The Housekeeping Gene Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Regulates Multiple Developmental and Metabolic Pathways of Murine Embryonic Stem Cell Neuronal Differentiation

Abstract

The mechanisms by which mutations of the purinergic housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) cause the severe neurodevelopmental Lesch Nyhan Disease (LND) are poorly understood. The best recognized neural consequences of HPRT deficiency are defective basal ganglia expression of the neurotransmitter dopamine (DA) and aberrant DA neuronal function. We have reported that HPRT deficiency leads to dysregulated expression of multiple DA-related developmental functions and cellular signaling defects in a variety of HPRT-deficient cells, including human induced pluripotent stem (iPS) cells. We now describe results of gene expression studies during neuronal differentiation of HPRT-deficient murine ESD3 embryonic stem cells and report that HPRT knockdown causes a marked switch from neuronal to glial gene expression and dysregulates expression of Sox2 and its regulator, genes vital for stem cell pluripotency and for the neuronal/glial cell fate decision. In addition, HPRT deficiency dysregulates many cellular functions controlling cell cycle and proliferation mechanisms, RNA metabolism, DNA replication and repair, replication stress, lysosome function, membrane trafficking, signaling pathway for platelet activation (SPPA) multiple neurotransmission systems and sphingolipid, sulfur and glycan metabolism. We propose that the neural aberrations of HPRT deficiency result from combinatorial effects of these multi-system metabolic errors. Since some of these aberrations are also found in forms of Alzheimer's and Huntington's disease, we predict that some of these systems defects play similar neuropathogenic roles in diverse neurodevelopmental and neurodegenerative diseases in common and may therefore provide new experimental opportunities for clarifying pathogenesis and for devising new potential therapeutic targets in developmental and genetic disease.

Consequences of impaired purine recycling on the proteome in a cellular model of Lesch–Nyhan disease

“…There are no prior proteomic studies of HGprt-deficient cells for comparisons with the current studies, but there are several published transcriptomic studies involving mouse MN9D neuroblastoma cells [26], human NT2 neuroblastoma cells [43], human SH-SY5Y neuroblastoma cells [27], mouse ESD3 embryonic stem cells [30], and human fibroblasts [60]. A common finding in all of these studies was an unexpectedly broad array of gene expression abnormalities.…”

“…Another study involving the shRNA knockdown strategy applied to human SH-SY5Y neuroblastoma cultures suggested abnormalities of the Wnt/beta-catenin pathways [27], and another showed changes in microRNAs that may influence levels of these transcription factors [40, 41]. A recent study of the transcriptome using the shRNA strategy to knock down HGprt in ESD3 mouse embryonic stem cells differentiated towards the dopaminergic phenotype confirmed prominent dysregulation of the dopaminergic phenotype and associated developmental transcription factors [30]. …”

“…Clinical studies have suggested that dysfunction of specific dopaminergic neurons projecting to the basal ganglia underlie many of the neurological and behavioral abnormalities seen in patients with LND [12, 13], and autopsy studies have revealed significant reductions in basal ganglia dopamine and related indices of the dopamine neuron phenotype [14-16]. Prominent abnormalities of dopamine neurons have been documented for an HGprt knockout mouse [17-20], and HGprt − neuron-like cells in culture [21-30]. These findings imply that HGprt deficiency causes a cell-intrinsic defect that can be modeled in vitro, in the absence of complex inter-cellular relationships in the developing nervous system.…”

MicroRNAs

“…Along with miRNAs, other sets of complex transcriptional aberrations have been documented in human and mouse in vitro models of HPRT deficiency. These findings have pointed to an intricate pattern of dysregulated functions and pathways that lead to a complex neuropathogenicity (Kang et al, 2013). It seems highly possible that miRNAs play a central role in the pathogenic mechanisms responsible for this monogenic disease.…”

“…Microarray and RNA-seq (transcriptome) analyses have been conducted on HPRT-deficient cells including LND-derived fibroblasts and stem cells as well as brains of HPRT-KO mice to determine how the disruption of purine metabolism affects gene expression and biochemical pathways (Ceballos-Picot et al, 2009;Kang & Friedmann, 2015;Kang, Park, Bader, & Friedmann, 2013;Song & Friedmann, 2007). A complex and yet compelling picture has started to emerge out of those genomics studies.…”