ON-bipolar cell gene expression during retinal degeneration: Implications for optogenetic visual restoration.
ON-bipolar cell gene expression during retinal degeneration: Implications for optogenetic visual restoration.
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视网膜变性过程中ON型双极细胞的基因表达：对光遗传学视觉修复的意义
DOI：
10.1016/j.exer.2021.108553
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发表时间：
2021-06
影响因子：
3.4
通讯作者：
Hankins MW
中科院分区：
文献类型：
--
作者：
Gilhooley MJ;Hickey DG;Lindner M;Palumaa T;Hughes S;Peirson SN;MacLaren RE;Hankins MW
在遗传性视网膜变性（IRD）的后期阶段，视网膜双极细胞依然能够存活，因此它们成为光遗传学视力恢复方法颇具吸引力的靶点。然而，目前尚不清楚这些细胞在变性过程中所经历的重塑在多大程度上会影响其功能。具体而言，尚不清楚它们是否不受代谢应激影响、是否能接受腺相关病毒载体、是否适合基于视蛋白的光遗传学工具，以及是否能够通过释放神经递质来传递信号。
采用荧光激活细胞分选（FACS）技术，从携带或不携带IRD突变Pde6brd1/rd1的同窝幼崽的解离视网膜中分离标记的双极细胞，这些幼崽在ON型双极细胞中选择性表达增强型黄色荧光蛋白（EYFP）作为标记。随后提取mRNA，利用Illumina®微阵列比较变性视网膜与野生型视网膜双极细胞中的基因表达。在变性过程中，使用视网膜免疫组织化学方法在蛋白质水平进一步研究四个候选基因的变化。
共有60个差异表达的转录本达到统计学显著性：其中不包括任何与天然初级双极细胞信号传导直接相关的基因，也没有与代谢应激相符的变化。四个显著改变的基因（Srm2、Slf2、Anxa7和Cntn1）与突触重塑、神经递质释放和病毒载体进入有关，其免疫组织化学染色与ON型双极细胞标记共定位，且在变性过程中有所变化。
我们的研究结果表明，在变性背景下基因表达变化相对较少：尽管发生了重塑，双极细胞仍可能是光遗传学视力恢复的可行靶点。此外，观察到变化的几个基因可为提高光遗传学治疗效果的研究提供基础。
双极细胞是IRD治疗性光遗传学颇具吸引力的靶点。
这是首次在IRD模型中对双极细胞进行的细胞特异性转录组分析。
双极细胞维持着作为光遗传学靶点所必需的基因表达。
利用免疫组织化学方法证实了与四个候选基因（Anxa7、Cntn1、Srm2、Sulf2）相关的蛋白质染色。
Retinal bipolar cells survive even in the later stages of inherited retinal degenerations (IRDs) and so are attractive targets for optogenetic approaches to vision restoration. However, it is not known to what extent the remodelling that these cells undergo during degeneration affects their function. Specifically, it is unclear if they are free from metabolic stress, receptive to adeno-associated viral vectors, suitable for opsin-based optogenetic tools and able to propagate signals by releasing neurotransmitter. Fluorescence activated cell sorting (FACS) was performed to isolate labelled bipolar cells from dissociated retinae of litter-mates with or without the IRD mutation Pde6brd1/rd1 selectively expressing an enhanced yellow fluorescent protein (EYFP) as a marker in ON-bipolar cells. Subsequent mRNA extraction allowed Illumina® microarray comparison of gene expression in bipolar cells from degenerate to those of wild type retinae. Changes in four candidate genes were further investigated at the protein level using retinal immunohistochemistry over the course of degeneration. A total of sixty differentially expressed transcripts reached statistical significance: these did not include any genes directly associated with native primary bipolar cell signalling, nor changes consistent with metabolic stress. Four significantly altered genes (Srm2, Slf2, Anxa7 & Cntn1), implicated in synaptic remodelling, neurotransmitter release and viral vector entry had immunohistochemical staining colocalising with ON-bipolar cell markers and varying over the course of degeneration. Our findings suggest relatively few gene expression changes in the context of degeneration: that despite remodelling, bipolar cells are likely to remain viable targets for optogenetic vision restoration. In addition, several genes where changes were seen could provide a basis for investigations to enhance the efficacy of optogenetic therapies. Bipolar cells are attractive targets for therapeutic optogenetics in IRDs. This is the first cell specific transcriptomic analysis of bipolar cells in an IRD model. Bipolar cells maintain expression of genes essential to act as targets for optogenetics. Protein staining relating to four candidate genes (Anxa7, Cntn1, Srm2, Sulf2) is confirmed using immunohistochemistry.
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影响因子：
11.1
作者：
Cronin T;Vandenberghe LH;Hantz P;Juttner J;Reimann A;Kacsó AE;Huckfeldt RM;Busskamp V;Kohler H;Lagali PS;Roska B;Bennett J
通讯作者：
Bennett J
影响因子：
46.9
作者：
Canales, Roger D.;Luo, Yuling;Goodsaid, Federico M.
通讯作者：
Goodsaid, Federico M.
DOI：
10.1111/j.2517-6161.1995.tb02031.x
发表时间：
1995-01-01
影响因子：
5.8
作者：
BENJAMINI, Y;HOCHBERG, Y
通讯作者：
HOCHBERG, Y
DOI：
10.1038/mtm.2013.5
发表时间：
2014-01-08
期刊：
Molecular therapy. Methods & clinical development
影响因子：
--
作者：
通讯作者：
--
影响因子：
4.6
作者：
Fairbank, Pamela D.;Lee, Chanjae;Wallingford, John B.
通讯作者：
Wallingford, John B.