Mechanism of alpha-synuclein aggregate clearance by Rab7

帕金森病的特征是错误折叠的蛋白质聚集，特别是α-突触核蛋白。我们的目的是通过诱导α-突触核蛋白聚集体的清除来阻止帕金森病的进展。聚集体通过自噬降解，胞浆碎片被膜吞噬。这些自噬小体随后与溶酶体融合，以降解其内容。在以前的工作中，我们可以证明α-突触核蛋白聚集体的自溶酶体降解是由Rab7的过度表达引起的。几种Rab蛋白促进聚集清除，但Rab7特别令人感兴趣，因为它介导粉色、parkin和LRRK2的影响。编码这些蛋白质的基因突变与家族性帕金森氏症有关。Rab7最重要的效应子是FYCO1(FYVE和包含1的螺旋结构域)。为了进一步将我们以前的工作发展成为帕金森病的治疗策略，我们将使用两种策略。首先，我们将在更接近临床情况的模型中验证我们在细胞系和黑腹果蝇中的发现。因此，我们将在原代小鼠神经元和脑内注射α-突触核蛋白纤维的小鼠模型中测试Rab7、FYCO1和进一步的干预。在第二种方法中，我们旨在了解Rab7诱导聚集清除的工作原理，以便为未来设计更好的操作。聚合文员涉及多个步骤和因素。为了集中我们的努力，我们在初步实验的基础上提出了两个可检验的假说：-自噬小体与溶酶体的融合是聚集清除的限速步骤。-胆固醇在自噬小体和内质网之间的接触部位的运输是聚集清除所必需的。我们还将研究细胞内运输和分泌与聚集清除的相关性。

Parkinson's disease is characterized by aggregates of misfolded proteins, in particular alpha-synuclein. Our aim is to stop progression of Parkinson's disease by inducing clearance of alpha-synuclein aggregates.Aggregates are degraded by autophagy where pieces of cytosol are engulfed by a membrane. These autophagosomes subsequently fuse with lysosomes to degrade their content. In previous work we could show that autolysosomal degradation of alpha-synuclein aggregates is induced by overexpression of Rab7. Several Rab proteins promote aggregate clearance, but Rab7 is of particular interest since it mediates effects of PINK, parkin and LRRK2. Mutations in the genes that encode these proteins are associated with familial Parkinson's disease. The most important effector of Rab7 is FYCO1 (FYVE and coiled-coiled domain containing 1).In order to further develop our previous work into a therapeutic strategy for Parkinson's disease we will use two strategies. First, we will validate our findings from cell lines and drosophila melanogaster in models that resemble the clinical situation mor closely. We will therefore test Rab7, FYCO1 and further interventions in primary mouse neurons and in a mouse model using intracerebral injection of alpha-synuclein fibrils.In a second approach we aim to understand how Rab7-induced aggregate clearance works in order to design better manipulations for the future. Many steps and factors are involved in aggregate clerance. To focus our efforts we therefoer formulated two testable hypotheses based on our preliminary experiments:- Fusion of autophagosomes with lysosomes is the rate-limiting step of aggregate clearance. - Cholesterol transport at contact sites between autophagosomes and the ER is necessary for aggregate clearance.We will also investigate the relevance of intracellular transport and secretion for aggregate clearance.