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. To better understand the pathology of this disease, we aimed to generate a MLIV disease model in zebrafish. Two putative zebrafish MCOLN1 co-orthologs have been identified, mcoln1.1 and mcoln1.2. By using specific Zinc Finger Nucleases (ZFN), we successfully created two independent mcoln1.1 knockout lines. Initial characterization of mcoln1.1 homozygous null embryos revealed noticeable cell death in the eye. Cell death was confirmed as cell apoptosis by TUNEL staining in both mcoln1.1 knockout lines. When mcoln1.1-/- fish embryos were injected with mcoln1.2 morpholino, the observed phenotype become even more apparent and increased apotosis was detected in the whole body of the mcoln1 null embryos, thus suggesting some level of redundancy between mcoln1.1 and mcoln1.2. To further confirm these observations we have made use of the recently developed CRISPR/Cas9-mediated genome editing technology to generate mcoln1.2-KO and mcoln1.1/mcoln1.2 double KO lines. Initial characterization of these zebrafish lines indicates that the mcoln1.1/mcoln1.2 double KO animals reproduce many of the abnormalities observed in MLIV patients, including eye abnormalities and muscle defects. These results indicate that our zebrafish line constitutes a valuable model for the study of MLIV. Moreover, this animal model has the potential to facilitate large-scale drug screening as well as preclinical drug evaluation for the treatment of MLIV.