Gut-microbiome composition in response to phenylketonuria depends on dietary phenylalanine in BTBR Pah^enu2 mice

Phenylketonuria (PKU) is a metabolic disorder caused by a hepatic enzyme deficiency causing high blood and brain levels of the amino acid Phenylalanine (Phe), leading to severe cognitive and psychological deficits that can be prevented, but not completely, by dietary treatment. The behavioral outcome of PKU could be affected by the gut-microbiome-brain axis, as diet is one of the major drivers of the gut microbiome composition. Gut-microbiome alterations have been reported in treated patients with PKU, although the question remains whether this is due to PKU, the dietary treatment, or their interaction. We, therefore, examined the effects of dietary Phe restriction on gut-microbiome composition and relationships with behavioral outcome in mice. Male and female BTBR Pah(enu2) mice received either a control diet (normal protein, “high” Phe), liberalized Phe-restricted (33% natural protein restriction), or severe Phe-restricted (75% natural protein restriction) diet with protein substitutes for 10 weeks (n = 14 per group). Their behavioral performance was examined in an open field test, novel and spatial object location tests, and a balance beam. Fecal samples were collected and sequenced for the bacterial 16S ribosomal RNA (rRNA) region. Results indicated that PKU on a high Phe diet reduced Shannon diversity significantly and altered the microbiome composition compared with wild-type animals. Phe-restriction prevented this loss in Shannon diversity but changed community composition even more than the high-Phe diet, depending on the severity of the restriction. Moreover, on a taxonomic level, we observed the highest number of differentially abundant genera in animals that received 75% Phe-restriction. Based on correlation analyses with differentially abundant taxa, the families Entereococacceae, Erysipelotrichaceae, Porphyromonadaceae, and the genus Alloprevotella showed interesting relationships with either plasma Phe levels and/or object memory. According to our results, these bacterial taxa could be good candidates to start examining the microbial metabolic potential and probiotic properties in the context of PKU. We conclude that PKU leads to an altered gut microbiome composition in mice, which is least severe on a liberalized Phe-restricted diet. This may suggest that the current Phe-restricted diet for PKU patients could be optimized by taking dietary effects on the microbiome into account

Long-term dietary intervention with low Phe and/or a specific nutrient combination improve certain aspects of brain functioning in phenylketonuria (PKU)

Introduction In phenylketonuria (PKU), a gene mutation in the phenylalanine metabolic pathway causes accumulation of phenylalanine (Phe) in blood and brain. Although early introduction of a Phe-restricted diet can prevent severe symptoms from developing, patients who are diagnosed and treated early still experience deficits in cognitive functioning indicating shortcomings of current treatment. In the search for new and/or additional treatment strategies, a specific nutrient combination (SNC) was postulated to improve brain function in PKU. In this study, a long-term dietary intervention with a low-Phe diet, a specific combination of nutrients designed to improve brain function, or both concepts together was investigated in male and female BTBR PKU and WT mice. Material & methods 48 homozygous wild-types (WT, +/+) and 96 PKU BTBRPah2 (-/-) male and female mice received dietary interventions from postnatal day 31 till 10 months of age and were distributed in the following six groups: high Phe diet (WT C-HP, PKU C-HP), high Phe plus specific nutrient combination (WT SNC-HP, PKU SNC-HP), PKU low-Phe diet (PKU C-LP), and PKU low-Phe diet plus specific nutrient combination (PKU SNC- LP). Memory and motor function were tested at time points 3, 6, and 9 months after treatment initiation in the open field (OF), novel object recognition test (NOR), spatial object recognition test (SOR), and the balance beam (BB). At the end of the experiments, brain neurotransmitter concentrations were determined. Results In the NOR, we found that PKU mice, despite being subjected to high Phe conditions, could master the task on all three time points when supplemented with SNC. Under low Phe conditions, PKU mice on control diet could master the NOR at all three time points, while PKU mice on the SNC supplemented diet could master the task at time points 6 and 9 months. SNC supplementation did not consistently influence the performance in the OF, SOR or BB in PKU mice. The low Phe diet was able to normalize concentrations of norepinephrine and serotonin; however, these neurotransmitters were not influenced by SNC supplementation. Conclusion This study demonstrates that both a long-lasting low Phe diet, the diet enriched with SNC, as well as the combined diet was able to ameliorate some, but not all of these PKU-induced abnormalities. Specifically, this study is the first long-term intervention study in BTBR PKU mice that shows that SNC supplementation can specifically improve novel object recognition

Blood and brain biochemistry and behaviour in NTBC and dietary treated tyrosinemia type 1 mice

