STUDY ON NEURODEGENERATION USING TDP-43 TRANSGENIC RATS

 描述(申请人提供)：在已知的肌萎缩侧索硬化症基因中，TDP-43特别令人感兴趣，因为它的蛋白在散发性肌萎缩侧索硬化症中高度磷酸化和泛素化。与基因突变相比，翻译后修饰在患者身上很难检测到，在动物模型中也很难复制。因此，了解TDP-43致病机制的关键一步是揭示突变型TDP-43导致ALS神经变性的途径。TDP-43与许多RNA和蛋白质结合，但致病突变如何影响TDP-43功能尚未确定，特别是在适当的动物模型中。出乎意料的是，WT和突变体TDP-43在啮齿类动物中的过度表达会导致无法区分的表型，表明过量的TDP-43具有神经毒性。由于转基因表达受位置效应的影响很大，转基因表达的模式和水平在不同的线路上有很大的差异。因此，使用现有的转基因模型无法确定突变的TDP-43比其WT形式的毒性更大还是更小。关于致病突变对TDP-43功能的影响的问题进一步混淆了观察到的TDP-43缺失导致TDP-43基因敲除和敲除小鼠的神经变性。由于TDP-43与其自身mRNA的3‘-非翻译区结合，从而调节自身的表达，因此，cDNA敲入(KI)方法破坏了TDP-43的自我调节机制，使TDP-43KI动物变得不太有用。即使在ALS中发现TDP-43突变7年后，突变的TDP-43是否通过功能的获得或丧失而导致ALS仍然是不确定的，这一悬而未决的问题是解开TDP-43疾病机制的障碍。为了解决这个关键问题，我们通过在大鼠基因组中引入一个与疾病相关的点突变来创造KI大鼠。通过前所未有的大鼠模型，我们将系统地确定致病突变如何影响TDP-43的功能。

 DESCRIPTION (provided by applicant): Among known ALS genes, TDP-43 is of particular interest because its protein is hyper-phosphorylated and ubiquitinated in sporadic ALS. Compared with genetic mutation, post-translational modification is difficult to detect in patients and hard to reproduce in animal models. Therefore, a critical step toward understanding TDP-43 pathogenesis is revealing the pathways by which mutant TDP-43 causes neurodegeneration in ALS. TDP-43 binds to many RNAs and proteins, but how pathogenic mutation impacts TDP-43 function has not yet been determined, particularly in an appropriate animal model. Unexpectedly, overexpression of both WT and mutant TDP-43 in rodents causes indistinguishable phenotypes, indicating that excessive TDP-43 is neurotoxic. Because transgene expression is heavily influenced by position effect, the patterns and levels of transgene expression vary greatly from line to line. Whether mutant TDP-43 is more or less toxic than its WT form thus cannot be determined using existing transgenic models. The question regarding the impact of pathogenic mutation on TDP-43 function is further confounded by observations that TDP-43 depletion induces neurodegeneration in TDP-43 knock-out and knock-down mice. As TDP-43 binds to the 3'-untranslated region of its own mRNA and thereby regulates its own expression, the cDNA knock-in (KI) approach destroys TDP-43 self-regulatory machinery, rendering TDP-43 KI animals not very useful. Even 7 years after the discovery of TDP-43 mutation in ALS, whether mutant TDP-43 causes ALS through a gain or loss of function is still uncertain, and this unresolved issue is a roadblock to unraveling TDP-43 disease mechanisms. To resolve this critical issue, we created KI rats by introducing a single disease-linked point mutation into the rat genome. With the unprecedented rat models, we will determine how pathogenic mutation impacts TDP-43 function in a systematic manner.