Regulation of Folate Transport at the Blood-brain Barrier: A Novel Strategy for the Treatment of Childhood Neurological Disorders Associated with Cerebral Folate Deficiency

Folates are crucial for the maintenance of central nervous system homeostasis. Folate transport is mediated by three major pathways, i.e., folate receptor alpha (FRα), proton-coupled folate transporter (PCFT) and reduced folate carrier (RFC), known to be regulated by transcription factors. Brain folate delivery occurs predominantly at the choroid plexus through concerted actions of FRα and PCFT; inactivation of these transport systems causes cerebral folate deficiency resulting in early childhood neurodegeneration. Increasing brain folate permeability or finding alternative routes for brain folate transport could lead to therapeutic benefits. The overall goal of this PhD thesis was to characterize folate transport at the blood-brain barrier (BBB) by examining the role and regulation of folate transporters (i.e., RFC) in brain folate uptake. The objectives of this thesis were to: i) determine RFC functional expression in several in vitro (primary or immortalized cultures of human/rodent brain microvessel endothelial cells) and ex vivo (isolated mouse brain capillaries) BBB models, ii) investigate the role of transcription factors, particularly vitamin D receptor (VDR) and nuclear respiratory factor 1 (NRF-1), in the regulation of RFC in various BBB model systems, and iii) examine in vivo, using Folr1 (FRα) knockout mice, the relative contribution of RFC in overall brain folate uptake. We initially demonstrated that RFC is functionally expressed in in vitro systems representative of human (hCMEC/D3 cells) and rodent (mouse brain capillaries) BBB, and that activation of VDR by its ligand, 1,25-dihydroxyvitamin D3 or calcitriol, significantly increased RFC mRNA and protein expression as well as function. We further showed in vivo, using Folr1 knockout mice, that loss of FRα substantially decreased folate delivery to the brain, but transport was restored through calcitriol administration. Additionally, we provided in vitro and in vivo evidence that RFC expression and transport activity is inducible by another transcription factor, NRF-1. These findings demonstrate that augmenting RFC functional expression through interaction with specific transcription factors could constitute a novel strategy for enhancing brain folate delivery. Modulating folate uptake at the BBB may have clinical significance due to the lack of established optimal therapy for neurometabolic disorders caused by loss of FRα or PCFT function.Ph.D