Use of Pentachloropsuedilin (PCIP) as a novel tool to assess in vivo the molecular mechanisms of Rhodopsin trafficking in mice.

Abstract

Purpose : Retinal degeneration is irreversible and can be seen with various defective pathways. Loss of the unconventional motor protein MYO1C demonstrated dysregulated interactions with rhodopsin in mouse retina, resulting in rhodopsin mislocalization and subsequent retinal phenotypes, similar to those observed in Retinitis Pigmentosa (RP) or Usher Syndrome (USH). To determine the impact of MYO1C inhibition in rhodopsin trafficking, we utilized a specific MYO1C inhibitor, PCIP, and inquired about its interaction with MYO1C, impact under in-vitro and in-vivo conditions in cell lines and WT/C57B6 mice.

Methods : MYO1C and Rhodopsin interaction was analyzed with Haddock software. PyRx software was used to assess PCIP interaction with MYO1C. Cos1 cells were co-transfected with GFP-Rhodopsin and mCherry-Myo1c plasmids, and then treated with PCIP or PBS. FRAP was used to study the kinetics of MYO1C in Rhodopsin foci movement. WT/C57B6 mice received intravitreal injections of either PCIP or PBS. Retinal function was tested with ERG, and Rhodopsin localization was monitored with immunofluorescence and confocal microscopy.

Results : Haddock software showed that the MYO1C Post-IQ region interacts with Rhodopsin. Similarly, Pyrx software showed PCIP interaction with the MYO1C ATPase-domain. PCIP-treated Cos1 cells demonstrated Rhodopsin aggregation and reduced overlap of fluorescent intensity, while also displaying a lower mobility on FRAP compared to PBS control. WT/C57 mice treated with PCIP demonstrated reduced a-wave and b-wave amplitudes in scotopic and PhNR ERGs, along with a reduced Rhodopsin response recovery on ERG. However, Oscillatory potential ERG were not significantly altered. Morphologically, PCIP exposure also resulted in significantly thinner ONL and OS layers compared to PBS exposure.

Conclusions : The limitations of non-genetically-modified models to study the retinal degeneration phenotypic of RP or USH necessitates the urgency to find the molecular mechanism of Rhodopsin mislocalization via allosteric inhibitors like PCIP. PCIP directly impairs MYO1C ATPase function and results in rhodopsin mistrafficking in photoreceptors and ability to recover and also imparts Rhodopsin aggregation in photoreceptor inner segments. Ultimately, PCIP can be used to discern the in vivo role of MYO1C in Rhodopsin trafficking.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.