STGD1型Stargardt病是一種遺傳性視網(wǎng)膜退行性疾病，與突變的ATP結(jié)合盒，亞科A，成員4(ABCA4)基因有關(guān)。STGD1是幼年黃斑變性最常見的形式，發(fā)病于兒童時期至成年早期或中期，可導(dǎo)致進(jìn)行性、不可逆的視力障礙和失明，然而目前尚無有效的治療方法。

2018年Tamara L. Lenisfa在線發(fā)表“Expression of ABCA4 in the retinal pigment epithelium and its implications for Stargardt macular deggeneration”的研究論文，該論文已被在國際知名醫(yī)學(xué)期刊PNAS（Q1,IF=10.2）接收并在線發(fā)表。

研究背景：

隱性Stargardt病(STGD1)是一種由Abca4基因突變引起的遺傳性致盲疾病，其臨床特征為發(fā)病早，RPE中熒光脂褐素堆積，光感受器變性甚至致盲。ABCA4是光感受器外段(OS)上的一種翻轉(zhuǎn)酶，它通過OS盤膜轉(zhuǎn)運(yùn)與磷脂酰乙醇胺結(jié)合的視黃醛。ABCA4 - / -小鼠和STGD1患者中ABCA4的缺失導(dǎo)致視網(wǎng)膜色素上皮(RPE)中脂褐素的積聚和光感受器的退化，導(dǎo)致失明。目前尚無針對STGD1的有效治療方法，本研究提出假設(shè)ABCA4通常存在于RPE細(xì)胞的內(nèi)溶酶體膜中。

研究結(jié)果：

圖1. ABCA4在RPE中內(nèi)源性表達(dá)。

注：ABCA4以前被稱為光感受器輻射蛋白RmP或ABCR，目前已知，人類的ABCA4基因有50個外顯子，2273個氨基酸蛋白，分子量為256kDa，ABCA4基因轉(zhuǎn)錄一個大的特定的視網(wǎng)膜蛋白，即ABCA4蛋白，該蛋白幾乎只存在于視網(wǎng)膜的視桿細(xì)胞的外節(jié)盤緣上，具有兩個串聯(lián)的區(qū)域，即氮端和碳端，每個區(qū)域各包含跨膜區(qū)（transmembrane domain，TMD）、糖基化胞外域（exocytoplasmic domain，EDC）和核苷酸結(jié)合結(jié)構(gòu)域（nucleotide binding domain，NBD）各一個。

圖2. ABCA4與內(nèi)溶酶體標(biāo)記共定位。

(A) 2歲野生型balb /c(上)和白化Abca4?/?(下)小鼠視網(wǎng)膜/RPE切片的代表性共聚焦圖像與Abca4(紅色)和LAMP1(綠色)抗體反應(yīng)。注意，ABCA4和LAMP1在野生型RPE中共定位，但在OS中沒有。在ABCA4?/?RPE細(xì)胞中也存在LAMP1，但不存在ABCA4的免疫反應(yīng)性。(B) 5個月大野生型(129/Sv)(上)、Abca4?/?(中)和Mertk?/?(下)小鼠視網(wǎng)膜切片的代表性合并共聚焦圖像，分別用Abca4(紅色)和Rab5(綠色)抗體進(jìn)行免疫染色。ABCA4和Rab5在129/Sv和Mertk?/?RPE細(xì)胞中均有共定位，如橙色信號所示。在Abca4?/?視網(wǎng)膜切片的RPE中，僅見Rab5免疫反應(yīng)。白色箭頭表示視網(wǎng)膜脫離，白色星號表示Mertk?/?視網(wǎng)膜由于光感受器變性而沒有OS。(C)固定hfRPE細(xì)胞的代表性共聚焦圖像，標(biāo)記ABCA4(紅色)(上)或內(nèi)體CAV1(綠色)(中)抗體。(下)ABCA4和CAV1的共聚焦合并圖像。

