STGD1型Stargardt病是一种遗传性视网膜退行性疾病，与突变的ATP结合盒，亚科A，成员4(ABCA4)基因有关。STGD1是幼年黄斑变性最常见的形式，发病于儿童时期至成年早期或中期，可导致进行性、不可逆的视力障碍和失明，然而目前尚无有效的治疗方法。

2018年Tamara L. Lenisfa在线发表“Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular deggeneration”的研究论文，该论文已被在国际知名医学期刊PNAS（Q1,IF=10.2）接收并在线发表。

隐性Stargardt病(STGD1)是一种由Abca4基因突变引起的遗传性致盲疾病，其临床特征为发病早，RPE中荧光脂褐素堆积，光感受器变性甚至致盲。ABCA4是光感受器外段(OS)上的一种翻转酶，它通过OS盘膜转运与磷脂酰乙醇胺结合的视黄醛。ABCA4 - / -小鼠和STGD1患者中ABCA4的缺失导致视网膜色素上皮(RPE)中脂褐素的积聚和光感受器的退化，导致失明。目前尚无针对STGD1的有效治疗方法，本研究提出假设ABCA4通常存在于RPE细胞的内溶酶体膜中。

图1. ABCA4在RPE中内源性表达。

注：ABCA4以前被称为光感受器辐射蛋白RmP或ABCR，目前已知，人类的ABCA4基因有50个外显子，2273个氨基酸蛋白，分子量为256kDa，ABCA4基因转录一个大的特定的视网膜蛋白，即ABCA4蛋白，该蛋白几乎只存在于视网膜的视杆细胞的外节盘缘上，具有两个串联的区域，即氮端和碳端，每个区域各包含跨膜区（transmembrane domain，TMD）、糖基化胞外域（exocytoplasmic domain，EDC）和核苷酸结合结构域（nucleotide binding domain，NBD）各一个。

图2. ABCA4与内溶酶体标记共定位。

(A) 2岁野生型balb /c(上)和白化Abca4−/−(下)小鼠视网膜/RPE切片的代表性共聚焦图像与Abca4(红色)和LAMP1(绿色)抗体反应。注意，ABCA4和LAMP1在野生型RPE中共定位，但在OS中没有。在ABCA4−/−RPE细胞中也存在LAMP1，但不存在ABCA4的免疫反应性。(B) 5个月大野生型(129/Sv)(上)、Abca4−/−(中)和Mertk−/−(下)小鼠视网膜切片的代表性合并共聚焦图像，分别用Abca4(红色)和Rab5(绿色)抗体进行免疫染色。ABCA4和Rab5在129/Sv和Mertk−/−RPE细胞中均有共定位，如橙色信号所示。在Abca4−/−视网膜切片的RPE中，仅见Rab5免疫反应。白色箭头表示视网膜脱离，白色星号表示Mertk−/−视网膜由于光感受器变性而没有OS。(C)固定hfRPE细胞的代表性共聚焦图像，标记ABCA4(红色)(上)或内体CAV1(绿色)(中)抗体。(下)ABCA4和CAV1的共聚焦合并图像。

图3.ABCA4在RPE- ABCA4-tg/ABCA4−/−小鼠的RPE中表达。

(A) BALB/c、Abca4-/-和RPE-Abca4-tg/Abca4-/-小鼠(均为白化)视网膜和RPE匀浆的代表性免疫印迹与抗Abca4或α-微管蛋白的抗血清反应。神经视网膜总蛋白负荷为10 μg, RPE/eyecup匀浆为25 μg。(B)ABCA4-/-和RPE-Abca4-Tg/ABCA4-/-匀浆中ABCA4蛋白水平归一化为α-微管蛋白，并相对于野生型BALB/c水平呈现;每组6龄小鼠7只。(C)BALB/C(左)、Abca4−/−(中)和RPE-Abca4-Tg/Abca4−/−(右)小鼠视网膜切片的代表性共聚焦图像。RPE中ABCA4的免疫反应性(红色)- ABCA4 - tg / ABCA4−/−显示主要针对RPE的特异性。Abca4-/-和RPE-Abca4-Tg/Abca4-/-小鼠的os层均未被Abca4抗体染色。DAPI核染色为蓝色。

