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. 2018 Jan;123(1):6-20.
doi: 10.1016/j.ymgme.2017.10.009. Epub 2017 Oct 19.

Blood Phenylalanine Reduction Corrects CNS Dopamine and Serotonin Deficiencies and Partially Improves Behavioral Performance in Adult Phenylketonuric Mice

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Free PMC article

Blood Phenylalanine Reduction Corrects CNS Dopamine and Serotonin Deficiencies and Partially Improves Behavioral Performance in Adult Phenylketonuric Mice

Shelley R Winn et al. Mol Genet Metab. .
Free PMC article

Abstract

Central nervous system (CNS) deficiencies of the monoamine neurotransmitters dopamine and serotonin have been implicated in the pathophysiology of neuropsychiatric dysfunction in human phenylketonuria (PKU). In this study, we confirmed the occurrence of brain dopamine and serotonin deficiencies in association with severe behavioral alterations and cognitive impairments in hyperphenylalaninemic C57BL/6-Pahenu2/enu2 mice, a model of human PKU. Phenylalanine-reducing treatments, including either dietary phenylalanine restriction or liver-directed gene therapy, initiated during adulthood were associated with increased brain monoamine content along with improvements in nesting behavior but without a change in the severe cognitive deficits exhibited by these mice. At euthanasia, there was in Pahenu2/enu2 brain a significant reduction in the protein abundance and maximally stimulated activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase 2 (TPH2), the rate limiting enzymes catalyzing neuronal dopamine and serotonin synthesis respectively, in comparison to levels seen in wild type brain. Phenylalanine-reducing treatments initiated during adulthood did not affect brain TH or TPH2 content or maximal activity. Despite this apparent fixed deficit in striatal TH and TPH2 activities, initiation of phenylalanine-reducing treatments yielded substantial correction of brain monoamine neurotransmitter content, suggesting that phenylalanine-mediated competitive inhibition of already constitutively reduced TH and TPH2 activities is the primary cause of brain monoamine deficiency in Pahenu2 mouse brain. We propose that CNS monoamine deficiency may be the cause of the partially reversible adverse behavioral effects associated with chronic HPA in Pahenu2 mice, but that phenylalanine-reducing treatments initiated during adulthood are unable to correct the neuropathology and attendant cognitive deficits that develop during juvenile life in late-treated Pahenu2/enu2 mice.

Keywords: Behavior; Cognition; Dopamine; Phenylalanine hydroxylase; Phenylketonuria; Serotonin; Tryptophan; Tryptophan hydroxylase; Tyrosine; Tyrosine hydroxylase.

