Genetic Studies of Optic Atrophy

描述（由申请人提供）：视神经萎缩基因1（OPA 1）是编码线粒体蛋白的核基因。OPA 1基因突变是常染色体显性视神经萎缩（DOA）最常见的病因。这种情况主要影响眼睛，其特征是逐渐视力丧失，色觉缺陷和颞部视觉苍白。先前的研究表明，OPA 1是普遍表达的，并作为细胞色素C释放的看门人，在线粒体融合和ATP产生中很重要。为了了解OPA 1突变导致视神经萎缩的分子机制，并促进有效治疗视神经萎缩的药物的开发，最近我们在果蝇中建立了Opa 1敲除模型。通过分析发育中和成年果蝇眼睛的表型，我们发现由P元件插入引起的dOpa 1（人类OPA 1的直系同源物）的杂合突变在显微镜下没有可辨别的眼睛表型，而是与寿命缩短相关，而纯合突变导致胚胎致死。利用强大的果蝇遗传技术，我们创造了眼睛特异性体细胞突变体。dOpa 1在眼睛中的体细胞纯合突变引起成年果蝇的粗糙（图案错误）和有光泽（减少透镜沉积）的眼睛表型，并且通过精确切除插入的P-元件是可逆的。此外，我们还表明，超氧化物歧化酶1（SOD 1），维生素E，和基因过表达的人SOD 1（hSOD 1）能够逆转dOPA 1突变体大克隆的光泽眼表型，进一步表明ROS在视锥细胞和色素细胞死亡中发挥重要作用。我们的初步结果还表明，一些OPA 1突变会导致视力、听力和其他器官异常。听力损失与非同步耳蜗传导有关，人工耳蜗植入物提供了显着的效果，以恢复听力。我们的中心假设是OPA 1的突变会对多个器官产生影响。在这项研究中，我们将对OPA 1进行突变分析，并在必要时招募更多的视神经萎缩患者。对于已知OPA 1突变的患者，我们将进行临床评估，以扩大临床表型，包括听力测试，耳蜗电位分析和运动感觉异常（目标1）。在目标2中，我们将进一步表征表型，并确定dOpa 1突变导致眼睛粗糙的分子机制。我们将分析眼睛表型中细胞凋亡的突变体，并测试半胱天冬酶抑制剂逆转粗糙眼表型的有效性。在目标3中，我们将测试OPA 1突变影响寿命的分子机制。我们的初步研究结果表明，dOpa 1突变导致ROS产生增加和对胁迫的耐受性差。我们将检查超氧化物歧化酶（SOD）的活性和线粒体呼吸速率在我们的dOpa 1苍蝇。最后，我们将测试抗氧化剂是否可以挽救缩短的寿命表型。我们预计，这项研究将提供新的见解视神经萎缩的发病机制，并协助开发新的治疗方法，以防止视力下降。通过平行研究动物模型和人类受试者，加强了拟议研究的临床相关性。许多退行性视网膜疾病具有相似的临床特征，因此，本研究的数据可以扩展到这些疾病。公共卫生相关性：视神经萎缩基因1（OPA 1）对线粒体（我们体内细胞的发电厂）中发现的蛋白质进行编程。例如，我们发现OPA 1的突变会导致视神经萎缩和听力损失。我们还惊讶地发现Opa 1突变会影响果蝇的寿命。在这项研究中，我们将对已知OPA 1突变的患者进行临床评估，以进一步扩大我们对该基因对其他器官影响的理解。我们将仔细观察在果蝇模型中视神经萎缩是如何引起的。此外，我们还可以利用该模型筛选治疗视神经萎缩的药物。通过了解Opa 1如何影响寿命，我们可以开发延长寿命的药物。这项研究将使我们能够了解OPA 1突变如何导致视神经萎缩，并探索其在生命周期中的新功能，并为我们开发该疾病的治疗方法提供潜在的机会。

DESCRIPTION (provided by applicant): Optic atrophy gene 1 (OPA1) is a nuclear gene encoding a mitochondrial protein. Mutation of OPA1 is the most common cause for autosomal dominant optic atrophy (DOA). This condition primarily affects eyes, and is characterized by gradual vision loss, color vision defects, and temporal optic pallor. Previous studies have shown that OPA1 is ubiquitously expressed and serves as a gatekeeper for cytochrome-c release, and is important in mitochondrial fusion and ATP production. To understand the molecular mechanism by which OPA1 mutations cause optic atrophy and to facilitate the development of an effective therapeutic agent for optic atrophies, recently we have generated an Opa1 knockout model in Drosophila. By analyzing phenotypes in the developing and adult Drosophila eyes, we found that the heterozygous mutation of dOpa1, ortholog of human OPA1, caused by a P-element insertion results in no discernable eye phenotype under a microscope, but is instead associated with a shortened lifespan, whereas the homozygous mutation results in embryonic lethality. Taking advantage of the powerful Drosophila genetic techniques, we created eye-specific somatic mutants. The somatic homozygous mutation of dOpa1 in the eyes caused rough (mispatterning) and glossy (decreased lens deposition) eye phenotypes in adult flies, and was reversible by precise excision of the inserted P-element. Moreover, we also show that Superoxide dismutase 1 (SOD1), Vitamin E, and genetically overexpressed human