Inositol 5-Phosphatases in Primary Cilia Formation in Lowe syndrome

Abstract

 

Purpose

 

Inositol phosphatases are important regulators of cell signaling, polarity, and vesicular trafficking. Mutations in OCRL, an inositol polyphosphate 5-phosphatase, result in Oculocerebrorenal syndrome of Lowe—an X-linked recessive disorder that presents with congenital cataracts, glaucoma, renal dysfunction and mental retardation[1]. The function of OCRL in causing cataracts and glaucoma is not understood.

 

Methods

 

Using human eyes previous enucleated for uveal melanoma, we examined the distribution of OCRL and INPP5B in trabecular meshwork, lens epithelium, and ciliary body epithelium. We also examined the subcellular distribution of INPP5B by immunofluorescence in HTM and RPE cells. Using zebrafish embryos, antisense morpholinos against INPP5B and OCRL were used to examine the function of these 5-phosphatases in cilia formation.

 

Results

 

We have showed that endogenous OCRL is localized in the trabecular meshwork and Schlemm’s canal endothelial cells in human and murine eyes. We also showed that INPP5B, a paralog of OCRL, is expressed in low levels in human trabecular meshwork but is highly expressed in murine trabecular meshwork. We show that the knockdown of INPP5B resulted in a decrease in primary cilia formation using INPP5B specific shRNA in hTERT-RPE1 cell line. In a zebrafish model, INPP5B morpholino knockdown morphants presented a dose-dependent phenotype of microphthalmia, body axis asymmetry and kinked tail, which is a phenotype caused by Kupffer’s vesicle defect and also found in OCRL morphants. The decreased cilia in Kupffer vesicles of INPP5B morphants are shorter than control morphants, and cilia-dependent melanosome transport of INPP5B morphants delayed much longer when embryos exposed to epinephine. All these cilia related phenotypes could be rescued by co-injected with mouse INPP5B mRNA.

 

Conclusions

 

OCRL and INPP5B are differentially expressed in the human and murine eyes, which may explain the ocular phenotypes of Lowe syndrome. 1. Luo N, West CC, Murga-Zamalloa CA, Sun L, Anderson RM, et al. (2012) OCRL localizes to the primary cilium: a new role for cilia in Lowe syndrome. Human molecular genetics.

 

 

Fig 2. Generation of zebrafish morphants of INPP5B. A) Knockdown of INPP5B in zebrafish. B) microphthalmia and anophthalmia of INPP5B zebrafish morphants.

 

 

Fig 1. Expression of OCRL and INPP5B in trabecular meshwork in human eyes.

 

Keywords: 421 anterior segment • 539 genetics • 497 development