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.

Mutations of the gene encoding the inositol polyphosphate 5-phosphatase OCRL1 are responsible for causing two disorders in humans: Lowe syndrome and type 2 Dent's disease (Dent-2). Lowe syndrome (oculocerebrorenal syndrome of Lowe) is an X-linked genetic disorder that causes multisystem defects affecting predominantly the eyes, brain and kidneys. Dent-2 disease is very similar to Lowe syndrome, but it affects primarily the kidneys with little or no symptoms in the brain and eyes. The enzymatic activity, structure and binding partners of the OCRL1 protein have been described and progress on the cellular functions of OCRL1 has been made. However the studies to date have not provided the necessary insight to explain the tissue-specific defects observed in Lowe syndrome and Dent-2 patients. In order to investigate the role of OCRL1 and the consequences of its deficiency in a physiological context an animal model is required. We have chosen the zebrafish for this study due to its suitability for investigating vertebrate early development and the abundance of research techniques available for this model organism. We have studied the expression of OCRL1 in zebrafish and its role in the early embryonic development. We have also investigated its role in the endocytic function of the zebrafish larval pronephric kidney. Finally we have investigated its role in ciliogenesis and function of pronephric cilia. Our studies show that OCRL1 depletion does not cause gross developmental defects, nor affects the development of pronephros, but impairs their endocytic activity. We have also shown, that efficient pronephric uptake requires OCRL1 interactions with clathrin, Rab GTPase family proteins, APPL1 and IPIP27A/B. Our studies link the reduced uptake with lowered levels of megalin receptor, which is responsible for the bulk of protein reabsorption in the kidney. Together our results strongly suggest that defects in this process are responsible for low molecular weight proteinuria present in Lowe syndrome and Dent-2 patients and zebrafish is a suitable model to study the renal aspect of these diseases.

Le gène OCRL1, responsable du syndrome de Lowe de transmission récessive liée à l'X, code pour une PIP2 5-phosphatase associée à l'appareil de Golgi. 107 mutations du gène OCRL1 ont été identifiées chez 146 familles de syndrome de Lowe et 6 mutations ont été identifiées chez 23 patients atteints de maladie de Dent sans mutation du gène CLCN5. L'haplotypage de 18 familles a mis en évidence 3 cas de mosaïcisme germinal et somatique. L'activité PIP2 5-phosphatase fibroblastique des patients est très abaissée par rapport à des fibroblastes normaux. L'analyse par Western blot de la protéine OCRL1 montre une diminution variable de la quantité protéique et pas forcément corrélée à l'activité résiduelle de la protéine. L'analyse des transcrits d'OCRL1 montre qu'il existe une variabilité quantitative chez les patients mais également chez les témoins. Les premières études cliniques chez 55 patients atteints de syndrome de Lowe ne montrent pas de corrélation génotype-phénotype évidente.

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