Danique van Vliet

Large Neutral Amino Acid Supplementation Exerts Its Effect through Three Synergistic Mechanisms: Proof of Principle in Phenylketonuria Mice

Background Phenylketonuria (PKU) was the first disorder in which severe neurocognitive dysfunction could be prevented by dietary treatment. However, despite this effect, neuropsychological outcome in PKU still remains suboptimal and the phenylalanine-restricted diet is very demanding. To improve neuropsychological outcome and relieve the dietary restrictions for PKU patients, supplementation of large neutral amino acids (LNAA) is suggested as alternative treatment strategy that might correct all brain biochemical disturbances caused by high blood phenylalanine, and thereby improve neurocognitive functioning. Objective As a proof-of-principle, this study aimed to investigate all hypothesized biochemical treatment objectives of LNAA supplementation (normalizing brain phenylalanine, non-phenylalanine LNAA, and monoaminergic neurotransmitter concentrations) in PKU mice. Methods C57Bl/6 Pah-enu2 (PKU) mice and wild-type mice received a LNAA supplemented diet, an isonitrogenic/isocaloric high-protein control diet, or normal chow. After six weeks of dietary treatment, blood and brain amino acid and monoaminergic neurotransmitter concentrations were assessed. Results In PKU mice, the investigated LNAA supplementation regimen significantly reduced blood and brain phenylalanine concentrations by 33% and 26%, respectively, compared to normal chow (p<0.01), while alleviating brain deficiencies of some but not all supplemented LNAA. Moreover, LNAA supplementation in PKU mice significantly increased brain serotonin and norepinephrine concentrations from 35% to 71% and from 57% to 86% of wild-type concentrations (p<0.01), respectively, but not brain dopamine concentrations (p = 0.307). Conclusions This study shows that LNAA supplementation without dietary phenylalanine restriction in PKU mice improves brain biochemistry through all three hypothesized biochemical mechanisms. Thereby, these data provide proof-of-concept for LNAA supplementation as a valuable alternative dietary treatment strategy in PKU. Based on these results, LNAA treatment should be further optimized for clinical application with regard to the composition and dose of the LNAA supplement, taking into account all three working mechanisms of LNAA treatment.

BH4 treatment in BH4-responsive PKU patients: preliminary data on blood prolactin concentrations suggest increased cerebral dopamine concentrations.

In phenylketonuria (PKU), cerebral neurotransmitter deficiencies have been suggested to contribute to brain dysfunction. Present treatment aims to reduce blood phenylalanine concentrations by a phenylalanine-restricted diet, while in some patients blood phenylalanine concentrations also respond to cofactor treatment with tetrahydrobiopterin (BH4). Recently, a repurposing approach of BH4 was suggested to increase cerebral neurotransmitter synthesis. To investigate whether BH4 may improve cerebral dopamine concentrations in PKU patients beyond its effect through lowering blood phenylalanine concentrations, we investigated blood prolactin concentrations-as a parameter of brain dopamine availability. We retrospectively compared blood prolactin in relation to blood phenylalanine concentrations of nine (male) BH4-responsive PKU patients, when being treated without and with BH4. Blood prolactin concentrations positively correlated to blood phenylalanine concentrations (p=0.002), being significantly lower with than without BH4 treatment (p=0.047). In addition, even in this small number of male patients, blood prolactin concentrations tended to be lower at increasing BH4 dose (p=0.054), while taking blood phenylalanine concentrations into account (p=0.002). In individual BH4-responsive patients, median blood prolactin concentrations were significantly lower while using BH4 than before using BH4 treatment (p=0.024), whereas median blood phenylalanine concentrations tended to be lower, but this did not reach statistical significance (p=0.107). Therefore, these data show that high blood phenylalanine in BH4-responsive PKU male patients seems to be associated with increased blood prolactin concentrations, suggesting reduced cerebral dopamine availability. Moreover, these data suggest that BH4 treatment in itself could decrease blood prolactin concentrations in a dose-responsive way, independent of blood phenylalanine concentrations. We conclude that these preliminary data indicate that BH4 treatment may improve cerebral dopamine concentrations in PKU patients beyond its effect through lowering blood phenylalanine concentrations, possibly in a dose-dependent manner, but further research would be warranted.

