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

Abstract

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 indicative of 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 rapidly depleted cells of intracellular tyrosine and DA. Compared to physiological levels (75 {micro}M), either low (35 {micro}M) or high concentrations of tyrosine (275 or 835 {micro}M) 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. Specifically, levels of the broad specificity transporter of neutral amino acids LAT1 and the branched-chain amino-acid-transporter BCAT2 were both similarly increased. 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 on the molecular level in neurometabolic disorders, such as PKU and TYRSN1. HighlightsTyrosinemia type I and phenylketonuria are rare metabolic disorders, characterized by extremely elevated plasma levels of the aromatic amino acids tyrosine and phenylalanine, respectively. To study the molecular consequences of dysregulated amino acid uptake on dopamine homeostasis, we simulated these conditions using rat pheochromocytoma (PC12) cells, an established model system for investigating catecholamine production. Non-physiological tyrosine levels induced a broad cellular response, including reduced protein level and phosphorylation stoichiometry for tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, which in turn resulted in decreases in both intra- and extracellular dopamine levels. Similarly, dopamine content was also decreased when cells were exposed to high phenylalanine levels characteristic of phenylketonuria, although the underlying molecular mechanism for this response appears to differ.