Pharmacological inhibition or genetic inactivation of ERAD effector VCP suppresses Rho^ΔI255-associated retinal degeneration

Abstract

Purpose : Deletion of the isoleucine (I) monomer at codon 255 within the rhodopsin gene (Rho^ΔI255) causes autosomal dominant retinitis pigmentosa (adRP). This study aimed to elucidate the pathomechanism associated with this adRP and study the effects of pharmacological inhibition or silencing expression of the ERAD effector, valosin-containing protein (VCP).

Methods : HEK293 and COS-7 cells were transfected with plasmids encoding Rho^WT and Rho^ΔI255 tagged with EGFP. Rhodopsin protein (RHO) degradation along the ERAD pathway was analyzed via immunostaining. RHO clearance following VCP inhibition by ML240 was assessed using Western blot. Retinal explants from heterozygous Rho^ΔI255 (Rho^ΔI255/+) mice were treated with ML240. As an alternative way to inhibit VCP, we downregulated protein expression by using reverse magnetofection-transduced siRNA. RHO localization was then evaluated by immunostaining. In retinal explants, photoreceptor (PR) survival was analyzed using TUNEL staining and cell row counting of the outer nuclear layer (ONL). Gliosis and retinal microglial activation before and after VCP inhibition were evaluated by GFAP and Iba-1staining in retinal explants.

Results : In contrast to normal RHO, the RHO^ΔI255 was found aggregated in the perinuclear region of transfecting cells, trapping the normal isoform in perinuclear aggregates. RHO^ΔI255 aggregates were colocalized with the ERAD markers calnexin, VCP, ubiquitin, and PSMB5. Inhibition of VCP impaired the clearance of RHO^ΔI255 by ERAD, increasing the generation of high-molecular-weight aggregates. In Rho^ΔI255/+ retinal explants, VCP inhibition and VCP silencing significantly increased ONL layers, reduced the number of TUNEL-positive cells (ML240 0.38 ± 0.15, control: 1.69 ± 0.43; P<0.01/ VCP siRNA 0.83 ± 0.16, scrambled siRNA 3.38 ± 1.25; P<0.01), improved the correct RHO targeting, and increased OS thickness (ML240 10.51 ± 3.80, control: 5.57± 1.19; P<0.001/ VCP siRNA 26.63 ± 4.83, scrambled siRNA 15.20 ± 2.59; P<0.001). Moreover, GFAP staining and microglial migration in the ONL layer were both reduced in response to VCP inhibition.

Conclusions : Mutant RHO^ΔI255 traps normal RHO at the ER and is degraded by ERAD with the requirement of VCP. Pharmacological or genetic inhibition of VCP releases RHO and rescues rod PR from cell death, enhances the correct distribution of RHO in the OS, and decreases microglia activation.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.