Chaperoning Drp1 mediated fission in neurons

线粒体是细胞的“发电厂”，它的问题最近被认为与多种神经退行性疾病有关。细胞需要能量来运作，线粒体将能量转化为一种可以为细胞过程提供燃料的形式。由于这一点和其他重要的细胞作用，健康的线粒体是必不可少的。在细胞中，线粒体经历连续的分裂(分裂)和融合周期，以调节其组织成网络。这种动态的性质是调节线粒体功能、维持健康线粒体和移除线粒体网络受损部分的关键。这在神经元中尤其重要，因为如果线粒体的大小错误，它们就不能有效地沿着细小的分支树突运输。事实上，有重要的证据表明，线粒体的结构和功能异常与衰老和与年龄相关的神经退行性疾病有关，如阿尔茨海默氏症、帕金森氏症、亨廷顿氏症和肌萎缩侧索硬化症。在这种情况下，我们理解神经元线粒体分裂的调控机制是至关重要的。这一过程的主要驱动力已被确定为动力素相关蛋白-1(Drp1)。为了发生裂变，Drp1必须被招募到线粒体分裂的未来位置，在那里它聚合成螺旋，从而收缩和掐断网络的一部分。在哺乳动物细胞中，Drp1的募集和激活还没有完全被了解。我们已经确定了Drp1和另一种称为Sacsin的蛋白质之间的相互作用。糖蛋白突变导致遗传性神经退行性疾病常染色体痉挛共济失调(ARSACS)。线粒体动力学和功能的缺陷是ARSACS的特征，糖蛋白的丢失导致线粒体网络变得更加相互连接。此外，缺乏糖蛋白的细胞即使在被诱导分裂后，与其线粒体相关的DRp1的发生率也降低了。Drp1还在另一种细胞器--过氧化物体的生物发生中发挥作用。与Sacsin在与Drp1的共同途径中的功能一致，我们观察到Sacsin的丢失与过氧化物体数量的减少相关。Sacsin是迄今发现的最大的蛋白质之一，它包含许多与其他已知功能的蛋白质具有同源性的区域。这些包括分子伴侣和辅伴侣蛋白中发现的结构域。伴侣和他们的伴侣共同作用于其他蛋白质的折叠和蛋白质复合体的组装。Sacsin中J结构域的存在表明它与Hsp70蛋白一起发挥功能，可能会招募Hsp70作用于特定的细胞角色。综上所述，这些数据表明在Drp1介导的裂变中需要伴侣活性。为了理解Drp1介导的裂变是如何被Sacsin调控的，我们寻找了其他与Sacsin相互作用的蛋白质。这确定了dynactin-6，它被认为在线粒体的生物发生中发挥作用，是参与将Drp1运输到线粒体的复合体的一部分。我们发现dynactin-6和Drp1也相互作用，降低细胞中dynactin-6的水平会导致更相互连接的线粒体网络，而增加Drp1的水平会导致线粒体的碎裂。结合这些数据，这些数据与Drp1介导的裂变是由一种利用Hsp70伴侣作用的新型蛋白质复合体调节的一致。我们假设，Sacsin是这种复合体的核心成分或支架，这是Drp1在神经元中充分活跃所必需的。这项提案中概述的研究将通过一系列全面的实验来测试这些概念，这些实验旨在确定Sacsin和dynactin-6如何在Drp1介导的分裂中发挥作用。这项研究的主要成果将是阐明控制线粒体分裂的细胞机制和分子伴侣作用的要求。

Problems with mitochondria, the cellular 'power plants', have recently been linked to multiple neurodegenerative diseases. Cells need energy to function and mitochondria convert energy into a form that can fuel cellular processes. Because of this and other important cellular roles healthy mitochondria are essential. In cells, mitochondria undergo continuous cycles of division (fission) and fusion that regulate their organisation into networks. This dynamic nature is key to regulating mitochondrial function, maintaining healthy mitochondria and removing damaged parts of the mitochondrial network. This is particularly important in neurons because if mitochondria are the wrong size they cannot be effectively transported along thin branching dendrites. Indeed, there is significant evidence that structural and functional abnormalities in mitochondria are involved in ageing and age-related neurodegenerative diseases, such as Alzheimer's, Parkinson's, Huntington's and amyotrophic lateral sclerosis.Given this context it is essential that we understand the mechanisms that regulate mitochondrial fission in neurons. The principle driver of this process has been identified as dynamin related protein-1 (Drp1). For fission to occur Drp1 must be recruited to future sites of mitochondrial division where it polymerises into spirals that act to constrict and pinch off parts of the network. In mammalian cells the recruitment and activation of Drp1 is not fully understood.We have identified an interaction between Drp1 and another protein called sacsin. Mutations in sacsin cause the inherited neurodegenerative disease Autosomal Spastic Ataxia of Charlevoix Saguenay (ARSACS). Defects in mitochondrial dynamics and function are features of ARSACS with loss of sacsin causing mitochondrial networks to become more interconnected. Moreover, cells lacking sacsin had a reduced incidence of Drp1 associated with their mitochondria even after fission is induced. Drp1 also functions in biogenesis of another organelle, the peroxisomes. Consistent with sacsin functioning in a common pathway with Drp1 we observed loss of sacsin correlates with a decrease in peroxisome number. Sacsin is one of the largest proteins ever identified and contains a number of regions with homology to other proteins of known function. These include domains that are found in molecular chaperone and cochaperone proteins. Chaperones and their cochaperone partners work together in the folding of other proteins and assembly of protein complexes. The presence of a J-domain in sacsin indicates that it functions with an Hsp70 protein, potentially recruiting Hsp70 action to a specific cellular role. Together these data suggest a requirement for chaperone activity in Drp1 mediated fission.To comprehend how Drp1 mediated fission is modulated by sacsin we looked for additional proteins that interact with sacsin. This identified dynactin-6, which has been suggested to play a role in mitochondrial biogenesis and is part of a complex involved in transport of Drp1 to mitochondria. We found dynactin-6 and Drp1 also interact and that reducing dynactin-6 levels in cells lead to a more interconnected mitochondrial network while increasing Drp1 levels caused mitochondrial fragmentation. In combination these data are consistent with Drp1 mediated fission being modulated by a novel protein complex that utilises Hsp70 chaperone action. We hypothesise that sacsin represents the core component or scaffold of such a complex, which is required for Drp1 to be fully active in neurons. The research outlined in this proposal will test these concepts through a comprehensive series of experiments designed to identify how sacsin and dynactin-6 function in Drp1 mediated fission. The main outcome of this research will be the elucidation of the cellular mechanisms controlling mitochondrial fission and the requirement for molecular chaperone action.