INVESTIGATION OF OCRL1 AND ITS INTERACTION PARTNERS IN ZEBRAFISH

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Abstract

Oculocerebrorenal syndrome of Lowe is a rare X-linked disorder caused bymutation of the inositol 5-phosphatase OCRL1. Lowe Syndrome manifests as renaltubular dysfunction, neurological and ocular defects. OCRL1 uses its catalyticdomain to hydrolyze two phosphoinositide species, PI(4,5)P2 and PI3,4,5)P3. It isinvolved in regulation of membrane trafficking, actin dynamics, cytokinesis andciliogenesis. OCRL1 interacts with IPIP27A and B, which have been shown to bekey players in endocytic trafficking in mammalian cells, specifically in the recyclingof proteins from early and recycling endosomes to both the plasma membrane andtrans-Golgi network. It has been proposed that defective endocytic trafficking maybe responsible for the renal tubulopathy seen in Lowe Syndrome patients,characterized by low molecular weight proteinuria and aminoaciduria, but thishypothesis has yet to be tested.Using zebrafish as a model for Lowe syndrome, we show that depletion ofOcrl1 can indeed cause defects in endocytosis in the renal tubule. This coincideswith a reduction in levels of the multi-ligand receptor megalin, reduced abundanceof the endocytic apparatus and increased numbers of enlarged lysosomes in thekidney tubular cells. We also show that knocking-down Pip5K in the Ocrl1 mutantsto rebalance PI(4,5)P2 levels can rescue the endocytic defect. This indicates thattight 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 targetfor patients with Lowe Syndrome.To further characterize the molecular mechanisms by which OCRL1 promotesendocytosis, we have focused on the recently identified Ocrl1 interaction partnersIPIP27A and B, which are known to function in endocytosis and receptor recycling.Here we report identification and characterization of the zebrafish Ipip27s, includinganalysis of conservation and expression profiles. To assess Ipip27s function invivo, KO zebrafish lines were generated using TALENs. This was successful forIpip27A, but so far not for Ipip27B. Functional analysis using the Ipip27A KO lineand KD with morpholinos revealed that both Ipip27s contribute to neuraldevelopment and may participate in ciliogenesis. Moreover, preliminary analysisindicates an important role for Ipip27A within the endocytic pathway in the kidneytubule, where its loss phenocopies many aspects of the Ocrl1 mutant phenotype.

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OCRL1; ZEBRAFISH

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