The effects of phenylalanine and tyrosine levels on dopamine production in rat PC12 cells. Implications for treatment of phenylketonuria, tyrosinemia type 1 and comorbid neurodevelopmental disorders.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene, resulting in phenylalanine accumulation and impaired tyrosine production. In Tyrosinemia type 1 (TYRSN1) mutations affect fumarylacetoacetate hydrolase, leading to accumulation of toxic intermediates of tyrosine catabolism. Treatment of TYRSN1 with nitisinone results in extreme tissue levels of tyrosine. Although PKU and TYRSN1 have opposite effects on tyrosine levels, both conditions have been associated with neuro-psychiatric symptoms typically present in ADHD, possibly indicating an impaired dopamine (DA) synthesis. However, concrete in vivo data on the possible molecular basis for disrupted DA production under disease mimicking conditions have been lacking. In pursuit to uncover associated molecular mechanisms, we exposed an established, DA producing cell line (PC12) to different concentrations of phenylalanine and tyrosine in culture media. We measured the effects on viability, proteomic composition, tyrosine, DA and tyrosine hydroxylase (TH) levels and TH phosphorylation. TH catalyzes the rate-limiting step in DA synthesis. High extracellular levels of phenylalanine depleted cells of intracellular tyrosine and DA. Compared to physiological levels (75 µM), either low (35 µM) or high concentrations of tyrosine (275 or 835 µM) decreased cellular DA, TH protein, and its phosphorylation levels. Using deep proteomic analysis, we identified multiple proteins, biological processes and pathways that were altered, including enzymes and transporters involved in amino acid metabolism. Using this information and published data, we developed a mathematical model to predict how extracellular levels of aromatic amino acids can affect the cellular synthesis of DA via different mechanisms. Together, these data provide new information about the normal regulation of neurotransmitter synthesis and how this may be altered in neurometabolic disorders, such as PKU and TYRSN1, with implications for the treatment of cognitive symptoms resulting from comorbid neurodevelopmental disorders.

Personalized Medicine to Improve Treatment of Dopa-Responsive Dystonia-A Focus on Tyrosine Hydroxylase Deficiency.

Dopa-responsive dystonia (DRD) is a rare movement disorder associated with defective dopamine synthesis. This impairment may be due to the fact of a deficiency in GTP cyclohydrolase I (GTPCHI, GCH1 gene), sepiapterin reductase (SR), tyrosine hydroxylase (TH), or 6-pyruvoyl tetrahydrobiopterin synthase (PTPS) enzyme functions. Mutations in GCH1 are most frequent, whereas fewer cases have been reported for individual SR-, PTP synthase-, and TH deficiencies. Although termed DRD, a subset of patients responds poorly to L-DOPA. As this is regularly observed in severe cases of TH deficiency (THD), there is an urgent demand for more adequate or personalized treatment options. TH is a key enzyme that catalyzes the rate-limiting step in catecholamine biosynthesis, and THD patients often present with complex and variable phenotypes, which results in frequent misdiagnosis and lack of appropriate treatment. In this expert opinion review, we focus on THD pathophysiology and ongoing efforts to develop novel therapeutics for this rare disorder. We also describe how different modeling approaches can be used to improve genotype to phenotype predictions and to develop in silico testing of treatment strategies. We further discuss the current status of mathematical modeling of catecholamine synthesis and how such models can be used together with biochemical data to improve treatment of DRD patients.

Structure of the ALS Mutation Target Annexin A11 Reveals a Stabilising N-Terminal Segment.

The functions of the annexin family of proteins involve binding to Ca2+, lipid membranes, other proteins, and RNA, and the annexins share a common folded core structure at the C terminus. Annexin A11 (AnxA11) has a long N-terminal region, which is predicted to be disordered, binds RNA, and forms membraneless organelles involved in neuronal transport. Mutations in AnxA11 have been linked to amyotrophic lateral sclerosis (ALS). We studied the structure and stability of AnxA11 and identified a short stabilising segment in the N-terminal end of the folded core, which links domains I and IV. The crystal structure of the AnxA11 core highlights main-chain hydrogen bonding interactions formed through this bridging segment, which are likely conserved in most annexins. The structure was also used to study the currently known ALS mutations in AnxA11. Three of these mutations correspond to buried Arg residues highly conserved in the annexin family, indicating central roles in annexin folding. The structural data provide starting points for detailed structure-function studies of both full-length AnxA11 and the disease variants being identified in ALS.

Serine 19 phosphorylation and 14-3-3 binding regulate phosphorylation and dephosphorylation of tyrosine hydroxylase on serine 31 and serine 40.

