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

最近，我们的研究小组在tau外显子10的5'剪接位点中鉴定出与17号染色体（FTDP-17）相关的额颞叶痴呆和帕金森综合征相关的突变，首次证明了含有4个和3个微管结合重复的Tau的比例对成年人脑中Tau的正确功能至关重要。 剪接位点突变（+3、+13、+14和+16）均使茎环结构不稳定，该结构可能通过抑制U1 snRNP结合和外显子定义参与外显子1-选择性剪接的调控。 这些突变被认为通过茎环的破坏引起U1 snRNP结合增加，这直接导致外显子10的剪接增加和4个重复Tau的增加。我们是第一个证明突变增加外显子10的剪接和增加4个重复的Tau。我们首次通过FTDP-17脑的RT-PCR分析和体外剪接测定证明突变增加了tau外显子10的剪接。 此外，我们已经证明，使用体外剪接测定法，确实是外显子10的5'剪接位点中的茎环结构被FTDP-17突变破坏，其在该外显子的选择性剪接的调节中起主要作用。使4个重复Tau的比例增加少至两倍的突变在FTDP-17中是致病性的事实表明具有3个和4个重复的Tau同种型的比例对Tau的正确功能的重要性。在其他物种中，4个重复与3个重复的Tau的比率可以广泛变化，此外，一段时间以来已知在胎儿脑中仅观察到3个重复的Tau，其中4个重复的同种型的产生直到出生后的一段时间才发生。 因此，4个重复与3个重复的Tau比率似乎几乎肯定反映了（如果不是基础的话）不同物种和发育过程中神经元作用的一些根本差异。 该项目旨在增加我们对4比3重复Tau比率的调节的理解，为什么这个比率在神经元的正确功能中很重要，为什么这个比率的破坏会导致神经退行性变。

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.