是哺乳动物细胞中关键生物合成过程所必需的水溶性维生素。叶酸在生物膜和组织中的转运主要通过三种途径介导：叶酸受体α (FRa)、质子偶联叶酸转运蛋白（PCFT）和还原性叶酸载体（RFC）。这些叶酸转运途径的功能表达可由多种转录因子调控，如维生素D受体（VDR）。叶酸对大脑发育和功能至关重要。FRa是血脑脊液屏障中叶酸的主要转运途径，其失活可通过功能缺失突变或FRa自身抗体的存在导致严重的脑叶酸缺乏，其特征为炎症、氧化应激、线粒体功能障碍和脑髓鞘形成异常。最近，作为2015年4月批准的NSERC拨款的一部分，我们证明了通过诱导特异性叶酸转运蛋白（即RFC）来调节血脑屏障（BBB）的叶酸摄取可以提供一种在FRa缺陷的情况下增强脑叶酸输送的新策略。我们提供的证据表明，通过暴露于其激活配体1,25-二羟基维生素D3（骨化三醇）激活VDR，可以诱导血脑屏障的RFC功能，并有效地恢复FRa缺乏小鼠大脑中的叶酸水平。虽然这些结果非常令人兴奋，但我们观察到，用骨化三醇治疗的动物出现了诸如体重减轻和血钙水平高的毒性。NRF-1是一种转录因子，主要以促进线粒体和呼吸功能而闻名。我们最近确定它也可以调节血脑屏障上的叶酸转运蛋白RFC。我们提出，在大脑FRa缺陷的情况下，NRF-1通过其特异性配体吡咯喹啉醌（PQQ）（一种天然无毒化合物）的激活可以增强RFC功能，增加叶酸进入大脑，逆转炎症、氧化应激和线粒体功能障碍。我们的工作将揭示与脑叶酸缺乏相关的线粒体功能障碍、脑炎症和氧化应激的基本机制，阐明当脑叶酸水平恢复时这些影响是如何逆转的，并为FRa功能丧失引起的神经代谢紊乱提供新的治疗方法。此外，由于越来越多的证据表明线粒体功能障碍与神经系统疾病有关，本项目的研究结果具有更广泛的应用前景，也可能被开发为几种已知具有非常差的中枢神经系统通透性的药物（即抗癌药物）的新递送途径。通过这项工作的开展，我们计划培养和指导高素质的人才，成为未来自然科学领域的领导者。

Folates (Vit B9) are water soluble vitamins required for key biosynthetic processes in mammalian cells. Folate transport across biological membranes and tissues is mediated by three major pathways: folate receptor alpha (FRa), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). The functional expression of these folate transport pathways can be regulated by several transcription factors such as the Vitamin D receptor (VDR). Folates are critical for brain development and function. Inactivation of FRa, the major folate transport pathway at the blood-cerebrospinal fluid barrier, through loss-of-function mutations or the presence of FRa autoantibodies, causes severe cerebral folate deficiency characterized by inflammation, oxidative stress, mitochondrial dysfunction and abnormal brain myelination. Recently, as part of the NSERC grant approved in April 2015, we demonstrated that modulating folate uptake at the blood-brain barrier (BBB) through induction of specific folate transporters (i.e., RFC) could present a novel strategy for enhancing brain folate delivery in the context of defective FRa. We provided evidence that activation of VDR through exposure to its activating ligand, 1,25-dihydroxyvitamin D3 (calcitriol), induced RFC function at the BBB and effectively restored folate levels in the brain of mice lacking FRa. Although these results are very exciting, we observed that the animals treated with calcitriol presented toxicities such as weight loss and high calcium blood levels. NRF-1 is a transcription factor primarily known for its role in promoting mitochondrial and respiratory function. We recently determined that it can also regulate the folate transporter, RFC at the BBB. We propose that, in the context of defective FRa in the brain, activation of NRF-1 by its specific ligand, pyrroloquinoline quinone (PQQ), a natural non-toxic compound, can enhance RFC function and increase folate delivery into the brain reversing inflammation, oxidative stress, as well as mitochondrial dysfunction. Our proposed work will uncover fundamental mechanisms involved in mitochondrial dysfunction, brain inflammation and oxidative stress related to cerebral folate deficiency, elucidate how these effects can be reversed when brain folate levels are restored, and provide novel treatment approaches for neurometabolic disorders caused by loss of FRa function. Further, the findings of this project present broader applications due to the growing body of evidence associating mitochondrial dysfunction to neurological disorders and may also be exploited as a novel delivery route for several pharmacological agents known to have very poor CNS permeability (i.e., anticancer drugs). With the undertaking of the proposed work, we plan to train and mentor highly qualified personnel to become future leaders in the natural sciences.