DEVELOPING A MOUSE RETINAL MODEL OF NEURODEGENERATIVE DISEASE

描述（由申请人提供）：这是一项开发神经变性视网膜模型的提案，该模型最终将比目前可能的更快、更有效地评估疾病的化学调节剂。我们正在使用亨廷顿蛋白（Htt）进行概念验证实验，当它含有一个细长的谷氨酰胺束时，会导致亨廷顿病。这项工作的意义是很高的，因为如果我们成功了，我们将表明有可能分离出中枢神经系统的一个离散部分，用于化学和遗传学研究，使用解剖学，生理学和功能。这有助于将神经退行性疾病的细胞研究与既昂贵又耗时的全动物研究联系起来，前者可以快速生成大量数据。视网膜模型允许独立测量每只眼睛的功能，使动物内部控制大大提高了统计能力。在前期工作中，我们已经证明在R6/2 HD小鼠模型中检测视网膜变性是可能的。我们在视网膜生理学（视网膜电图）测试中进一步证明，这与功能的进行性丧失有关。我们已经证明，可以使用脂质体介导的药物递送递送微量的疾病修饰化合物，并观察到对视网膜生理学的有益影响。我们还开发了一种行为试验，可以让我们独立地测量一只不受约束的老鼠的右眼和左眼的视力。最后，我们已经证明，在单次注射修饰的腺相关病毒后，有可能转导大量视网膜神经元。这项工作使我们处于一个很好的位置，可以测试我们是否可以建立一个基于各种Htt蛋白表达的视网膜变性模型，这些蛋白具有对HD领域广泛感兴趣的突变。目的1：表征视网膜内Htt蛋白的病毒转导。我们将使用AAV2来表达各种形式的Htt，并确定它们对视网膜神经元的影响。目的2：利用ERG测量视网膜生理学的影响并比较突变体。我们将使用视网膜电图来测量各种Htt蛋白对视网膜生理的影响。目标3。使用验光测量对视网膜神经元功能的影响，并比较突变体。我们将使用专门的行为测定来测量各种Htt蛋白对视觉功能的影响。这项工作的基础是使用重组腺相关病毒，将这种病毒微注射到野生型小鼠的眼内，并确定病毒介导的Htt基因在眼内表达的后果。我们还将使用最先进的技术来监测视网膜病理、生理和功能。如果成功，这项工作将为更快地在体内分析神经变性的化学和遗传修饰剂奠定基础。这可能对人类健康产生巨大影响，因为神经退行性疾病是美国面临的最大健康问题之一。

DESCRIPTION (provided by applicant): This is a proposal to develop a retinal model of neurodegeneration that will ultimately enable much more rapid and efficient evaluation of chemical modifiers of disease than is currently possible. We are carrying out proof-of-concept experiments using the huntingtin (Htt) protein, which causes Huntington disease when it contains an elongated tract of glutamines. The significance of this work is high, because if we are successful we will show that it is possible to isolate a discrete portion of the CNS for chemical and genetic studies, using anatomy, physiology, and function. This could help bridge cellular studies of neurodegeneration, which can rapidly generate much data, with whole animal studies, which are expensive and time consuming. The retinal model allows independent measurement of function in each eye, enabling internal controls within an animal that vastly improve statistical power. In preliminary work, we have shown that it is possible to detect retinal degeneration in the R6/2 mouse model of HD. We have further demonstrated that this is associated with progressive loss of function in tests of retinal physiology (electroretinography). We have shown that it is possible to deliver a disease-modifying compound in tiny amounts using liposome-mediated drug delivery, and to observe a beneficial effect on retinal physiology. We have also developed a behavior assay that lets us independently measure vision in the right vs. left eye of an unrestrained mouse. Finally, we have shown that it is possible to transduce large numbers of retinal neurons after a single injection of a modified adeno-associated virus. This work puts us in an excellent position to test whether we can build a model of retinal degeneration based on expression of various Htt proteins with mutations of broad interest to the HD field. Aim 1: Characterize viral transduction of Htt proteins within the retina. We will use AAV2 to express various forms of Htt and determine their effects on retinal neurons over time. Aim 2: Use ERG to measure effects on retinal physiology and compare mutants. We will use electroretinography to measure effects of various Htt proteins on retinal physiology. Aim 3. Use optomotry to measure effects on retinal neuron function and compare mutants. We will use a specialized behavior assay to measure the effects of various Htt proteins on visual function. The work is based on the use of recombinant adeno-associated virus, micro-injection of this virus into the intraocular space of wild-type mice, and the determination of the consequences of virus-mediated Htt gene expression within the eye. We will also use state-of-the-art techniques to monitor retinal pathology, physiology, and function. If successful, this work will set the stage for much more rapid in vivo analyses of chemical and genetic modifiers of neurodegeneration. This could have an enormous impact on human health, since neurodegenerative disease is one of the single biggest health problems faced by the United States. PUBLIC HEALTH RELEVANCE: It is very difficult to translate cellular studies of neurodegenerative disease into animal models. Cell models are very useful for identifying early drug leads that could be translated into patient use, as well as for the identification of genes that modify the neurodegenerative disease process. However, it is very difficult, time consuming, and expensive to evaluate experimental compounds in whole animals, and to test genetic modifiers. Evaluation of small molecules is difficult because it requires large amounts of compound, and any early compound must be safe, and easily administered, with good bioavailability. Evaluation of genetic modifiers is difficult because this requires creating hybrid mice that express multiple genes of interest. This is a proposal to develop a mouse retinal model of neurodegenerative disease that could be useful for evaluating experimental compounds and modifier genes. It is based on the idea that experimental compounds can be safely administered locally to each individual eye via injection, and the genetic modifications can be delivered by a single injection of a specialized virus. If we are successful, we will show that it is possible to direct expression of a disease-associated gene to the eye, and then use highly quantitative measures of visual function to determine the relative toxicity of various mutants. We will be studying huntingtin, the protein that causes Huntington disease, a devastating neurodegenerative condition. In Aim 1 we will create specialized viruses that allow us to efficiently express various forms of the huntingtin protein within the retina. We will determine that we are getting good expression, and will monitor animals over long periods to determine any effects of gene expression on the integrity of retinal neurons. In Aim 2 we will use a technique called electroretinography to measure the effects of the various forms of the huntingtin protein on the physiology of neurons in the mouse retina. We will correlate any changes with pathology observed in Aim 1. We will independently measure the physiologic changes in the left vs. right eyes of the animals. This will enable much more effective studies of relative effects of the various huntingtin mutations. In Aim 3 we will use a state-of-the-art behavior technique to measure the effects of huntingtin protein expression on visual acuity in the injected mice. This allows us to independently measure the function of the left vs. right retina, which enables much more effective studies of the various huntingtin mutations. Relevance to public health Neurodegenerative diseases represent an enormous cost to society, and afflict millions of people. There are no effective therapies, and the methods to develop new treatments and to study these diseases are relatively slow and cumbersome. If successful, this work will develop a new method to study neurodegenerative disease that could vastly improve our ability to develop new drugs and to study the basic mechanisms of disease. This will help speed the development of more effective treatments.