Examining the role of PC2 ciliary interactors and folding therapies for PC1 missense mutants in the context of ADPKD

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Autosomal dominant polycystic kidney disease (ADPKD) is the most common potentially lethal monogenic disorder, affecting one in 600 to 1000 live births. ADPKD is characterized by extensive cystic enlargement in the kidney that results in progressive loss of functional renal tissue. Human disease-based gene discovery has identified mutations in polycystin-1 (PC1) and polycystin-2 (PC2) accounting for ADPKD. ADPKD is part of a spectrum of inherited cystic diseases that also includes autosomal dominant polycystic liver disease (ADPLD). ADPLD is mainly characterized by cysts in the liver and is caused by mutations in the genes PRKCSH and SEC63, which affect PC1 protein translocation and maturation in the endoplasmic reticulum. Recently, Professor Somlo’s laboratory (Nephrology, Yale University, USA) has shown that heterologous upregulation of the transcription factor XBP1s may improve the cellular and in vivo phenotype associated with ADPLD by augmenting functional PC1 activity on a Prkcsh gene mutation background. The effects of PC1 dosage in ADPKD have been implied by recent findings that ADPKD patients with missense mutations in PC1 typically have a milder course than patients with truncating mutations in PC1. This project is thus based on the hypothesis that ADPLD and ADPKD share the common underlying molecular genetic mechanism of reduced PC1 activity resulting from missense mutations in PC1. We therefore hypothesize that due to missense mutations in PC1, ADPLD and ADPKD share a common underlying molecular genetic mechanism based on reduced activity of PC1.) As enhanced protein folding via XBP1s may improve PC1 biogenesis in ADPLD, we wish to investigate whether that effect is also applicable in ADPKD due to PC1 missense mutations. We used the CRISPR/Cas9-system to selectively introduce missense mutations into PC1 in epithelial cell lines and mouse models. We will determine whether upregulation of transcription factor XBP1s that was effective in ADPLD mutations can impact the cellular and in vivo phenotypes of ADPKD missense mutations. Based on those results, we will then investigate whether pharmaceutical activation of Ire1α, the upstream inducer of XBP1, can decrease the burden of unfolded PC1 in ADPKD models and hence impact the ADPKD phenotype resulting from missense PC1 mutations.A second aim is to validate a list of ciliary proteins that have been proposed to interact with PC2 in a Yeast Two-Hybrid Screen. We will first confirm the result in a co-immunoprecipitation experiment and subsequently investigate the effect of the candidate protein on translocation/folding homeostasis.In summary, the results of this project should contribute to a better understanding of the therapeutic potential of chaperone therapies to ameliorate disease in patients with missense mutations in PKD genes.