Dissecting the molecular mechanisms of lysosome:ER contact site formation and their relevance to neurodegenerative disease

人类细胞包含膜封闭的隔间，称为细胞器，执行专门的任务。膜接触位点，桥接相邻细胞器的膜，为细胞器之间的通信提供平台。胆固醇是膜的重要组成部分。细胞有两种来源的胆固醇：它可以来自我们的饮食或新合成的细胞。膳食胆固醇被吸收到细胞中的一个称为内体的隔室中，从那里它被运输到溶酶体，再向前运输到另一个隔室，内质网（ER）。ER是细胞内胆固醇合成的位点，来自溶酶体的膳食胆固醇的到来关闭了新胆固醇的合成。当膳食胆固醇不能从溶酶体转运到ER时，它在溶酶体中积累，而ER中的胆固醇合成仍在继续。由此产生的细胞胆固醇失衡对细胞有毒，并与神经退行性疾病有关。我们最近发现，ER和溶酶体之间的膜接触位点通过将细胞胆固醇转运到正确的位置来调节胆固醇平衡。这项研究计划的目的是确定这些接触部位是如何形成的，如果他们运输其他脂质以及胆固醇，如果他们可以在神经退行性疾病的治疗效益为目标。尼曼皮克病C型（NPC）是一种罕见的，但破坏性的进行性神经退行性疾病，往往开始于儿童早期。我们知道大多数（95%）NPC病例是由编码NPC蛋白NPC 1的基因突变引起的。NPC 1位于溶酶体膜上，是膳食胆固醇离开溶酶体运输到ER所必需的。我们小组以前的工作发现，NPC 1形成了胆固醇可以穿过的溶酶体-ER桥的一部分。在NPC患者的细胞中，溶酶体未能与ER正确连接。结果，胆固醇在溶酶体中积累，溶酶体停止正常工作。在这项工作中，我们打算找出NPC 1如何桥接这两个细胞器，以及它与ER上的伴侣的相互作用是否受到溶酶体中膳食胆固醇水平的影响。除了胆固醇，其他脂质也在NPC的溶酶体中积累，这些脂质被认为有助于疾病的进展。事实上，目前唯一获批的NPC治疗药物（麦格司他）可抑制这些脂质的合成。尽管麦格司他不是一种治愈方法，但它减缓疾病进展的事实表明，这些脂质的不平衡对细胞具有毒性。我们的初步数据表明，这些脂质中的一些也可能通过接触部位转运到ER，在那里它们被降解。在这个项目中，我们将提高我们对膜接触位点和脂质代谢之间的关系的理解。我们最近发现，人工桥接溶酶体的ER可以逆转胆固醇积累的细胞缺乏NPC 1。这是令人兴奋的，因为它可能对NPC有治疗意义。我们将评估在NPC细胞模型中扩大膜接触部位的不同方法，并在这些研究的基础上观察在动物模型中增加接触部位的效果。斑马鱼是研究NPC的一个很好的模型系统。NPC斑马鱼模型模拟许多哺乳动物NPC表型，包括脂质积累和运动缺陷。因此，我们将生成NPC的斑马鱼模型，在其中研究ER-溶酶体接触位点并测试我们的假设，即扩大这些接触位点将拯救脂质积累和下游神经变性/运动缺陷。这可能会产生新的治疗策略，用于治疗NPC和其他神经退行性疾病与脂质储存缺陷。

Human cells contain membrane enclosed compartments, called organelles, that perform specialised tasks. Membrane contact sites, that bridge the membranes of neighbouring organelles, provide platforms for communication between organelles. Cholesterol is an important component of membranes. Cells have two sources of cholesterol: it can either be derived from our diet or newly synthesised by the cell. Dietary cholesterol is taken up into the cell into a compartment called the endosome, from where it is trafficked to the lysosome for onward transport to another compartment, the endoplasmic reticulum (ER). The ER is the site of cholesterol synthesis within the cell and the arrival of dietary cholesterol from the lysosome switches off the synthesis of new cholesterol. When dietary cholesterol fails to be transported from the lysosome to the ER, it accumulates in the lysosome, while cholesterol synthesis in the ER still continues. The resulting imbalance in cellular cholesterol is toxic to the cell and is associated with neurodegenerative disease. We have recently shown an important role for membrane contact sites between the ER and lysosomes in regulating the cholesterol balance by transporting cellular cholesterol to the right location. The aim of this research proposal is to determine exactly how these contact sites are formed, if they transport other lipids as well as cholesterol and if they can be targeted for therapeutic benefit in neurodegenerative disease.Niemann Pick disease type-C (NPC) is a rare but devastating progressive neurodegenerative disease that often starts in early childhood. We know that most (95%) cases of NPC are caused by mutations in a gene that encodes the NPC protein NPC1. NPC1 is on the lysosome membrane and is required for dietary cholesterol to exit the lysosome for transport to the ER. Previous work by our group has found that NPC1 forms part of the lysosome-ER bridge that cholesterol can travel across. In cells from NPC patients, the lysosome fails to connect properly with the ER. As a result, cholesterol accumulates in the lysosome and the lysosome stops working properly. In this proposed work we intend to find out how NPC1 bridges the two organelles and if its interactions with partners on the ER are affected by levels of dietary cholesterol in the lysosome.As well as cholesterol, other lipids also accumulate in lysosomes in NPC that are believed to contribute to the disease progression. Indeed, the only current licensed therapeutic for NPC (miglustat) inhibits the synthesis of these lipids. Although miglustat is not a cure, the fact that it slows progression of the disease shows that imbalance of these lipids is toxic to the cell. Our preliminary data suggests that some of these lipids might also be transported across contact sites, to the ER where they are degraded. In this project we will improve our understanding of the relationship between membrane contact sites and lipid metabolism. We recently showed that artificially bridging lysosomes to the ER can reverse cholesterol accumulation in cells lacking NPC1. This is exciting as it could have therapeutic implications for NPC. We will assess different ways to expand membrane contact sites in cellular models of NPC and build on these studies to see the effect of increased contact sites in animal models. Zebrafish are an excellent model system for studying NPC. NPC zebrafish models mimic many mammalian NPC phenotypes, including lipid accumulation, and movement defects. We will therefore generate zebrafish models of NPC in which to study ER-lysosome contact sites and test our hypothesis that expanding these contact sites will rescue both lipid accumulation and downstream neurodegeneration /movement defects. This may yield novel therapeutic strategies for the treatment of NPC and other neurodegenerative diseases with lipid storage defects.