Figures

Figure 1
Figure 1. Phenylketonuria, effects of hyperphenylalaninemia upon brain monoamine neurotransmitter metabolism, and phenylalanine-lowering treatments applied in this work
Pahenu2/enu2 mice lack phenylalanine hydroxylase (PAH) activity in liver and cannot convert L-phenylalanine (Phe) to L-tyrosine (Tyr). Tyr and L-tryptophan (Trp) are the substrates for dopamine (DA) and serotonin synthesis respectively in brain. Both amino acids cross the blood brain barrier and enter neurons via diffusion facilitated by the LAT1 neutral amino acid transporter in competition with Phe and other large neutral amino acids. Tyr is converted to L-DOPA by tyrosine hydroxylase (TH), the rate-limiting enzyme in DA synthesis. TH activity is stimulated by Tyr, but inhibited through Phe-mediated competition, DA or L-DOPA-mediated feedback inhibition, and via a DA-activated, G-protein coupled, synthesis-modulating autoreceptor. TH activity is also regulated through reversible phosphorylation triggered by a variety of stimuli (not shown). DA is synthesized from L-DOPA by aromatic amino acid decarboxylase (AADC), taken up into secretory vesicles via a monoamine specific transporter (VMAT2) and secreted into the synapse following Ca+2 influx in response to a nerve stimulus. Neurotransmission terminates through DA reuptake via a specific transporter (DAT) or through degradation of DA to homovanillic acid (HVA), hence brain HVA content can be taken as a measure of DA turnover. Extracellular DA can also act via another G protein-coupled autoreceptor to suppress further synaptic DA release. Analogous mechanisms regulate the synthesis and secretion of serotonin from Trp followed by degradation to 5-hydroxyindoleacetic acid (5-HIAA). The present work has been based upon the hypothesis that correction of serum and consequently brain phenylalanine concentrations following administration of either DIETARY PHENYLALANINE RESTRICTION or AAV8-mediated LIVER GENE THERAPY to restore liver PAH activity would lead to functionally increased brain TH and TPH2 activity and correction of brain dopamine and serotonin content in hyperphenylalaninemic Pahenu2/enu2 mice. Furthermore, we proposed that correction of brain monoamine neurotransmitter content would be associated with improvement in the behavioral and cognitive phenotypes of hyperphenylalaninemic Pahenu2/enu2 mice.
Figure 2
Figure 2. Effects of genotype upon amino acids and monoamine neurotransmitters in C57BL/6-Pahenu2/enu2 mice
Serum and brain amino acids and brain monoamine neurotransmitter content in female (F) and male (M) Pah+/+, Pah+/−, Pah−/− mice. Data expressed as mean ± SEM. The data were analyzed using two-way ANOVA with post hoc intergroup comparisons by Tukey’s multiple comparison test. Horizontal bars and asterisks above the columns indicate the probability (p) from the Tukey’s test that the means of the selected groups are identical. **** p < 0.0001, ** p < 0.01, * p < 0.05. Serum amino acid concentrations are expressed as μM, brain amino acid content as nmol/gm brain wet weight, and brain monoamine neurotransmitters and their metabolites are expressed relative to the mean of untreated Pah−/− mice (both genders combined). (A) Serum phenylalanine, (B) brain phenylalanine. (C) Plot of brain phenylalanine vs. serum phenylalanine combining data from all groups of mice (including Pah−/− mice undergoing Phe-reducing treatment). Linear regression analysis reveals a probability of p < 0.0001 that the slope of the curve is equal to zero and suggests a strong correlation between the serum and brain phenylalanine concentrations. (D) Serum tyrosine, (E) brain tyrosine, (F) relative brain dopamine, (G) relative brain homovanillic acid (HVA), (H) serum tryptophan, (I) brain tryptophan, (J) relative brain serotonin, (K) relative brain 5-hydroxyindoleacetic acid (5-HIAA).
Figure 3
Figure 3. Behavioral performance of C57BL/6-Pahenu2/enu2 mice
Results of behavioral (open field, fear conditioning, and nesting behavior) assessments in female (F) and male (M) Pah+/+, Pah+/−, and Pah−/− mice. The results of the behavioral performance in the open field are presented in panel (A) open field activity (total distance moved) and (B) open field anxiety (% time in center). Baseline activity while in the fear conditioning enclosures is shown in panel (C). Nest building (qualitative nest rating scale) is presented in panel (D). All data are mean ± SEM. Pah+/− mice showed higher activity levels than Pah+/+ mice and Pah−/− mice in the open field. Pah+/+ and Pah+/− mice both showed higher baseline activity than Pah−/− mice in the fear conditioning enclosure. Pah−/− mice build less elaborate nests than Pah+/− mice and Pah+/+ mice. In Pah−/− mice, females built more elaborate nests than males. Pah+/+ and Pah+/− mice showed better nesting behavior than Pah−/− mice (p < 0.05, p < 0.01, respectively). Vertical bars and asterisks above the columns indicate the probability (p) from the Tukey’s test that the means of the selected groups are identical. **** p < 0.0001, ** p < 0.01, * p < 0.05.
Figure 4
Figure 4. Cognitive performance of C57BL/6-Pahenu2/enu2 mice
Performance in the water maze is presented with both sexes combined. The results are shown in panel (A) average velocity (cm/second), (B) latency to the target platform (seconds) (C) cumulative distance to the target (cm) and (D) total distance moved (cm). The data are represented as mean ± SEM for each experimental group at each individual session. (A). There were no differences in swim speeds over the five sessions (p > 0.05). (B). Latency to locate the target platform in the water maze. Pah+/+ mice showed better performance than Pah+/− (p = 0.020) and Pah−/− mice (p = 0.001) and located the platform in less time. In addition, Pah+/− mice showed better performance than Pah−/− mice (p < 0.001). (C) Similar results were seen when cumulative distance to the target location was analyzed. Pah+/− mice showed better performance than Pah−/− mice (p < 0.001) and swam on average closer to the platform location. There was a genotype × session interaction (F(8,224) = 3.057, p = 0.003). There was an effect of genotype in all Sessions (Session 1: Pah+/+ vs Pah−/−, p < 0.001; Pah+/+ vs Pah+/− p = 0.009; Session 2: Pah+/+ vs Pah−/−, p < 0.001; Pah+/− vs Pah−/−, p = 0.003; Session 3: Pah+/+ vs Pah−/−, p = 0.000; Pah+/− vs Pah−/−, p < 0.001; Session 4: Pah+/+ vs Pah−/−, p < 0.001; Pah+/− vs Pah−/−, p < 0.001; Session 5: Pah+/+ vs Pah−/−, p < 0.001; Pah+/− vs Pah−/−, p < 0.001). In the Pah−/− mice, females showed a higher cumulative distance to the platform than males. (D) Analysis of the total distance moved confirmed the results of the other outcome measures. Both Pah+/+ and Pah+/− mice showed better performance than Pah−/− mice (p < 0.001). The asterisks denote sessions in which performance of the experimental group (Pah+/− or Pah−/−) differed significantly from those of Pah+/+ mice.
