Saturday, May 31, 2025
5/31/2025
Molecular Mechanisms of TULP1-Mediated Photoreceptor Degeneration - Abstract. Mutations in the TULP1 gene underlie a severe, early-onset form of autosomal recessive retinitis pigmentosa (RP). Our long-term objectives are to understand the cellular function of TULP1 and the pathogenic mechanism responsible for retinal degeneration. TULP1 is a photoreceptor-specific protein involved in vesicular trafficking in two photoreceptor compartments. In tulp1-/- mice, several phototransduction proteins are mislocalized, indicating that TULP1 is necessary for protein transport to the outer segment (OS). These mice also have a synaptic malformation with few intact ribbons, indicating a defect in neurotransmitter vesicular cycling. Our preliminary data indicates that TULP1 has two distinct interactomes. We postulate that TULP1 functions as an adapter protein linking OS-bound protein transport vesicles to the axoneme of the transition zone and neurotransmitter-filled vesicles to the ribbon complex. Preliminary studies done in vitro suggest that mutant TULP1 proteins cause ER stress and leads to induction of the unfolded protein response (UPR) pathway, suggesting a possible mechanism for cell death. Our overall hypothesis is that TULP1 is a phospholipid-sensing adapter protein required for the vesicular transport of proteins and that mutant forms of TULP1 cause protein mistrafficking which induces chronic ER stress leading to photoreceptor cell death through overload of protein degradation pathways. Experiments in Aim 1 will identify TULP1 compartment-specific protein-protein interactions and whether these interacting networks are disrupted by RP- associated TULP1 mutations. This will be accomplished by performing immunoprecipitations, localization experiments, high-resolution imaging, proximity proteomics and quantitative cross-linking mass spectroscopy methods. Experiments in Aim 2 will determine the molecular signatures, temporal contributions and mechanism of stress response pathway(s) activation throughout disease progression in Tulp1 mutant models of RP by analyzing molecular markers unique to each pathway. For these experiments, we generated two novel knock-in mouse lines each expressing an RP-associated TULP1 missense mutation. WT, tulp1-/-, tulp1D94Y and tulp1F491L mice will be crossed with three different mouse lines each expressing an in vivo reporter gene relevant to UPR, proteasome activity, or autophagy. Comprehensive phenotypic analyses will be performed and compared across mice to determine in vivo whether mutant Tulp1 proteins induce chronic ER stress leading to activation of stress response pathways causing cell death by overwhelming the photoreceptors proteostasis network capacity. By clarifying the proteins involved in vesicular trafficking, this project will significantly impact an important aspect of photoreceptor biology relevant to human retinal function and disease. Importantly, determining the pathogenic mechanism behind TULP1-associated RP and identifying the activated pathways will aid in discovering therapeutic targets aimed at slowing this blinding condition.
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