Perturbation of endocytic trafficking in rods causes retinitis pigmentosa-like phenotypes and non-A2E lipofuscin accumulation

Abstract

Purpose : The mislocalization of photoreceptor components is a hallmark of several retinitis pigmentosa (RP) mouse models. We know little about how mistrafficked components are degraded in rods and the consequences of the undigestible waste buildup. To test whether the impaired endolysosomal system in rods causes RP-like phenotypes, we characterized a novel mouse line with the Vps35 gene deleted in rods. VPS35 is the core of the retromer complex, which is expressed in the early endosome and serves as a hub that centrally controls several intracellular trafficking pathways.

Methods : Rod-specific VPS35 knockout mouse line was generated by crossing rod-specific Cre line iCre75 and the Vps35-floxed mice. Age-matched iCre75+/-; VPS35f/f (KO) and iCre75-/-; VPS35f/f (Ctrl) littermates were analyzed. Micron III-OCT2 and Spectralis HRA+OCT were used for fundus imaging. Espion e2 was used for electroretinogram (ERG). Focused Ion-beam scanning electron microscopy (FIB-SEM) was used for ultrastructural characterization. Retinoids were quantified using HPLC. Histological, immunochemical, qPCR and immunoblotting assays were also used to analyze these mice.

Results : The rod-specific loss of VPS35 in KO mice was confirmed by immunostaining. KO mice exhibited reduced ERG signals and underwent early retinal degeneration. VPS35-KO rods had significantly increased protein degradation but accumulated lipid-membrane wastes. The KO terminals released abundant lipofuscin granules and multilaminar bodies. Microglia had lipofuscins engulfed were activated, migrated to the subretinal space, and expressed bright autofluorescence. Superhigh-resolution 3D scanning EM did not detect any ingested outer segments in the subretinal microglia. The activated microglia in KO were detected as bright fluorescent dots by fundoscopy, whereas Ctrl mice only had weak and diffuse AF signals. HPLC quantification showed Ctrl and KO mouse eyes expressed comparable levels of A2E, ruling out A2E as a major contributor to the increased AF in the KO mouse retinas.

Conclusions : Rods with perturbed endosomes have early synaptic terminal loss preceding cell death. VPS35 deficiency in rods causes dysregulated protein trafficking, outer segment genesis, autophagic flux, and lipid degradation. Lipofuscins accumulated in microglia, rather than increased bisretinoids, represent the fundoscopy-detected AF dots in these mice.

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