Purinergic signaling in human pluripotent stem cells is regulated by ...

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Purinergic signaling in human pluripotent stem cells is regulated by the housekeeping gene encoding hypoxanthine guanine phosphoribosyltransferase Lina Mastrangeloa, Ji-Eun Kimb, Atsushi Miyanoharaa, Tae Hyuk Kanga, and Theodore Friedmanna,1 a Department of Pediatrics, Center for Neural Circuits and Behavior, University of California at San Diego School of Medicine, La Jolla, CA 92093; and bNeurobiology Section, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093

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

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omplete genetic deficiency of the enzyme hypoxanthine guanine phosphoribosyltransferase (HPRT) causes Lesch– Nyhan 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 (7, 8). However, the mechanisms linking HPRT deficiency with the neurologic dysfunctions are little understood. Recently, evidence has accumulated identifying key roles for

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purine derivatives in modulating neurotransmitter release and demonstrating the involvement of purinergic receptors in neuronal growth and survival (9–13). A role of purinergic receptors in neuronal differentiation has been reported as well (14, 15). Recent studies also have established roles of nucleotides in controlling several aspects of neurogenesis, neural differentiation, and migration in the developing mammalian CNS through interactions with two classes of purinergic P2 receptors, the ionotropic P2X receptors and the P2Y class of G protein-coupled receptors (GPCRs) (16–18). Other studies have shown that ATP, acting via P2X and P2Y receptors, stimulates increased proliferation of embryonic stem cells (19). Moreover, P2Y receptor activation in adult neural progenitor cells occurs at least in part through effects on nucleotide-induced expression and on the function of transcription factors ERK1/2 and CREB phosphorylation (17). P2Y receptors also affect the vital Wnt/β-catenin signaling pathway, which includes a major class of growth factors with potent effects on stem cells and developmental processes (20, 21). Recent evidence shows that P2Y receptors mediate phosphorylation of glycogen synthase kinase (GSK-3β), a key mediator of Wnt signaling and nuclear transport of the transcription factor β-catenin (22). 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). Our present study on the effect of HPRT deficiency on purinergic signaling are based on the working hypothesis that alterations in purine pools resulting from defective salvage purine biosynthesis might affect the function of purinergic receptors, thereby leading to aberrant development of the CNS dopaminerelated pathways and aberrant dopaminergic neurogenesis. We have chosen to study mechanisms associated with that interaction in human induced pluripotent stem (iPS) and embryonic stem (ES) cells because of the growing importance of such cells in delineating the early molecular and cellular events in the defective neural development characteristic of LND and other neurodegenerative and neurodevelopmental diseases (25). A more detailed understanding of the effects of HPRT deficiency during embryogenesis is likely to clarify the developmental outcomes of LND and identify potential new therapeutic targets.

Author contributions: L.M. designed research; L.M. and T.H.K. performed research; L.M., J.-E.K., A.M., and T.H.K. contributed new reagents/analytic tools; L.M., J.-E.K., A.M., T.H.K., and T.F. analyzed data; and L.M. and T.F. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1

To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1118067109/-/DCSupplemental.

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Edited by Susan G. Amara, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved January 19, 2012 (received for review November 3, 2011)

Results HPRT Knockdown in CVB iPS Cells. Expression of the shRNA tar-

geted to HPRT profoundly reduces HPRT expression but does not produce major disturbances in the pluripotency of iPS cells. Fig. 1A shows mRNA levels of HPRT in CVB iPS cells transduced with the control anti-luciferase (shLux) and with the antiHPRT vector (shHPRT). HPRT mRNA expression was virtually undetectable in this assay and was reduced by >99% in cells transduced by shHPRT compared with control luciferase-knockdown cells. Similarly, HPRT protein expression was markedly reduced in the HPRT-knockdown cells (Fig. 1B). Finally, TLC-based HPRT enzyme assays confirmed the virtual complete loss of HPRT enzymatic activity in HPRT-knockdown CVB cells, as indicated by absence of IMP in the chromatography assay (Fig. 1C). The process of transduction of cells with the knockdown vectors does not lead to major changes in the pluripotency state of the cells, as measured by expression of the pluripotency markers Nanog and Oct-4 (Fig. 1 D and E). Expression of Nucleotide Signaling Components. Transcription of nucleotide signaling pathway components was examined by quantitative PCR assays (Fig. 2). Total mRNA was extracted from undifferentiated CVB iPS cells transduced with the control shLux or the shHPRT vectors, and real-time PCR analyses were performed for the purinergic receptors P2Y1, P2Y2, P2X3, and adenosine A2a and the ectonucleotidase enzyme NTPase. Fig. 2A demonstrates that HPRT knockdown in CVB cells is accompanied by a marked (∼90%) reduction of mRNA expression for the GPCR purinergic receptor P2Y1, a significant but less marked reduction of the ligand-gated receptor P2X3, and a significant up-regulation of NTPase but no change in the expression of P2Y2 or adenosine receptor A2a (Fig. 2C). Given that previous studies have demonstrated an important role of P2Y1mediated ATP signaling in neuronal migration (18), we used Western blot analysis to assess P2Y1 protein levels in control and HPRT-knockdown CVB cells. Fig. 2B shows that P2Y1 protein

