HPRT-Deficiency Dysregulates cAMP-PKA Signaling and Phosphodiesterase 10A Expression : Mechanistic Insight and Potential Target for Lesch-Nyhan Disease?

The authors thank Dr. Hyder Jinnah from Emory University School of Medicine for providing us with MN9D cells. The authors also thank Dr. Atsushi Miyanohara, Director of the vector development laboratory from the Department of Pediatrics, UCSD Gene Therapy Program for the preparation of viral vectors. Funding: This work is supported by a grant from DK082840 from the National Institute of Health, USA, and by a research grant from the University of California San Diego Academic Senate. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

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

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

Schematic and summary model of HPRT-deficiency role in dysregulating striatal gene expression and signaling.

<p>HPRT-deficiency alters the purine pool; and via mechanisms that are still ill-defined, these alterations affect the gene expression of keys striatal-enriched transcription factors such as Ascl1, Bcl11b and Foxp1, notably during neurodevelopment. Later in mature neurons, these transcription factors control (directly or indirectly) the expression of several genes among them, <i>Darpp-32</i> and <i>Bdnf</i>, whose the dysregulation affects striatal neurons signaling and neurotransmission, thus contributing to LNS neurological phenotype.</p

Effects of hydrogen peroxide-mediated cellular toxicity in HPRT-deficient striatal cells:

<p>(<b>A</b>) (Y705/706) TrkB phosphoprotein expression in response to hydrogen peroxide H₂O₂ treatment (100 µM). Immuno-blot and quantification of Y705/706 TrkB phosphoprotein in control and HPRT-deficient striatal STHdh cells. The quantification bar graphs are shown as means ± SEM (n = 6, *P<0.05). (<b>B</b>) Enhanced neuroprotective effects against reactive oxygen species (ROS)-mediated cell death in HPRT-deficient striatal cells. Control (CTL) and HPRT-knock-down cells (HPRTKD) were treated with hydrogen peroxide H₂O₂ for 4 hours and then exposed to Sytox green for 10 min. Figure shows microscopy images of DAPI staining and green fluorescence which is a measure of the overall cell death level. There is reduced green fluorescence in HPRT-deficient cells relative to control after stimulation with H₂O₂. (Bar scale, 100 µm). This is confirmed by the quantification of the number of Sytox green fluorescent cells as illustrated in the appended graph. Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisks (*) represent statistical significance between H₂O₂ treated cells (p<0.05, t-test).</p

Gene & Protein expression for HPRT and ASCL1.

<p>(<b>A</b>) Gene expression for HPRT and ASCL1 in control (CTL, open bars) and HPRT knockdown (HPRTKD, closed bars) striatal STHdh cells. Bars represent mean ±SEM of duplicate PCR measurements carried out independently twice (n = 4). mRNA expression is normalized to GAPDH level (*P<0.05, t-test). (<b>B</b>) Immuno-blot and quantification of HPRT and ASCL1 protein expression in striatal STHdh cells. The quantification bar graphs are shown as means ± SEM (n = 3, *P<0.05, t-test). (<b>C</b>) Gene expression for DLX2, FOXG1 and GSX1 in control (CTL, open bars) and HPRT knockdown (HPRTKD, closed bars) Striatal cells. Bars represent mean ±SEM (n = 4) *P<0.05, t-test.</p

Striatal Neurodevelopment Is Dysregulated in Purine Metabolism Deficiency and Impacts DARPP-32, BDNF/TrkB Expression and Signaling: New Insights on the Molecular and Cellular Basis of Lesch-Nyhan Syndrome

<div><p>Lesch-Nyhan Syndrome (LNS) is a neurodevelopmental disorder caused by mutations in the gene encoding the purine metabolic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). This syndrome is characterized by an array of severe neurological impairments that in part originate from striatal dysfunctions. However, the molecular and cellular mechanisms underlying these dysfunctions remain largely unidentified. In this report, we demonstrate that HPRT-deficiency causes dysregulated expression of key genes essential for striatal patterning, most notably the striatally-enriched transcription factor B-cell leukemia 11b (Bcl11b). The data also reveal that the down-regulated expression of Bcl11b in HPRT-deficient immortalized mouse striatal (STH<i>dh</i>) neural stem cells is accompanied by aberrant expression of some of its transcriptional partners and other striatally-enriched genes, including the gene encoding dopamine- and cAMP-regulated phosphoprotein 32, (DARPP-32). Furthermore, we demonstrate that components of the BDNF/TrkB signaling, a known activator of DARPP-32 striatal expression and effector of Bcl11b transcriptional activation are markedly increased in HPRT-deficient cells and in the striatum of HPRT knockout mouse. Consequently, the HPRT-deficient cells display superior protection against reactive oxygen species (ROS)-mediated cell death upon exposure to hydrogen peroxide. These findings suggest that the purine metabolic defect caused by HPRT-deficiency, while it may provide neuroprotection to striatal neurons, affects key genes and signaling pathways that may underlie the neuropathogenesis of LNS.</p></div

Gene and Protein Expression for BDNF and TRKB.

