Compromised phagosome maturation underlies RPE pathology in cell culture and whole animal models of Smith-Lemli-Opitz Syndrome

Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed.

使用胆固醇通路抑制剂AY9944处理大鼠，可构建史密斯-莱姆利-奥皮茨综合征（Smith-Lemli-Opitz syndrome, SLOS）动物模型——该疾病是一种由胆固醇合成缺陷引发的常染色体隐性遗传病。该SLOS大鼠模型会出现进行性且不可逆的神经视网膜变性，并伴随视网膜色素上皮（retinal pigmented epithelium, RPE）的病理改变。本研究进一步阐明了RPE病理发生的潜在机制。与未处理的对照组动物相比，SLOS大鼠模型的RPE顶端自发荧光显著增强，且该现象与A2E及其他双类视黄醇物质的水平升高呈显著相关。利用体外培养的人类诱导多能干细胞（induced pluripotent stem cell, iPSC）分化获得的SLOS RPE细胞进行实验，我们发现其稳态7-脱氢胆固醇（7-dehydrocholesterol, 7DHC）水平显著升高，而胆固醇水平降低——这正是SLOS的关键生化标志性特征。蛋白质免疫印迹（Western blot）分析结果显示，巨自噬/自噬（macroautophagy/autophagy）标志物MAP1LC3B-II与SQSTM1/p62的表达水平发生改变，同时泛素化蛋白出现蓄积。与iPSC来源的正常人类对照RPE细胞相比，SLOS RPE细胞中未成熟自噬体的蓄积伴随吞噬的外源性视网膜杆状外节降解效率低下，该现象可通过视紫红质（rhodopsin, RHO）C端1D4表位的持续存在得以证实。SLOS RPE细胞的溶酶体pH与组织蛋白酶D（CTSD）活性均处于正常生理区间内，由此可排除溶酶体功能异常参与该病理过程的可能性。此外，在SLOS的药理学与遗传学啮齿类动物模型中，RPE内蓄积的1D4阳性吞噬体均无法与溶酶体发生融合。综合上述实验结果，本研究表明吞噬体成熟缺陷是导致RPE病理改变的核心机制。本文还探讨了本研究发现与SLOS的潜在关联，以及胆固醇对吞噬体成熟的必要性。