expression is both qualitatively and quantitatively reduced in HPRT-deficient CVB cells. P2Y1 Expression in HPRT-Deficient HUES11 Human ES Cells. To determine whether aberrant P2Y1 expression is specific to CVB iPS cells or is a more general consequence of HPRT deficiency in pluripotent stem cells, we used real-time PCR to examine P2Y1 expression in HPRT-knockdown human HUES11 ES cells. We found that in these cells, efficient (>95%) HPRT knockdown with vector shHPRT measured by real-time PCR (Fig. 3A), Western immunoblot analysis (Fig. 3B), and HPRT enzymatic activity (Fig. 3C) leads to a significantly (∼60%) reduced expression of P2Y1 mRNA levels (Fig. 3D). Our results are consistent with changes seen in the HPRT-deficient CVB iPS cells. P2Y1 Regulates CREB Expression and ERK1/2 Phosphorylation in CVB iPS Cells. Recent studies with murine adult neural progenitor cells

have shown that the P2Y1 receptor agonists ADPβS and UTP stimulate the expression of phosphorylated CREB and ERK1/2, and that this stimulation is inhibited by the P2Y1 receptor antagonist MRS2179 (17). Because HPRT knockdown markedly down-regulates P2Y1 expression (Fig. 2 A and B), we examined the effect of HPRT knockdown on phospho-CREB and phospho-ERK1/2 expression in CVB cells. Our Western blot analyses (Fig. 4A) demonstrate, as expected, that in control CVB cells the P2Y1 agonist ATP up-regulates pCREB expression (lane b) compared with cells not exposed to ATP (lane a), and that the P2Y1 inhibitor MRS2179 abrogates that effect (lane c). In contrast, HPRT-knockdown CVB cells show little or no detectable basal level pCREB expression (lane d) or induction of pCREB (lane e) after exposure to the P2Y1 agonist ATP. Paradoxically, treatment of the CVB knockdown cells with the P2Y1 inhibitor MRS2179 leads to detectable expression of pCREB (lane f). ERK1/2 Signaling. As shown in Fig. 4B, Western blot analysis of CVB control HPRT-positive cells (lane a) demonstrates constitutively up-regulated pERK1/pERK2 expression after treatment

Fig. 1. HPRT knockdown of pluripotent CVB iPS cells. (A) HPRT mRNA expression was reduced by >99% compared with control luciferase-knockdown cells. (B) Whole-cell lysates (10 μg of protein per lane) were used for Western blot analysis to detect expression of HPRT. The blot shows the down-regulation of HPRT compared with the control sample. The β-actin level was determined by stripping and reprobing the membrane. (C) Chromatography showed undetectable levels of IMP in HPRT-knockdown CVB cells, and an 87% reduction in HPRT enzyme activity was measured by scintillation counting. (D and E) Expression of pluripotent markers, such as Nanog and Oct-4, was observed in CVB iPS CTL and HPRT-knockdown cells.

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with the P2Y1 agonist ATP (lane b). In contrast to the results obtained for pCREB, however, expression of pERK1/2 in ATPtreated cells is essentially unchanged in the presence of the P2Y1 inhibitor MRS2179 (lane c). Strikingly, in contrast, HPRTknockdown cells show constitutive activation of pERK1/2 expression, even in the absence of the agonist ATP (lane d), and no added effect of ATP (lanes e) or reduced expression in response to the P2Y1 inhibitor MRS2179 (lane f). These qualitative results were confirmed by quantitation of the Western blot analysis results shown in Fig. 4B. Increased GSK-3β Phosphorylation in HPRT-Deficient CVB iPS Cells.

Previous studies have identified a role for purinergic receptors in GSK-3β phosphorylation and β-catenin nuclear translocation in granule neurons (22). Accordingly, we used Western blot analysis to determine the levels of pGSK-3β in control and HPRTknockdown CVB cells. Fig. 5A shows that pGSK-3β protein expression in HPRT-knockdown CVB cells (lane d) is up-regulated compared with expression in control HPRT-positive CVB cells (lane a). Moreover, CVB cells transduced with the control antiluciferase vector show increased levels of pGSK-3β in response to treatment with the purinergic receptor agonist ATP (lane b), whereas in contrast, HPRT-knockdown CVB cells are refractory to the inducing effects of ATP (lane e). In both control and HPRT-knockdown CVB cells, ATP-induced pGSK-3β expression is not affected by exposure of cells to the P2Y1 inhibitor MRS2179 (lanes c and f). Decreased β-Catenin and β-Catenin Phosphorylation in HPRTDeficient CVB iPS Cells. pGSK-3β is a key determinant of canoni-

