Kuny Sharee, Cho Woo Jung, Dimopoulos Ioannis S, Sauvé Yves
Department of Ophthalmology and Visual Sciences, University of Alberta, Edmonton, Alberta, Canada.
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada.
Invest Ophthalmol Vis Sci. 2015 Nov;56(12):7109-21. doi: 10.1167/iovs.15-17567.
We investigated the interplay between photoreceptors expressing mutant ELOVL4 (responsible for Stargardt-like disease, STGD3) and RPE in the initial stages of retinal degeneration.
Using electron microscopy and electroretinogram (ERG), we assessed RPE and photoreceptor ultrastructure and function in transgenic ELOVL4 (TG1-2 line; TG) and wild-type (WT) littermates. Experiments were done at P30, 1 month before photoreceptor loss in TG and at P90, a time point with approximately 30% rod loss. To further elucidate the mechanism underlying our ultrastructural and functional results, we undertook Western blotting and immunohistochemistry of key proteins involved in phagocytosis of outer segments by RPE cells.
Firstly, we showed that in TG mouse photoreceptors, endogenous ELOVL4 protein is not mislocalized in the presence of the mutated ELOVL4 protein. Secondly, we found evidence of RPE toxicity at P30, preceding any photoreceptor loss. Pathology in RPE cells was exacerbated at P90. Furthermore, higher proportions of phagosomes remained at the apical side of RPE cells. Subretinal lysosomal deposits were immunopositive for phagocytic proteins. Ultrastructural analysis of photoreceptor (rod) outer segments showed disrupted surface morphology consisting of disc spacing irregularities. Finally, rods and RPE exhibited signs of dysfunction as measured by the ERG a-wave leading edge (P30) and c-wave (P90), respectively.
The presence of human mutant ELOVL4 in transgenic mouse photoreceptors leads to early outer segment disc pathology and RPE cytotoxicity. Defective processing of these abnormal discs by RPE cells ultimately may be responsible for outer segment truncation, photoreceptor death, and vision loss.
我们研究了表达突变型ELOVL4（与类Stargardt病，即STGD3相关）的光感受器与视网膜色素上皮（RPE）在视网膜变性初始阶段的相互作用。
我们利用电子显微镜和视网膜电图（ERG），评估了转基因ELOVL4（TG1-2系；TG）小鼠和野生型（WT）同窝小鼠的RPE和光感受器的超微结构及功能。实验分别在P30（TG小鼠光感受器丧失前1个月）和P90（此时杆状细胞损失约30%）进行。为进一步阐明我们超微结构和功能结果背后的机制，我们对RPE细胞吞噬外节所涉及的关键蛋白进行了蛋白质免疫印迹和免疫组织化学分析。
首先，我们发现，在TG小鼠光感受器中，内源性ELOVL4蛋白在突变型ELOVL4蛋白存在的情况下并未出现错误定位。其次，我们发现在P30时，在任何光感受器丧失之前就有RPE毒性的证据。在P90时，RPE细胞的病理学变化加剧。此外，更高比例的吞噬体滞留在RPE细胞的顶端。视网膜下溶酶体沉积物对吞噬蛋白呈免疫阳性。光感受器（杆状细胞）外节的超微结构分析显示，其表面形态破坏，包括盘间距不规则。最后，分别通过ERG的a波前沿（P30）和c波（P90）测量发现，杆状细胞和RPE均表现出功能障碍的迹象。
转基因小鼠光感受器中人类突变型ELOVL4的存在导致早期外节盘病理学变化和RPE细胞毒性。RPE细胞对这些异常盘的处理缺陷最终可能导致外节截断、光感受器死亡和视力丧失。