Figure 5
Figure 5. Effects of phenylalanine-reducing treatments upon amino acids and monoamine neurotransmitters in C57Bl/6-Pahenu2/enu2 mice
Serum and brain amino acids and brain monoamine neurotransmitter content in female (F) and male (M) untreated Pah−/− mice (Pah−/−), Pah−/− mice receiving a phenylalanine-restricted diet (Pah −/− low Phe diet), and Pah−/− mice following AAV-mediated liver-directed gene therapy (Pah−/− AAV8). Data expressed as mean ± SEM. The data were analyzed using two-way ANOVA with post hoc intergroup comparisons by Tukey’s multiple comparison test. Horizontal bars and asterisks above the columns indicate the probability (p) from the Tukey’s test that the means of the selected groups are identical. **** p < 0.0001, ** p < 0.01, * p < 0.05. Serum amino acid concentrations are expressed as μM, brain amino acid content as nmol/gm brain wet weight, and brain monoamine neurotransmitters and their metabolites are expressed relative to the mean of untreated Pah−/− mice (both genders combined). The gray line labeled +/+ represents the mean of wild type Pah+/+ mice (both genders combined) for comparison. (A) Serum phenylalanine, (B) brain phenylalanine, (D) serum tyrosine, (E) brain tyrosine, (F) relative brain dopamine, (G) relative brain homovanillic acid (HVA), (H) serum tryptophan, (I) brain tryptophan, (J) relative brain serotonin, (K) relative brain 5-hydroxyindoleacetic acid (5-HIAA). (C) Photograph of an untreated Pah−/− mouse (Pahenu2) and a Pah−/− mouse eight weeks after receiving rAAV2/8-mediated liver-directed gene therapy (AAV8) demonstrating the change in coat color associated with normalization of blood Phe concentration.
Figure 6
Figure 6. Effects of Phe-reducing treatments on behavioral testing in C57BL/6-Pahenu2/enu2 mice
Results of behavioral performance in the open field, fear conditioning chamber, and nesting behavior of female (F) and male (M) untreated Pah−/− mice, Pah−/− mice receiving a phenylalanine-restricted diet (PAH −/− low Phe diet), and Pah−/− mice following AAV-mediated liver-directed gene therapy (PAH−/− AAV8). The results of the behavioral performance in the open field are presented in panel (A) activity in the open field (total distance moved) and (B) measures of anxiety in the open field (% time in center). Baseline activity while in the fear conditioning enclosure is shown in panel (C). Nest building (qualitative nest rating scale) is presented in panel (D). All data are expressed as mean ± SEM. (A) In Pah−/− mice, higher activity levels were seen in mice on a low Phe diet compared to those on a regular diet in the open field enclosure. (B) There were no effects of treatment on measures of anxiety in the center of the open field. (C) Baseline activity in the fear conditioning enclosure showed similar treatment effects. Pah−/− mice moved less than Pah−/− mice treated with low Phe diet and Pah−/− treated with AAV8. (D) Pah−/− mice treated with AAV8 showed more complex nest building than Pah−/− mice treated with low Phe diet. Vertical bars and asterisks above the columns indicate the probability (p) from the Tukey’s test that the means of the selected groups are identical. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.
Figure 7
Figure 7. Effects of Phe-reducing treatments on cognitive testing in C57BL/6-Pahenu2/enu2 mice
Results of cognitive performance in the Morris water maze of female and male untreated Pah−/− mice, Pah−/− mice receiving a phenylalanine-restricted diet (PAH −/− low Phe diet), and Pah−/− mice following AAV-mediated liver-directed gene therapy (PAH−/− AAV8). The results of water maze testing are presented in panel (A) average velocity (cm/second), (B) latency to the target platform (seconds), (C) cumulative distance to the target (cm), and (D) total distance moved (cm) with results shown for both sexes combined. (A) PAH−/− mice that received AAV8 swam faster than both Pah−/− untreated and Pah−/− low Phe diet mice. (B) When latency to locate the platform was analyzed, there was a significant treatment effect in the second session. (C) Pah−/− mice that received AAV8 showed better performance than genotype-matched mice that received no treatment (p = 0.020) or a low Phe diet (p = 0.006) and significant treatment effects were seen in the first and second sessions. (D) Analyzing total distance moved did not reveal any differences between the treatment groups. The data are represented as mean ± SEM for each experimental group at each individual session. For the treatment effects, asterisks denote sessions in which results in the treatment groups differed significantly from that of untreated Pah−/− mice.
Figure 8
Figure 8. Expression of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH2) in brain of C57BL/6-Pahenu2/enu2 mice and effects of phenylalanine reducing treatments
The abundance of (A) TH and (C) TPH2 proteins in half brain homogenates from wild type mice (5 male, 5 female), untreated Pah−/− mice (5 male, 5 female), and Pah−/− mice (4 male and 4 female in each group) treated with either dietary phenylalanine restriction or AAV8 liver gene therapy were measured relative to GAPDH by Western blotting. The data are presented as the mean ± SEM TH/GAPDH or TPH/GAPDH ratios. The maximally stimulated TH and TPH activities (panels (B) and (D) respectively) in half brain homogenates were measured in vitro and the data were expressed as mean ± SEM pmol reaction product produced/min/mg brain protein. Differences across groups were explored using one-way ANOVA with post hoc Holm-Sidak’s multiple comparisons test. Vertical bars and asterisks above the columns indicate the probability (p) from the Tukey’s test that the means of the selected groups are identical. **** p < 0.0001, ** p < 0.01, * p < 0.05. TH (panel E, 200 × magnification) abundance in microscopic sections of brain striatum was detected by immunohistology and fluorescence microscopy (representative images shown).

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