Targeted inactivation of synaptic HRG4 (UNC119) causes dysfunction in the distal photoreceptor and slow retinal degeneration, revealing a new function
Targeted inactivation of synaptic HRG4 (UNC119) causes dysfunction in the distal photoreceptor and slow retinal degeneration, revealing a new function
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DOI：
10.1016/j.exer.2006.10.016
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发表时间：
2007-03-01
影响因子：
3.4
通讯作者：
Inana, George
中科院分区：
文献类型：
--
作者：
Ishiba, Yasutsugu;Higashide, Tornorni;Inana, George
HRG4(UNC119)是一种光感受器蛋白，主要定位于光感受器突触，少量定位于内节。在一名迟发性视锥-视杆细胞营养不良患者中发现了HRG4杂合截断突变，表达相同突变蛋白的转基因(TG)小鼠发生了迟发性视网膜变性，证实了HRG4的致病潜力。最近，在TG模型中，主要的负性致病机制是截断突变体HRG4与其靶标ARL2亲和力增加，导致其下游靶标、线粒体抗、线粒体应激、突触变性、跨突触变性和整个光感受器通过细胞凋亡而延迟下降。本研究克隆了小鼠高密度脂蛋白4(MRG4)基因，并以此为靶点构建了高密度脂蛋白4基因敲除(KO)小鼠模型，以研究完全灭活该蛋白的效果。采用基因组Southern杂交、Western blotting、免疫荧光、眼底镜、光镜和电镜、ERG和TUNEL等方法对KO模型进行检测。KO模型发展为缓慢进展的视网膜变性，特征是眼底斑驳，光感受器层轻度变薄，6个月时细胞凋亡增加，17个月时急剧加速，20个月时光感受器几乎完全消失。与TG模型中的视网膜变性相比，在KO模型中存在显著的差异，包括更严重的和早期的光感受器死亡，而没有像在TG中所看到的早期突触和跨突触变性的证据，通过光镜和EM组织病理学，ERG和突触蛋白的Western blotting证实。结果表明，光感受器远端突触外的KO功能障碍，MRG4也在那里定位。在KO和TG模型中，视网膜变性表型的差异反映了光感受器两端，即远端内/外节和近端突触的功能障碍，表明MRG4在远端光感受器中具有第二功能和MRG4的双重功能。因此，通过基因靶向灭活MRG4导致了一种与以前在TG中看到的完全不同的视网膜变性表型，这证明了MRG4功能的多样性，以及该蛋白对正常视网膜功能的重要性。这些模型将有助于阐明HRG4/MRG4的功能和慢性视网膜变性的机制。(C)2006爱思唯尔有限公司。保留所有权利。
HRG4 (UNC 119) is a photoreceptor protein predominantly localized to the photoreceptor synapses and to the inner segments to a lesser degree. A heterozygous truncation mutation in HRG4 was found in a patient with late onset cone-rod dystrophy, and a transgenic (TG) mouse expressing the identical mutant protein developed late onset retinal degeneration, confirming the pathogenic potential of HRG4. Recently, the dominant negative pathogenic mechanism in the TG model was shown to involve increased affinity of the truncated mutant HRG4 for its target, ARL2, which leads to a delayed decrease in its downstream target, mitochondrial ANTI, mitochondrial stress, synaptic degeneration, trans-synaptic degeneration, and whole photoreceptor degeneration by apoptosis. In this study, the mouse HRG4 (MRG4) gene was cloned and targeted to construct a knock-out (KO) mouse model of HRG4 in order to study the effects of completely inactivating this protein. The KO model was examined by genomic Southern blotting, Western blotting, immunofluorescence, funduscopy, LM and EM histopathology, ERG, and TUNEL analyses. The KO model developed a slowly progressive retinal degeneration, characterized by mottling in the fundus, mild thinning of the photoreceptor layer, and increase in apoptosis as early as 6 months, dramatic acceleration at similar to 17 months, and virtual obliteration of the photoreceptors by 20 months. When compared to retinal degeneration in the TG model, significant differences existed in the KO consisting of more severe and early photoreceptor death without evidence of early synaptic and trans-synaptic degeneration as seen in the TG, confirmed by LM and EM histopathology, ERG, and Western blotting of synaptic proteins. The results indicated a dysfunction in the KO outside the synapses in the distal end of photoreceptors where MRG4 is also localized. Differences in the phenotypes of retinal degeneration in the KO and TG models reflect a dysfunction in the two opposite ends of photoreceptors, i.e., the distal inner/outer segments and proximal synapses, respectively, indicating a second function of MRG4 in the distal photoreceptor and dual functionality of MRG4. Thus, inactivation of MRG4 by gene targeting resulted in a retinal degeneration phenotype quite different from that previously seen in the TG, attesting to the multiplicity of MRG4 function, in addition to the importance of this protein for normal retinal function. These models will be useful in elucidating the functions of HRG4/MRG4 and the mechanism of slow retinal degeneration. (c) 2006 Elsevier Ltd. All rights reserved.