<p>(<b>A</b>) Gene expression for <i>Bdnf</i> and <i>Trkb</i> in control (CTL, open bars) and HPRT knockdown (HPRTKD, closed bars) striatal STHdh cells. Bars represent mean ±SEM (n = 4). mRNA expression is normalized to GAPDH RNA level (*P<0.05, t-test). (<b>B</b>) Immuno-blot and quantification of BDNF and TRKB protein expression in striatal cells. The quantification bar graphs are shown as means ± SEM (n = 3, *P<0.05, t-test). (<b>C</b>) Gene expression for <i>Bdnf</i> and <i>Trkb</i> in the striatum of wild-type (WT, open bars) and HPRT knockout (HPRTKO, closed bars) mice. Bars represent mean ±SEM of duplicate PCR measurements (n = 4). mRNA expression is normalized to GAPDH mRNA level (*P<0.05, t-test). (<b>D</b>) Immuno-blot and quantification of BDNF and TrkB protein expression in the striatum of WT and HPRTKO mice. The quantification bar graphs are shown as means ± SEM (n = 3, *P<0.05, t-test). (<b>E</b>) (Y705/706) TrkB phosphoprotein expression in response to BDNF treatment (10 ng/ml). Immuno-blot and quantification of Y705/706) TrkB phosphoprotein in control and HPRT-deficient striatal cells, the quantification bar graphs are shown as means ± SEM (n = 6, *P<0.05).</p

Restoration of Bcl11b expression in HPRT-deficient striatal cells:

<p>(<b>A</b>) Immuno-blot and quantification of Bcl11b expression in control (CTL) and HPRT-deficient striatal cells transfected with plasmid encoding Bcl11b (Bcl11b) and GFP (GFP). The quantification bar graphs are shown means± SEM (n = 3, *P<0.05, ANOVA, Tukey post-hoc test). (<b>B</b>) Immuno-blot and quantification of Y705/706 TrkB after H₂O₂ exposure for control (CTL), HPRT-deficient cells transfected with Bcl11b (Bcl11b) and GFP (GFP). The quantification bar graphs are shown as means ± SEM (n = 4, *P<0.05, ANOVA, Tukey post-hoc test). (<b>C</b>) quantification of the number of Sytox green fluorescent cells upon treatment of control (CTL) and HPRT-deficient cells transfected with Bcl11b (BCL11b) and GFP (GFP) plasmid. Data show that the rescue of Bcl11b expression in HPRT-deficient cells does not lead to added protection from cell death triggered by H₂O₂ (ns =  non significant, *p<0.05, ANOVA). (<b>D</b>) Gene expression of DARPP-32 in control (CTL), GFP and Bcl11b (rescued) transfected cells. Data show that the rescued expression of Bcl11b in HPRT-deficient cells leads to restoration of DARPP-32 expression in level similar to control, the data are shown as means ± SEM ((ns =  non significant, n = 4, *P<0.05, ANOVA, Tukey post-hoc test). (<b>E</b>) Gene expression for Actn2, Arpp19, Foxp1, Ngef, Pde10a, and Rgs9 in control (CTL, open bars), GFP (GFP, closed bars) and Bcl11b (Bcl11b, grey bars) transfected striatal cells. Bars represent mean ±SEM (n = 4). *P<0.05, ANOVA.</p

Gene and Protein Expression for <i>Ppp1r1b</i>/DARPP-32.

<p>(<b>A</b>) Gene expression for <i>Ppp1r1b</i>/DARPP-32 in control striatal STHdh cells (CTL, open bars) and HPRT-knockdown (HPRTKD, closed bars). Bars represent mean ±SEM (n = 4). mRNA expression is normalized to GAPDH mRNA level (*P<0.05, t-test). (<b>B</b>) Immuno-blot and quantification of DARPP-32 protein expression in striatal cells. The quantification bar graphs are shown as means ± SEM (n = 3, *P<0.05, t-test). (<b>C</b>) Gene expression for <i>Ppp1r1b</i>/DARPP-32 in the striatum of wild-type (WT, open bars) and HPRT knockout (HPRTKO, closed bars) mice. Bars represent mean ±SEM (n = 4). mRNA expression is normalized to GAPDH RNA level (*P<0.05, t-test). (<b>D</b>) Immuno-blot and quantification of DARPP-32 protein expression in the striatum of WT and HPRTKO mice. The quantification bar graphs are shown as means ± SEM (n = 3, *P<0.05, t-test). (<b>E</b>) DARPP-32 phosphoprotein (Thr34) expression in response to D1R (SKF393298) agonist treatment (50 µM). Immuno-blot and quantification of Thr34-DARPP-32 in control and HPRT-deficient striatal cells, the quantification bar graphs are shown as means ± SEM (n = 6, *P<0.05). (<b>F</b>) Thr34-DARPP-32 phosphoprotein expression in striatal tissue of WT (open bar) and HPRTKO (closed bar) mice (n = 3, *P<0.05).</p