Re-expression of miR-183/96/182 in Photoreceptors Rescues Their Functional Defects in miR-183/96/182 Knockout Mice

Abstract

Purpose : The miR-183/96/182 cluster (miR-183/96/182) is expressed in all sensory organs. In retina, it is expressed in all photoreceptors after birth, peaking in the adult, suggesting importance in photoreceptor postnatal functional maturation. Inactivation of miR-183/96/182 in mice (miR-183C^GT/GT) resulted in congenital syndromic retinal dystrophy affecting both rods and cones. We hypothesize that defects in rods and cones are intrinsic to loss of their endogenous miR-183/96/182, but not secondary to each other nor to systemic metabolic defect. The purpose of current study is to test this hypothesis.

Methods : Cone-specific knockout (ko) mice (LMOP-cre;miR-183C^GT/GT) were created by breeding miR-183C^GT/+ with rod-specific Cre mice, long mouse opsin promoter (LMOP)-cre (kindly provided by Dr. Yun-zheng Le at UOHSC). Rod- and M-cone-specific ko (BP-Cre;miR-183C^GT/GT) were produced by breeding S cone-specific Cre (blue pigment gene promoter (BP)-cre (kindly provided by Dr. Anand Swaroop, NEI) with miR-183C^GT/+; while rod- and S-cone-specific ko mice, HRGP-cre;miR-183C^GT/GT, by breeding human red/green pigment gene promoter (HRGP)-cre (provided by Dr. Le) with miR-183C^GT/+. Electroretinogram (ERG) was performed on 5-wk-old mice. Total retinal RNA was isolated for qRT-PCR. Retinal sections were prepared for immunohistological studies.

Results : 1) In LMOP-cre;miR-183C^GT/GT mice, rod-specific Cre induced rod-specific inversion of the genetrap (GT) construct in the miR-183C^GT allele and re-expression of miR-183/96/182 in rods, and rescued defects in scotopic ERG of miR-183C^GT/GT mice, but had no effect on photopic ERG; 2) In BP-Cre;miR-183C^GT/GT and HRGP-cre;miR-183C^GT/GT mice, S- and M-cone-specific expression of Cre partially rescued defects of photopic ERG, with little impact on scotopic ERG; 3) In all double transgenic mice, functional defects in other sensory organs persisted; 4) photoreceptor-specific expression of Cre appeared to have negative impact on ERG b-wave amplitudes.

Conclusions : miR-183/96/182 is intrinsically required for normal development and functions of both rods and cones. Defects in photoreceptors in miR-183C^GT/GT mice are results of loss of their endogenous miR-183/96/182, but not due to systemic metabolic abnormality. Caution should be taken when using cell-type specific ko created by crossing with Cre transgenic lines.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.