Proteasome-Mediated Regulation of Cdhr1a by Siah1 Modulates Photoreceptor Development and Survival in Zebrafish.
Proteasome-Mediated Regulation of Cdhr1a by Siah1 Modulates Photoreceptor Development and Survival in Zebrafish.
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DOI：
10.3389/fcell.2020.594290
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发表时间：
2020
影响因子：
5.5
通讯作者：
Famulski JK
中科院分区：
文献类型：
--
作者：
Piedade WP;Titialii-Torres K;Morris AC;Famulski JK
先天性视网膜营养不良是世界范围内无法预防和无法治愈的失明的主要原因。CDHR1（一种视网膜特异性钙粘蛋白）的突变与锥杆营养不良有关。泛素蛋白酶体系统（UPS）负责调节有序和精确的蛋白质降解目标，以维持生物稳态和协调适当的发育，包括视网膜发育。最近，我们的实验室发现E3泛素连接酶的7 in absentia （Siah）家族在视裂隙融合中发挥作用，并确定Cdhr1a是Siah的潜在靶点。利用双色原位杂交和免疫组化技术，我们检测了siah1和cdhr1a在受精后3-5天视网膜外核层（ONL）的共表达和蛋白定位，更准确地说，在杆状体和锥体的连接纤毛中（dpf）。我们在细胞培养中通过共转染和共免疫沉淀证实Siah1靶向Cdhr1a进行蛋白酶体降解。为了分析这种相互作用的功能重要性，我们创建了两个转基因斑马鱼系，在热休克启动子的控制下表达siah1或不活跃的siah1 (siah1ΔRING)，以调节光感受器发育过程中的Siah活性。siah1过表达，而siah1ΔRING不表达，导致受精后72 h杆状体和球果数量减少。Siah1过表达不影响视网膜神经节细胞、无突细胞和双极细胞的数量，Siah1过表达视网膜的增殖细胞无明显减少。然而，我们确实检测到细胞死亡增加，证实了ONL中TUNEL +细胞数量的增加，这是蛋白酶体依赖性的，因为蛋白酶体抑制挽救了细胞死亡表型。此外，注射cdhr1a mRNA可以挽救因Siah1表达增加而导致的视杆细胞和视锥细胞的减少，甚至更大程度上可以通过注射Siah1不敏感的cdhr1a变体mRNA来挽救。最后，CRISPR诱导Cdhr1a功能缺失，表型Siah1过表达导致杆状细胞和视锥细胞显著减少。综上所述，我们的工作提供了Cdhr1a在早期光感受器发育过程中发挥作用的第一个证据，并且Cdhr1a通过UPS受到Siah1的调节。
Congenital retinal dystrophies are a major cause of unpreventable and incurable blindness worldwide. Mutations in CDHR1, a retina specific cadherin, are associated with cone-rod dystrophy. The ubiquitin proteasome system (UPS) is responsible for mediating orderly and precise targeting of protein degradation to maintain biological homeostasis and coordinate proper development, including retinal development. Recently, our lab uncovered that the seven in absentia (Siah) family of E3 ubiquitin ligases play a role in optic fissure fusion and identified Cdhr1a as a potential target of Siah. Using two-color whole mount in situ hybridization and immunohistochemistry, we detected siah1 and cdhr1a co-expression as well as protein localization in the retinal outer nuclear layer (ONL), and more precisely in the connecting cilium of rods and cones between 3–5 days post fertilization (dpf). We confirmed that Siah1 targets Cdhr1a for proteasomal degradation by co-transfection and co-immunoprecipitation in cell culture. To analyze the functional importance of this interaction, we created two transgenic zebrafish lines that express siah1 or an inactive siah1 (siah1ΔRING) under the control of the heat shock promoter to modulate Siah activity during photoreceptor development. Overexpression of siah1, but not siah1ΔRING, resulted in a decrease in the number of rods and cones at 72 h post fertilization (hpf). The number of retinal ganglion cells, amacrine and bipolar cells was not affected by Siah1 overexpression, and there was no significant reduction of proliferating cells in the Siah1 overexpressing retina. We did, however, detect increased cell death, confirmed by an increase in the number of TUNEL + cells in the ONL, which was proteasome-dependent, as proteasome inhibition rescued the cell death phenotype. Furthermore, reduction in rods and cones resulting from increased Siah1 expression was rescued by injection of cdhr1a mRNA, and to an even greater extent by injection of a Siah1-insensitive cdhr1a variant mRNA. Lastly, CRISPR induced loss of Cdhr1a function phenocopied Siah1 overexpression resulting in a significant reduction of rods and cones. Taken together, our work provides the first evidence that Cdhr1a plays a role during early photoreceptor development and that Cdhr1a is regulated by Siah1 via the UPS.
影响因子：
3.7
作者：
Buch PK;Mihelec M;Cottrill P;Wilkie SE;Pearson RA;Duran Y;West EL;Michaelides M;Ali RR;Hunt DM
通讯作者：
Hunt DM