Endocytic Trafficking and Human Diseases

黏脂质沉积症IV型（MLIV）是一种常染色体隐性遗传病，其特征为急性精神运动迟缓、失速和视觉异常，包括视网膜变性、角膜混浊、视神经萎缩和斜视。在大多数MLIV患者的组织中发现溶酶体包涵体。储存材料的组成是不均匀的，包括脂质和粘多糖形成特征的多同心片层，以及可溶性的颗粒状蛋白质。MLIV是由粘磷脂-1 （MCOLN1，也称为TRPML1）突变引起的，粘磷脂-1是一种内溶酶体阳离子通道，属于瞬时受体电位（TRP）离子通道超家族。全细胞膜片钳和天然内溶酶体膜的记录表明，MCOLN1作为一个向内（从管腔到细胞质）的整流通道，可渗透到Ca2+、Na+、K+和Fe2+/ Mn2+，其活性在低pH下增强。

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.