Understanding the molecular mechanisms underlying retina degeneration in INPP5E-Joubert Syndrome

Abstract

Purpose : Mutations in INPP5E are associated with Joubert syndrome and MORM disease (mental retardation, truncal obesity, retinal dystrophy and micropenis). Joubert syndrome is a syndromic ciliopathy that includes ataxia, hyperpnea, abnormal eye and tongue movements, polydactyly and retinitis pigmentosa. To date, the function of INPP5E is unclear. Germline deletion of INPP5E is embryonically lethal in mouse, therefore, we sought to study INPP5E function by generating retina- and photoreceptor-specific knockouts. Our goal is to identify the onset of degeneration and devise gene-based therapies to ameliorate or cure the retina disease.

Methods : Conditional Inpp5e knockout mice were generated by mating Inpp5e^flox/flox mice with Six3-Cre, iCre75, or inducible ET-Cre transgenic mice. Mating of floxed mice with various Cre-expressing mice excises Inpp5e exons 2-6 in photoreceptor progenitors, rods or mature photoreceptors, respectively. Deletion of exons 2-6 removes much of the INPP5E phosphatase domain. Retina phenotypes are subsequently characterized by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT), rotarod performance test, OptoMotry, confocal immunohistochemistry using a battery of outer segment-related antibodies, transmission electron microscopy and immunoblotting.

Results : INPP5E specifically converts PI(3,4,5)P₃-->PI(3,4)P₂ and PI(4,5)P₂ to PI(4)P. PI(3,4,5)P₃ and PI(4,5)P₂ are plasma membrane enriched, whereas PI(4)P is enriched at the Golgi and at the primary cilium. Using qRT/PCR, we found that INPP5E is abundantly expressed in the retina, ten- to twenty-fold higher than in trachea or testis. INPP5E is farnesylated at the C-terminus, a posttranslational modification that provides membrane anchoring. In WT mouse rods, INPP5E immunolocalizes to inner segments and is absent in the outer segment, connecting cilium and basal body. As absence of INPP5E in rod outer segments is controversial, we expressed EGFP-INPP5E in rods by neonatal electroporation. The results show that INPP5E is excluded from WT outer segments and distributes to the ER surrounding the nucleus and Golgi apparatus, colocalizing with the Golgi marker, anti-giantin.

Conclusions : The role of INPP5E at the Golgi is unclear. While we hypothesize that INPP5E may be required for endosomal trafficking and ciliogenesis, INPP5E substrates PI(3,4,5)P₃ and PI(4,5)P₂ may be involved in actin remodeling.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.