Inhibition of VCP structurally and functionally reconstitutes photoreceptor cells in Rh^P23H organotypic retina cultures

Abstract

Purpose : Due to high production rates in visual pigment and high metabolic activity, photoreceptor neurons (PN) are especially vulnerable to defects in proteostasis. The structurally misfolded rhodopsin (Rh)^P23H exerts excessive stress on rods, causing adRP. The ATPase valosin-containing protein (VCP) acts as a quality control checkpoint of membrane proteins and binds to Rh before its release from the ER. This study evaluated the effect of VCP inhibition in the Rh^P23H rat in vitro

Methods : Retinal organ cultures from heterozygous Rh^P23H transgenic rats (SD-Tg(P23H)1Lav, RRRC) with the RPE attached, as previously described (Arango-Gonzalez et al. 2010), were incubated in supplemented serum-free medium and treated either with Eeyarestatin I (EerI) or with ML240. PN survival, as well as functional and structural integrity, was evaluated by cell row quantification, TUNEL assay, high-resolution light microscopy and electron microscopy (EM). One-way ANOVA followed by Tukey’s HSD test was used for statistical analysis. The impact of the treatment on the retinal visual responses was investigated by light stimulation and simultaneous ganglion cell recordings, utilizing multi-electrode arrays (MEA)

Results : The percentage of TUNEL(+) cells in the ONL indicates that VCP inhibition significantly reduced the number of dying cells (EerI: 1,70%±0,39, P<0,001; ML240: 2,23%±0,36, P<0,0001; control: 5,08%±0,41). More importantly, the ONL of EerI and ML240-treated Rh^P23H retinas contained more cell rows than the controls (EerI: 9,48±0,21, P<0.0222; ML240; 9,49±0,93, P<0.0063; control: 8,08±0,54). VCP inhibition also corrected the aberrant distribution of Rh, arrestin, and transducin in the Rh^P23H transgenic rat retina, restoring their physiological localization similar to that of the WT phenotype. Ultrastructural analysis by EM confirmed preservation of proper morphology within treated Rh^P23H rod outer segments (OS) and electrophysiological MEA recordings demonstrated improved responses to light stimulation in the treated Rh^P23H retina

Conclusions : VCP inhibition strongly rescues degenerating Rh^P23H rods and preserve the functional properties of the retina, suggesting VCP as a target to treat adRP. Protection correlates with restoration of physiological Rh trafficking to rod OS. This results in improved rod OS ultrastructure as well as an electrophysiological response in treated Rh^P23H retinas

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.