Endocytic Trafficking and Human Diseases

内吞贩运与人类疾病

• 批准号: 9157301
• 负责人: Rosa Puertollano-Moro
• 金额: $ 47.24万
• 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/9157301
• 关键词: Acute; Animal Model; Animals; Antigen Presentation; Apoptosis; Birth; Bone remodeling; Cations; Cell Death; Cell membrane; Cell physiology; Cells; Characteristics; Cholesterol Homeostasis; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Cornea; Cytoplasm; Defect; Disease; Disease model; Down-Regulation; Embryo; Eye; Eye Abnormalities; Fishes; Functional disorder; Ganglioside Sialidase Deficiency Disease; Glycosaminoglycans; In Situ Nick-End Labeling; Ion Channel; Knock-out; Laboratories; Lipids; Lysosomes; Mediating; Membrane; Metabolic Diseases; Molecular; Muscle; Mutation; Optic Atrophy; Organelles; Organism; Orthologous Gene; Pathology; Patients; Phenotype; Preclinical Drug Evaluation; Prevalence; Proteins; Retinal Degeneration; Staining method; Stains; Strabismus; Study models; Surface; Technology; Tissues; Variant; Visual; Zebrafish; Zinc Fingers; base; genome editing; human disease; nuclease; patch clamp; receptor; repaired; trafficking

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.

IV 型粘脂沉积症 (MLIV) 是一种常染色体隐性遗传疾病，其特征为急性精神运动迟缓、胃酸缺乏和视觉异常，包括视网膜变性、角膜混浊、视神经萎缩和斜视。 MLIV 患者的大多数组织中均发现溶酶体包涵体。储存材料的成分是异质的，包括形成特征性多同心片层的脂质和粘多糖，以及可溶性颗粒状蛋白质。 MLIV 是由 mucolipin-1（MCOLN1，也称为 TRPML1）突变引起的，粘蛋白-1 是一种内溶酶体阳离子通道，属于离子通道瞬时受体电位 (TRP) 超家族。全细胞膜片钳以及天然内溶酶体膜的记录表明，MCOLN1 充当可渗透 Ca2+、Na+、K+ 和 Fe2+/Mn2+ 的内部（从腔到细胞质）整流通道，其活性因低 pH 值而增强。 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建立 MLIV 疾病模型。已鉴定出两种假定的斑马鱼 MCOLN1 直系同源物：mcoln1.1 和 mcoln1.2。通过使用特定的锌指核酸酶 (ZFN)，我们成功创建了两个独立的 mcoln1.1 敲除系。 mcoln1.1 纯合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 染色确认细胞死亡为细胞凋亡。当 mcoln1.1-/- 鱼胚胎注射 mcoln1.2 吗啉时，观察到的表型变得更加明显，并且在 mcoln1 无效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，我们利用最近开发的 CRISPR/Cas9 介导的基因组编辑技术来生成 mcoln1.2-KO 和 mcoln1.1/mcoln1.2 双 KO 系。这些斑马鱼系的初步特征表明，mcoln1.1/mcoln1.2 双 KO 动物再现了 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系构成了 MLIV 研究的有价值的模型。此外，该动物模型有可能促进大规模药物筛选以及治疗 MLIV 的临床前药物评价。