The tumor suppressor PTEN dephosphorylates PtdIns(3,4,5)P3 into PtdIns(4,5)P2. Here, we make the unexpected discovery that in Drosophila melanogaster PTEN reduces PtdIns(4,5)P2 levels on endosomes, independently of its phosphatase activity. This new PTEN function requires the enzymatic action of dPLCXD, an atypical phospholipase C. Importantly, we discovered that this novel PTEN/dPLCXD pathway can compensate for depletion of dOCRL, a PtdIns(4,5)P2 phosphatase. Mutation of OCRL1, the human orthologue of dOCRL, causes oculocerebrorenal Lowe syndrome, a rare multisystemic genetic disease. Both OCRL1 and dOCRL loss have been shown to promote accumulation of PtdIns(4,5)P2 on endosomes and cytokinesis defects. Here, we show that PTEN or dPLCXD overexpression prevents these defects. In addition, we found that chemical activation of this pathway restores normal cytokinesis in human Lowe syndrome cells and rescues OCRL phenotypes in a zebrafish Lowe syndrome model. Our findings identify a novel PTEN/dPLCXD pathway that controls PtdIns(4,5)P2 levels on endosomes. They also point to a potential new strategy for the treatment of Lowe syndrome.
The zebrafish is an important animal system for modelling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. Zebrafish also have a high fecundity, with females capable of laying 200-300 eggs per week, thereby facilitating chemical and mutation screening. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is lacking. Coupling the advantages of the zebrafish system with a tool to measure proteinuria will advance the scope for testing the efficacy of drugs to treat kidney diseases. Here, we generated a stable transgenic zebrafish line using the l-fabp10 liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated-Protein (RAP) to create NL-D3. In the healthy state, NL-D3 is excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 presence in the urine increased. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, col4a4, and col4a5) also induced NL-D3 proteinuria. Furthermore, we found that treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria. Our findings confirm the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria and thereby prioritise candidates for further translational studies.
Endocytosis is a fundamentally important process through which material is internalized into cells from the extracellular environment. In the renal proximal tubule, endocytosis of the abundant scavenger receptor megalin and its co-receptor cubilin play a vital role in retrieving low molecular weight proteins from the renal filtrate. Although we know much about megalin and its ligands, the machinery and mechanisms by which the receptor is trafficked through the endosomal system remain poorly defined. In this study, we show that Ipip27A, an interacting partner of the Lowe syndrome protein OCRL, is required for endocytic traffic of megalin within the proximal renal tubule of zebrafish larvae. Knockout of Ipip27A phenocopies the endocytic phenotype seen upon loss of OCRL, with a deficit in uptake of both fluid-phase and protein cargo, which is accompanied by a reduction in megalin abundance and altered endosome morphology. Rescue and co-depletion experiments indicate that Ipip27A functions together with OCRL to support proximal tubule endocytosis. The results therefore identify Ipip27A as a new player in endocytic traffic in the proximal tubule in vivo and support the view that defective endocytosis underlies the renal tubulopathy in Lowe syndrome and Dent-2 disease.
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