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Abstract：
OBJECTIVE: Bietti crystalline dystrophy (BCD) is an inherited retinal degeneration disease caused by mutations in the CYP4V2 gene. Currently, there is no clinical therapy approach available for BCD patients. Previous research has suggested that polyunsaturated fatty acids (PUFAs) may play a significant role in the development of BCD, implicating the involvement of ferroptosis in disease pathogenesis. In this work, we aimed to investigate the interplay between ferroptosis and BCD and to detect potential therapeutic strategies for the disease.
METHODS: Genetic-edited RPE cell line was first established in this study by CRISPR-Cas9 technology. Cyp4v3 (the homologous gene of human CYP4V2) knock out (KO) mice have also been used. Lipid profiling and transcriptome analysis of retinal pigment epithelium (RPE) cells from Cyp4v3 KO mice have been conducted. Ferroptosis phenotypes have been first investigated in BCD models in vitro and in vivo, including lipid peroxidation, mitochondrial changes, elevated levels of reactive oxygen species (ROS), and altered gene expression. Additionally, an iron chelator, deferiprone (DFP), has been tested in vitro and in vivo to determine its efficacy in suppressing ferroptosis and restoring the BCD phenotype.
RESULTS: Cyp4v3 KO mice exhibited progressive retinal degeneration and lipid accumulation, similar to the BCD phenotype, which was exacerbated by a high-fat diet (HFD). Increased levels of PUFAs, such as EPA (C22:5) and AA (C20:4), were observed in the RPE of Cyp4v3 KO mice. Transcriptome analysis of RPE in Cyp4v3 KO mice revealed changes in genes involved in iron homeostasis, particularly an upregulation of NCOA4, which was confirmed by immunofluorescence. Ferroptosis-related characteristics, including mitochondrial defects, lipid peroxidation, ROS accumulation, and upregulation of related genes, were detected in the RPE both in vitro and in vivo. Abnormal accumulation of ferrous iron was also detected. DFP, an iron chelator administration suppressed ferroptosis phenotype in CYP4V2 mutated RPE. Oral administration of DFP also restored the retinal function and morphology in Cyp4v3 KO mice.
CONCLUSIONS: This study represented the first evidence of the substantial role of ferroptosis in the development of BCD. PUFAs resulting from CYP4V2 mutation may serve as substrates for ferroptosis, potentially working in conjunction with NCOA4-regulated iron accumulation, ultimately leading to RPE degeneration. DFP administration, which chelates iron, has demonstrated its ability to reverse BCD phenotype both in vitro and in vivo, suggesting a promising therapeutic approach in the future.
METHODS: Genetic-edited RPE cell line was first established in this study by CRISPR-Cas9 technology. Cyp4v3 (the homologous gene of human CYP4V2) knock out (KO) mice have also been used. Lipid profiling and transcriptome analysis of retinal pigment epithelium (RPE) cells from Cyp4v3 KO mice have been conducted. Ferroptosis phenotypes have been first investigated in BCD models in vitro and in vivo, including lipid peroxidation, mitochondrial changes, elevated levels of reactive oxygen species (ROS), and altered gene expression. Additionally, an iron chelator, deferiprone (DFP), has been tested in vitro and in vivo to determine its efficacy in suppressing ferroptosis and restoring the BCD phenotype.
RESULTS: Cyp4v3 KO mice exhibited progressive retinal degeneration and lipid accumulation, similar to the BCD phenotype, which was exacerbated by a high-fat diet (HFD). Increased levels of PUFAs, such as EPA (C22:5) and AA (C20:4), were observed in the RPE of Cyp4v3 KO mice. Transcriptome analysis of RPE in Cyp4v3 KO mice revealed changes in genes involved in iron homeostasis, particularly an upregulation of NCOA4, which was confirmed by immunofluorescence. Ferroptosis-related characteristics, including mitochondrial defects, lipid peroxidation, ROS accumulation, and upregulation of related genes, were detected in the RPE both in vitro and in vivo. Abnormal accumulation of ferrous iron was also detected. DFP, an iron chelator administration suppressed ferroptosis phenotype in CYP4V2 mutated RPE. Oral administration of DFP also restored the retinal function and morphology in Cyp4v3 KO mice.
CONCLUSIONS: This study represented the first evidence of the substantial role of ferroptosis in the development of BCD. PUFAs resulting from CYP4V2 mutation may serve as substrates for ferroptosis, potentially working in conjunction with NCOA4-regulated iron accumulation, ultimately leading to RPE degeneration. DFP administration, which chelates iron, has demonstrated its ability to reverse BCD phenotype both in vitro and in vivo, suggesting a promising therapeutic approach in the future.
