The Pah-R261Q mouse reveals oxidative stress associated with amyloid-like hepatic aggregation of mutant phenylalanine hydroxylase

Oscar Aubi,Arve Ulvik,Ann Kari Grindheim,Beat Thöny,Aurora Martinez,Ming Ying,Adrian Mccann,Endy Spriet,Kunwar Jung-Kc,Tanja Scherer,Tie-Jun Sten Shi,Karina S Prestegård

doi:10.1038/s41467-021-22107-1

Abstract

Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels. A homozygous Pah-R261Q mouse, with a highly prevalent misfolding variant in humans, reveals the expected hepatic PAH activity decrease, systemic L-Phe increase, L-tyrosine and L-tryptophan decrease, and tetrahydrobiopterin-responsive hyperphenylalaninemia. Pah-R261Q mice also present unexpected traits, including altered lipid metabolism, reduction of liver tetrahydrobiopterin content, and a metabolic profile indicative of oxidative stress. Pah-R261Q hepatic tissue exhibits large ubiquitin-positive, amyloid-like oligomeric aggregates of mutant PAH that colocalize with selective autophagy markers. Together, these findings reveal that PKU, customarily considered a loss-of-function disorder, can also have toxic gain-of-function contribution from protein misfolding and aggregation. The proteostasis defect and concomitant oxidative stress may explain the prevalence of comorbid conditions in adult PKU patients, placing this mouse model in an advantageous position for the discovery of mutation-specific biomarkers and therapies.

Highlights

Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels
The custom-made mouse model with the p.R261Q-PAH mutation was generated by CRISPR/Cas[9] genome editing technology, as schematically represented in Supplementary Fig. 2a, and proven to have the correct genotype (Supplementary Fig. 2b, c)
Pah-R261Q knock-in mouse that carries a frequent mutation in HPA/PKU patients exhibits reduced total hepatic PAH activity and presents phenotypic traits characteristic of homozygous patients with the R261Q:R261Q genotype, such as increased L-Phe and decreased L-Trp and L-Tyr in blood compared to WT, sensitivity to L-Phe challenge, and responsiveness to BH4 supplementation[8,16]

Summary

Introduction

Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels. Pah-R261Q hepatic tissue exhibits large ubiquitin-positive, amyloid-like oligomeric aggregates of mutant PAH that colocalize with selective autophagy markers Together, these findings reveal that PKU, customarily considered a loss-of-function disorder, can have toxic gain-of-function contribution from protein misfolding and aggregation. PAH is a tetrameric, non-heme iron aromatic amino acid hydroxylase that catalyzes the hydroxylation of L-Phe to L-tyrosine (L-Tyr) using molecular oxygen as additional substrate and the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4)[1,2]. This is the rate-limiting step in the catabolic degradation of L-. These required studies would greatly benefit from the availability of useful model organisms

Methods

Results

Conclusion