TBK1-Related Glaucoma

 描述(申请人提供)：青光眼是一种常见的视神经疾病，影响着全球6000多万人，是美国失明和视力残疾的主要原因。然而，导致青光眼的生物学途径还不是很清楚，这阻碍了对这种疾病的早期发现和治疗。 因此，从分子水平上阐明青光眼的病因是非常必要的。我们发现水箱结合激酶1(TBK1)基因的重复与青光眼的发生有关，这种青光眼没有眼压升高--称为正常眼压性青光眼(NTG)。TBK1在眼睛中的功能还没有被研究过，尽管已经证明TBK1在非眼组织的自噬中起到了作用。自噬是一种细胞过程，它将胞浆蛋白、细胞器甚至细胞内病原体运送到溶酶体进行清除。值得注意的是，三个已知的NTG基因(TBK1、OPTN、TLR4)都相互作用，并在激活自噬中发挥重要作用，这表明自噬可能是NTG发病过程中的一条重要的共同途径。此外，在青光眼动物模型中，自噬也与视网膜神经节细胞死亡和视神经损伤有关。基于这些数据，我们推测，TBK1基因复制改变了自噬，导致了NTG的视网膜神经节细胞死亡和视力丧失。我们创建了一套独特的资源来研究TBK1、自噬和青光眼：培养的视网膜神经节细胞(使用诱导干细胞技术)具有额外剂量的TBK1基因-与我们的TBK1相关青光眼患者相同的缺陷。对这些细胞的初步研究提供了更多证据表明，TBK1基因的复制激活了自噬并导致青光眼。为了验证我们的假设并研究自噬在青光眼中的作用，我们提出了这个由两部分组成的目标：使用IPSC衍生的RGC样神经元来确定TBK1基因复制和自噬在NTG发病机制中的作用·目标1A：通过显微镜、生化和自噬通量分析，测试因TBK1基因复制而导致NTG患者的NTG细胞(皮肤成纤维细胞、iPSC和iPSC衍生的RGC样神经元)自噬分子的变化。目的1B：用免疫沉淀法和免疫印迹法检测TBK1基因复制对其底物OPTN在自噬中所必需的关键残基的磷酸化的影响。通过这些实验，我们将开始描述TBK1基因缺陷改变自噬并导致青光眼的生物学途径。这些研究还将为开发新的青光眼疗法提供基础，这些疗法针对自噬和不升高眼压的青光眼。

 DESCRIPTION (provided by applicant): Glaucoma is a common disease of the optic nerve that affects over 60 million people worldwide and is a leading cause of blindness and visual disability in the United States. However, the biological pathways that lead to glaucoma are not well understood, and this has hindered efforts for early detection and treatment of this condition. Consequently, there is great need to clarify the causes of glaucoma at the molecular level. We discovered that duplication of the TANK binding kinase 1 (TBK1) gene is associated with glaucoma that occurs without elevation of eye pressure - termed normal tension glaucoma (NTG). TBK1 function hasn't been studied in the eye although TBK1 has been shown to have a role in autophagy in non-ocular tissue. Autophagy is a cellular process that delivers cytosolic proteins, organelles, and even intracellular pathogens to lysosomes for clearance. Remarkably, three known NTG genes (TBK1, OPTN, TLR4) all interact with each other and have important roles in activating autophagy, which suggests that autophagy may be an important common pathway in NTG pathogenesis. Moreover, autophagy has also been implicated in the retinal ganglion cell death and optic nerve damage in animal models of glaucoma. Based on these data, we hypothesize that TBK1 gene duplication alters autophagy and leads to the retinal ganglion cell death and vision loss of NTG. We have created a unique set of resources to study TBK1, autophagy, and glaucoma: cultured retinal ganglion cells (using induced stem cell technology) that have an extra dose of the TBK1 gene - the same defect as our patients with TBK1-associated glaucoma. Preliminary studies of these cells have provided more evidence that duplication of TBK1 gene activates autophagy and causes glaucoma. To test our hypothesis and investigate the role of autophagy in glaucoma we propose this two-part aim: DETERMINE THE ROLE OF TBK1 GENE DUPLICATION AND AUTOPHAGY IN THE PATHOGENESIS OF NTG USING IPSC-DERIVED RGC-LIKE NEURONS • Aim 1A: Test cells cultured from patients with NTG due to a TBK1 gene duplication (skin fibroblasts, iPSCs and iPSC-derived RGC-like neurons) for altered autophagy molecules with microscopic, biochemical, and autophagic flux assays. • Aim 1B: Determine the effect of TBK1 gene duplication on the phosphorylation of its substrate OPTN at key residues necessary for its function in autophagy with immunoprecipitation and western blot analysis with anti-phospho-OPTN antibodies. With these experiments, we will begin to characterize the biological pathway by which defects in the TBK1 gene alter autophagy and lead to glaucoma. These studies will also provide the basis for development of novel glaucoma therapies that are targeted to autophagy and glaucoma that occurs without elevation of eye pressure.