Rescue of defective ATP8B1 trafficking by CFTR correctors as a therapeutic strategy for familial intrahepatic cholestasis

Background & aims: ATP8B1 deficiency is an autosomal recessive liver disease characterized by intrahepatic cholestasis. ATP8B1 mutation p.I661T, the most frequent mutation in European patients, results in protein misfolding and impaired targeting to the plasma membrane. Similarly, mutations in cystic fibrosis transmembrane conductance regulator (CFTR), associated with cystic fibrosis, impair protein folding and trafficking. The aim of this study was to investigate whether compounds that rescue CFTR F508del trafficking are capable of improving p.I661T-ATP8B1 plasma membrane expression.

Methods: The effect of CFTR corrector compounds on plasma membrane expression of p.I661T-ATP8B1 was evaluated by cell surface biotinylation and immunofluorescence. ATPase activity was evaluated of a purified analogue protein carrying a mutation at the matching position (p.L622T-ATP8A2).

Results: The clinically used compounds, 4-phenylbutyric acid (4-PBA), suberoylanilide hydroxamic acid (SAHA) and N-butyldeoxynojirimycin (NB-DNJ) improved p.I661T-ATP8B1 plasma membrane targeting. Compounds C4, C5, C13 and C17 also significantly increased plasma membrane expression of p.I661T-ATP8B1. SAHA and compound C17 upregulated ATP8B1 transcription. p.I661T-ATP8B1 was partly targeted to the canalicular membrane in polarized cells, which became more evident upon treatment with SAHA and/or C4. p.L622T-ATP8A2 showed phospholipid-induced ATPase activity, suggesting that mutations at a matching position in ATP8B1 do not block functionality. Combination therapy of SAHA and compound C4 resulted in an additional improvement of ATP8B1 cell surface abundance.

Conclusions: This study shows that several CFTR correctors can improve trafficking of p.I661T-ATP8B1 to the plasma membrane in vitro. Hence, these compounds may be suitable to be part of a future therapy for ATP8B1 deficiency and other genetic disorders associated with protein misfolding.

Lay summary: Compounds that improve the cellular machinery dealing with protein homeostasis (proteostasis) and allow for proper folding of proteins with (mild) missense mutations are called proteostasis regulators (Balch, Science 2008). Such compounds are potentially of high therapeutic value for many (liver) diseases. In this manuscript, we investigated whether compounds identified in screens as CFTR folding correctors are actually proteostasis regulators and thus have a broader application in other protein folding diseases. Using these compounds, we could indeed show improved trafficking to the (apical) plasma membrane of a mutated ATP8B1 protein, carrying the p.I661T missense mutation. This is the most frequently identified mutation in this rare cholestatic disorder. Importantly, ATP8B1 shows no similarity to CFTR. These data are important in providing support for the concept that rare, genetic liver diseases can potentially be treated using a generalized strategy.