摘要：
目的：Bietti晶体营养不良（BCD）是一种由CYP4V2基因突变引起的遗传性视网膜变性疾病。目前,目前尚无适用于BCD患者的临床治疗方法.先前的研究表明，多不饱和脂肪酸（PUFAs）可能在BCD的发展中起重要作用。暗示铁性凋亡参与疾病的发病机制。在这项工作中,我们旨在研究铁细胞凋亡与BCD之间的相互作用,并检测该疾病的潜在治疗策略.
方法：本研究首先通过CRISPR-Cas9技术建立了基因编辑的RPE细胞系。Cyp4v3(人CYP4V2的同源基因)敲除(KO)小鼠也已被使用。已经进行了来自Cyp4v3KO小鼠的视网膜色素上皮(RPE)细胞的脂质谱分析和转录组分析。首次在体外和体内BCD模型中研究了Ferroptosis表型，包括脂质过氧化,线粒体变化,活性氧（ROS）水平升高，和改变基因表达。此外,一种铁螯合剂,去铁酮(DFP)，已在体外和体内进行了测试，以确定其在抑制铁细胞凋亡和恢复BCD表型方面的功效。
结果：Cyp4v3KO小鼠表现出进行性视网膜变性和脂质积聚，类似于BCD表型，高脂饮食（HFD）加剧了这种情况。增加PUFA的水平，如EPA(C22:5)和AA(C20:4)，在Cyp4v3KO小鼠的RPE中观察到。Cyp4v3KO小鼠RPE的转录组分析揭示了铁稳态基因的变化，特别是NCOA4的上调，免疫荧光证实了这一点。铁凋亡相关特征，包括线粒体缺陷,脂质过氧化，ROS积累，以及相关基因的上调，在体外和体内均在RPE中检测到。还检测到亚铁的异常积累。DFP,在CYP4V2突变的RPE中,施用铁螯合剂抑制了铁凋亡表型。口服DFP也恢复了Cyp4v3KO小鼠的视网膜功能和形态。
结论：这项研究首次证明了铁细胞凋亡在BCD发展中的重要作用。CYP4V2突变产生的PUFA可以作为铁凋亡的底物，可能与NCOA4调节的铁积累相结合，最终导致RPE退化。DFP管理,螯合铁，已经证明了其在体外和体内逆转BCD表型的能力，提出了未来有希望的治疗方法。
方法：本研究首先通过CRISPR-Cas9技术建立了基因编辑的RPE细胞系。Cyp4v3(人CYP4V2的同源基因)敲除(KO)小鼠也已被使用。已经进行了来自Cyp4v3KO小鼠的视网膜色素上皮(RPE)细胞的脂质谱分析和转录组分析。首次在体外和体内BCD模型中研究了Ferroptosis表型，包括脂质过氧化,线粒体变化,活性氧（ROS）水平升高，和改变基因表达。此外,一种铁螯合剂,去铁酮(DFP)，已在体外和体内进行了测试，以确定其在抑制铁细胞凋亡和恢复BCD表型方面的功效。
结果：Cyp4v3KO小鼠表现出进行性视网膜变性和脂质积聚，类似于BCD表型，高脂饮食（HFD）加剧了这种情况。增加PUFA的水平，如EPA(C22:5)和AA(C20:4)，在Cyp4v3KO小鼠的RPE中观察到。Cyp4v3KO小鼠RPE的转录组分析揭示了铁稳态基因的变化，特别是NCOA4的上调，免疫荧光证实了这一点。铁凋亡相关特征，包括线粒体缺陷,脂质过氧化，ROS积累，以及相关基因的上调，在体外和体内均在RPE中检测到。还检测到亚铁的异常积累。DFP,在CYP4V2突变的RPE中,施用铁螯合剂抑制了铁凋亡表型。口服DFP也恢复了Cyp4v3KO小鼠的视网膜功能和形态。
结论：这项研究首次证明了铁细胞凋亡在BCD发展中的重要作用。CYP4V2突变产生的PUFA可以作为铁凋亡的底物，可能与NCOA4调节的铁积累相结合，最终导致RPE退化。DFP管理,螯合铁，已经证明了其在体外和体内逆转BCD表型的能力，提出了未来有希望的治疗方法。
