Protective effects of pyrroloquinoline quinone in brain folate deficiency.

BACKGROUND: Folates (Vitamin B9) are critical for normal neurodevelopment and function, with transport mediated by three major pathways: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Cerebral folate uptake primarily occurs at the blood-cerebrospinal fluid barrier (BCSFB) through concerted actions of FRα and PCFT, with impaired folate transport resulting in the neurological disorder cerebral folate deficiency (CFD). Increasing evidence suggests that disorders associated with CFD also present with neuroinflammation, oxidative stress, and mitochondrial dysfunction, however the role of brain folate deficiency in inducing these abnormalities is not well-understood. Our laboratory has identified the upregulation of RFC by nuclear respiratory factor 1 (NRF-1) at the blood-brain barrier (BBB) once indirectly activated by the natural compound pyrroloquinoline quinone (PQQ). PQQ is also of interest due to its anti-inflammatory, antioxidant, and mitochondrial biogenesis effects. In this study, we examined the effects of folate deficiency and PQQ treatment on inflammatory and oxidative stress responses, and changes in mitochondrial function. METHODS: Primary cultures of mouse mixed glial cells exposed to folate-deficient (FD) conditions and treated with PQQ were analyzed for changes in gene expression of the folate transporters, inflammatory markers, oxidative stress markers, and mitochondrial DNA (mtDNA) content through qPCR analysis. Changes in cellular reactive oxygen species (ROS) levels were analyzed in vitro through a DCFDA assay. Wildtype (C57BL6/N) mice exposed to FD (0 mg/kg folate), or control (2 mg/kg folate) diets underwent a 10-day (20 mg/kg/day) PQQ treatment regimen and brain tissues were collected and analyzed. RESULTS: Folate deficiency resulted in increased expression of inflammatory and oxidative stress markers in vitro and in vivo, with increased cellular ROS levels observed in mixed glial cells as well as a reduction of mitochondrial DNA (mtDNA) content observed in FD mixed glial cells. PQQ treatment was able to reverse these changes, while increasing RFC expression through activation of the PGC-1α/NRF-1 signaling pathway. CONCLUSION: These results demonstrate the effects of brain folate deficiency, which may contribute to the neurological deficits commonly seen in disorders of CFD. PQQ may represent a novel treatment strategy for disorders associated with CFD, as it can increase folate uptake, while in parallel reversing many abnormalities that arise with brain folate deficiency.

Novel localization of folate transport systems in the murine central nervous system.

BACKGROUND: Folates are a family of B9 vitamins that serve as one-carbon donors critical to biosynthetic processes required for the development and function of the central nervous system (CNS) in mammals. Folate transport is mediated by three highly specific systems: (1) folate receptor alpha (FRα; FOLR1/Folr1), (2) the reduced folate-carrier (RFC; SLC19A1/Slc19a1) and (3) the proton-coupled folate transporter (PCFT; SLC46A1/Slc46a1). Folate transport into and out of the CNS occurs at the blood-cerebrospinal fluid barrier (BCSFB), mediated by FRα and PCFT. Impairment of folate transport at the BCSFB results in cerebral folate deficiency in infants characterized by severe neurological deficiencies and seizures. In contrast to the BCSFB, CNS folate transport at other brain barriers and brain parenchymal cells has not been extensively investigated. The aim of this study is to characterize folate transport systems in the murine CNS at several known barriers encompassing the BCSFB, arachnoid barrier (AB), blood-brain barrier (BBB) and parenchymal cells (astrocytes, microglia, neurons). METHODS: Applying immunohistochemistry, localization of folate transport systems (RFC, PCFT, FRα) was examined at CNS barriers and parenchymal sites in wildtype (C57BL6/N) mice. Subcellular localization of the folate transport systems was further assessed in an in vitro model of the mouse AB. Gene and protein expression was analyzed in several in vitro models of brain barriers and parenchyma by qPCR and western blot analysis. RESULTS: RFC, PCFT, and FRα expression was localized within the BCSFB and BBB consistent with previous reports. Only RFC and PCFT expression was detected at the AB. Varied levels of RFC and PCFT expression were detected in neuronal and glial cells. CONCLUSIONS: Localization of RFC and PCFT within the AB, described here for the first time, suggest that AB may contribute to folate transport between the peripheral circulation and the CSF. RFC and PCFT expression observed in astrocytes and microglia is consistent with the role that one or both of these transporters may play in delivering folates into cells within brain parenchyma. These studies provide insights into mechanisms of folate transport in the CNS and may enhance our understanding of the critical role folates play in neurodevelopment and in the development of novel treatment strategies for disorders of brain folate deficiency due to impaired transporter function.

Transporters in Drug Development: International Transporter Consortium Update on Emerging Transporters of Clinical Importance.