圖3.ABCA4在RPE- ABCA4-tg/ABCA4?/?小鼠的RPE中表達(dá)。

(A) BALB/c、Abca4-/-和RPE-Abca4-tg/Abca4-/-小鼠(均為白化)視網(wǎng)膜和RPE勻漿的代表性免疫印跡與抗Abca4或α-微管蛋白的抗血清反應(yīng)。神經(jīng)視網(wǎng)膜總蛋白負(fù)荷為10 μg, RPE/eyecup勻漿為25 μg。(B)ABCA4-/-和RPE-Abca4-Tg/ABCA4-/-勻漿中ABCA4蛋白水平歸一化為α-微管蛋白，并相對于野生型BALB/c水平呈現(xiàn);每組6齡小鼠7只。(C)BALB/C(左)、Abca4?/?(中)和RPE-Abca4-Tg/Abca4?/?(右)小鼠視網(wǎng)膜切片的代表性共聚焦圖像。RPE中ABCA4的免疫反應(yīng)性(紅色)- ABCA4 - tg / ABCA4?/?顯示主要針對RPE的特異性。Abca4-/-和RPE-Abca4-Tg/Abca4-/-小鼠的os層均未被Abca4抗體染色。DAPI核染色為藍(lán)色。

圖4.RPE-Abca4- tg /Abca4 - / -小鼠RPE中的類雙維甲酸、自身熒光和脂褐素水平降低。

(A-D)從3齡白化小鼠視網(wǎng)膜和RPE勻漿中提取類雙維甲酸，采用正相高效液相色譜法進(jìn)行分析。RPE/eyecup和神經(jīng)視網(wǎng)膜提取物的代表性HPLC色譜圖見SI附錄，圖S3。注意，RPE-Abca4- tg /Abca4 - / -小鼠RPE中所有類雙維甲酸水平較低。(A)總A2E (A2E和反A2E之和)以每只眼皮摩爾表示。(B -D)全反式視黃醛二聚體PE (atRAL-Dimer-PE) (B)、A2PE- h2 (C)和A2PE (D)以每只眼的毫吸光度單位(mAU)表示。數(shù)據(jù)以mean±SD表示;每組5只;* p < 0.0001， ** p < 0.001;N /s，不顯著。(E)使用488 nm激發(fā)激光器和500- 545 nm發(fā)射濾光片捕獲的rpe -脈絡(luò)膜-鞏膜平面支架的代表性共聚焦圖像。注意，與Abca4- / - RPE平片相比，RPE-Abca4- tg /Abca4?/?平片的自熒光強(qiáng)度(AF，綠色)降低?？箊o1染色顯示RPE細(xì)胞邊界(藍(lán)色);細(xì)胞核用DAPI染色(藍(lán)色);每組6齡小鼠3或4只。(比例尺，20 μm.) (F) 1歲BALB/c(左)、Abca4?/?(中)和RPE-Abca4- tg /Abca4?/?(右)白化病小鼠RPE細(xì)胞的代表性電鏡圖。箭頭指向RPE細(xì)胞質(zhì)內(nèi)電子密度不均勻的多態(tài)脂褐質(zhì)顆粒。BM，布魯氏膜;N,細(xì)胞核。(比尺，2 μm.) (G)測量每100 μm2細(xì)胞面積的脂褐素顆粒分?jǐn)?shù)，并從每只眼睛10個相鄰的電鏡圖像中取平均值。數(shù)據(jù)以mean±SD表示;N = 5 ~ 9只/組;* p = 0.0186;** p < 0.001。

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

(A)通過光學(xué)顯微鏡獲得的1歲白化病小鼠的代表性視網(wǎng)膜圖像。(B)每100 μm2細(xì)胞面積計(jì)算光感受器細(xì)胞核總數(shù)。注意，與Abca4-/-小鼠相比，RPE -Abca4- tg /Abca4 - / -小鼠的ONL細(xì)胞數(shù)量增加，表明光感受器變性部分恢復(fù)。數(shù)據(jù)以mean±SD表示;N = 5 - 9只/組;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。

圖6所示。ABCA4在RPE內(nèi)溶酶體膜中的功能。

(A)正常RPE細(xì)胞。在紫紅質(zhì)蛋白水解過程中釋放的11cRAL在光腔表面與PE縮合形成11-順式- n -視黃醛-磷脂酰乙醇胺(11c-N-ret-PE)，后者經(jīng)過異構(gòu)化形成全反式(at)和11c-N-ret-PE的混合物。兩種N-ret-PE異構(gòu)體都被ABCA4翻轉(zhuǎn)到細(xì)胞質(zhì)表面，其中Nret-PE的水解是由細(xì)胞視黃醛結(jié)合蛋白(CRALBP)結(jié)合11cRAL或視黃醛脫氫酶11型(RDH11)將atRAL還原為atROL的質(zhì)量作用驅(qū)動的。atROL由RPE視覺循環(huán)處理，通過卵磷脂視黃醇?；D(zhuǎn)移酶(LRAT)酯化生成全反式視黃醇酯，如全反式視黃醇棕櫚酸酯(atRP)， RPE65異構(gòu)化生成11-順式視黃醇(11cROL)，視黃醇脫氫酶5型(RDH5)氧化生成11cRAL，并與CRALBP結(jié)合。11cRAL使RPE細(xì)胞在鄰近的光感受器OS中再生視覺色素。(B)Abca4?/?突變型RPE細(xì)胞。ABCA4 - / -小鼠或STGD1患者的RPE內(nèi)溶酶體中缺乏ABCA4會導(dǎo)致視黃醛的清除延遲，從而導(dǎo)致游離視黃醛和N-ret-PE濃度升高。這導(dǎo)致atRAL或11cRAL與N-ret-PE二次縮合形成類雙維甲酸。

