The molecular cell biology jigsaw of Parkinson's disease: bringing the pieces together

PD是第二大最常见的神经退行性疾病，影响65岁以上人群的2-3%。迄今为止，有17个基因与PD风险增加有关（“PARK基因”）。该领域最大的挑战是将这些基因排列成可能支持病理的共同途径。共同的主题逐渐出现，将几个最具特征的PD相关基因与两个总体过程联系起来：内溶酶体运输和有丝分裂。这两个过程都是两个合作主办实验室的重点研究领域。内溶酶体的运输对于自噬处理有缺陷的细胞器和有毒的蛋白质聚集体是至关重要的。PD通常与含有α -突触核蛋白的聚集体（称为“路易体”）在大脑离散区域的积累有关。线粒体自噬涉及通过选择性自噬控制有缺陷的线粒体的处置。由此产生的自噬体与溶酶体融合，在那里发生封闭物质的降解。受损线粒体和黑质多巴胺能神经元聚集物的清除失败被认为会导致活性氧积累和钙失衡，最终导致细胞死亡。我们对最具特征的PD相关基因（LRRK2, VPS35， PINK1和Parkin）之间的功能联系感兴趣。Parkin是一种泛素E3连接酶，由PINK1激活，在线粒体自噬中起关键作用。有趣的是，我们已经证明Parkin可以使VPS35泛素化，VPS35是一种参与囊泡运输的蛋白质。VPS35突变体导致新合成的溶酶体酶分选错误和溶酶体生物发生缺陷。PD相关的VPS35突变形式已被发现激活LRRK2激酶，这是与家族性PD相关的最常见突变基因之一。LRRK2最重要的底物属于小gtpase的Rab家族，是囊泡运输和VPS35功能的关键调节因子。该项目将结合活细胞和固定细胞成像技术以及使用模型组织培养细胞系统的生化方法。预计将有蛋白质组学成分，并将提供质谱样品制备方面的培训，以及蛋白质组学数据集的分析和解释。我们将使用CRISPR/Cas9基因编辑来生成等基因细胞系，以便对突变型和野生型PD相关基因进行比较。该项目的一个组成部分将需要使用由神经前体细胞（npc）产生的多巴胺能神经元，这些神经元可以从患者来源的成纤维细胞中提取。与iPSC模型相比，这种创新系统具有优势，因为npc保留了与衰老相关的表观遗传特征。

PD is the second most common neurodegenerative disorder affecting 2-3% of the population over the age of 65. Seventeen genes have been associated with increased PD risk to date ("PARK genes"). The big challenge in the field is to order these genes into common pathways that may underpin the pathology. Common themes are gradually emerging, linking several of the best characterised PD associated genes to two overarching processes: endo-lysosomal trafficking and mitophagy. Both these processes are areas of intense research in the two collaborating host laboratories. Endo-lysosomal traffic is critical for the disposal of defective organelles and toxic protein aggregates by autophagy. PD is commonly associated with the accumulation of alpha-synuclein containing aggregates, called "Lewy bodies" in discrete regions of the brain. Mitophagy concerns the controlled disposal of defective mitochondria by selective autophagy. The resultant autophagosomes fuse with lysosomes where the degradation of enclosed material takes place. Failure to clear damaged mitochondria and aggregates in the dopaminergic neurons of the substantia nigra is thought to result in accumulation of reactive oxygen species and a calcium imbalance ultimately leading to cell death. We are interested in the functional links between the best characterised PD associated genes (LRRK2, VPS35, PINK1 and Parkin). Parkin is a ubiquitin E3 ligase that is activated by PINK1 and plays a critical role in mitophagy. Interestingly, we have shown that Parkin can ubiquitylate VPS35, a protein involved in vesicular trafficking. VPS35 mutants cause mis-sorting of newly sythesised lysosomal enzymes and defects in lysosome biogenesis. A PD associated mutant form of VPS35 has been found to activate the LRRK2 kinase, one of the most commonly mutated genes associated with familial PD. The most prominent substrates of LRRK2 belong to the Rab family of small GTPases, key regulators of vesicular trafficking and VPS35 function. The project will incorporate live and fixed cell imaging techniques and biochemical methods using model tissue culture cell systems. A proteomic component is anticipated and training in the preparation of samples for mass spectrometry as well as the analysis and interpretation of proteomic datasets will be provided. We will use CRISPR/Cas9 gene editing to generate isogenic cell lines that will allow for comparison between mutant and wild-type forms of PD associated genes. A component of the project will entail the use of dopaminergic neurons generated from neuronal precursor cells (NPCs) that can be derived from patient derived fibroblasts. This innovative system is advantageous compared with iPSC models as NPCs retain the epigenetic signatures associated with ageing.