Multisite phosphorylation and structural flexibility allow for complex regulation of proteins through cellular signaling. Tyrosine hydroxylase (TH), a key enzyme of catecholamine synthesis, is regulated by multiple neuronal signaling pathways through phosphorylation at serine 19 (Ser19), serine 31 (Ser31), and serine 40 (Ser40) located in the flexible, far N-terminal region of the regulatory domain. Phosphorylated Ser19 (pSer19) provides a binding site for 14-3-3 proteins, a family of multi-target binding adaptor proteins. We hypothesized that pSer19 and 14-3-3 binding can regulate access to the Ser31 and Ser40 sites and modulate the dynamics of their phosphorylation state. To avoid complications from upstream signal interactions and have good control of TH-phosphorylation and 14-3-3 binding stoichiometry, we used purified recombinant human TH and 14-3-3 dimer types. We found that pSer19 strongly stimulated Ser31 phosphorylation (4.6-fold), but inhibited pSer31 dephosphorylation (3.4-fold). Binding of 14-3-3ζ counteracted the stimulatory effect of pSer19 on phosphorylation at Ser31, but amplified the effect on its dephosphorylation. In contrast, phosphorylation at Ser19 had moderate effect on pSer40 dephosphorylation, but 14-3-3ζ binding inhibited dephosphorylation, an effect that was consistent across different homo- and heterodimeric 14-3-3s. Additional phosphorylation of Ser31 or Ser40 had little impact on the binding affinity of pSer19 TH to 14-3-3s. Mathematical modeling was performed to elucidate possible physiological implications of these observations. We propose a role of Ser19 and 14-3-3 proteins as modulators of TH phosphorylation in response to neuronal co-signaling events. These mechanisms add to our understanding of the multifaceted roles of phosphorylation and adaptor proteins in cellular signaling.

Tyrosinemia Type 1 and symptoms of ADHD: Biochemical mechanisms and implications for treatment and prognosis.

Hereditary tyrosinemia Type 1 (HT-1) is a rare metabolic disease where the enzyme catalyzing the final step of tyrosine breakdown is defect, leading to accumulation of toxic metabolites. Nitisinone inhibits the degradation of tyrosine and thereby the production of harmful metabolites, however, the concentration of tyrosine also increases. We investigated the relationship between plasma tyrosine concentrations and cognitive functions and how tyrosine levels affected enzyme activities of human tyrosine hydroxylase (TH) and tryptophan hydroxylase 2 (TPH2). Eight Norwegian children between 6 and 18 years with HT-1 were assessed using questionnaires measuring Attention Deficit Hyperactivity Disorder (ADHD)-symptoms and executive functioning. Recent and past levels of tyrosine were measured and the enzyme activities of TH and TPH2 were studied at conditions replicating normal and pathological tyrosine concentrations. We observed a significant positive correlation between mean tyrosine levels and inattention symptoms. While TH exhibited prominent substrate inhibition kinetics, TPH2 activity also decreased at elevated tyrosine levels. Inhibition of both enzymes may impair syntheses of dopamine, noradrenaline, and serotonin in brain tissue. Inattention in treated HT-1 patients may be related to decreased production of these monoamines. Our results support recommendations of strict guidelines on plasma tyrosine levels in HT-1. ADHD-related deficits, particularly inattention, should be monitored in HT-1 patients to determine whether intervention is necessary.

The quaternary structure of human tyrosine hydroxylase: effects of dystonia-associated missense variants on oligomeric state and enzyme activity.

Tyrosine hydroxylase (TH) is a multi-domain, homo-oligomeric enzyme that catalyses the rate-limiting step of catecholamine neurotransmitter biosynthesis. Missense variants of human TH are associated with a recessive neurometabolic disease with low levels of brain dopamine and noradrenaline, resulting in a variable clinical picture, from progressive brain encephalopathy to adolescent onset DOPA-responsive dystonia (DRD). We expressed isoform 1 of human TH (hTH1) and its dystonia-associated missense variants in E. coli, analysed their quaternary structure and thermal stability using size-exclusion chromatography, circular dichroism, multi-angle light scattering, transmission electron microscopy, small-angle X-ray scattering and assayed hydroxylase activity. Wild-type (WT) hTH1 was a mixture of enzymatically stable tetramers (85.6%) and octamers (14.4%), with little interconversion between these species. We also observed small amounts of higher order assemblies of long chains of enzyme by transmission electron microscopy. To investigate the role of molecular assemblies in the pathogenesis of DRD, we compared the structure of WT hTH1 with the DRD-associated variants R410P and D467G that are found in vicinity of the predicted subunit interfaces. In contrast to WT hTH1, R410P and D467G were mixtures of tetrameric and dimeric species. Inspection of the available structures revealed that Arg-410 and Asp-467 are important for maintaining the stability and oligomeric structure of TH. Disruption of the normal quaternary enzyme structure by missense variants is a new molecular mechanism that may explain the loss of TH enzymatic activity in DRD. Unstable missense variants could be targets for pharmacological intervention in DRD, aimed to re-establish the normal oligomeric state of TH.