研究小結(jié)：

ABCA4在RPE細(xì)胞中表達(dá)，并且至少部分ABCA4?/?表型是由RPE表達(dá)的ABCA4的缺失引起的。除了光感受器外，RPE細(xì)胞也應(yīng)該作為abca4介導(dǎo)的視網(wǎng)膜變性的治療靶點(diǎn)。

重要性：

隱性Stargardt黃斑變性(STGD1)和錐桿營養(yǎng)不良是由ABCB4基因突變引起的。ABCA4蛋白是光感受器細(xì)胞中的一種翻轉(zhuǎn)酶，有助于消除視黃醛，一種有毒的視覺光產(chǎn)物。

在這里，我們發(fā)現(xiàn)ABCA4在小鼠視網(wǎng)膜色素上皮(RPE)中也存在，其豐度約為神經(jīng)視網(wǎng)膜中的1%。在RPE中表達(dá)ABCA4而在光感受器細(xì)胞中不表達(dá)ABCA4的轉(zhuǎn)基因小鼠顯示，在ABCA4 - / -小鼠和inSTGD1患者中觀察到的脂褐素積累和光感受器變性均部分恢復(fù)。這些觀察結(jié)果表明，theRPE中的ABCA4可以阻止ABCA4?/?小鼠以及STGD1患者的光感受器變性。

相關(guān)信息：

光感受器細(xì)胞外段膜盤中包含視色素，正常膜盤不段更新，在視網(wǎng)膜色素變性和某些視網(wǎng)膜病變時，外段膜盤的更新可能出現(xiàn)障礙。

信源:

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.

參考文獻(xiàn)

1. Wald G (1968) Molecular basis of visual excitation. Science 162:230–239.

2. Rattner A, Smallwood PM, Nathans J (2000) Identification and characterization of all-trans-retinol dehydrogenase from photoreceptor outer segments, the visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol. J Biol Chem 275:11034–11043.

3. Quazi F, Lenevich S, Molday RS (2012) ABCA4 is an N-retinylidene-phosphatidylethanolamine

and phosphatidylethanolamine importer. Nat Commun 3:925.

4. Sun H, Nathans J (1997) Stargardt’s ABCR is localized to the disc membrane of retinal rod outer segments. Nat Genet 17:15–16.

5. Allikmets R (1997) A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat Genet 17:122.

6. Maugeri A, et al. (2000) Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy. Am J Hum Genet 67:960–966.

7. Burke TR, et al. (2014) Quantitative fundus autofluorescence in recessive Stargardt disease. Invest Ophthalmol Vis Sci 55:2841–2852.

8. Young RW, Bok D (1969) Participation of the retinal pigment epithelium in the rod outer segment renewal process. J Cell Biol 42:392–403.

9. Young RW, Bok D (1970) Autoradiographic studies on the metabolism of the retinal pigment epithelium. Invest Ophthalmol 9:524–536.

10. Mata NL, Weng J, Travis GH (2000) Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR-mediated retinal and macular degeneration. Proc Natl Acad Sci USA 97:7154–7159.

11. Boyer NP, et al. (2012) Lipofuscin and N-retinylidene-N-retinylethanolamine (A2E)

accumulate in retinal pigment epithelium in absence of light exposure: Their origin is 11-cis-retinal. J Biol Chem 287:22276–22286.

12. LaVail MM (1976) Rod outer segment disk shedding in rat retina: Relationship to cyclic lighting. Science 194:1071–1074.

13. Stempel AJ, Morgans CW, Stout JT, Appukuttan B (2014) Simultaneous visualization and cell-specific confirmation of RNA and protein in the mouse retina. Mol Vis 20:1366–1373.

