HPRT Deficiency Coordinately Dysregulates Canonical Wnt and Presenilin-1 Signaling: A Neuro-Developmental Regulatory Role for a Housekeeping Gene?

Abstract

We have used microarray-based methods of global gene expression together with quantitative PCR and Western blot analysis to identify dysregulation of genes and aberrant cellular processes in human fibroblasts and in SH-SY5Y neuroblastoma cells made HPRT-deficient by transduction with a retrovirus stably expressing an shRNA targeted against HPRT. Analysis of the microarray expression data by Gene ontology (GO) and Gene Set Enrichment Analysis (GSEA) as well as significant pathway analysis by GeneSpring GX10 and Panther Classification System reveal that HPRT deficiency is accompanied by aberrations in a variety of pathways known to regulate neurogenesis or to be implicated in neurodegenerative disease, including the canonical Wnt/β-catenin and the Alzheimer's disease/presenilin signaling pathways. Dysregulation of the Wnt/β-catenin pathway is confirmed by Western blot demonstration of cytosolic sequestration of β-catenin during in vitro differentiation of the SH-SY5Y cells toward the neuronal phenotype. We also demonstrate that two key transcription factor genes known to be regulated by Wnt signaling and to be vital for the generation and function of dopaminergic neurons; i.e., Lmx1a and Engrailed 1, are down-regulated in the HPRT knockdown SH-SY5Y cells. In addition to the Wnt signaling aberration, we found that expression of presenilin-1 shows severely aberrant expression in HPRT-deficient SH-SY5Y cells, reflected by marked deficiency of the 23 kDa C-terminal fragment of presenilin-1 in knockdown cells. Western blot analysis of primary fibroblast cultures from two LND patients also shows dysregulated presenilin-1 expression, including aberrant proteolytic processing of presenilin-1. These demonstrations of dysregulated Wnt signaling and presenilin-1 expression together with impaired expression of dopaminergic transcription factors reveal broad pleitropic neuro-regulatory defects played by HPRT expression and suggest new directions for investigating mechanisms of aberrant neurogenesis and neuropathology in LND and potential new targets for restoration of effective signaling in this neuro-developmental defect.

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.…”

“…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.…”

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

“…Furthermore, purinergic signaling has been reported to regulate GSK-3β and nuclear translocation of β-catenin in astrocytes (23). In addition, we recently reported evidence of major disruption of phosphorylated β-catenin expression and impaired nuclear transport in HPRT-deficient human fibroblasts (24).…”