Purinergic signaling in human pluripotent stem cells is regulated by the housekeeping gene encoding hypoxanthine guanine phosphoribosyltransferase

Abstract

Lesch-Nyhan disease (LND) is an X-linked genetic disorder caused by mutations of the hypoxanthine guanine phosphoribosyltransferase (HPRT) purine biosynthesis gene and characterized by aberrant purine metabolism, deficient basal ganglia dopamine levels, dystonia, and severe neurobehavioral manifestations, including compulsive self-injurious behavior. Although available evidence has identified important roles for purinergic signaling in brain development, the mechanisms linking HPRT deficiency, purinergic pathways, and neural dysfunction of LND are poorly understood. In these studies aimed at characterizing purinergic signaling in HPRT deficiency, we used a lentivirus vector stably expressing an shRNA targeted to the HPRT gene to produce HPRT-deficient human CVB induced pluripotent stem cells and human HUES11 embryonic stem cells. Both CVB and HUES11 cells show >99% HPRT knockdown and demonstrate markedly decreased expression of the purinergic P2Y1 receptor mRNA. In CVB cells, P2Y1 mRNA and protein down-regulation by HPRT knockdown is refractory to activation by the P2Y1 receptor agonist ATP and shows aberrant purinergic signaling, as reflected by marked deficiency of the transcription factor pCREB and constitutive activation of the MAP kinases phospho-ERK1/2. Moreover, HPRT-knockdown CVB cells also demonstrate marked reduction of phosphorylated β-catenin. These results indicate that the housekeeping gene HPRT regulates purinergic signaling in pluripotent human stem cells, and that this regulation occurs at least partly through aberrant P2Y1-mediated expression and signaling. We propose that such mechanisms may play a role in the neuropathology of HPRT-deficiency LND and may point to potential molecular targets for modulation of this intractable neurological phenotype.neurodevelopment | neurotransmitters C omplete genetic deficiency of the enzyme hypoxanthine guanine phosphoribosyltransferase (HPRT) causes LeschNyhan disease (LND), an intractable neurobehavioral disorder characterized by hyperuricemia, cognitive impairment, dystonia with spasticity choreoathetosis and compulsive self-mutilation (1). The enzyme HPRT plays a major role in salvage purine biosynthesis by catalyzing the conversion of hypoxanthine and guanine to the nucleotides inosine monophosphate (IMP) and guanosine monophosphate (1, 2). HPRT deficiency leads to the hallmark biochemical defect in LND: greatly increased de novo purine synthesis, with resulting elevated levels of oxypurines and tissue accumulation of uric acid, gout, and life-threatening nephrolithiasis (3, 4). Although treatment with the xanthine oxidase inhibitor allopurinol effectively prevents hyperuricemia and renal damage in LND and thereby markedly extends the lifespan of patients, neither allopurinol nor any other treatment produces lasting improvement in neurological manifestations (5, 6).PET scanning and postmortem studies of brains from patients with LND have demonstrated decreased levels of dopamine, dopamine biosynthetic enzymes, and dopamine uptake in the basal ganglia (...

Impairment of adenylyl cyclase 2 function and expression in hypoxanthine phosphoribosyltransferase-deficient rat B103 neuroblastoma cells as model for Lesch–Nyhan disease: BODIPY–forskolin as pharmacological tool

“…Implications of our study for understanding LND HPRT deficiency has been repeatedly associated with defects in neuronal differentiation (Ma et al 2001;Connolly 2001;Jinnah 2009;Erdorf et al 2011a;Kang et al 2011;Guibinga et al 2010Guibinga et al , 2012Mastrangelo et al 2012). We could confirm this differentiation defect since HPRT − cells underwent much less striking morphological changes upon exposure to FS and BODIPY-FS than control cells (Fig.…”

“…In the present study, we used a rat neuroblastoma cell line as model. Future studies will have to examine abnormalities in AC isoform function and expression in human neuroblastoma cell culture models (Shirley et al 2007), pluripotent human stem cells (Mastrangelo et al 2012) and in the HPRT − mouse model (Jinnah et al 1992;Song and Friedmann 2007). Currently, we are establishing a HPRT − mouse colony in our group on a clearly defined genetic background for such studies.…”

“…Currently, we are establishing a HPRT − mouse colony in our group on a clearly defined genetic background for such studies. A strong argument in favor of B103 cells being a relevant cell culture model for LND comes from the fact that in both HPRT − B103 cells and pluripotent human stem cells abnormalities in purinoceptor function have been observed (Erdorf et al 2011a;Mastrangelo et al 2012). Thus, we anticipate that altered AC function and expression will also be detected in human stem cells.…”

“…5). The differentiation defect of HPRT − cells is seen under different experimental conditions and involves multiple signal transduction pathways including purinoceptors, microRNAs and the canonical Wnt pathway (Ma et al 2001;Connolly 2001;Jinnah 2009;Erdorf et al 2011a;Kang et al 2011;Guibinga et al 2010Guibinga et al , 2012Mastrangelo et al 2012).…”

Transcriptomic Approach to Lesch-Nyhan Disease

“…Recent studies also have established roles of nucleotides in controlling several aspects of neurogenesis, neural differentiation, [27–29] and migration in the developing mammalian brain through interactions with two classes of purinergic P2 receptors. [30,31] Because LND is a classical inborn error of purine metabolism, and because we and other groups have demonstrated aberrant neurogenesis and neural pathway development in LND [10,11,16,21,22,32] , we have based much of our recent work on human iPS cells obtained from LND patients' fibroblasts for studying the development of dopaminergic pathways.…”