Investigation of the mechanism by which huntingtin fragments are produced and their pathogenic relevance to Huntington's disease

亨廷顿病(HD)是一种平均发病年龄为40岁的神经退行性疾病。受影响的个人失去控制运动的能力，精神能力受损，出现精神问题，体重减轻。这种疾病会发展15-20年，直到死亡。对于一些精神问题(如抑郁症)，有有效的治疗方法，但没有办法阻止或减缓疾病的进展。HD是遗传性的，受影响的人的后代有50%的机会患上这种疾病。在受感染的人和没有患病的人中，Huntingtin基因(HTT)的唯一区别是DNA序列CAG的长度增加到接近其开始的长度。这导致亨廷顿蛋白(HTT)中有一条超长的谷氨酸(构成蛋白质的构件)。额外的谷氨酸导致HTT蛋白以一种异常的方式与自身和其他蛋白质相互作用，但这导致HD的机制仍然知之甚少。已经在HD患者和HD小鼠模型的大脑中检测到HTT片段，大量研究表明，HTT片段在疾病过程中比全长蛋白更重要。大多数对这些片段来源的研究都集中在确定相关的蛋白酶(分解蛋白质的酶)上，但尽管如此，大多数片段的来源仍然未知。我们最近发现，最小的HTT片段是通过HTT基因的改变处理而不是通过蛋白酶消化而产生的。大多数基因由内含子隔开的外显子(编码蛋白质的DNA序列)组成。当基因被转录成信使RNA(信使RNA)时，内含子在蛋白质生产之前通过剪接被移除。HTT基因由67个外显子组成，我们已经发现，当存在HD突变时，HTT的外显子1并不总是与外显子2拼接，产生一个包含外显子1和内含子1序列的新的小mRNA，并终止于标记末端mRNA的Polya尾巴。这被翻译为产生一个小的外显子1 HTT蛋白，已被证明在多种模型系统中具有高致病性。这些结果很重要，因为了解这种HTT片段是如何产生的，将使我们能够制定策略来防止它的形成，从而确定它在疾病过程中的作用程度。了解HTT基因的加工和HTT片段的产生是解开引起HD的致病过程的基本必要条件。在这个建议中，我们将：1.进一步研究HD突变(超长CAG序列)导致HTT外显子1到外显子2不完全剪接的机制。我们已经确定了一个剪接因子(SRSF6)，它可以识别CAG重复序列，调节基因的剪接，我们已经证明它与HTT基因的起始结合。我们将研究SRSF6影响HTT剪接的机制，并确定可能涉及的其他剪接因素。在过去的十年里，我们对HD的各种小鼠模型进行了广泛的表征。我们将使用一种新的技术(锌指核酸酶)来操纵HD小鼠的htt基因，以防止通过这种错接机制产生外显子1 htt蛋白。这将最终使我们能够确定外显子1 HTT蛋白在HD疾病过程中的作用程度。我们已经在HD小鼠的大脑中发现了一些额外的HTT片段。除了外显子1 HTT，这些片段的身份和来源尚不清楚。我们将使用化学修饰和蛋白质测序的组合来识别尽可能多的这些其他片段的末端。这可能揭示了防止特定片段的形成并确定它们在发病中的作用的策略。

Huntington's disease (HD) is a neurodegenerative disorder with an average age of onset of 40 years. Affected individuals lose their ability to control movement, develop an impaired mental capacity and psychiatric problems and lose weight. The disease progresses for 15-20 years until death. There are effective treatments for some psychiatric problems (e.g. depression) but there is no way to halt or slow the disease progression. HD is inherited and offspring of affected individuals have a 50% chance of developing the disease. The only difference between the huntingtin gene (HTT) in people who become affected and those who remain disease free is the increased length of the DNA sequence CAG close to its beginning. This results in an extra long tract of glutamines (a building block for proteins) in the huntingtin protein (HTT). The extra glutamines cause the HTT protein to interact with itself and other proteins in an aberrant fashion but the mechanism by which this causes HD remains poorly understood. Fragments of HTT have been detected in the brains of HD patients and HD mouse models and a considerable amount of research indicates that HTT fragments are more important for the disease process than the full-length protein. Most research into the origin of these fragments has focussed on identifying the relevant proteases (enzymes that cleave proteins), but despite this, the origin of most fragments remains unknown. We have recently found that the smallest HTT fragment is generated by altered processing of the HTT gene and not by protease digestion. Most genes are composed of exons (DNA sequences that code for the protein) that are separated by introns. As the gene is transcribed into messenger RNA (mRNA), the introns are removed by splicing prior to protein production. The HTT gene is comprised of 67 exons and we have discovered that when the HD mutation is present, exon 1 of HTT does not always splice to exon 2, producing a small novel mRNA that contains exon 1 and intron 1 sequences and terminates in a polyA tail, that marks the end mRNA. This is translated to produce a small exon 1 HTT protein that has been shown to be highly pathogenic in multiple model systems. These results are important because understanding how this HTT fragment is generated will allow us to devise strategies to prevent its formation and thereby determine the extent to which it contributes to the disease process. An understanding of the processing of the HTT gene and the production of HTT fragments is a basic requisite to unraveling the pathogenic process that causes HD. In this proposal we shall:1. Further investigate the mechanism by which the HD mutation (extra long CAG sequence) results in the incomplete splicing of exon 1 HTT to exon 2. We have identified a splicing factor (SRSF6) that recognizes CAG repeat sequences, is known to modulate the splicing of genes and we have shown that it binds to the beginning of the HTT gene. We shall investigate the mechanism by which SRSF6 influences HTT splicing and identify other splicing factors that may be involved.2. Over the past ten years, we have performed an extensive characterization of various mouse models of HD. We shall use a new technology (zinc finger nucleases) to manipulate the Htt gene in HD mice in order to prevent the exon 1 HTT protein from being generated via this mis-splicing mechanism. This will ultimately allow us to determine the extent to which the exon 1 HTT protein contributes to the HD disease process.3. We have identified a number of additional HTT fragments in the brains of the HD mice. With the exception of exon 1 HTT, the identity and origin of these fragments remains unknown. We shall use a combination of chemical modification and protein sequencing to identify the ends of as many of these other fragments as possible. This may reveal strategies by which the formation of specific fragments can be prevented and their contribution to pathogenesis determined.