Retinal gene expression analysis of mouse models of RP59

Abstract

Purpose : RP59 is due to mutations in the essential gene, dehydrodolichyl diphosphate synthase (DHDDS), which is ultimately required for protein N-glycosylation. A subset of DHDDS mutations cause non-syndromic RP. To identify genes and pathways that are affected by disruption of the Dhdds locus, we analyzed the retina RNA profiles of two RP59 mouse models, in comparison to controls.

Methods : Total RNA was isolated from the retinas (2/sample) of N=3 mice/genotype: Dhdds^K42E/K42E knock-in (KI, 4 wks.; an RP59 mutation); Dhdds^flx/flx Rho iCre (KO; rod-specific Dhdds knockout, 4 wks.); Dhdds^flx/flx without iCre littermates; and WT (C57Bl/6J) controls. RNA was analyzed using the NanoString platform by the UAB Nanostring Core Laboratory. Data from three panels were analyzed: Neuropathology (770 genes), Glial (770 genes), and a Custom Panel (479 genes) with retina- and glycosylation pathway-related genes. Data were combined and analyzed using NanoString software nSolver 4.0 and advanced analysis 2.0, setting a fold-change cutoff of 2.0. In Situ Hybridization (ISH) and cytokine array analysis were performed to validate transcript/protein level increases of selected target genes. Statistical analysis: Student’s t-test; significance threshold, P<0.001.

Results : Excluding overlap among panels, 1466 unique genes were analyzed, resulting in 11 up- (range: 2- to 6-fold) and 31 down-regulated (range: -2 to -131-fold) transcripts identified in KI retinas and 89 up- (2- to 15-fold) and 28-down-regulated (range: -2 to -8-fold) transcripts found in KO retinas. Pathway analysis showed significant changes in the adaptive immune system, intracellular signaling by second messengers and cytokines, toll receptor cascades, extracellular matrix reorganization, and neutrophil degranulation genes. In KI retinas, significant changes were seen in neuronal systems, signaling by nuclear receptors and rho GTPases, and vesicle-mediated transport pathways genes. ISH analysis suggested upregulation of C1qb, an inflammatory marker, in activated microglia.

Conclusions : The results are consistent with the previously observed early degenerative changes, neuroinflammation and cytokine upregulation observed in rod-specific Dhdds KO retinas and the physiologic changes previously observed in DhddsK42E/K42E KI mice. Since several of the identified genes yield proteins that become glycosylated, they may be candidates for primary and secondary effects relevant to altered Dhdds function.

This is a 2021 ARVO Annual Meeting abstract.