ANALYSIS OF THE ROLE OF 3 AND 4 REPEAT TAU IN VITRO AND IN VIVO

我们最近在17号染色体（FTDP-17）上发现了与额颞叶痴呆和帕金森病相关的tau外显子10的5'剪接位点突变，首次证明了tau含有4和3微管结合重复的比例对成人大脑中tau的正确功能至关重要。剪接位点突变（+3,+13，+14和+16）都破坏了茎环结构的稳定性，该结构可能通过抑制U1 snRNP结合和外显子定义参与调节外显子1-选择性剪接。突变被认为通过破坏茎环导致U1 snRNP结合增加，这直接导致外显子10剪接增加和4重复Tau增加。我们首先证明了突变增加了外显子10的剪接并增加了4个重复Tau。通过对FTDP-17脑的RT-PCR分析和体外剪接试验，我们首次证明了突变增加了tau外显子10的剪接。此外，通过体外剪接实验，我们已经证明，确实是FTDP-17突变破坏了外显子10的5'剪接位点的茎环结构，在调节该外显子的选择性剪接中起主要作用。在FTDP-17中，将4重复Tau比例增加两倍的突变是致病性的，这一事实表明，具有3和4重复的Tau亚型的比例对Tau的正确功能至关重要。在其他物种中，4重复Tau蛋白与3重复Tau蛋白的比例差异很大，此外，一段时间以来人们已经知道，在胎儿大脑中只观察到3重复Tau蛋白，直到出生后一段时间才产生4重复亚型。因此，4次重复与3次重复的Tau比率似乎几乎肯定反映了不同物种和发育过程中神经元作用的一些根本差异。这个项目旨在增加我们对4比3重复Tau比例调节的理解，为什么这个比例对神经元的正确功能很重要，为什么这个比例的破坏会导致神经变性。

The recent identification by our group of mutations in the 5' splice site of tau exon 10 that are associated with Fronto-temporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) demonstrated for the first time that the ratio of Tau containing 4 and 3 microtubule binding repeat is crucial to the correct functioning of Tau in the adult Human brain. The splice site mutations (+3, +13, +14 and +16) all destabilize a stem-loop structure that is likely involved in regulation of exon 1- alternative splicing by inhibiting U1 snRNP binding and exon definition. The mutations are thought to cause increased U1 snRNP binding, through disruption of the stem-loop, which directly leads to increased splicing of exon 10 and increased 4 repeat Tau. We were the first to demonstrate that the mutations increasing splicing of exon 10 and increased 4 repeat Tau. We were the first demonstrate that the mutations increased splicing of tau exon 10 by RT-PCR analysis of FTDP-17 brains and also through the use of an in vitro splicing assay. Further we have since demonstrated, using the in vitro splicing assay, that it is indeed the stem-loop structure in the 5' splice site of exon 10 that is disrupted by the FTDP-17 mutations that which plays a major role in the regulation of alternative splicing of this exon. The fact that mutations that increase the proportion of 4 repeat Tau by as little as two fold are pathogenic in FTDP-17 indicates the importance of the ratio of Tau isoforms with 3 and 4 repeats to the correct functioning of Tau. In other species the ratio of 4 repeat to 3 repeat Tau can vary widely and in addition it has been known for some time that in fetal brain only 3 repeat Tau is observed with the generation of 4 repeat isoforms not occurring until some time after birth. Thus the 4 repeat to 3 repeat Tau ratio seems virtually certain to reflect if not to underlie, some fundamental differences in the role of neurons in different species and during development. This project is designed to increase our understanding of the regulation of the ratio of 4 to 3 repeat Tau, why this ratio is important in the correct functioning of neurons and why disruption of this ratio can result in neurodegeneration.