Abstract
Purpose:
A challenge in the field of inherited retinal degenerations is identifying mechanisms of photoreceptor cell death related to specific genetic mutations. Our in vitro data suggest that TULP1 missense mutations produce misfolded protein products that accumulate in the endoplasmic reticulum (ER) and induce cellular apoptosis via the unfolded protein response (UPR) pathway. In this study, we generated an in vivo animal model to directly test whether mutant forms of TULP1 induce the ER-UPR stress response complex leading to photoreceptor cell death.
Methods:
Full-length TULP1 was amplified from human retinal RNA and cloned into the mammalian expression vector pEGFP-N1. The wild type (wt) TULP1 plasmid was used as a template to engineer two known TULP1 missense mutations (D94Yand F491L) by site directed mutagenesis. The D94Y mutation is the only known missense mutation located outside of the C-terminal tubby domain; whereas the F491L mutation is located within the highly conserved tubby domain and is the most prevalent in patients with retinitis. P1 wt mice underwent a subretinal injection of wt or individual mutant TULP1 plasmid followed by electroporation. Eyes were harvested at P16 and evaluated by histology and immunohistochemistry.
Results:
Flatmount preparations of electroporated retinas indicate a transfection rate of ~15%. Histologically, transfected wt and mutant TULP1 proteins localize to all photoreceptor compartments. In comparison to mice injected with wt TULP1, mutant TULP1 forms aggregates within the IS. This suggests that the accumulation of mutant protein is involved in the pathogenesis of retinal degeneration caused by TULP1 missense mutations.
Conclusions:
We have established an animal model to evaluate TULP1 mutant proteins in vivo. Consistent with our in vitro studies, preliminary data suggest that mutant TULP1 protein accumulates in the IS, the location of the photoreceptor ER. Therefore, TULP1 aggregates may induce ER stress and activate the ER-UPR pathway. Further studies are ongoing to determine whether ER-UPR activation is the mechanism of photoreceptor cell death in TULP1-associated RP.