Endocytic Trafficking and Human Diseases

IV型黏液脂蛋白沉积症(MLIV)是一种常染色体隐性遗传病，以急性精神运动迟缓、精神萎缩症和视觉异常为特征，包括视网膜变性、角膜混浊、视神经萎缩和斜视。MLIV患者的大部分组织中均可见溶酶体包涵体。储存材料的组成是异质的，包括形成特征的多同心片层的脂类和粘多糖，以及可溶的颗粒状蛋白质。MLIV是由粘蛋白-1(MCOLN1，也称为TRPML1)突变引起的，MCOLN1是一种内溶酶体阳离子通道，属于瞬时受体电位(Trp)超家族离子通道。全细胞膜片钳和天然内溶酶体膜的记录表明，MCOLN1是一种内向(从管腔到细胞质)的整流通道，可以通向钙、钠、钾和Fe2+/Mn2+，其活性在低pH值下被增强。 为了更好地了解这种疾病的病理，我们的目标是在斑马鱼中建立MLIV疾病模型。已经确定了两个可能的斑马鱼MCOLN1共同源基因，mcoln1.1和mcoln1.2。通过使用特定的锌指核酸酶(ZFN)，我们成功地创建了两个独立的mcoln1.1基因敲除系。Mcoln1.1纯合子空胚胎的初步特征显示眼睛有明显的细胞死亡。两种mcoln1.1基因敲除系的TUNEL染色证实细胞死亡为细胞凋亡。当mcoln1.1-/-FISH胚胎被注射mcoln1.2吗啉时，观察到的表型变得更加明显，并在mcoln1缺失的胚胎全身检测到更多的细胞凋亡，这表明mcoln1.1和mcoln1.2之间存在一定程度的冗余。为了进一步证实这些观察结果，我们利用最近发展起来的CRISPR/Cas9介导的基因组编辑技术来产生mcoln1.2-KO和mcoln1.1/mcoln1.2双KO系。对这些斑马鱼品系的初步特征表明，mcoln1.1/mcoln1.2双重KO动物复制了MLIV患者中观察到的许多异常，包括眼睛异常和肌肉缺陷。这些结果表明我们的斑马鱼品系为MLIV的研究提供了一个有价值的模型。此外，该动物模型具有促进大规模药物筛选以及治疗MLIV的临床前药物评估的潜力。

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.