图4.RPE-Abca4- tg /Abca4 - / -小鼠RPE中的类双维甲酸、自身荧光和脂褐素水平降低。

(A-D)从3龄白化小鼠视网膜和RPE匀浆中提取类双维甲酸，采用正相高效液相色谱法进行分析。RPE/eyecup和神经视网膜提取物的代表性HPLC色谱图见SI附录，图S3。注意，RPE-Abca4- tg /Abca4 - / -小鼠RPE中所有类双维甲酸水平较低。(A)总A2E (A2E和反A2E之和)以每只眼皮摩尔表示。(B -D)全反式视黄醛二聚体PE (atRAL-Dimer-PE) (B)、A2PE- h2 (C)和A2PE (D)以每只眼的毫吸光度单位(mAU)表示。数据以mean±SD表示;每组5只;* p < 0.0001， ** p < 0.001;N /s，不显著。(E)使用488 nm激发激光器和500- 545 nm发射滤光片捕获的rpe -脉络膜-巩膜平面支架的代表性共聚焦图像。注意，与Abca4- / - RPE平片相比，RPE-Abca4- tg /Abca4−/−平片的自荧光强度(AF，绿色)降低。抗zo1染色显示RPE细胞边界(蓝色);细胞核用DAPI染色(蓝色);每组6龄小鼠3或4只。(比例尺，20 μm.) (F) 1岁BALB/c(左)、Abca4−/−(中)和RPE-Abca4- tg /Abca4−/−(右)白化病小鼠RPE细胞的代表性电镜图。箭头指向RPE细胞质内电子密度不均匀的多态脂褐质颗粒。BM，布鲁氏膜;N,细胞核。(比尺，2 μm.) (G)测量每100 μm2细胞面积的脂褐素颗粒分数，并从每只眼睛10个相邻的电镜图像中取平均值。数据以mean±SD表示;N = 5 ~ 9只/组;* p = 0.0186;** p < 0.001。

图5.在RPE-Abca4-Tg/Abca4−/−和Abca4−/−小鼠中保留光感受器。

(A)通过光学显微镜获得的1岁白化病小鼠的代表性视网膜图像。(B)每100 μm2细胞面积计算光感受器细胞核总数。注意，与Abca4-/-小鼠相比，RPE -Abca4- tg /Abca4 - / -小鼠的ONL细胞数量增加，表明光感受器变性部分恢复。数据以mean±SD表示;N = 5 - 9只/组;RPE-Abca4-Tg/Abca4−/−vs. Abca4−/−，*P = 0.0319;Abca4−/−vs. BALB/c， **P < 0.0001;RPE-Abca4-Tg/Abca4−/−vs. BALB/c, P = 0.0061。

图6所示。ABCA4在RPE内溶酶体膜中的功能。

(A)正常RPE细胞。在紫红质蛋白水解过程中释放的11cRAL在光腔表面与PE缩合形成11-顺式- n -视黄醛-磷脂酰乙醇胺(11c-N-ret-PE)，后者经过异构化形成全反式(at)和11c-N-ret-PE的混合物。两种N-ret-PE异构体都被ABCA4翻转到细胞质表面，其中Nret-PE的水解是由细胞视黄醛结合蛋白(CRALBP)结合11cRAL或视黄醛脱氢酶11型(RDH11)将atRAL还原为atROL的质量作用驱动的。atROL由RPE视觉循环处理，通过卵磷脂视黄醇酰基转移酶(LRAT)酯化生成全反式视黄醇酯，如全反式视黄醇棕榈酸酯(atRP)， RPE65异构化生成11-顺式视黄醇(11cROL)，视黄醇脱氢酶5型(RDH5)氧化生成11cRAL，并与CRALBP结合。11cRAL使RPE细胞在邻近的光感受器OS中再生视觉色素。(B)Abca4−/−突变型RPE细胞。ABCA4 - / -小鼠或STGD1患者的RPE内溶酶体中缺乏ABCA4会导致视黄醛的清除延迟，从而导致游离视黄醛和N-ret-PE浓度升高。这导致atRAL或11cRAL与N-ret-PE二次缩合形成类双维甲酸。

ABCA4在RPE细胞中表达，并且至少部分ABCA4−/−表型是由RPE表达的ABCA4的缺失引起的。除了光感受器外，RPE细胞也应该作为abca4介导的视网膜变性的治疗靶点。

隐性Stargardt黄斑变性(STGD1)和锥杆营养不良是由ABCB4基因突变引起的。ABCA4蛋白是光感受器细胞中的一种翻转酶，有助于消除视黄醛，一种有毒的视觉光产物。

在这里，我们发现ABCA4在小鼠视网膜色素上皮(RPE)中也存在，其丰度约为神经视网膜中的1%。在RPE中表达ABCA4而在光感受器细胞中不表达ABCA4的转基因小鼠显示，在ABCA4 - / -小鼠和inSTGD1患者中观察到的脂褐素积累和光感受器变性均部分恢复。这些观察结果表明，theRPE中的ABCA4可以阻止ABCA4−/−小鼠以及STGD1患者的光感受器变性。

光感受器细胞外段膜盘中包含视色素，正常膜盘不段更新，在视网膜色素变性和某些视网膜病变时，外段膜盘的更新可能出现障碍。

Tamara L, Lenis,Jane, Hu,Sze Yin, Ng et al. Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular degeneration.[J] .Proc Natl Acad Sci U S A, 2018, 115: 0.

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