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。 我们和其他人提出，MCOLN1在细胞中的主要作用是介导晚期内小体和溶酶体的钙外流。细胞内小泡之间的融合和细胞器的动态平衡需要这些酸性物质的局部钙释放。事实上，我们发现在MCOLN1基因缺陷的细胞中，自噬小体与溶酶体的融合受到损害，从而导致蛋白质聚集体积累和细胞器受损。我们的工作促成了目前的观点，即缺陷自噬在许多LSD的发病机制中发挥着重要作用。为了深入了解调控MCOLN1活性的分子机制，我们通过下拉试验和裂解泛素酵母双杂交筛选寻找与MCOLN1结合的蛋白质。这些实验使五个EF-Hand蛋白ALG-2和LAPTM溶酶体转运蛋白家族被确定为MCOLN1的新的相互作用因子。 在Andrea Ballabio的合作中，我们描述了MCOLN1的表达受到TFEB的调控，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MCOLN1介导的溶酶体胞吐，并导致几个LSD中异常溶酶体的清除，进一步证实了MCOLN1在细胞器融合中的作用。最近，我们发现TFE3也显著上调了MCOLN1的表达。 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丢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，我们目前正在使用CRISPR-CAS9系统来产生mcoln1.2动物。总体而言，我们的结果表明mcoln1在早期胚胎发育中扮演了一个新的和意想不到的角色。 这些和其他重要的问题可以通过我们的实验设计来解决，从而对MCOLN1的分子功能提供了无与伦比的见解，提高了我们对MLIV的理解，并为开发这种疾病的治疗方法开辟了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 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. More recently we showed that the expression of MCOLN1 is also significantly upregulated by TFE3. 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 lost embryos, thus suggesting some level of redundancy between mcoln1.1 and mcoln1.2. To further confirm these observations we are currently using the CRISPR-Cas9 system to generate of mcoln1.2 animals. Overall, our results indicate a novel and unexpected role of mcoln1 during early embryonic development. 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.