Endocytic Trafficking and Human Diseases

IV型粘脂沉积症（MLIV）是一种常染色体隐性遗传疾病，其特征为急性精神发育迟滞、瞳孔缺乏和视觉异常，包括视网膜变性、角膜混浊、视神经萎缩和斜视。在MLIV患者的大多数组织中发现溶酶体包涵体。储存材料的组成是异质的，包括形成特征性多同心薄片的脂质和粘多糖，以及可溶性颗粒蛋白质。MLIV是由粘磷脂-1（MCOLN 1，也称为TRPML 1）突变引起的，MCOLN 1是一种属于离子通道瞬时受体电位（TRP）超家族的内溶酶体阳离子通道。全细胞膜片钳以及天然内溶酶体膜的记录表明，MCOLN 1作为一个向内（从管腔到细胞质）的整流通道，可渗透Ca 2+，Na+，K+和Fe 2 +/Mn 2+，其活性通过低pH增强。 我们和其他人提出，MCOLN 1在细胞中的主要作用是介导晚期内体和溶酶体的钙外流。从这种酸性储存中释放的局部钙是内吞囊泡之间融合和维持细胞器稳态所必需的。事实上，我们发现自噬体与溶酶体的融合在MCOLN 1缺陷细胞中受损，从而导致蛋白质聚集体的积累和受损的细胞器。我们的工作有助于目前的观点，即缺陷性自噬在许多LSD的疾病发病机制中起着重要作用。为了深入了解调节MCOLN 1活性的分子机制，我们通过下拉试验和分裂泛素酵母双杂交筛选来寻找结合MCOLN 1的蛋白。这些实验允许鉴定penta-EF-hand蛋白ALG-2和溶酶体转运蛋白的LAPTM家族作为MCOLN 1的新型相互作用物。 与Andrea Ballabio小组合作，我们最近描述了MCOLN 1的表达受TFEB调节，TFEB是一种促进自噬和溶酶体基因转录的转录因子。TFEB的过表达导致MCOLN 1介导的溶酶体胞吐作用，并导致几种LSD中异常溶酶体的清除，进一步证实了MCOLN 1在细胞器融合中的作用。 为了进一步表征MCOLN 1的生理作用，我们已经开始开发体内动物模型。特别是，我们已经使用特定的锌指核酸酶（ZFN）来创建MCOLN 1基因缺失（敲除）的稳定遗传工程斑马鱼。我们将重点关注的具体问题的一些例子将是：（a）什么是MCOLN 1在不同组织和发育阶段的表达模式，（B）什么是消耗MCOLN 1在各种器官的功能和发育的影响，（c）在MCOLN 1敲除斑马鱼，我们可以复制MLIV表型，如果是这样，我们可以了解疾病的病理学。这些和其他重要问题可以通过我们的实验设计来解决，从而为MCOLN 1的分子功能提供无与伦比的见解，提高我们对MLIV的理解，并为开发该疾病的治疗开辟新的令人兴奋的场所。

Mucolipidosis type IV (MLIV) is an autosomal recessive disorder characterized by acute psychomotor delays, achlorydria, and visual abnormalities including retinal degeneration, corneal clouding, optic atrophy, and strabismus. Lysosomal inclusions are found in most tissues in MLIV patients. The composition of the storage material is heterogeneous and includes lipids and mucopolysaccharides forming characteristic multiconcentric lamellae, as well as soluble, granulated proteins. MLIV is caused by mutations in mucolipin-1 (MCOLN1, also known as TRPML1), an endo-lysosomal cation channel belonging to the transient receptor potential (TRP) superfamily of ion channels. Whole cell patch clamp, as well as recording of native endolysosomal membranes, suggest that MCOLN1 functions as an inwardly (from lumen to cytoplasm) rectifying channel permeable to Ca2+, Na+, K+ and Fe2+/ Mn2+ whose activity is potentiated by low pH. We and others have proposed that the primary role of MCOLN1 in cells is to mediate calcium efflux from late endosomes and lysosomes. Localized calcium release from such acidic stores is required for fusion between endocytic vesicles and to maintain organelle homeostasis. In fact, we found that fusion of autophagosomes with lysosomes is impaired in MCOLN1-deficient cells, thus leading to accumulation of protein aggregates and damaged organelles. Our work contributed to the current view that defective autophagy plays an important role in the disease pathogenesis of many LSDs. To gain insight into the molecular mechanisms that regulate MCOLN1 activity we searched for proteins that bind MCOLN1 though pull-down assays and split-ubiquitin yeast-two hybrid screening. These experiments allowed the identification of the penta-EF-hand protein ALG-2 and the LAPTM family of lysosomal transporters as novel interactors of MCOLN1. In collaboration with the group of Andrea Ballabio, we have recently described that the expression of MCOLN1 is regulated by TFEB, a transcription factor that promotes transcription of autophagic and lysosomal genes. Over-expression of TFEB leads to MCOLN1-mediated exocytosis of lysosomes and results in clearance of abnormal lysosomes in several LSDs, further confirming the role of MCOLN1 in organelle fusion. To further characterize the physiological role of MCOLN1, we have initiated the development of in vivo animal models. In particular, we have used specific Zinc Finger Nucleases (ZFN) to create stable genetically engineered zebrafish with gene deletion (knockouts) for MCOLN1. Some examples of specific questions we will focus on will be: (a) what is the pattern of expression of MCOLN1 in different tissues and stages of development, (b) what is the effect of depleting MCOLN1 in the function and development of various organs, (c) in the MCOLN1 knockout zebrafish, can we reproduce the MLIV phenotype and if so, what can we learn about the pathology of the disease. These and other important questions can be addressed by our experimental design, thus providing unparalleled insight in to the molecular function of MCOLN1, improving our understanding of MLIV, and opening new and exciting venues for the development of a treatment for the disease.