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

人体细胞包含被称为细胞器的膜封闭隔间，它们执行特定的任务。膜接触点是连接相邻细胞器的膜，为细胞器之间的交流提供平台。胆固醇是细胞膜的重要组成部分。细胞中的胆固醇有两种来源：一种来自我们的饮食，另一种是由细胞新合成的。膳食胆固醇被吸收到细胞内一个叫做核内体的小室，从那里它被运送到溶酶体，然后再运输到另一个小室——内质网（ER）。内质网是细胞内胆固醇合成的地方，从溶酶体到达的膳食胆固醇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内质网时，它在溶酶体中积累，而内质网中的胆固醇合成仍在继续。由此导致的细胞胆固醇失衡对细胞是有毒的，并与神经退行性疾病有关。我们最近表明，内质网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，具有重要作用。这项研究计划的目的是确定这些接触位点是如何形成的，它们是否运输其他脂质和胆固醇，以及它们是否可以作为治疗神经退行性疾病的目标。Niemann Pick病c型（NPC）是一种罕见但破坏性的进行性神经退行性疾病，通常始于儿童早期。我们知道大多数（95%）鼻咽癌病例是由编码鼻咽癌蛋白NPC1的基因突变引起的。NPC1位于溶酶体膜上，是膳食胆固醇离开溶酶体转运到内质网所必需的。我们小组之前的工作已经发现NPC1形成了溶酶体-内质网桥的一部分，胆固醇可以通过这个桥。在鼻咽癌患者的细胞中，溶酶体不能与内质网正常连接。结果，胆固醇在溶酶体中积累，溶酶体停止正常工作。在这项拟议的工作中，我们打算找出NPC1如何连接两个细胞器，以及它与内质网伙伴的相互作用是否受到溶酶体中膳食胆固醇水平的影响。除胆固醇外，其他脂质也在鼻咽癌溶酶体中积累，被认为有助于疾病进展。事实上，目前唯一许可的鼻咽癌治疗药物（米卢司他）抑制这些脂质的合成。尽管米卢司他不能治愈，但它减缓疾病进展的事实表明，这些脂质失衡对细胞是有毒的。我们的初步数据表明，其中一些脂质也可能通过接触部位运输到内质网，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关系的理解。我们最近发现，人工桥接溶酶体到内质网可以逆转缺乏NPC1细胞中的胆固醇积累。这是令人兴奋的，因为它可能对鼻咽癌有治疗意义。我们将评估在鼻咽癌细胞模型中扩大膜接触点的不同方法，并在这些研究的基础上观察动物模型中增加接触点的效果。斑马鱼是研究NPC的优秀模型系统。斑马鱼鼻咽癌模型模拟了许多哺乳动物鼻咽癌表型，包括脂质积累和运动缺陷。因此，我们将生成鼻窦炎的斑马鱼模型，用于研究er溶酶体接触位点，并验证我们的假设，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。这可能为鼻咽癌和其他具有脂质储存缺陷的神经退行性疾病的治疗提供新的治疗策略。

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.