Wang, JoanneSun, Austin2023-08-142023-08-142023Sun_washington_0250E_25129.pdfhttp://hdl.handle.net/1773/50519Thesis (Ph.D.)--University of Washington, 2023The blood-cerebrospinal fluid barrier (BCSFB) is formed by the choroid plexus epithelial (CPE) cells, which express several polyspecific membrane transporters that contribute towards clearance of xenobiotic and endogenous compounds from the cerebrospinal fluid (CSF). However, BCSFB transporters are poorly characterized with respect to function, activity, and pharmacokinetic significance. Previous approaches using fluorescence microscopy to study BCSFB transport in intact choroid plexus tissues were poorly validated and more qualitative in nature due to the lack of real time quantitative methods for image analysis. This dissertation research is aimed to develop and validate an approach to study BCSFB transport activity through live tissue imaging and quantitative fluorescence microscopy, and subsequently utilize this approach and other biochemical approaches to elucidate the molecular mechanisms and functional significance of organic anion transporting polypeptides (OATPs), breast cancer resistance protein (Bcrp), and P-glycoprotein (P-gp) at the BCSFB. To better assess the transepithelial transport process at the blood-CSF barrier, I developed and validated a quantitative confocal microscopy approach to study CSF-to-blood organic anion and organic cation transport processes at the murine BCSFB in real time. Real time quantification of fluorescence occurring at different tissue compartments can enable the deconvolution of transport kinetics occurring at the apical (CSF-facing) and basolateral (blood-facing) membranes of the BCSFB. This approach was demonstrated to be consistent, reproducible, and capable of tracking transepithelial transport at the BCSFB with temporal and spatial resolution. I showed that large organic anion probes, 8-fluorescein-cAMP (fluo-cAMP) and fluorescein methotrexate (FL-MTX), are efficiently transported from the CSF compartment into the CPE cells and subsequently effluxed into the subepithelial space. Transport of the large organic anion probes were rate limited by the apical uptake transport, presumed to be mediated by OATPs. In contrast, the small organic cation and plasma membrane monoamine transporter (PMAT) substrate IDT307, was transported into CPE cells and retained. A novel parameter, choroid plexus efflux index (CPEI), was proposed to distinguish between transepithelial flux and CPE cell accumulation. The approach presented is valuable for the characterization of compartment specific accumulation of substrates, perpetrator drug interactions, and rate-determining steps in transepithelial transport at the BCSFB. To elucidate the molecular mechanisms of OATP-mediated organic anion clearance at the blood-CSF barrier, I utilized the recently developed quantitative fluorescence microscopy approach alongside other biochemical approaches to probe OATP1A expression, localization, and function. Using RT-PCR and supporting literature data, we found that OATP1A5 is the primary OATP1A isoform expressed on the apical, CSF-facing membrane Using quantitative fluorescence microscopy, I demonstrated that the fluorescent organic anions, sulforhodamine101 (SR101), FL-MTX, and fluo-cAMP were efficiently transported across the blood-CSF barrier. Transepithelial transport of these compounds across the CPE cells was abolished in Oatp1a/1b-/- mice, suggesting OATP1A5 is the primary contributor to large organic anion uptake in mice. Using transporter-expressing cell lines, the fluorescent probes were confirmed to be substrates of mouse OATP1A5 and its human homolog OATP1A2, corroborating our findings in the isolated CP and suggesting an overlap in function between mouse OATP1A5 and human OATP1A2. Immunofluorescence staining revealed the presence of OATP1A2 protein at the apical membrane in human CP tissues. Based on these data, we proposed that large organic anions in the CSF are actively transported into CPE cells by apical OATP1A2 (OATP1A5 in mice), then subsequently effluxed into the blood by basolateral multidrug resistance associated proteins (MRPs). As OATP1A2 transports a wide range of xenobiotics and endogenous compounds, the presence of this transporter at the BCSFB apical membrane may imply a novel route for removing neurohormones, drugs, and toxins from the CSF.Previous studies report that P-gp and Bcrp are expressed apically or subapically at the blood-CSF barrier, implying a paradoxical function to mediate blood-to-CSF transport of xenobiotics. To probe P-gp and BCRP function at the BCSFB, I utilized the approach described in Chapter 2 alongside selective inhibitors and knockout models to functionally evaluate the activity, mechanisms, and potential interplay of P-gp and Bcrp BCSFB transport. Using qRT-PCR I identified the relative mRNA expression of P-gp and Bcrp isoforms at the murine BCSFB. Through quantitative fluorescence microscopy in isolated CP tissues of wild-type and Bcrp-/- mice, I demonstrated BODIPY FL-prazosin was actively transported by Bcrp, indicating functional activity of apical Bcrp efflux. Furthermore, I detected an additional Bcrp-independent, elacridar-sensitive apical efflux mechanism at the BCSFB, suggested, but not confirmed, to be P-gp. As Bcrp and P-gp at the BCSFB apical (CSF-facing) membrane may contribute to the entry of endogenous compounds and nutrients into the CNS and may alter the disposition of drugs within the CNS, this study highlights the need for further research on characterizing the role of these transporters towards endobiotic and xenobiotic transport at the BCSFB. This dissertation research has contributed greatly to our understanding of the molecular mechanisms mediating drug transport at the blood-CSF barrier. Notably, I revealed a functional role of OATP1A5 in clearing large organic anions at the BCSFB apical membrane from the CSF in mice and suggested a similar role for OATP1A2 at the human BCSFB. In addition, I have provided supporting evidence towards Bcrp and P-gp functional efflux activity at the BCSFB apical membrane in mice. Taken together, this research established an uptake transport mechanism of large organic anions at the BCSFB and provides novel mechanistic insights into several poorly defined transport pathways at the BCSFB. Knowledge gained from this dissertation research contributes to a mechanistic understanding of several major BCSFB drug transporters in regulating CSF drug concentrations and suggests potential roles of these transporters in modulating CNS disposition of drugs and endogenous substances. This knowledge can benefit our understanding of the relationship between CSF and unbound brain concentrations for transported drug substrates, which can subsequently improve the predictions of CNS drug disposition, efficacy, and toxicity in humans.application/pdfen-USCC BY-NCBlood-cerebrospinal fluid barrierCNS pharmacokineticsDrug transportersFluorescence microscopyOrganic anion transporting polypeptides (OATPs)Pharmaceutical sciencesPharmaceuticsThesis