Activation of the ER-UPR Pathways in TULP1 Mutant Mice

Abstract

Purpose : Mutations in TULP1 are associated with early-onset retinitis pigmentosa (RP). Tulp1 plays a role in assisting vesicle mediated transportation of newly synthesized photoreceptor outer segment (OS) proteins. Our previous work identified activation of endoplasmic reticulum (ER)-unfolded protein response (UPR) pathway in cultured hTERT-RPE-1 cells expressing mutant forms of Tulp1 carrying RP-associated missense mutations. To better dissect the disease mechanism, we analyzed activation of ER stress and UPR pathways in two Tulp1 mutant mouse models, both of which show early-onset progressive photoreceptor degeneration: the tulp1^−/− mice and the tulp1^F491L knockin mice which carry a missense mutation in the conserved TUBBY domain.

Methods : The ERAI reporter mouse model is a transgenic line that produces GFP in response to ER stress, allowing evaluation of ER stress levels in retinas by funduscopic imaging. ERAI mice were crossed to both tulp1^−/− and tulp1^F491L mice and ER stress in the retinas of transgenic mice were quantified by in vivo live imaging with confocal scanning laser ophthalmoscope (cSLO). GFP signal from photoreceptor cells was detected using blue light autofluorescence (BAF) filter. Fundus images were collected and the BAF signals were quantified using ImageJ software. Biomarkers of ER stress were further analyzed using IHC and Western blot to assess the activation of specific ER-UPR pathway proteins in the tulp1^−/− and tulp1^F491L retinas.

Results : ER stress was seen in retinas of both Tulp1 mutant mouse models starting at P21. Here we report temporally increased ER stress from photoreceptors in Tulp1 mutant retinas via live imaging of the fluorescent ER stress signal, and IHC staining and immunoblot analysis of GFP expression. We detected activation of key components of the major apoptotic branches of the ER-UPR pathway, PERK and IRE1, similar to that previously seen in vitro. When quantified, the ER stress signal progressively increased with age from P21 to P45, aligning the degeneration process in both Tulp1 mutant models.

Conclusions : Our results clearly demonstrate the involvement of ER stress in the retinas of Tulp1 mutant mice starting from an early stage of retinal degeneration. Our findings show that the ER-UPR pathway, a known mechanism of apoptosis, is involved in Tulp1-associated photoreceptor degeneration, suggesting a potential therapeutic target for this form of RP.

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