Endocytic Trafficking and Human Diseases

内吞贩运与人类疾病

• 批准号: 8939746
• 负责人: Rosa Puertollano-Moro
• 金额: $ 42.87万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8939746
• 关键词: Acute; Address; Animals; Antigen Presentation; Apoptosis; Autophagocytosis; Autophagosome; Biological Assay; Birth; Bone remodeling; Calcium; Cations; Cell Death; Cell membrane; Cell physiology; Cells; Characteristics; Cholesterol Homeostasis; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Cornea; Cytoplasm; Development; Disease; Disease model; Down-Regulation; EF Hand Motifs; Embryo; Embryonic Development; Endocytic Vesicle; Exocytosis; Experimental Designs; Eye; Family; Fishes; Functional disorder; Ganglioside Sialidase Deficiency Disease; Genes; Genetic Transcription; Glycosaminoglycans; Homeostasis; In Situ Nick-End Labeling; Ion Channel; Knock-out; Laboratories; Lipids; Lysosomes; Mediating; Membrane; Metabolic Diseases; Molecular; Mutation; Optic Atrophy; Organelles; Organism; Orthologous Gene; Pathogenesis; Pathology; Patients; Pentas; Phenotype; Play; Prevalence; Protein Binding; Proteins; Retinal Degeneration; Role; Staining method; Stains; Strabismus; Surface; System; TFE3 gene; Tissues; Ubiquitin; Variant; Visual; Work; Yeasts; Zebrafish; Zinc Fingers; base; human disease; improved; insight; late endosome; novel; nuclease; patch clamp; protein aggregate; receptor; repaired; research study; screening; trafficking; transcription factor; yeast two hybrid system

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.

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。最近，我们发现MCOLN 1的表达也被TFE 3显著上调。 为了更好地了解这种疾病的病理学，我们的目标是在斑马鱼中产生MLIV疾病模型。已经鉴定了两种假定的斑马鱼MCOLN 1共同直系同源物，mcoln1.1和mcoln1.2。通过使用特异性锌指核酸酶（ZFN），我们成功地创建了两个独立的mcoln1.1敲除系。mcoln1.1纯合子无效胚胎的初步表征显示眼睛中有明显的细胞死亡。在两个mcoln1.1敲除系中通过TUNEL染色证实细胞死亡为细胞凋亡。当用mcoln1.2吗啉代注射mcoln1.1-/-鱼胚胎时，观察到的表型变得更加明显，并且在mcoln 1丢失胚胎的整个身体中检测到细胞凋亡增加，从而表明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，我们目前正在使用CRISPR-Cas9系统来产生mcoln1.2动物。总的来说，我们的研究结果表明，一个新的和意想不到的作用mcoln 1在早期胚胎发育。 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。