Understanding the role of DNA repair in Huntington's Disease pathogenesis: towards new therapeutic targets

背景和目的：亨廷顿舞蹈病（HD）是一种遗传性脑退行性疾病，患者会出现多种症状，包括不自主运动、情绪和行为改变以及痴呆。该病进展缓慢，但持续15-20年，并缩短生命。我们没有任何治疗方法可以预防或减缓它。患者在很长一段时间内往往有复杂的护理需求，这给他们的护理人员和家庭以及一般的医疗保健资源带来了相当大的压力。虽然HD是一种罕见的疾病（约8000人中有1人患病），但它是由基因中DNA重复部分扩增引起的30多种疾病之一。据估计，这些疾病加起来影响全世界300多万人（2000年1人），造成了巨大的人力和经济代价。1993年发现了导致HD的基因突变，但目前尚不清楚这是如何导致特定的神经损伤和大脑损失的。突变包括亨廷顿蛋白基因DNA中重复“CAG”序列的扩展。未受影响的人有9到35个CAG重复；HD患者至少有36个，通常重复次数越多，疾病开始得越早。受这种疾病影响最严重的脑神经细胞比同一患者体内的其他细胞含有更多的重复序列。最近一项涉及我们小组和其他人的基因研究发现，各种DNA修复因子是HD症状开始年龄的决定因素。我们认为，DNA修复过程可能直接导致易损细胞中CAG重复序列的增加（扩增），并导致细胞死亡和疾病的发生。这个项目的目的是描述这个过程是如何发生的，并试图找出它可能被阻止的方式。项目将如何实施：研究活的HD患者大脑中的细胞过程是困难的，因此我们将使用各种实验方法来测试我们的想法。遗传学。我们将寻找与CAG重复序列长度预测相比，在早期或晚期发生HD的患者的DNA修复基因中的变异。我们希望这能识别出新的变体，从而了解高清是如何以及何时开始的。细胞培养。我们将开发一个模型系统，用于在实验室培养的神经细胞中测试CAG的扩增。一旦这个系统建立，我们将测试DNA修复变异体对CAG扩增的影响。导致重复扩增大大增强的因素可能代表新的治疗靶点：如果我们能够阻断这一过程，理论上我们就可以延缓疾病的发作。生物化学。为了弄清楚DNA修复蛋白是如何与DNA中的CAG重复序列相互作用并导致它们膨胀的，我们将纯化不同的成分并在实验室分析它们的反应。我们将能够把基因筛选中最有趣的突变插入生化分析中，以揭示有关机制的更多细节。重复扩增抑制剂。我们将在CAG扩增系统中测试能够阻断DNA修复蛋白的化合物。如果它们能够阻断CAG的扩张，那么它们可能代表着新的治疗线索，具有改变疾病发病的潜力。因此，该项目将增加对HD突变如何导致疾病的重要见解，将遗传学与细胞和分子生物学联系起来。它可以阐明重复扩增的机制，并确定新的药物靶点，为从实验到临床提供明确的联系。鉴于潜在的致病机制可能相似，这些发现可能对其他DNA重复疾病以及HD有益。更广泛地说，对HD细胞机制的深入了解可能会揭示神经元生物学的新方面，这对其他类型的神经系统疾病和痴呆的研究很有用。

Background and aims:Huntington's disease (HD) is an inherited degenerative brain condition in which patients develop a mixture of symptoms including involuntary movements, changes in mood and behaviour, and dementia. The disease progresses slowly but relentlessly over 15-20 years, and is life-shortening. We have no treatments that can prevent or slow it. Patients often have complex care needs over a long period of time and these put a considerable strain on their carers and families, as well as on healthcare resources in general. Although HD is a rare condition (affecting about 1 in 8000 people), it is one of a family of over 30 diseases caused by expansion of repeating sections of DNA in genes. Together these conditions are estimated to affect over 3 million people worldwide (1 in 2000) at considerable human and economic cost. The genetic mutation causing HD was identified in 1993 but it is still unclear exactly how this leads to specific nerve damage and loss in the brain. The mutation consists of expansion of a repeating 'CAG' sequence in the DNA of the huntingtin gene. Unaffected people have between 9 and 35 CAG repeats; HD patients have at least 36, and generally the greater the number of repeats, the earlier the disease starts. The brain nerve cells that are most affected by the disease harbour more repeats than other cells in the same patient.A recent genetic study involving our group and others identified various DNA repair factors as determinants of the age at which HD symptoms start. We think that DNA repair processes might directly cause an increased number (expansion) of CAG repeats in vulnerable cells and lead to cell death and the onset of disease. The aims of this project are to characterise how this process occurs, and to try to identify ways in which it could be blocked.How the project will be carried out:It is difficult to study cellular processes in the brains of living HD patients and so we will use a variety of experimental methods to test our ideas.1. Genetics. We will look for variants in the DNA repair genes of patients who have developed HD at an unexpectedly early or late age compared to that predicted from their CAG repeat length. We expect that this will identify new variants that will give insight into how and when HD starts.2. Cell culture. We will develop a model system for testing CAG expansion in nerve cells grown in the laboratory. Once this system is established we will test the effects of the DNA repair variants on CAG expansion. Factors that lead to greatly enhanced repeat expansion could represent novel therapeutic targets: if we can block this process we could theoretically delay disease onset.3. Biochemistry. To work out exactly how DNA repair proteins interact with CAG repeats in DNA and cause them to expand, we will purify the different components and analyse their reactions in the laboratory. We will be able to plug the most interesting mutations from our genetic screen into the biochemical analysis to uncover more detail about mechanism.4. Inhibitors of repeat expansion. We will test compounds that can block DNA repair proteins in our CAG expansion system. If they can block CAG expansion then they could represent new therapeutic leads with the potential to alter disease onset. Therefore this project will add important insight as to how the HD mutation leads to disease, linking genetics with cellular and molecular biology. It could elucidate mechanisms of repeat expansion and identify new drug targets, giving a clear link from bench to bedside. These findings are likely to be beneficial in other DNA repeat diseases as well as HD given that the underlying pathogenic mechanisms are probably similar. More broadly, insights into the cellular mechanisms underlying HD may well reveal novel aspects of neuronal biology that are useful in the investigation of other types of neurological disease and dementia.