Long-term proline exposure alters nucleotide catabolism and ectonucleotidase gene expression in zebrafish brain

Abstract

Hyperprolinemia is an inherited disorder of proline metabolism and hyperprolinemic patients can present neurological manifestations, such as seizures cognitive dysfunctions, and psychotic disorders. However, the underlying mechanisms of these symptoms are still unclear. Since adenine nucleotides play crucial roles in neurotransmission and neuromodulation, we evaluated the in vivo and in vitro effects of proline on ectonucleotidase activities and gene expression in zebrafish brain. For the in vivo studies, animals were exposed at two proline concentrations (1.5 and 3.0mM) during 1h or 7days (short- or long-term treatments, respectively). For the in vitro assays, different proline concentrations (ranging from 3.0 to 1000μM) were tested. Short-term proline exposure did not promote significant changes on the ectonucleotidase activities and gene expression. Long-term proline exposure significantly increased ATP catabolism in both concentrations tested (14% and 22%, respectively), whereas ADP and AMP hydrolysis were increased only at 3.0mM proline (21% and 17%, respectively) when compared to control. Moreover, the relative gene expression of enpd3 increased in both treated groups after long-term proline, whereas enptd1 increased only at 3.0mM proline. Proline in vitro did not promote significant changes on ectonucleotidase activities. Altogether, these data indicate that the enzymes responsible for the control of extracellular nucleotides levels might be altered after proline exposure in zebrafish, contributing to better understand the pathophysiology of this disease. Moreover, such findings might facilitate the use of the zebrafish as a complementary vertebrate model for studying inborn errors of amino acid metabolism.