During its fourth transporter workshop in 2021, the International Transporter Consortium (ITC) provided updates on emerging clinically relevant transporters for drug development. Previously highlighted and new transporters were considered based on up-to-date clinical evidence of their importance in drug-drug interactions and potential for altered drug efficacy and safety, including drug-nutrient interactions leading to nutrient deficiencies. For the first time, folate transport pathways (PCFT, RFC, and FRα) were examined in-depth as a potential mechanism of drug-induced folate deficiency and related toxicities (e.g., neural tube defects and megaloblastic anemia). However, routine toxicology studies conducted in support of drug development appear sufficient to flag such folate deficiency toxicities, whereas prospective prediction from in vitro folate metabolism and transport inhibition is not well enough established to inform drug development. Previous suggestion of a retrospective study of intestinal OATP2B1 inhibition to explain unexpected decreases in drug exposure were updated. Furthermore, when the absorption of a new molecular entity is more rapid and extensive than can be explained by passive permeability, evaluation of the OATP2B1 transport may be considered. Emerging research on hepatic and renal OAT2 is summarized, but current understanding of the importance of OAT2 was deemed insufficient to justify specific consideration for drug development. Hepatic, renal, and intestinal MRPs (MRP2, MRP3, and MRP4) were revisited. MRPs may be considered when they are suspected to be the major determinant of drug disposition (e.g., direct glucuronide conjugates); MRP2 inhibition as a mechanistic explanation for drug-induced hyperbilirubinemia remains justified. There were no major changes in recommendations from previous ITC whitepapers.

Nuclear respiratory factor 1 (NRF-1) upregulates the expression and function of reduced folate carrier (RFC) at the blood-brain barrier.

Folates are important for neurodevelopment and cognitive function. Folate transport across biological membranes is mediated by three major pathways: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Brain folate transport primarily occurs at the choroid plexus through FRα and PCFT; inactivation of these transport systems results in suboptimal folate levels in the cerebrospinal fluid (CSF) causing childhood neurological disorders. Our group has reported that upregulation of RFC at the blood-brain barrier (BBB) through interactions with specific transcription factors, that is, vitamin D receptor (VDR) could increase brain folate delivery. This study investigates the role of nuclear respiratory factor 1 (NRF-1) in the regulation of RFC at the BBB. Activation of NRF-1/PGC-1α signaling through treatment with its specific ligand, pyrroloquinoline quinone (PQQ), significantly induced RFC expression and transport activity in hCMEC/D3 cells. In contrast, transfection with NRF-1 or PGC-1α targeting siRNA downregulated RFC functional expression in the same cell system. Applying chromatin immunoprecipitation (ChIP) assay, we further demonstrated that PQQ treatment increased NRF-1 binding to putative NRF-1 binding sites within the SLC19A1 promoter, which encodes for RFC. Additionally, in vivo treatment of wild type mice with PQQ-induced RFC expression in isolated mouse brain capillaries. Together, these findings demonstrate that NRF-1/PGC-1α activation by PQQ upregulates RFC functional expression at the BBB and could potentially enhance brain folate uptake.

Identification and cloning of the kinin receptor in the Chagas disease vector, Rhodnius prolixus.

Within invertebrates, the kinin family of neuropeptides is responsible for the modulation of a host of physiological and behavioural processes. In Rhodnius prolixus, kinins are primarily responsible for eliciting myotropic effects on various feeding and diuresis-related tissues. Here, the R. prolixus kinin receptor (RhoprKR) has been identified, cloned and sequenced from the central nervous system (CNS) and hindgut of R. prolixus. Sequence analyses show high similarity and identity between RhoprKR and other cloned invertebrate kinin receptors. The expression profile of RhoprKR shows the RhoprKR transcript throughout the R. prolixus gut, with highest expression in the hindgut, suggesting a role of Rhopr-kinins in various aspects of feeding and digestion. RNA interference (RNAi)-mediated knockdown of the RhoprKR transcript resulted in a significant reduction of hindgut contractions in response to Rhopr-kinin 2 and an Aib-containing kinin analog. dsRhoprKR- injected insects also consumed a significantly larger meal, suggesting a role of Rhopr-kinins in satiety.

Physiological effects of biostable kinin and CAPA analogs in the Chagas disease vector, Rhodnius prolixus.

In the Chagas disease vector Rhodnius prolixus, the kinin and CAPA family of neuropeptides are implicated in feeding and diuresis-related behaviours, with Rhopr-kinins stimulating contractions of the midgut, salivary glands, and hindgut, and RhoprCAPA-2 functioning as an anti-diuretic hormone. The current study examined the effects of kinin and CAPA neuropeptides and their analogs on feeding and diuresis, and on hindgut contractions and MT fluid secretion in R. prolixus. The biostable Aib-containing kinin analog 2139[Φ1]wp-2 was found to have antifeedant effects, and to be more potent than Rhopr-kinin 2 in stimulating hindgut contractions. The CAPA analog 2129-SP3[Φ3]wp-2 induced the intake of a larger blood meal, and increased the rate of post-prandial rapid diuresis. RhoprCAPA-2, but not its analog, potentiated hindgut contractions induced by Rhopr-kinin 2. Potentiation was observed with the CAPA analog on 5-HT-stimulated increases in frequency of hindgut contractions, whereas RhoprCAPA-2 inhibited this 5-HT-mediated stimulation. The CAPA analog induced hindgut contractions and prevented the inhibition induced by RhoprCAPA-2 on 5-HT-stimulated MT secretion. These results demonstrate novel interactions between Rhopr-kinin and RhoprCAPA-2 on the hindgut, possibly influencing post-feeding excretion. The kinin analog is a potent agonist of the kinin receptor, and the CAPA analog an antagonist of the CAPA receptor. The use of neuropeptide mimetics is a promising approach to vector control as they can disrupt behaviours, and the effects of these neuropeptide analogs highlight their value as lead compounds, given their ability to interfere with epidemiologically-relevant behaviours.