Endocytic Trafficking and Human Diseases

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

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.