Single amino acid supplementation in aminoacidopathies: a systematic review

Aminoacidopathies are a group of rare and diverse disorders, caused by the deficiency of an enzyme or transporter involved in amino acid metabolism. For most aminoacidopathies, dietary management is the mainstay of treatment. Such treatment includes severe natural protein restriction, combined with protein substitution with all amino acids except the amino acids prior to the metabolic block and enriched with the amino acid that has become essential by the enzymatic defect. For some aminoacidopathies, supplementation of one or two amino acids, that have not become essential by the enzymatic defect, has been suggested. This so-called single amino acid supplementation can serve different treatment objectives, but evidence is limited. The aim of the present article is to provide a systematic review on the reasons for applications of single amino acid supplementation in aminoacidopathies treated with natural protein restriction and synthetic amino acid mixtures.

Heterogeneous clinical spectrum of DNAJC12-deficient hyperphenylalaninemia: from attention deficit to severe dystonia and intellectual disability

Background Autosomal recessive mutations in DNAJC12, encoding a cochaperone of HSP70 with hitherto unknown function, were recently described to lead to hyperphenylalaninemia, central monoamine neurotransmitter (dopamine and serotonin) deficiency, dystonia and intellectual disability in six subjects affected by homozygous variants. Objective Patients exhibiting hyperphenylalaninemia in whom deficiencies in hepatic phenylalanine hydroxylase and tetrahydrobiopterin cofactor metabolism had been excluded were subsequently analysed for DNAJC12 variants. Methods To analyse DNAJC12, genomic DNA from peripheral blood (Sanger sequencing), as well as quantitative messenger RNA (Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)) and protein expression (Western blot) from primary skin fibroblasts were performed. Results We describe five additional patients from three unrelated families with homozygosity/compound heterozygosity in DNAJC12 with three novel variants: c.85delC/p.Gln29Lysfs*38, c.596G>T/p.*199Leuext*42 and c.214C>T/p.(Arg72*). In contrast to previously reported DNAJC12-deficient patients, all five cases showed a very mild neurological phenotype. In two subjects, cerebrospinal fluid and primary skin fibroblasts were analysed showing similarly low 5-hydroxyindolacetic acid and homovanillic acid concentrations but more reduced expressions of mRNA and DNAJC12 compared with previously described patients. All patients responded to tetrahydrobiopterin challenge by lowering blood phenylalanine levels. Conclusions DNAJC12 deficiency appears to result in a more heterogeneous neurological phenotype than originally described. While early identification and institution of treatment with tetrahydrobiopterin and neurotransmitter precursors is crucial to ensure optimal neurological outcome in DNAJC12-deficient patients with a severe phenotype, optimal treatment for patients with a milder phenotype remains to be defined.

Voluntary Exercise Prevents Oxidative Stress in the Brain of Phenylketonuria Mice

BACKGROUND High phenylalanine levels in phenylketonuria (PKU) have been associated with brain oxidative stress and amino acid imbalance. Exercise has been shown to improve brain function in hyperphenylalaninemia and neurodegenerative diseases. This study aimed to verify the effects of exercise on coordination and balance, plasma and brain amino acid levels, and brain oxidative stress markers in PKU mice. METHODS Twenty wild-type (WT) and 20 PAH(enu2) (PKU) C57BL/6 mice were placed in cages with (exercise, Exe) or without (sedentary, Sed) running wheels during 53 days. At day 43, a balance beam test was performed. Plasma and brain were collected for analyses of amino acid levels and the oxidative stress parameters superoxide dismutase (SOD) activity, sulfhydryl and reduced glutathione (GSH) contents, total radical-trapping antioxidant potential (TRAP), and total antioxidant reactivity (TAR). RESULTS SedPKU showed poor coordination (p < 0.001) and balance (p < 0.001), higher plasma and brain phenylalanine (p < 0.001), and increased brain oxidative stress (p < 0.05) in comparison to SedWT. ExePKU animals ran less than ExeWT (p = 0.018). Although no improvement was seen in motor coordination and balance, exercise in PKU restored SOD, sulfhydryl content, and TRAP levels to controls. TAR levels were increased in ExePKU in comparison to SedPKU (p = 0.012). Exercise decreased plasma and brain glucogenic amino acids in ExePKU, but did not change plasma and brain phenylalanine in both WT and PKU. CONCLUSIONS Exercise prevents oxidative stress in the brain of PKU mice without modifying phenylalanine levels. Hence, exercise positively affects the brain, demonstrating its value as an intervention to improve brain quality in PKU.

