P450 oxidoreductase regulates barrier maturation by mediating retinoic acid metabolism in a model of the human BBB

Abstract

The blood-brain barrier (BBB) selectively regulates the entry of molecules into the central nervous system (CNS). A crosstalk between brain microvascular endothelial cells (BMECs) and resident CNS cells promotes the acquisition of functional tight junctions (TJs). Retinoic acid (RA), a key signaling molecule during embryonic development, is used to enhance in vitro BBB models' functional barrier properties. However, its physiological relevance and affected pathways are not fully understood. P450 oxidoreductase (POR) regulates the enzymatic activity of microsomal cytochromes. POR-deficient (PORD) patients display impaired steroid homeostasis and cognitive disabilities. Here, we used both patient-specific POR-deficient and CRISPR-Cas9-mediated POR-depleted induced pluripotent stem cell (iPSC)-derived BMECs (iBMECs) to study the role of POR in the acquisition of functional barrier properties. We demonstrate that POR regulates cellular RA homeostasis and that POR deficiency leads to the accumulation of RA within iBMECs, resulting in the impaired acquisition of TJs and, consequently, to dysfunctional development of barrier properties.

Keywords: BBB; P450 oxidoreductase; POR; blood-brain barrier; cytochrome; disease modeling; iPSCs; metabolism; retinoic acid; tight junctions.

Graphical abstract

Figure 1

POR is expressed in human and mouse NVU and in iBMECs (A) POR expression in acutely purified human brain cells from the Barres lab database (Zhang et al., 2016). (B) IHC analysis of mouse cortical brain section shows that CD31 (red)-positive endothelial cells express Por (green). Nuclei were stained using DAPI (blue). White arrowheads denote the co-localization of CD31 and Por. (C) Por (green) shows a perinuclear pattern of expression. (D) Schematic illustration of iBMEC differentiation. EC, endothelial cells; bFGF, basic fibroblast growth factor; RA, all-trans retinoic acid. (E) ICC analysis confirms that CTR iBMECs express GLUT-1, ZO-1, and CLDN-5. (F) ICC analysis of iBMECs shows that POR is localized to the perinuclear area. (G) WB analysis showed expression of POR as a 75 kDa band both in undifferentiated iPSCs and iBMECs. Ponceau S staining shows total protein levels. See also Figure S1.

Figure 2

POR is enzymatically active in CTR and heterozygous iBMECs and severely attenuated in PORD and PORM iBMECs (A) WB analysis of POR in undifferentiated iPSCs and iBMECs. CTR, POR^01het, and POR^G539R (PORD) cells express POR as a 75 kDa band in both undifferentiated iPSCs and iBMECs. CTR^mut1 (PORM) did not express POR, and CTR^mut2 produced a shorter, 55 kDa band, as predicted in Figure S2. (B) ICC showed POR (green) expression in POR^01het and POR^02G539R but not in CTR^mut1 iBMECs. (C) POR enzymatic activity is decreased in PORD and further reduced in the PORM lines compared with the CTR and POR^01het lines. One-way ANOVA; ^∗∗p < 0.005, ^∗∗∗∗p < 0.0001, activity was normalized to the maximum level of each experiment. Different symbols refer to different cell lines (n = 4–10). See also Figures S2 and S3 and Table S1.

Figure 3

POR is necessary for the RA-dependent development of functional TJs (A) ICC analysis of the TJ relevant markers ZO-1 (red), GLUT-1 (green), and CLDN-5 (green) for iBMECs differentiated with 10 μM RA from the healthy (POR^01het), PORD, and PORM lines. White arrowheads denote gaps between cells in which ZO-1 was expressed. (B) Scatterplots of maximum (max) TEER values for the CTR, PORD, and PORM lines differentiated with 0 (vehicle [Ve]), 0.25, 1, or 10 μM RA (CTR n = 28; PORD, n = 27; PORM, n = 9). Two-way ANOVA with Tukey’s multiple comparison test; ^∗∗p < 0.005, ^∗∗∗p < 0.0001. (C) Quantification of the paracellular permeability of the fluorescent tracer sodium fluorescein. When iBMECs were differentiated with 10 μM RA, the PORD (n = 8) and PORM (n = 5) lines showed a significant increase in paracellular permeability compared with CTR lines (n = 9). PORM iBMECs had higher permeability compared with PORD iBMECs. Two-way ANOVA with Tukey’s multiple comparison test; ^∗p < 0.05, ^∗∗p < 0.005, ^∗∗∗p < 0.0001. Bars represent 95% confidence intervals; the solid red line represents the median. See also Figure S4.

Figure 4

POR regulates RA-dependent gene expression (A) Venn diagrams showing significant DEG profiles for each line in response to the RA concentration gradient. The size of the circle is proportional to the number of DEGs indicated within each line. (B) Heatmap of hierarchical cluster analysis for genes that were defined as DEGs in at least one condition in any cell line. (C) A PCA plot of normalized counts. Each dot represents a different sample. Technical repeats (a and b) were averaged and merged (ab). All lines cluster along the PC2 axis when differentiated without RA (encircled in blue). POR-deficient lines differentiated with all RA concentrations clustered along PC2 with the CTR line differentiated with 10 μM (encircled in red). C, CTR; M, PORM; P, PORD. 0, 0.25, 1, 10: RA concentrations in μM. See also Figure S5.

Figure 5

Expression of the CYP26 gene family in response to RA Log₂(fold change) of each CYP26 gene in response to RA for CTR, PORD, and PORM iBMECs. Significance threshold, market with asterisks was taken as adjusted p < 0.1. See Figure S6 and supplemental experimental procedures for details.

Figure 6

RA dose response in CTR iBMECs To detect gene sets that are altered in response to RA in a dose-response manner, gene set enrichment analysis (GSEA) was performed on all CTR samples with all RA concentrations (continuous GSEA). (A) Upregulated RA-related genes (literature curated gene set). (B) Upregulated blood-brain-barrier-related genes (gene set taken from PathCards database). (C) Downregulated genes of cell-cycle checkpoints (gene set taken from REACTOME database). Fifteen top altered genes are presented in each heatmap. (D and E) GSEA analysis using the MsigDB hallmark collection was used to compare gene expression within each cell line in response to the various RA concentrations. The gene set of TNFα signaling via NF-κB is upregulated in (D) PORD and (E) PORM iBMECs. NES, normalized enrichment signal; FDR, false discovery rate.

Figure 7

Schematic description of the suggested mechanism (A) POR mediates the activity of CYP26s, which regulate cellular RA levels by catabolizing RA. In turn, RA regulates the expression of CYP26s, TJs, and integrin (ITGB) genes, which leads to the formation of a functional barrier. (B) When POR is malfunctioning, CYP26s are inactive, causing accumulation of cellular RA. In turn, this leads to transcriptional alterations and an inflammatory response. As a result, TJ formation is impaired, leading to a malfunctioning barrier.