Lowe syndrome is a rare X linked disorder, characterized by renal, ocular and cerebral defects, caused by mutation in the protein OCRL1. OCRL1 has been implicated in a plethora of cellular functions, and loss of its catalytic conversion of PtdIns(4,5)P2 into PtdIns(4)P is proposed to underly many of the cellular phenotypes associated with lack of OCRL1. The interaction with other proteins such as IPIP27A are also required for the correct function of OCRL1. Renal tubular dysfunction similar to that seen in Lowe syndrome patients is seen in zebrafish models of ocrl1 and ipip27a mutation. Using zebrafish as a model of Lowe syndrome, a reduction of increased PtdIns(4,5)P2 levels in ocrl1-/- embryos is shown to alleviate the renal tubular dysfunction. This demonstrates that targeting PtdIns(4,5)P2 is a viable option for therapeutic treatment of Lowe syndrome. Novel transgenic zebrafish lines are also described, that provide megalin specific, fluorescent and luminescent readouts of proximal tubular endocytic function. These will be an important tool to perform high throughput screens for compounds that alleviate the symptoms of Lowe syndrome. The importance of the binding of IPIP27A to its interaction partners OCRL1 and SH3 containing proteins such as PACSIN2 is demonstrated by rescue of the ipip27a-/- mutant with ipip27a with mutated binding sites. The phenotype of ipip27a-/- mutant embryos is further characterised to demonstrate there is no long term growth defect or defect in tubular polarity, however tubular dilation is seen, suggesting possible mild ciliary defects. In the zebrafish proximal tubule in fish with no functional IPIP27A or OCRL1, a more severe defect in 10 kDa dextran endocytosis is seen, as well as hydrocephaly and curved body axis, cilia impairment related phenotypes. This indicates that IPIP27A and OCRL1 are acting in the same pathway, and therefore depletion of both exacerbates phenotypes. Finally, transgenic lines expressing ubiquitous or pronephric tubule specific fluorescently tagged Rab proteins as markers of membrane compartments in zebrafish are described.
|Date of Award||1 Aug 2017|
- The University of Manchester
|Supervisor||Martin Lowe (Supervisor) & Adam Hurlstone (Supervisor)|