The Behavioral Consequence of Phenylketonuria in Mice Depends on the Genetic Background.

To unravel the role of gene mutations in the healthy and the diseased state, countless studies have tried to link genotype with phenotype. However, over the years, it became clear that the strain of mice can influence these results. Nevertheless, identical gene mutations in different strains are often still considered equals. An example of this, is the research done in phenylketonuria (PKU), an inheritable metabolic disorder. In this field, a PKU mouse model (either on a BTBR or C57Bl/6 background) is often used to examine underlying mechanisms of the disease and/or new treatment strategies. Both strains have a point mutation in the gene coding for the enzyme phenylalanine hydroxylase which causes toxic concentrations of the amino acid phenylalanine in blood and brain, as found in PKU patients. Although the mutation is identical and therefore assumed to equally affect physiology and behavior in both strains, no studies directly compared the two genetic backgrounds to test this assumption. Therefore, this study compared the BTBR and C57Bl/6 wild-type and PKU mice on PKU-relevant amino acid- and neurotransmitter-levels and at a behavioral level. The behavioral paradigms were selected from previous literature on the PKU mouse model and address four domains, namely (1) activity levels, (2) motor performance, (3) anxiety and/or depression-like behavior, and (4) learning and memory. The results of this study showed comparable biochemical changes in phenylalanine and neurotransmitter concentrations. In contrast, clear differences in behavioral outcome between the strains in all four above-mentioned domains were found, most notably in the learning and memory domain. The outcome in this domain seem to be primarily due to factors inherent to the genetic background of the mouse and much less by differences in PKU-specific biochemical parameters in blood and brain. The difference in behavioral outcome between PKU of both strains emphasizes that the consequence of the PAH mutation is influenced by other factors than Phe levels alone. Therefore, future research should consider these differences when choosing one of the genetic strains to investigate the pathophysiological mechanism underlying PKU-related behavior, especially when combined with new treatment strategies.

A specific nutrient combination attenuates the reduced expression of PSD-95 in the proximal dendrites of hippocampal cell body layers in a mouse model of phenylketonuria

The inherited metabolic disease phenylketonuria (PKU) is characterized by increased concentrations of phenylalanine in the blood and brain, and as a consequence neurotransmitter metabolism, white matter, and synapse functioning are affected. A specific nutrient combination (SNC) has been shown to improve synapse formation, morphology and function. This could become an interesting new nutritional approach for PKU. To assess whether treatment with SNC can affect synapses, we treated PKU mice with SNC or an isocaloric control diet and wild-type (WT) mice with an isocaloric control for 12 weeks, starting at postnatal day 31. Immunostaining for post-synaptic density protein 95 (PSD-95), a post-synaptic density marker, was carried out in the hippocampus, striatum and prefrontal cortex. Compared to WT mice on normal chow without SNC, PKU mice on the isocaloric control showed a significant reduction in PSD-95 expression in the hippocampus, specifically in the granular cell layer of the dentate gyrus, with a similar trend seen in the cornus ammonis 1 (CA1) and cornus ammonis 3 (CA3) pyramidal cell layer. No differences were found in the striatum or prefrontal cortex. PKU mice on a diet supplemented with SNC showed improved expression of PSD-95 in the hippocampus. This study gives the first indication that SNC supplementation has a positive effect on hippocampal synaptic deficits in PKU mice.

