The thioredoxin encoded by the Rod-derived Cone Viability Factor gene protects cones against oxidative stress

Abstract

Purpose: Rod-derived cone viability factor (RdCVF) is an alternative splice product of the Nucleoredoxin-like-1 (Nxnl1) gene which also encodes for an enzymatically active thioredoxin RdCVFL. RdCVF is secreted by rods and protects cones in several rodent models of retinitis pigmentosa. While the action of RdCVF on cones relies on its cell surface receptor, it is unclear if Nxnl1 has any cell autonomous role in cones.

Methods: By homologous recombination, we produced the [Cone-Cre : Nxnl1^cd/cd] mouse model, with Nxnl1 recombination specifically in cones. [Cone-Cre : Nxnl1^cd/cd] mice and their littermates [Cone-Cre : Nxnl^+/+] were housed 1 week in 75% or 21% O₂ at 1-month-old or alternatively housed in 21% O₂ to 8-month-old of age. Cone photopic and flicker electroretinogram (ERG) were then recorded. All mice were sacrificed, their eyes enucleated, retinas dissected to proceed to immunolabeling for cone counting. [Cone-Cre : Nxnl1^cd/cd] mice at postnatal 20 days (PN20) were sub-retinally injected AAV2.8-RdCVFL or AAV2.8-RdCVF or AAV2.8-GFP, housed at PN35 for 5 days in 75% O₂. The ERG photopic, flicker activities and cone density were measured at PN40. In parallel, adult [Cone-Cre : Nxnl1^+/+] mice were intravitreally injected AAV2-7m8 Mopsin-eGFP. Forty days later, retinas were dissociated to pick single GFP positive cone cells using a micropipette. The expression of genes in single cone was analyzed by RT-PCR. Cone-enriched chicken cultures made from chicken embryos were electroporated by siRdCVFL or siScrumble. After 7 days, cell survival was measured using a RFP reporter.

Results: In [Cone-Cre : Nxnl1^cd/cd] mice, we observed an age-related reduction of the cone function, correlated with cone loss. This loss of cone function was accelerated by maintaining the animals under hyperoxic condition (75% O₂). The loss of cone function in [Cone-Cre : Nxnl1^cd/cd] induced by hyperoxia was rescued by sub-retinal injection of AAV2.8-RdCVFL, but not by AAV2.8-RdCVF. Single cell RT-PCR confirmed that RdCVFL is expressed by cones. In addition, in cone-enriched chicken cultures made from chicken embryos, silencing of RdCVFL reduced cell viability.

Conclusions: Our results demonstrate that RdCVFL expression by cones is essential for their resistance against age-dependent oxidation.