Oculocerebrorenal syndrome of Lowe is a rare X-linked disorder caused by mutation of the inositol 5-phosphatase OCRL1. Lowe Syndrome manifests as renal tubular dysfunction, neurological and ocular defects. OCRL1 uses its catalytic domain to hydrolyze two phosphoinositide species, PI(4,5)P2 and PI3,4,5)P3. It is involved in regulation of membrane trafficking, actin dynamics, cytokinesis and ciliogenesis. OCRL1 interacts with IPIP27A and B, which have been shown to be key players in endocytic trafficking in mammalian cells, specifically in the recycling of proteins from early and recycling endosomes to both the plasma membrane and trans-Golgi network. It has been proposed that defective endocytic trafficking may be responsible for the renal tubulopathy seen in Lowe Syndrome patients, characterized by low molecular weight proteinuria and aminoaciduria, but this hypothesis has yet to be tested. Using zebrafish as a model for Lowe syndrome, we show that depletion of OCRL1 can indeed cause defects in endocytosis in the renal tubule. This coincides with a reduction in levels of the multi-ligand receptor megalin, reduced abundance of the endocytic apparatus and increased numbers of enlarged lysosomes in the kidney tubular cells. We also show that knocking-down Pip5K in the OCRL1 mutants to rebalance PI(4,5)P2 levels can rescue the endocytic defect. This indicates that tight control of PI(4,5)P2 level is essential for efficient endocytic trafficking in vivo. Importantly, this finding suggests that Pip5K may be a valuable therapeutic target for patients with Lowe Syndrome. To further characterize the molecular mechanisms by which OCRL1 promotes endocytosis, we have focused on the recently identified OCRL1 interaction partners IPIP27A and B, which are known to function in endocytosis and receptor recycling. Here we report identification and characterization of the zebrafish Ipip27s, including analysis of conservation and expression profiles. To assess Ipip27s function in vivo, KO zebrafish lines were generated using TALENs. This was successful for Ipip27A, but so far not for Ipip27B. Functional analysis using the Ipip27A KO line and KD with morpholinos revealed that both Ipip27s contribute to neural development and may participate in ciliogenesis. Moreover, preliminary analysis indicates an important role for Ipip27A within the endocytic pathway in the kidney tubule, where its loss phenocopies many aspects of the OCRL1 mutant phenotype.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764293 |
| Date | January 2014 |
| Creators | Oltrabella, Francesca |
| Contributors | Lowe, Martin ; Hurlstone, Adam |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/investigation-of-ocrl1-and-its-interaction-partners-in-zebrafish(77a97cd8-f030-4c4e-93cc-fe4e2ea44ee6).html |
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