In vivo pathway analysis to determine the mechanistic role of susceptibility genes for Parkinson's disease

帕金森氏病(PD)是一种常见且持续进行性的脑部疾病。它的主要症状是运动缓慢，这是由神经细胞(神经元)死亡引起的，神经细胞在大脑的黑质部分产生一种名为多巴胺的化学物质。目前可用的治疗方法是不够的：首先，它们只治疗疾病的部分症状，但不是全部。其次，它们不会改变潜在的疾病过程，因此无法减缓疾病的发展。这一失败的部分原因是我们对引起帕金森病的机制了解有限。在过去的20年里，在识别导致帕金森病的基因和机制方面取得了巨大的进展，这种帕金森病是由于基因的缺陷(突变)而遗传的(家族性帕金森病)。然而，在英国，只有大约5%-10%的帕金森病患者携带这种家族性帕金森病突变。该项目旨在阐明遗传因素如何导致更为常见的散发性帕金森病的风险。所谓的全基因组关联研究(GWAS)现在已经确定了我们基因组中的24个区域，这些区域包含导致散发性帕金森病风险的基因。然而，目前还不清楚这些基因如何导致帕金森病的风险，在某些情况下，这种区域内的哪个特定基因是罪魁祸首。我们的目的是研究那些最有可能导致帕金森病风险的基因(从此以后称为PD GWA基因)。我们和其他人认为，这些基因将参与三种不同的机制，即：1)由称为线粒体的细胞组件调节能量产生和能量维持(动态平衡)。2)通过称为自噬的过程和称为溶酶体的细胞组件来聚集和分解蛋白质。3)炎症(细胞对损伤和感染的反应)。我们建议利用斑马鱼携带的基因缺陷来使这些PD GWA基因失活，用于我们的研究。斑马鱼是脊椎动物，因此与蠕虫或果蝇等其他低等动物模型相比，与人类的亲缘关系要密切得多。它们还提供了研究PD GWA基因与衰老过程相互作用的机会。这一点很重要，因为老龄化是帕金森病最重要的风险因素。斑马鱼幼体在体外发育，是透明的。这使得研究神经元和免疫细胞之间的相互作用变得相对容易。斑马鱼也是药物发现的优秀动物模型。它们已经被用于广泛的其他人类疾病(包括肝脏、心脏和凝血障碍)，以阐明在全基因组关联研究中首次发现的基因是如何导致这些疾病的。我们和其他人已经在用斑马鱼来研究家族性PD基因。现在，我们希望利用我们的专业知识和斑马鱼作为人类疾病模型的主要优势来研究PD GWA基因，以更好地了解它们如何导致散发性PD的风险。使用一种名为CRISPR/CAS的革命性新基因编辑策略，我们已经制作了一系列斑马鱼品系，这些品系携带10个最重要的PD GWA风险基因的突变。这项试点工作将极大地加快我们拟议的研究进度。作为这项研究的一部分，我们将研究这些PD GWA风险基因对所谓的“全球基因表达”的影响。这包括量化大脑在多大程度上使用了基因组中的所有基因，从而洞察哪些生物系统过度活跃或不活跃。我们还将研究PD GWA风险基因是否与家族性PD基因相互作用。我们的工作将把重点放在识别这些系统中的“可用药”目标上。未来，我们计划针对这些目标进行药物筛查。这将有望帮助我们找到有前景的药物，然后可以用于帕金森病患者的临床试验。

Parkinson's disease (PD) is a common and relentlessly progressive brain disease. Its key symptom, slowness of movement, is caused by the death of nerve cells (neurons) that produce a chemical called dopamine in a part of the brain called the substantia nigra. Currently available therapies are inadequate: Firstly, they treat only some, but not all, of the symptoms of the disease. Secondly, they do not modify the underlying disease processes and therefore fail to slow down disease progression. This failure is partially due to our limited knowledge of the mechanisms that cause PD. Over the last 20 years, there has been huge progress with the identification of genes and mechanisms which cause PD that is inherited due to faults (mutations) in genes (familial PD). However, only about 5-10% of PD patients in the UK carry such a familial PD mutation. This project aims to elucidate how inherited factors contribute to the risk of the much more common sporadic form of PD. So-called genome-wide association studies (GWAS) have now identified 24 regions within our genome which contain genes that contribute to the risk of sporadic PD. However, it is not clear how these genes contribute to the risk of PD and, in some cases, which particular gene within such a region is responsible. Our aim is to study those genes which are most likely to contribute to the risk of PD (from hereon called PD GWA genes). We and others believe that these genes will be involved in 3 distinct mechanisms, namely:1) The regulation of energy production and energy maintenance (homeostasis) by cellular components called mitochondria.2) The aggregation and breakdown of proteins by processes called autophagy and cellular components called lysosomes.3) Inflammation (the cellular response to injury and infection). We propose to use zebrafish carrying gene defects which will inactivate these PD GWA genes for our studies. Zebrafish are vertebrates and therefore much more closely related to humans then other lower animal models such as worms or fruit flies. They also offer the opportunity to study the interaction of PD GWA genes with ageing processes. This is important since ageing is the most important risk factor for PD. Zebrafish larvae develop outside the body and are transparent. This makes it relatively easy to study the interaction between neurons and immune cells. Zebrafish are also an excellent animal model for drug discovery. They have already been used for a wide range of other human diseases (including liver, heart and clotting disorders) to elucidate how genes first identified in genome-wide association studies contribute to these disorders. We and others have already been using zebrafish to study familial PD genes. We now want to use our expertise and the major advantages of zebrafish as a model for human diseases to study the PD GWA genes to better understand how they contribute to the risk of sporadic PD. Using a revolutionary new gene editing strategy called 'CRISPR/Cas', we have already made a range of zebrafish lines which carry mutations in 10 of the most important PD GWA risk genes. This pilot work will greatly accelerate the progress of our proposed research. As part of this, we will be studying the effect of these PD GWA risk genes on so-called 'global gene expression'. This involves quantifying how much the brain is using all of the genes in the genome and thereby getting an insight into which biological systems are over or under active. We will also investigate whether PD GWA risk genes interact with familial PD genes. Our work will have a strong focus on the identification of "druggable" targets within these systems. In the future, we are planning to undertake a drug screen against these targets. This will hopefully help us to identify promising drugs which can then be taken into clinical trials for patients with PD.