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议，该模型最终将能够比目前可能的更快速和有效地评估疾病的化学修饰剂。我们正在使用亨廷顿蛋白（Htt）进行概念验证实验，当它包含一条细长的谷氨酰胺时，就会导致亨廷顿病。这项工作的意义是很高的，因为如果我们成功的话，我们将表明有可能分离CNS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。这可以帮助桥接神经变性的细胞研究，可以快速生成大量数据，整个动物研究既昂贵又耗时。视网膜模型允许对每只眼睛的功能进行独立测量，从而实现动物内部的控制，极大地提高了统计能力。在初步工作中，我们已经表明，有可能在HD的R6/2小鼠模型中检测到视网膜变性。我们已经进一步证明，这与视网膜生理学测试（视网膜电图）中的功能进行性丧失有关。我们已经证明，使用脂质体介导的药物递送可以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作用。我们还开发了一种行为测定，使我们能够独立地测量不受限制的小鼠的右眼与左眼的视力。最后，我们已经证明，在单次注射修饰的腺相关病毒后，可以使大量的视网膜神经元凋亡。这项工作使我们处于一个很好的位置，以测试我们是否可以建立一个视网膜变性模型的基础上表达的各种Htt蛋白与突变的广泛兴趣的HD领域。目的1：表征视网膜内Htt蛋白的病毒转导。我们将使用AAV 2表达各种形式的Htt，并确定它们随时间对视网膜神经元的影响。目的2：使用ERG测量对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蛋白对视网膜生理学的影响。目标3.使用光学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。这项工作是基于使用重组腺相关病毒，显微注射这种病毒到眼内的野生型小鼠的空间，并确定病毒介导的Htt基因表达的后果在眼睛内。我们还将使用最先进的技术来监测视网膜病理学，生理学和功能。如果成功的话，这项工作将为更快速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.