Abstract
Purpose :
Retinitis pigmentosa (RP) is an inherited retinal neurodegenerative disease impacting rod photoreceptors. RP is often caused by rod photoreceptor loss due to rhodopsin (Rho) mistrafficking, which has been observed in multiple models of RP, including P23H-Rho and rd10 mouse models. In rods from these mice, Rho is mislocalized throughout the cell, including in the presynaptic spherules; however, the impact of this mislocalization on synaptic function and morphology is not fully understood. We hypothesize that Rho mislocalization to the spherules disrupts the normal secretory system for synaptic proteins leading to localized presynaptic defects in RP mutant rods.
Methods :
We used a combination of structured illumination microscopy (SIM), transmission electron microscopy (TEM), quantitative confocal microscopy, quantitative western blotting, and tandem mass tag spectrometry (TMT) to examine the morphology and synaptic protein expression levels in mutant rods from P23H-hRho-tagRFP-T/+ mice, which have the P23H human rhodopsin gene fused to a RFP, and from rd10 mice.
Results :
SIM analysis of P23H–RFP/+ mice showed mislocalized P23H-Rho aggregated within the cytoplasm of rod presynaptic spherules at postnatal day (P) 30, as well as defects in synaptic ribbon morphology, which persist until P90. Synaptic ribbon defects were also observed with TEM at P30. Protein quantification methods revealed differences in rod synaptic protein levels in P23H-RFP/+ mice: dystroglycan and dystrophin levels were higher in P23H-RFP/+ rods at age P90, while Elfn1 was decreased. Imaging of rd10 retinas revealed Rho mislocalization along the rod spherule plasma membrane, and there were no differences in synaptic protein levels between rd10 and wild type rods at ages prior to and during rod degeneration.
Conclusions :
Our results show Rho mislocalization to the presynaptic compartment in both P23H-RFP/+ mice and rd10 mice, but this mislocalization differs between models and impacts the rod spherules differently. Rod synaptic protein expression levels are altered in P23H-RFP/+ mice but not in rd10 mice, which is likely due to mutant P23H-Rho aggregating in the ER and disrupting the normal ER secretory system. Future studies will focus on this under-investigated rod synaptic secretory pathway to determine its role in synaptic protein regulation and RP disease progression.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.