The <i>epg5</i> knockout zebrafish line: a model to study Vici syndrome

The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human <i>EPG5</i> gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish <i>epg5^-/-</i> mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in <i>epg5^<i>-</i>/-</i> mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in <i>epg5^<i>-</i>/-</i> than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved <i>epg5^<i>-</i>/-</i> mutants. By aging, <i>epg5^<i>-</i>/-</i> mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, <i>epg5^<i>-</i>/-</i> adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish <i>epg5</i> mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.EPG5蛋白是一种RAB7A效应蛋白，在巨自噬（macroautophagy/autophagy）过程中介导自噬体与晚期内体或溶酶体之间的融合特异性。人类EPG5基因发生突变会引发一种罕见且严重的多系统疾病，名为维克西综合征（Vici syndrome）。本研究证实，来自杂合子交配以及纯合子互交交配的斑马鱼epg5^-/-突变体均可存活，并能发育至性成熟阶段，外观无明显异常。与维克西综合征的自噬功能异常相符，蛋白质免疫印迹（western blot）结果显示，epg5^-/-突变体中自噬标志物Lc3-II的水平相较于野生型（WT）对照更高。此外，饥饿诱导后，epg5^-/-斑马鱼幼体的Lc3-II积累量较野生型更高，同时骨骼肌双折射显著降低。相应地，肌肉超微结构分析显示，饥饿处理的epg5^-/-突变体体内积累了降解缺陷型自噬溶酶体。随着年龄增长，epg5^-/-突变体出现运动能力受损、肌肉变薄，同时伴随非降解型自噬空泡的积累。此外，epg5^-/-成鱼的性腺与心脏出现形态学改变。上述研究结果表明，斑马鱼epg5突变体可作为研究EPG5相关疾病的理想模型，为解析EPG5在维克西综合征发病与进展中的作用提供了新的实验工具，同时也可用于筛选自噬激活类药物。 本研究涉及的缩写术语定义如下： ATG：自噬相关（autophagy related）； cDNA：互补DNA（complementary DNA）； DIG：地高辛（digoxigenin）； dpf：受精后天数（days post-fertilization）； EGFP：增强型绿色荧光蛋白（enhanced green fluorescent protein）； EPG：异位P颗粒（ectopic P granules）； GFP：绿色荧光蛋白（green fluorescent protein）； hpf：受精后小时数（hours post-fertilization）； IL1B：白细胞介素1β（interleukin 1 beta）； Lc3-II：脂化Lc3（lipidated Lc3）； mpf：受精后月数（months post-fertilization）； mRNA：信使RNA（messenger RNA）； NMD：无义介导的mRNA降解（nonsense-mediated mRNA decay）； PCR：聚合酶链式反应（polymerase chain reaction）； qPCR：实时聚合酶链式反应（real time-polymerase chain reaction）； RAB7A/RAB7：RAS癌基因家族成员RAB7a（RAB7a, member RAS oncogene family）； RACE：cDNA末端快速扩增技术（rapid amplification of cDNA ends）； RFP：红色荧光蛋白（red fluorescent protein）； RT-PCR：逆转录聚合酶链式反应（reverse transcriptase-polymerase chain reaction）； SEM：均值标准误（standard error of the mean）； sgRNA：向导RNA（guide RNA）； UTR：非翻译区（untranslated region）； WMISH：全标本原位杂交（whole mount in situ hybridization）； WT：野生型（wild type）。