Cellular Processing of Optineurin, the Product of a Glaucoma Gene

青光眼基因产物 Optineurin 的细胞加工

• 批准号: 7759139
• 负责人: BEATRICE Y.J.T. YUE
• 金额: $ 34.97万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7759139
• 关键词: Adolescent; Adult; Affect; Alzheimer' s Disease; Animals; Autophagocytosis; Binding; Biology; Cell Death; Cell Line; Cell physiology; Cells; Dependovirus; Disease; Doxycycline; Eukaryotic Cell; Eye; Fluorescence; Genes; Glaucoma; Hypoxia; Impairment; In Vitro; Injection of therapeutic agent; Label; Laboratories; Lead; Link; Lysosomes; Modality; Modeling; Molecular Chaperones; Monitor; Mutate; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Open-Angle Glaucoma; Oxidative Stress; Parkinson Disease; Pathway interactions; Physiologic pulse; Play; Process; Proteins; Rattus; Research; Retinal Ganglion Cells; Role; Route; Sirolimus; System; Techniques; Testing; Transfection; Ubiquitin; Ubiquitination; Virus; design; in vivo; inhibitor/antagonist; insight; multicatalytic endopeptidase complex; mutant; myocilin; novel therapeutics; overexpression; public health relevance; research study

DESCRIPTION (provided by applicant): Glaucoma is a major blinding disease. We propose a new study on optineurin, a gene identified in 2002 that is associated principally with low tension or normal tension glaucoma. In particular, we will examine the cellular processing of optineurin in neuronal cells. Proper processing of cellular proteins is of vital importance. In eukaryotic cells, the ubiquitin-proteasome system (UPS) and autophagy pathway are two major routes for protein clearance. Optineurin protein has ubiquitin binding domain. Preliminary studies performed also suggest that the endogenous optineurin is ubiquitinated and processed through UPS in retinal ganglion RGC5 cells. We have also noted that overexpressed wild type or mutant optineurin may be associated with autophagy and cell death. The central hypotheses are: i) in normal homeostatic situation, the turnover of endogenous optineurin involves UPS; ii) when optineurin is overexpressed or mutated, proteasome activity is impaired and autophagy comes into play, similar to that observed in neurodegenerative Alzheimer's and Parkinson's diseases; and iii) overexpressed and mutated optineurin leads to retinal ganglion cell (RGC) death. Three specific aims are proposed. Both in vitro culture and in vivo animal systems will be used. In Specific Aim 1, the turnover of endogenous optineurin in RGC5 cells and its ubiquitination will be studied. Specific proteasome and autophagy inhibitors/stimulator will be used in experiments including pulse chase to determine whether the degradation rate of optineurin would be affected. Molecular chaperones associated with endogenous optineurin will be identified. In Specific Aim 2, the involvement of UPS and autophagy in RGC5 cells after transfection to overexpress wild type and E50K optineurin and in inducible PC12 or RGC5 cell lines will be investigated. Experiments will be designed to determine whether proteasome activity is impaired with optineurin overexpression or mutation, whether autophagy is induced, and whether cell death is a consequence. The rescuing studies will be performed in Specific Aim 3. We will also deliver optineurin gene using adeno-associated viruses into the retinal ganglion cells of rat eyes to examine whether impairment of UPS, induction of autophagy and cell death take place in vivo, whether the overexpressed optineurin can be efficiently cleared, and whether cell death is reduced by rescuing treatments. We believe that the results obtained through the proposed studies will be valuable, providing not only basic information regarding biology of optineurin but also suggesting a neurodegenerative disease paradigm for glaucoma. Moreover, the rescuing efforts will potentially have high translational significance. PUBLIC HEALTH RELEVANCE Glaucoma is a major blinding disease. We propose a new study on optineurin, a gene associated with normal tension glaucoma. In particular, we will examine the processing of optineurin protein in neuronal cells. Results obtained from the proposed studies will provide insights into the basic biology of optineurin and help illustrate how glaucoma is developed. In addition, the new information may lead to novel therapeutic modalities.

描述（由申请人提供）：青光眼是一种主要的盲目疾病。我们提出了一项关于Optineurin的新研究，Opineurin，该研究在2002年鉴定出，该基因主要与低张力或正常张力青光眼有关。特别是，我们将检查神经元细胞中Optineurin的细胞加工。正确处理细胞蛋白至关重要。在真核细胞中，泛素 - 蛋白酶体系统（UPS）和自噬途径是蛋白质清除率的两种主要途径。 Optineurin蛋白具有泛素结合结构域。进行的初步研究还表明，内源性Optineurin是泛素化的，并通过视网膜神经节RGC5细胞中的UPS进行处理。我们还指出，过表达的野生型或突变型果蛋白可能与自噬和细胞死亡有关。中心假设是：i）在正常的稳态情况下，内源性openineurin的营业额涉及UPS； ii）当Optineurin过表达或突变时，蛋白酶体的活性受损并自噬起作用，类似于在神经退行性阿尔茨海默氏症和帕金森氏病中观察到的蛋白酶体。和iii）过表达和突变的Opineurin蛋白会导致视网膜神经节细胞（RGC）死亡。提出了三个具体目标。都将使用体外培养和体内动物系统。在特定的目标1中，将研究RGC5细胞中内源性Optineurin及其泛素化的营业额。特定的蛋白酶体和自噬抑制剂/刺激剂将用于包括脉冲追逐在内的实验，以确定果蛋白的降解速率是否会受到影响。将鉴定与内源性果蛋白有关的分子伴侣。在特定的目标2中，将研究转染过表达野生型和E50K Optineurin以及诱导型PC12或RGC5细胞系后，UPS和自噬在RGC5细胞中的参与。将设计实验以确定蛋白酶体的活性是否因果蝇过表达还是突变，是否诱导自噬以及细胞死亡是否是结果。救援研究将在特定的目标3中进行。我们还将使用与腺相关病毒相关的病毒传递Optineurin基因到大鼠眼睛的视网膜神经节细胞中，以检查UPS损伤，自噬和细胞死亡的损害是否在体内发生是否会有效地清除，以及是否有效地清除了细胞死亡，并且是否会减少过表达的Optineurin。我们认为，通过拟议的研究获得的结果将是有价值的，不仅提供有关Opineurin生物学的基本信息，而且还提出了针对青光眼的神经退行性疾病范式。此外，救援工作可能具有很高的翻译意义。公共卫生相关性青光眼是一种主要的盲目疾病。我们提出了一项关于与正常张力青光眼相关的Opineurin的新研究。特别是，我们将检查神经元细胞中果毒蛋白蛋白的加工。从拟议的研究中获得的结果将提供对Optineurin的基本生物学的见解，并有助于说明如何发展青光眼。此外，新信息可能导致新的治疗方式。