Departments of Ophthalmology and Biochemistry and Neuroscience Graduate Program, Jacobs School of Medicine and Biomedical Sciences, State University of New York- University at Buffalo, Buffalo, NY, USA.
Research Service, VA Western Ny Healthcare System, Buffalo, NY, USA.
Autophagy. 2022 May;18(5):1187-1201. doi: 10.1080/15548627.2021.1969634. Epub 2021 Oct 21.
We describe the utility of a tandem-tagged autophagy reporter mouse model (CAG-RFP-EGFP-MAP1LC3B) in investigating basal macroautophagic/autophagic flux in the neural retina. Western blot, hybridization, immunohistochemistry, and confocal microscopy showed that CAG promoter-driven expression of RFP-EGFP-MAP1LC3B increased "cytosolic" RFP-EGFP-LC3B-I levels, whereas RFP-EGFP-LC3B-II decorates true phagosomes. We verified that the electroretinographic (ERG) responses of tandem-tagged LC3B mice were comparable to those of age-matched controls. Optimized microscope settings detected lipofuscin autofluorescence in retinas of mice. The majority of retinal phagosomes in the reporter mice exhibited only RFP (not EGFP) fluorescence, suggesting rapid maturation of phagosomes. Only ~1.5% of the total phagosome population was EGFP-labeled; RFP-labeled (mature) phagosomes colocalized with lysosomal markers LAMP2 and CTSD. In the outer retina, phagosome sizes were as follows (in µm, ave ± SEM): RPE, 0.309 ± 0.015; photoreceptor inner segment-myoid, 0.544 ± 0.031; and outer nuclear layer, 0.429 ± 0.011. Detection of RPE phagosomes by fluorescence microscopy is challenging, due to the presence of melanin. Increased lipofuscin autofluorescence, such as observed in the mouse model of Stargardt disease, is a strong confounding factor when attempting to study autophagy in the RPE. In addition to RPE and photoreceptor cells, phagosomes were detected in inner retinal cell types, microglia, astrocytes, and endothelial cells. We conclude that the tandem-tagged LC3B mouse model serves as a useful system for studying autophagy in the retina. This utility, however, is dependent upon several technical and biological factors, including microscope settings, transgene expression, choice of fluorophores, and lipofuscin autofluorescence.: ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATG: autophagy related; CTSD: cathepsin D; DAPI: (4',6-diamido-2-phenylindole); DIC: differential interference contrast; EGFP: enhanced green fluorescent protein; ELM: external limiting membrane; ERG: electroretinography; GCL: ganglion cell layer; GLUL: glutamine-ammonia ligase (glutamine synthetase); INL: inner nuclear layer; IS-E/M: inner segment - ellipsoid/myoid; ISH: ; LAMP2: lysosomal-associated membrane protein 2; L.I.: laser Intensity; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; O.C.T.: optimal cutting temperature; OS: outer segment; ONL: outer nuclear layer; PE: phosphatidylethanolamine; RFP: red fluorescent protein; R.O.I.: region of interest; RPE: retinal pigment epithelium.
我们描述了串联标记自噬报告小鼠模型（CAG-RFP-EGFP-MAP1LC3B）在研究神经视网膜中基础巨自噬/自噬流中的效用。Western blot、杂交、免疫组织化学和共聚焦显微镜显示，CAG 启动子驱动的 RFP-EGFP-MAP1LC3B 的表达增加了“胞质”RFP-EGFP-LC3B-I 水平，而 RFP-EGFP-LC3B-II 则修饰真正的吞噬体。我们验证了串联标记 LC3B 小鼠的视网膜电图（ERG）反应与年龄匹配的对照相当。优化的显微镜设置可检测到小鼠视网膜中脂褐素的自发荧光。报告小鼠中的大多数视网膜吞噬体仅显示 RFP（而非 EGFP）荧光，表明吞噬体迅速成熟。只有约 1.5%的总吞噬体群体被 EGFP 标记；RFP 标记的（成熟）吞噬体与溶酶体标记物 LAMP2 和 CTSD 共定位。在外视网膜中，吞噬体的大小如下（µm，ave ± SEM）：RPE，0.309 ± 0.015；光感受器内节-类晶体，0.544 ± 0.031；和外核层，0.429 ± 0.011。由于黑色素的存在，用荧光显微镜检测 RPE 吞噬体具有挑战性。在 Stargardt 病的 小鼠模型中观察到的脂褐素自发荧光增加是尝试研究 RPE 自噬时的一个强烈混杂因素。除了 RPE 和光感受器细胞外，还在视网膜内细胞类型、小胶质细胞、星形胶质细胞和内皮细胞中检测到吞噬体。我们得出结论，串联标记的 LC3B 小鼠模型是研究视网膜自噬的有用系统。然而，这种效用取决于几个技术和生物学因素，包括显微镜设置、转基因表达、荧光团的选择和脂褐素的自发荧光。：ACTB：肌动蛋白，β；AIF1：同种异体炎症因子 1；ATG：自噬相关；CTSD：组织蛋白酶 D；DAPI：（4'，6-二脒基-2-苯基吲哚）；DIC：微分干涉对比；EGFP：增强型绿色荧光蛋白；ELM：外部限制膜；ERG：视网膜电图；GCL：节细胞层；GLUL：谷氨酰胺-氨连接酶（谷氨酰胺合成酶）；INL：内核层；IS-E/M：内节-椭圆体/类晶体；ISH：；LAMP2：溶酶体相关膜蛋白 2；MTOR：雷帕霉素靶蛋白激酶；O.C.T.：最佳切割温度；OS：外节；ONL：外核层；PE：磷脂酰乙醇胺；RFP：红色荧光蛋白；R.O.I.：感兴趣区域；RPE：视网膜色素上皮。
