Analysis of pathways and cells mediating optineurin glaucoma pathology

描述(申请人提供)：青光眼是全世界导致失明的主要原因之一，但其细胞和分子机制大多尚不清楚。对这种疾病的见解来自对动物模型和人类的基因研究。在一组青光眼患者中，多达13.5%的原发性开角型青光眼患者与视神经磷酸酶(OPTN)基因E50K的一个点突变有关。通过体外研究，OPTN功能涉及两个不同的细胞过程：核因子？B信号和囊泡运输。然而，目前尚不清楚野生型OPTN或E50K突变是否会在体内改变这些途径，特别是在眼睛内。此外，当OPTN突变时，哪些细胞类型负责产生青光眼相关病理仍是一个谜。因此，这项提案将对这些细胞过程进行广泛的调查，以确定OPTN或E50K是否可以改变它们在视网膜细胞类型中的功能。这将通过产生表达野生型OPTN或E50K变体(在斑马鱼中为E32K)的斑马鱼转基因品系来实现。首先，OPTN或E32K导致青光眼相关病理的细胞独立性将通过比较这些细胞普遍表达时的表型和在视网膜神经节细胞或胶质细胞中表达的表型来检验，这两种细胞类型与青光眼的发病机制高度相关。其次，将测试这些蛋白质抑制脊椎动物眼睛中的核因子-B信号的能力，以及这种抑制可能如何导致青光眼的病理。第三，将检查OPTN或E32K是否有能力改变谷氨酸受体的囊泡运输，以改变视网膜神经节细胞的钙动力学，可能导致兴奋性毒性。这项工作应该有助于了解OPTN在这些细胞类型中影响的细胞过程。这项研究还将确定E50K突变可能导致青光眼相关病理的机制。

DESCRIPTION (provided by applicant): Glaucoma is one of the leading causes of blindness throughout the world, but the cellular and molecular mechanisms are mostly unknown. Insights into this disease have come from genetic studies in animal models and humans. One point mutation in the gene optineurin (optn), E50K, has been implicated in causing disease in as many as 13.5% of primary open angle glaucoma cases for one population of glaucoma patients. Through in vitro studies, optn function has been implicated in two diverse cellular processes: NF-?B signaling and vesicle trafficking. However, it is not known whether wild-type optn or the E50K mutation alters these pathways in vivo and specifically within the eye. Furthermore, which cell types are responsible for generating glaucoma-associated pathology when optn is mutated remains a mystery. Therefore, this proposal will conduct a broad survey of these cellular processes to determine whether optn or E50K can alter their function in retinal cell types. This will be accomplished by generating zebrafish transgenic lines that express wild-type optn or the E50K variant (E32K in zebrafish). First, the cell autonomy of optn or E32K to cause glaucoma- associated pathology will be examined by comparing the phenotype when these cells are expressed ubiquitously versus expressed within retinal ganglion cells or glia, two cell types that are highly relevant to glaucoma pathogenesis. Second, the ability of these proteins to inhibit NF-?B signaling in the vertebrate eye will be tested, as well as how this inhibition may be contributing to glaucoma pathologies. Third, optn or E32K will be examined for their ability to alter vesicle trafficking of glutamate receptors to alter calcium dynamics in retinal ganglion cells, possibly leading to excitotoxicity. This work should provide an understanding of the cellular processes that optn affects in those cell types. This research will also determine potential mechanisms by which the E50K mutation can cause glaucoma-associated pathology.