Analysis of pathways and cells mediating optineurin glaucoma pathology

青光眼是世界范围内致盲的主要原因之一，但其细胞和分子机制尚不清楚。对这种疾病的深入了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，视神经蛋白（optn）基因E50K的一个点突变与多达13.5%的原发性开角型青光眼病例有关。通过体外研究，optn的功能涉及两种不同的细胞过程：NF-？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然是一个谜。因此，本提案将对这些细胞过程进行广泛的调查，以确定optn或E50K是否可以改变其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（斑马鱼中的E32K）的斑马鱼转基因系来实现。首先，通过比较optn或E32K细胞普遍表达与在视网膜神经节细胞或神经胶质细胞中表达时的表型，研究optn或E32K细胞引起青光眼相关病理的细胞自主性。这两种细胞类型与青光眼发病密切相关。其次，这些蛋白质抑制NF-？脊椎动物眼睛中的B信号将被测试，以及这种抑制如何可能导致青光眼病理。第三，我们将研究optn或E32K是否能够改变谷氨酸受体的囊泡运输，从而改变视网膜神经节细胞中的钙动力学，从而可能导致兴奋性毒性。这项工作应该提供对optn在这些细胞类型中影响的细胞过程的理解。这项研究还将确定E50K突变导致青光眼相关病理的潜在机制。

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.