Examining the role of PC2 ciliary interactors and folding therapies for PC1 missense mutants in the context of ADPKD
Final Report Abstract
Polycystin-2 (PC2/TRPP2), the product of one of the genes mutated in ADPKD, is expressed in the endoplasmic reticulum (ER) and ciliary membranes. The COOH-terminal tail of PC2 contains a potential Ca2+ binding EF-hand motif. A mouse model termed Pkd2TEAA, in which critical residues at the EF-hand were substituted with alanine (T769A, E772A) to inactivate the Ca2+-binding properties of PC2-EF, was generated via CRISPR/Cas9 methodology. The Pkd2TEAA mutant does not result in loss of PC2 function as evidenced by the lack of cysts in Pkd2TEAA/TEAA animals. As a next step we assessed the impact of the Pkd2TEAA allele on Pkd1 derived cysts. The Pkd1R2220W human REJ mutant (Pkd1RW) is a representative candidate of a PC1 hypomorphic missense mutation as it leads to a clear yet partial defect in PC1 biogenesis and cleavage. Mutant alleles were further modified by insertion of a V5 epitope tag inframe at the C-terminus to detect the mutant proteins (Pkd1RW-V5 and Pkd2TEAA-V5). The Pkd2TEAA mouse was crossed onto a Pkd1RW/flox background under the control of Pkhd1-Cre (collecting duct-specific). The kidneys were examined by histological and morphological parameters. Compared to Pkd1RW/flox; Pkhd1-Cre mice, the kidneys at P24 from Pkd1RW/flox; Pkd2TEAA/+; Pkhd1-Cre mice displayed a decreased KW/BW ratio and BUN. We observed a marked increase in apoptosis specifically in cyst-lining epithelia expressing Pkd2TEAA. No significant difference in the protein levels of PC1 or PC2 proteins could be detected between the different genotypes. Inactivation of the Ca2+-binding properties of PC2 EF-hand on a partially PC1 deficient background results in improved cystic phenotype possibly via specific apoptosis of PC1 mutant cyst-lining epithelial cells. Modulators of PC2 EF-hand function may alter PC2 function in vivo to slow cyst progression in the setting of some PC1 missense mutations. Polycystin-1 (PC1), the product of the other gene mutated in ADPKD accounts for ~85% of autosomal dominant polycystic kidney disease (ADPKD). Around 30% of mutations in PC1 are missense predicted to result in reduced function of the protein. XBP1 encodes the main chaperone modulator of the ER unfolded protein response. Here we investigated the role of XBP1 as a “genetic” chaperone therapy which may affect the levels of functional PC1 carrying patient derived missense mutations using the p.R2220W human REJ mutant (p.R2216W in mouse) as a representative candidate. The effect of XBP1 on the expression and trafficking of the PC1-R2220W-V5 mutant was determined in vitro. A Pkd1R2216W knock-in mouse model was generated. Using this backbone, Pkd1R2216W/fl; Pkhd1-Cre and Pkd1R2216W/fl; Pkhd1-Cre; XBP1-Rosa-floxstop-TG mice were examined by morphological, functional and biochemical analyses. Expression of XBP1 in transiently cells expressing PC1-R2220W-V5 leads to increased expression and GPS cleavage of the mutant protein. Ciliary trafficking of PC1-R2220W was markedly improved by coexpression of XBP1 as compared with PC1-R2220W alone. At P16, Pkd1R2216W/fl; Pkhd1-Cre mice developed cystic disease compared with Pkd1R2220W/+ animals as seen via a significant increase in renal parameters. Expression of the conditional XBP1 transgene in Pkd1R2216W/fl; XBP1-TG; Pkhd1-Cre mice at P16 led to a significant decrease in the cystic burden compared to the controls. Using TUNEL and Ki67 assays we found that induction of XBP1 in the cyst lining cells led to a significant reduction in proliferation with no impact on apoptosis suggesting that the improved cystic phenotype in the Pkd1R2216W/fl; XBP1-TG; Pkhd1-Cre animals is due to a reduction in cyst growth. The results of this project raise the possibility that in vivo chaperone therapy for the treatment of ADPKD may have a beneficial role for a subset of PC1 missense mutations.
Publications
- (2016) Is it Time to Fold the Cysts Away? Trends of Molecular Medicine. Volume 22, Issue 12, p997–999
Krappitz M, Gallagher AR, Fedeles S
(See online at https://doi.org/10.1016/j.molmed.2016.10.001) - Disruption of Ca2+-binding in the EF-hand domain of Polycystin-2 does not result in a cystic phenotype; New Haven/USA, (TH-PO597), Kidney Week (ASN) /Chicago, November 2017
M. Krappitz, K. Dong, S. Fedeles, A. Gallagher, Y. Cai, S. Somlo
- Enhanced protein folding via XBP1 activation ameliorates ADPKD due to PC1 misfolding, (TH-OR105), Kidney Week (ASN)/San Diego, October 2018
M. Krappitz, T. Hollman, D. Ruemmele, P. Westergerling, C. Roosendaal, Anna- Rachel Gallagher, S. Somlo, and S. Fedeles
- New Haven/USA; Disruption of the EF-hand domain of Polycystin-2 ameliorates cystic disease caused by Polycystin-1 deficiency, (TH-PO672), Kidney Week (ASN)/San Diego, October 2018
M. Krappitz, T. Staudner, P. Westergerling, K. Dong, S. Fedeles, A. Gallagher Y. Cai1, S. Somlo
- New Haven/USA; Verminderung der Überlebensfähigkeit von Zystenepithelzellen durch Inaktivierung von Xbp1 als Behandlungsstrategie von ADPKD, (P066), Jahreskongress der Deutschen Gesellschaft für Nephrologie/Berlin, September 2018
M. Krappitz, D. Rümmele, T. A. Hollmann, T. Staudner, K. Dong, S. Fedeles, S. Somlo