14. Hu J, Bok D (2010) Culture of highly differentiated human retinal pigment epithelium for analysis of the polarized uptake, processing, and secretion of retinoids. Methods Mol Biol 652:55–73.

15. Feng W, Yasumura D, Matthes MT, LaVail MM, Vollrath D (2002) Mertk triggers uptake of photoreceptor outer segments during phagocytosis by cultured retinal pigment epithelial cells. J Biol Chem 277:17016–17022.

16. Duncan JL, et al. (2003) An RCS-like retinal dystrophy phenotype in mer knockout mice. Invest Ophthalmol Vis Sci 44:826–838.

17. Sethna S, et al. (2016) Regulation of phagolysosomal digestion by caveolin-1 of the retinal pigment epithelium is essential for vision. J Biol Chem 291:6494–6506.

18. Haeseleer F, et al. (2002) Dual-substrate specificity short chain retinol dehydrogenases from the vertebrate retina. J Biol Chem 277:45537–45546.

19. Kaylor JJ, et al. (2017) Blue light regenerates functional visual pigments in mammals through a retinyl-phospholipid intermediate. Nat Commun 8:16.

20. Quazi F, Molday RS (2014) ATP-binding cassette transporter ABCA4 and chemical isomerization protect photoreceptor cells from the toxic accumulation of excess 11-cis-retinal. Proc Natl Acad Sci USA 111:5024–5029.

21. Anderson DMG, et al. (2017) Bis(monoacylglycero)phosphate lipids in the retinal pigment epithelium implicate lysosomal/endosomal dysfunction in a model of Stargardt disease and human retinas. Sci Rep 7:17352.

22. Chen C, Thompson DA, Koutalos Y (2012) Reduction of all-trans-retinal in vertebrate rod photoreceptors requires the combined action of RDH8 and RDH12. J Biol Chem 287:24662–24670.

23. Harper WS, Gaillard ER (2001) Studies of all-trans-retinal as a photooxidizing agent. Photochem Photobiol 73:71–76.

24. Radu RA, et al. (2008) Accelerated accumulation of lipofuscin pigments in the RPE of a mouse model for ABCA4-mediated retinal dystrophies following Vitamin A supplementation. Invest Ophthalmol Vis Sci 49:3821–3829.

25. Weng J, et al. (1999) Insights into the function of Rim protein in photoreceptors and etiology of Stargardt’s disease from the phenotype in abcr knockout mice. Cell 98:13–23.

26. Calame M, et al. (2011) Retinal degeneration progression changes lentiviral vector cell targeting in the retina. PLoS One 6:e23782.

27. Natkunarajah M, et al. (2008) Assessment of ocular transduction using single-stranded

and self-complementary recombinant adeno-associated virus serotype 2/8. Gene Ther15:463–467.

28. Carr AJ, et al. (2009) Protective effects of human iPS-derived retinal pigment epithelium cell transplantation in the retinal dystrophic rat. PLoS One 4:e8152.

29. Schwartz SD, et al. (2015) Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt’s macular dystrophy: Follow-up of two open-label phase 1/2 studies. Lancet 385:509–516.

30. Boulanger A, Liu S, Henningsgaard AA, Yu S, Redmond TM (2000) The upstream region of the Rpe65 gene confers retinal pigment epithelium-specific expression in vivo and in vitro and contains critical octamer and E-box binding sites. J Biol Chem 275:31274–31282.

31. Stec DE, Morimoto S, Sigmund CD (2001) Vectors for high-level expression of cDNAs controlled by tissue-specific promoters in transgenic mice. Biotechniques 31:256–258,260.

32. Illing M, Molday LL, Molday RS (1997) The 220-kDa rim protein of retinal rod outer segments is a member of the ABC transporter superfamily. J Biol Chem 272:10303–10310.

33. Radu RA, et al. (2011) Complement system dysregulation and inflammation in the retinal pigment epithelium of a mouse model for Stargardt macular degeneration. J Biol Chem 286:18593–18601.

34. Parish CA, Hashimoto M, Nakanishi K, Dillon J, Sparrow J (1998) Isolation and onestep preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium. Proc Natl Acad Sci USA 95:14609–14613.

35. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675.

36. Lenis TL, et al. (2017) Complement modulation in the retinal pigment epithelium rescues photoreceptor degeneration in a mouse model of Stargardt disease. Proc Natl Acad Sci USA 114:3987–3992.

END

文案 | 劉衛(wèi)勤

排版 | 姜笑南

審核 | 姜笑南

發(fā)布｜姜笑南

世界生命科學(xué)大會

RECRUIT