UC San Diego

Differentiation to the neuronal cell type requires the temporal and spatial specification of neuronal transcription factors. The dysregulation of expression of these neuronal transcription factors during differentiation may lead to neuronal dysfunction or neurological disease. In Lesch-Nyhan Disease (LND), mutations in the gene encoding the purine salvaging and purine biosynthesis enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) result in a characteristic syndrome of hyperuricemia, severe dystonia, and self-injurious behavior. LND is associated with a defect in basal ganglia dopaminergic (DA) pathways. However, the mechanisms linking the purine metabolism defect to disorders seen in the CNS are poorly understood. We have hypothesized that the "simple," monogenic defect in LND is associated with a complex network of genetic and protein interactions that affect development of DA neurons. In this study, we examined HPRT-deficiency in human embryonic carcinoma cells, NTera2 cl. D/1 (NT2), which provides an in vitro model of neurogenesis. In these studies we used a retrovirus expressing a small hairpin RNA (shRNA) to knock down HPRT expression in NT2 cells, and the resulting cells were induced to differentiate along neuronal pathways by retinoic acid (RA) treatment. We report here that several key neuronal transcription factors and dopamininergic neuron gene markers in the HPRT -deficient NT2 cells exhibit aberrant gene expression before and during differentiation. Differentiated HPRT- deficient neurons also showed morphological differences in neurite outgrowth, but retained wild-type electrophysiological properties. These results directly connect aberrant neurogenesis to HPRT deficiency and suggest that HPRT, a housekeeping gene, plays a role in neurodevelopment