Fig 1: DFP alleviated ferroptosis in CYP4V2 mt RPE. A. DFP increased cell viability in a dose-dependent manner in CYP4V2 mt RPE. B&E. Decreased lipid peroxidation level in CYP4V2 mt RPE with 50µM DFP pre-treatment (n = 3). C&F. Decreased ferrous iron level in CYP4V2 mt RPE with 50µM DFP pre-treatment (n = 3). D&G. Decreased ROS level in CYP4V2 mt RPE with 50µM DFP pre-treatment (n = 3). H. Increased cell viability in CYP4V2 mt RPE with siNCOA4 (n = 3). DFP, deferiprone; Mt, mutated; CR, control; ROS, reactive oxygen species. **P < 0.01, ***P < 0.001,****P < 0.0001
Fig 2: Diagnosis of a BCD patient and iPSCs generation. (A) Fundus image showing characteristic crystalline deposits. (B) OCT images show hyper-reflective crystalline deposits above the RPE in the BCD patients. Arrows indicate crystalline deposits. (C) Sanger sequencing showed a point mutation of CYP4V2 in the BCD patient (highlighted in pink). (D) The level of CYP4V2 mRNA in skin fibroblasts was significantly greater in the control than in the cells from the BCD patient. (E) Markers (including OCT-4, TRA-1–60, NESTIN, AFP and SMA) for iPSCs immunostaining. Data are presented as mean ± SEM. *p < 0.05.
Fig 3: CYP4V2 expression in human RPE and plasmids CYP4V2-encoding plasmids. (A) CYP4V2 is highly expressed in human RPE cells (TPM: transcripts per million) and ranks 4th among other CYP family genes, as determined by RNA-seq. (B) Schematic diagrams of plasmids encoding wild-type or codon-optimized CYP4V2 transgenes driven by a CAG (CMV-enhanced chicken beta-actin) promoter. (C and D) Strong CYP4V2 expression was observed in HEK293 cells transfected with p.coCYP4V2 compared to those transfected with p.wtCYP4V2, assessed by western blot and immunocytochemistry analysis, respectively. (E) CYP enzyme activity was measured in HEK293 cells transfected with p.eGFP, p.Exon7Del, p.992A > C p.CYP4X1, p.CYP1B1, p.wtCYP4V2 and p.coCYP4V2. The latter four plasmid transfections were exposed to a dose response experiment with HET0016, a selective inhibitor of CYP4V2. Data are presented as the mean ± SEM (n = 6). ****p < 0.00001. ns, not significant.
Fig 4: CYP4V2 expression following AAV2 transduction of cultured cells. (A and B) CYP4V2 expression in HEK293 and ARPE19 cells transduced with AAV2.wtCYP4V2 and AAV2.coCYP4V2, respectively, at multiplicities of infection (MOI) from 10E3 to 10E5, assessed by Western blot. (C and D) CYP4V2 expression in HEK293 and ARPE19 cells transduced with AAV2.wtCYP4V2 and AAV2.coCYP4V2, respectively at MOI of 10E5, assessed by immunocytochemistry. (E and F) CYP4V2 enzyme activity was quantified by Multi-CYP assay in HEK293 and ARPE19 cells transduced with AAV2.wtCYP4V2, AAV2.coCYP4V2 and control AAV2.eGFP. Mean ± SEM (n = 6). **p < 0.01. ****p < 0.00001. ns, not significant.
Fig 5: CYP4V2 expression in BCD iPSCs-derived RPE cells. (A) A schematic summary of the process of generation of iPSCs-RPE cells. (B and C) mRNA and protein expression of CYP4V2 was lower in BCD iPSCs-derived RPE (CYP4V2mt) cells than in control (CYP4V2wt) cells, as assessed by qPCR and western blot, respectively. (D) Immunocytochemistry showed stronger expression of CYP4V2 in BCD iPSCs-derived RPE cells transduced with AAV2.coCYP4V2 compared to those transduced with AAV2.wtCYP4V2 and untransduced controls. (E) CYP4V2 enzyme activity was quantified in BCD iPSCs-derived RPE cells transduced with AAV2.wtCYP4V2, AAV2.coCYP4V2 and control AAV2.eGFP, and data are presented as mean ± SEM (n = 12). ***p < 0.0001. ns, not significant.
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