Tyrosinemia type 1 (TT1) is a rare metabolic disease caused by a defect in the tyrosine degradation pathway. Neurocognitive deficiencies have been described in TT1 patients, that have, among others, been related to changes in plasma large neutral amino acids (LNAA) that could result in changes in brain LNAA and neurotransmitter concentrations. Therefore, this project aimed to investigate plasma and brain LNAA, brain neurotransmitter concentrations and behavior in C57 Bl/6 fumarylacetoacetate hydrolase deficient (FAH-/-) mice treated with 2-(2-nitro-4-trifluoromethylbenoyl)-1,3-cyclohexanedione (NTBC) and/or diet and wild-type mice. Plasma and brain tyrosine concentrations were clearly increased in all NTBC treated animals, even with diet (p <0.001). Plasma and brain phenylalanine concentrations tended to be lower in all FAH-/- mice. Other brain LNAA, were often slightly lower in NTBC treated FAH-/- mice. Brain neurotransmitter concentrations were usually within a normal range, although serotonin was negatively correlated with brain tyrosine concentrations (p <0.001). No clear behavioral differences between the different groups of mice could be found. To conclude, this is the first study measuring plasma and brain biochemistry in FAH-/- mice. Clear changes in plasma and brain LNAA have been shown. Further research should be done to relate the biochemical changes to neurocognitive impairments in TT1 patients

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

Phenylketonuria in a microbial world: Gut-microbiome-brain interactions shaping behavior in PKU

Phenylketonuria (PKU) is a metabolic disorder in which a deficiency in the conversion of phenylalanine leads to severe intellectual disability. Early and continuous treatment with a protein-restricted diet has been very successful, although several behavioral deficits still remain. In this thesis, we examined two elements that might be associated with this suboptimal outcome; the microbiome and sleep. The microbiota as a community will adapt to the elevated phenylalanine concentrations and/or the current dietary treatment. This can lead to changes in metabolites of the microbiome that can communicate with the brain via the “gut-microbiome-brain” (GMB) axis. We showed that the microbiome of the PKU mouse has indeed adapted to the PKU context, in which both diversity and composition are altered, depending on the severity of protein restriction. A pilot for follow-up research showed that both repeated oral gavage and cohousing were sufficient to transplant the microbiome into another mouse. The effects of sleep loss in PKU could directly underlie the suboptimal outcome. Our results confirmed increased fragmentation and diurnality in untreated PKU mice, although this did not seem related to the increased microglia activation in the hippocampus. Moreover, PKU mice showed disruptions in circadian stability and rhythmicity, which suggests a weakened circadian “time-keeping” system that might be a causal factor of sleep disturbances found at a behavioral level. Finally, a study in European PKU patients confirmed an increased prevalence of sleep disorders and other disturbances in men. The findings in this thesis demonstrate that PKU challenges the microbial community and the circadian system. These insights could provide opportunities for new treatment strategies, focusing on all facets of the disorder

Supplementary data for: Phenylketonuria in a microbial world, chapter 3

This folder contains the supplementary data visualizations of Phenylketonuria in a microbial world, chapter 3: Gut-microbiome composition in response to phenylketonuria depends on dietary phenylalanine in BTBR Pahenu2 mice

Hippocampal microglia modifications in C57Bl/6 <i>Pah^enu2</i> and BTBR <i>Pah^enu2</i> phenylketonuria (PKU) mice depend on the genetic background, irrespective of disturbed sleep patterns

Toxic levels of phenylalanine in blood and brain is a characteristic of (untreated) phenylketonuria (PKU), leading to cognitive deficits in PKU mice. In addition, our recent findings showed that PKU mice (as well as PKU patients) have a disturbed sleep/wake cycle. As a consequence, sleep loss may contribute to cognitive deficits in PKU. Sleep loss has been linked to increased activation of microglia in the hippocampus. In this study, we set out to examine morphological features of the microglia population in the hippocampus of the mouse PKU model, using both the C57Bl/6 and the BTBR strain and their wild-type controls (age 5.3 +/- 0.5 months; n=16 per group, both males and females; n=8 each). Microglial activation is reflected by retraction and thickening of the dendritic branches and an increase in cell body size of a microglial cell. Such morphological changes of microglia were studied by way of immunohistochemical staining for Iba-1, a microglia-specific calcium binding protein. We measured the number of microglia in seven subregions of the dorsal hippocampus. The level of microglial activation was determined, based on the ratio between the soma size and total cell size (soma size plus the area covered by the dendritic branches). Results showed subtle but statistical significant activation of hippocampal microglia in the C57Bl6, but not in the BTBR, PKU mice when compared with their wild-type controls. Also the total number of microglia was higher in the C57Bl/6 PKU (compared to the wild-type) mouse, but not in the BTBR PKU mouse. It is concluded that the C57Bl/6 PKU mouse has mildly higher microglia activity, which may support rather than hamper hippocampal homeostasis. The results further indicate that high levels of phenylalanine or disturbed sleep patterns do not consequently cause hippocampal microglial activation in the PKU mouse. It is currently unknown why the two PKU mouse strains show these differences in number and activation level of their hippocampal microglia, and to what extent it influences hippocampal functioning. Further scrutinizing the role of microglia functioning in the context of PKU is therefore warranted