Tetrahydrobiopterin (BH4) treatment stabilizes tyrosine hydroxylase: Rescue of tyrosine hydroxylase deficiency phenotypes in human neurons and in a knock-in mouse model.

Proteostatic regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, is crucial for maintaining proper brain neurotransmitter homeostasis. Variants of the TH gene are associated with tyrosine hydroxylase deficiency (THD), a rare disorder with a wide phenotypic spectrum and variable response to treatment, which affects protein stability and may lead to accelerated degradation, loss of TH function and catecholamine deficiency. In this study, we investigated the effects of the TH cofactor tetrahydrobiopterin (BH4) on the stability of TH in isolated protein and in DAn- differentiated from iPSCs from a human healthy subject, as well as from THD patients with the R233H variant in homozygosity (THDA) and R328W and T399M variants in heterozygosity (THDB). We report an increase in TH and dopamine levels, and an increase in the number of TH+ cells in control and THDA cells. To translate this in vitro effect, we treated with BH4 a knock-in THD mouse model with Th variant corresponding to R233H in patients. Importantly, treatment with BH4 significantly improved motor function in these mice, as demonstrated by increased latency on the rotarod test and improved horizontal activity (catalepsy). In conclusion, our study demonstrates the stabilizing effects of BH4 on TH protein levels and function in THD neurons and mice, rescuing disease phenotypes and improving motor outcomes. These findings highlight the therapeutic potential of BH4 as a treatment option for THDA patients with specific variants and provide insights into the modulation of TH stability and its implications for THD management.

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

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.

Phenylalanine hydroxylase variants interact with the co-chaperone DNAJC12.

DNAJC12, a type III member of the HSP40/DNAJ family, has been identified as the specific co-chaperone of phenylalanine hydroxylase (PAH) and the other aromatic amino acid hydroxylases. DNAJ proteins work together with molecular chaperones of the HSP70 family to assist in proper folding and maintenance of intracellular stability of their clients. Autosomal recessive mutations in DNAJC12 were found to reduce PAH levels, leading to hyperphenylalaninemia (HPA) in patients without mutations in PAH. In this work, we investigated the interaction of normal wild-type DNAJC12 with mutant PAH in cells expressing several PAH variants associated with HPA in humans, as well as in the Enu1/1 mouse model, homozygous for the V106A-Pah variant, which leads to severe protein instability, accelerated PAH degradation and mild HPA. We found that mutant PAH exhibits increased ubiquitination, instability, and aggregation compared with normal PAH. In mouse liver lysates, we showed that DNAJC12 interacts with monoubiquitin-tagged PAH. This form represented a major fraction of PAH in the Enu1/1 but was also present in liver of wild-type PAH mice. Our results support a role of DNAJC12 in the processing of misfolded ubiquitinated PAH by the ubiquitin-dependent proteasome/autophagy systems and add to the evidence that the DNAJ proteins are important players both for proper folding and degradation of their clients.