Fig 2: Neurotransmitter and BH4 content, and BH4-responsiveness in the Pah-R261Q mouse model.a Monoamine neurotransmitter content in brain lysates; data are presented as mean ± SD, individual values are plotted as circles (n = 5 WT and 5 Pah-R261Q mice). Abbreviations (from left to right): levodopa, 3-ortho-methyldopa, dopamine, homovanillic acid, norepinephrine, 3-methyl-4-hydroxyphenylglycol, epinephrine, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid. b BH4 determination in whole brain and liver lysates, presented as mean ± SD, individual values are plotted as circles (n = 6 WT and 5 Pah-R261Q). Statistical significance for the difference in brain BH4 content between both groups was calculated by two-tailed unpaired t test; p < 0.0001 (****). c Blood L-Phe concentration after L-Phe challenge in placebo-control (black) and BH4-treated (pink) Pah-R261Q mice (n = 5 placebo and 6 treated mice). L-Phe (200 µg L-Phe/g body weight) was administered by i.p. at time 0 and L-Phe concentration was monitored at 0, 35, 90, 150, and 300 min. The BH4 treated mice received (by i.p.) 20 mg/kg BH4 in 2% ascorbic acid and 10% DMSO, for 4 days, twice a day, previous to L-Phe administration, and the placebo control received the same solution without BH4. Data are presented as mean ± SD. Inset, area under the curve (AUC) for the time dependence of L-Phe concentration between 0 and 300 min for placebo and BH4-treated groups. Individual values are represented by circles. Statistical significance for the difference between both groups was calculated by two-tailed unpaired t test; p = 0.0299 (*). In panels a and b the data for WT are depicted in purple and for Pah-R261Q in ochre. Source data are provided as a Source Data file.

Fig 3: Physiological and metabolic characterization of Pah-R261Q compared with WT mice.a Bodyweight distribution by sex and genotype. The weight of WT mice (controls) was in agreement with averaged registered data (https://www.jax.org/strain/000664). Data are presented as mean ± SD, with individual values plotted as circles (females) and triangles (males) (n = 10 WT male, 14 WT female, 31 Pah-R261Q male, 26 Pah-R261Q female mice). Statistical significance for the weight difference for males in the two groups was calculated by two-tailed unpaired t test; p = 0.0031 (**). b–f Metabolic cage experiments, performed for 48 h, with 12 h of acclimation followed by 36 h of recordings. n = 3 WT and 5 Pah-R261Q mice in independent experiments, with one mouse per cage and 121 observations/animal. b Cumulative feed consumption (g). c Mice activity with continuous recording, expressed as mean ± SD. Inset, total activity for each mouse group presented as mean ± SD, individual values are plotted as circles. d Total Volume of O2 consumed and volume of CO2 produced for each mice type, obtained from the integration of the area under the curve (AUC) from data in Supplementary Fig. 3. Data are presented as the mean AUC ± SD, with individual values plotted as circles. Statistical significance for the difference between both mice groups was calculated by two-tailed unpaired t test; p = 0.0011 (**) for O2 and p < 0.0001 (****) for CO2. e Respiratory exchange ratio (RER) along the recording time. Inset: averaged RER presented as mean ± SD; the circles represent mean for the group at each time point. Statistical significance for differences between both groups was calculated by two-tailed unpaired t test; p < 0.0001 (****). f Energy expenditure obtained by indirect calorimetry expressed as mean ± SD. In all panels, the data for WT are depicted in purple and Pah-R261Q in ochre. Source data are provided as a Source Data file.

Fig 4: PAH content in liver lysates of homozygous and heterozygous Pah-R261Q mice.a Western blots for immunodetection of PAH (a-PAH) (a) and ubiquitinated protein (a-Ub) (b) showing the decrease in non-ubiquitinated PAH (non-Ub-PAH; ~51 kDa band) and increase of mono-ubiquitinated PAH (mono-Ub-PAH; ~56 kDa) from genotype PahWT/WT to PahR261Q/WT to PahR261Q/R261Q. The blots are representative from n = 3 replicates for each mice group. GAPDH was used as loading control. c Overview of relative PAH specific activity normalized to activity in PahWT/WT liver lysates (23.2 ± 2.4 nmol L-Tyr/min/mg protein) (n = 4 mice for each genotype) and non-Ub-PAH protein (51 kDa) levels from densitometric analysis normalized to both PahWT/WT liver lysates as well as to GAPDH loading control (n = 3 mice for each genotype). Data are presented as mean ± SD for PahWT/WT (purple), PahR261Q/WT (green), and PahR261Q/R261Q (ochre), individual values are represented as circles. Differences between genotypes were analyzed by one-way ANOVA followed by Tukey test; differences in PAH activity, p = 0.0005 (***) for PahWT/WT vs. PahR261Q/WT, p < 0.0001 (****) for both PahWT/WT vs. PahR261Q/R261Q and PahR261Q/WT vs. PahR261Q/R261Q; differences in PAH level, p = 0.0080 (**) for PahWT/WT vs. PahR261Q/R261Q. Source data are provided as a Source Data file.

Fig 5: Nuclear distribution of mutant PAH protein in Pah-R261Q and Enu1 mice liver.a Immunofluorescence of PAH (green) and the nuclear pore marker Nup98 (red) in hepatic tissue of Enu1 and Pah-R261Q mice (left panels), and 3D-rendering of stacks of confocal images using the surface tool in Imaris software at two different magnifications (middle and right panels). The images reveal the subcellular distribution of PAH in the nucleus and cytoplasm of Enu1 mice, whereas in Pah-R261Q mice PAH is distributed in the cytoplasm. Hoechst was used for nuclear staining (blue). b Immunohistochemically DAB-stained hepatocytes from WT, Pah-R261Q, and Enu1 mice at low (left panels) and high (right panels) magnification. Arrows indicate PAH immunoreactive hepatocytes in Pah-R261Q and Enu1 mice. a, b The micrographs are representative of n = 3 biological replicates in each mice group. c Two representative PAH immunoreactive hepatocytes at higher magnification in Pah-R261Q (left) and Enu1 (right) mice are shown, where arrowheads point to PAH-positive particle-like structures. Measurement of PAH particle size was performed in 30 µm-thick liver sections (n = 10) for each mice. At least 400 single PAH-positive particles in randomly selected regions of liver sections from each mice group were analyzed, and the size distribution of PAH-positive particles in Pah-R261Q and Enu1 hepatocytes is shown (lowest panel). Data are presented as mean ± SD and each dot represents a PAH-positive particle. The difference in size is statistically significant, as calculated by the two-tailed unpaired t test; P value < 0.0001 (****). Source data are provided as a Source Data file.