Large neutral amino acid supplementation as an alternative to the phenylalanine-restricted diet in adults with phenylketonuria : evidence from adult Pah-enu2 mice

Phenylketonuria treatment mainly consists of a phenylalanine-restricted diet but still results in suboptimal neuropsychological outcome, which is at least partly based on cerebral monoamine deficiencies, while, after childhood, treatment compliance decreases. Supplementation of large neutral amino acids (LNAAs) was previously demonstrated in young phenylketonuria mice to target all three biochemical disturbances underlying brain dysfunction in phenylketonuria. However, both its potential in adult phenylketonuria and the comparison with the phenylalanine-restricted diet remain to be established. To this purpose, several LNAA supplements were compared with a severe phenylalanine-restricted diet with respect to brain monoamine and amino acid concentrations in adult C57Bl/6 Pah-enu2 mice. Adult phenylketonuria mice received a phenylalanine-restricted diet, unrestricted diet supplemented with several combinations of LNAAs or AIN-93M control diet for 6 weeks. In addition, adult wild-type mice on AIN-93M diet served as controls. The severe phenylalanine-restricted diet in adult phenylketonuria mice significantly reduced plasma and brain phenylalanine and restored brain monoamine concentrations, while brain concentrations of most nonphenylalanine LNAAs remained subnormal. Supplementation of eight LNAAs was similarly effective as the severe phenylalanine-restricted diet to restore brain monoamines, while brain and plasma phenylalanine concentrations remained markedly elevated. These results provide biochemical support for the effectiveness of the severe phenylalanine-restricted diet and showed the possibilities of LNAA supplementation being equally effective to restore brain monoamines in adult phenylketonuria mice. Therefore, LNAA supplementation is a promising alternative treatment to phenylalanine restriction in adult phenylketonuria patients to further optimize neuropsychological functioning.

Presumptive brain influx of large neutral amino acids and the effect of phenylalanine supplementation in patients with Tyrosinemia type 1

Introduction Hereditary Tyrosinemia type 1 (HT1) is a rare metabolic disease caused by a defect in the tyrosine degradation pathway. Current treatment consists of 2-(2-nitro-4-trifluoromethylbenoyl)-1,3-cyclohexanedione (NTBC) and a tyrosine and phenylalanine restricted diet. Recently, neuropsychological deficits have been seen in HT1 patients. These deficits are possibly associated with low blood phenylalanine concentrations and/or high blood tyrosine concentrations. Therefore, the aim of the present study was threefold. Firstly, we aimed to calculate how the plasma amino acid profile in HT1 patients may influence the presumptive brain influx of all large neutral amino acids (LNAA). Secondly, we aimed to investigate the effect of phenylalanine supplementation on presumptive brain phenylalanine and tyrosine influx. Thirdly, we aimed to theoretically determine minimal target plasma phenylalanine concentrations in HT1 patient to ensure adequate presumptive brain phenylalanine influx. Methods Data of plasma LNAA concentrations were obtained. In total, 239 samples of 9 HT1 children, treated with NTBC, diet, and partly with phenylalanine supplementation were collected together with 596 samples of independent control children. Presumptive brain influx of all LNAA was calculated, using Michaelis-Menten parameters (Km) and Vmax-values obtained from earlier articles. Results In HT1 patients, plasma concentrations and presumptive brain influx of tyrosine were higher. However, plasma and especially brain influx of phenylalanine were lower in HT1 patients. Phenylalanine supplementation did not only tend to increase plasma phenylalanine concentrations, but also presumptive brain phenylalanine influx, despite increased plasma tyrosine concentrations. However, to ensure sufficient brain phenylalanine influx in HT1 patients, minimal plasma phenylalanine concentrations may need to be higher than considered thus far. Conclusion This study clearly suggests a role for disturbed brain LNAA biochemistry, which is not well reflected by plasma LNAA concentrations. This could play a role in the pathophysiology of the neuropsychological impairments in HT1 patients and may have therapeutic implications.