SOD1 (hSOD1) is able to reverse the glossy eye phenotype of dOPA1 mutant large clones, further suggesting that ROS play an important role in cone and pigment cell death. Our preliminary results also show that some OPA1 mutations cause loss of vision, hearing, and other organ abnormalities. The hearing loss is associated with asynchronous cochlear conduction and cochlear implants provide remarkable results to restore hearing. Our central hypothesis is that mutations in OPA1 cause effects in multiple organs. In this study, we will perform mutation analysis on OPA1 with a readily available large patient population and recruit additional patients with optic atrophy if necessary. For patients with known OPA1 mutations, we will perform clinical evaluations to expand the clinical phenotypes, including hearing test, cochlear potential analysis and motor sensory abnormalities (Aim 1). In Aim 2, we will further characterize phenotypes and identify the molecular mechanism by which dOpa1 mutations cause rough eyes. We will analyze the mutants for apoptosis in eye phenotypes and test the effectiveness of caspase inhibitor in reversing rough eye phenotypes. In Aim 3, we will test the molecular mechanisms by which mutation of OPA1 affects the lifespan. Our preliminary results showed that mutation of dOpa1 causes increased production of ROS and poor tolerance to stress. We will examine superoxide dismutase (SOD) activity and mitochondrial respiration rate in our dOpa1 flies. Finally, we will test if antioxidants can rescue the shortened lifespan phenotype. We anticipate that this study will provide novel insights into the pathogenesis of optic atrophy and assist in the development of novel therapies to prevent vision loss. The clinical relevance of the proposed research is strengthened by studying an animal model and human subjects in parallel. Many degenerative retinal diseases share similar clinical features and therefore, the data from this study may be extended to those diseases. PUBLIC HEALTH RELEVANCE: Optic atrophy gene 1 (OPA1) programs proteins found in mitochondria, the power plant of cells in our body. For example we have found that mutations of OPA1 cause optic atrophy as well as hearing loss. We were also surprised to find that Opa1 mutation affects the life span in fly. In this study, we will clinically evaluate patients with known mutation of OPA1 to further expand our understanding of the effects of this gene on other organs. We will closely discern how optic atrophy is caused in fly model. In addition, we can also use this model to screen drug for treatments of optic atrophy. By understanding how Opa1 affect the life span, we may develop a drug to prolong the life span. This study will allow us to understand how mutations of OPA1 cause optic atrophy and explore its novel function in lifespan and provide us a potential opportunity to develop treatments for the disease.