cal Wnt signaling through its role in regulating the phosphorylation and nuclear translocation of β-catenin (26). To characterize mechanisms associated with β-catenin signaling in HPRT-deficient CVB cells, we used Western blot analysis to measure total and phosphorylated β-catenin in control and HPRT-knockdown CVB iPS cells. We found that HPRT-knockdown CVB cells have a marked reduction of total cellular β-catenin and a virtual absence of pβ-catenin (Fig. 5B). Mastrangelo et al.

Discussion Most concepts of pathogenesis in LND have centered on the disruption of normal purine metabolism resulting from impaired reutilization and excessive de novo purine biosynthesis and the putative neurodevelopmental and metabolic effects of the resulting aberrant purine pools (3). The present study has identified an alternative potential mechanism for the dysregulation of purine and purine nucleotide metabolism in HPRT deficiency, involving severe disturbances in function of the purinergic receptor P2Y1 and its important role in neural development of the mammalian CNS. Recent studies have identified major roles for purines, purine nucleotides, and purinergic signaling in neurogenesis and the development of neural pathways during embryonic CNS development (16–19). It is now recognized that in cells of the developing mammalian CNS, binding of extracellular nucleotides to purinergic receptors initiates protein kinase cascades that stimulate downstream signaling pathways, which in turn lead to cell proliferation, migration, differentiation, neurite outgrowth, and synapse formation (10). Our demonstration of aberrant P2Y1 receptor expression complements previous demonstrations of disturbed expression of other purines and related receptors, such as adenosine, dopamine, and serotonin receptors, in HPRT deficiency (27). Before the present study, there was no evidence suggesting that purinergic receptors play a role in the HPRT-deficiency phenotype. Purinergic receptors are divided into two major families, P1 adenosine receptors and P2 receptors, which include ionotropic P2X and metabotropic P2Y receptors. To date, four subtypes of P1 receptors, seven subtypes of P2X receptors, and eight subtypes of P2Y receptors have been identified (11). The GPCR P2Y receptors are activated by a variety of nucleotides (e.g., ATP, ADP, UTP, UDP, NAD+) or nucleotide sugars and in turn activate several cellular signaling pathways. Evidence has accumulated indicting the involvement of purinergic mechanisms in such pathological conditions as brain trauma and ischemia, neurodegenerative diseases, and neuropsychiatric disorders, including depression and schizophrenia (28, 29). Important putative developmental functions of purinergic receptors have been PNAS | February 28, 2012 | vol. 109 | no. 9 | 3379

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Fig. 2. (A and C) Total RNA was isolated from CVB iPS control and HPRT-knockdown cells, and the relative quantities of P2Y1, P2X3, NTPase, P2Y2, and A2:00 AMRNA were determined by real-time PCR. Experiments were performed in triplicate. Data are presented as mean ± SD. *P < 0.05. (B) Decreased P2Y1 protein expression (10 μg protein per lane) in HPRT-knockdown cells (shHPRT) compared with CTL. A change in the level of desired protein was determined by densitometric scanning of the immunoreactive band using ImageJ software and corrected for GAPDH loading control. Data are presented as mean ± SD. *P < 0.05.

Fig. 3. Effect of HPRT knockdown on P2Y1 mRNA expression in human HUES11 ES cells. (A) HPRT mRNA levels were reduced by >99% in HPRTknockdown HUES11 cells. (B) Western blot analysis was performed to detect expression of HPRT, and β-actin level was determined by stripping and reprobing the membrane. (C) Scintillation counting revealed an 88% reduction in HPRT enzyme activity. (D) Effect of HPRT knockdown on P2Y1 mRNA expression. *P < 0.05.

inferred from in vivo studies showing transient and temporal expression of defined purinergic receptor subtypes during development (12, 30); for instance, P2Y1 purinergic receptors have

been detected in rat brain between embryonic day 10 and postnatal day 10 (9). Up-regulation of P2Y1 mRNA expression has been reported in rat brain during late embryonic development stages, and P2Y1 protein expression has been detected in postnatal developmental stages (30). Moreover, in chick brain, the P2Y1 receptor is regulated in a developmental manner during embryogenesis (31). These findings indicate that this class of receptors represents potentially suitable markers in human iPS and ES studies to model the neurodevelopmental consequences of HPRT deficiency and the neurological phenotype of LND (32). However, identifying the precise role of purinergic receptors in HPRT deficiency is complicated by the fact that in vitro studies have shown variable cell-dependent changes—both increases and decreases—in intracellular ATP concentrations in HPRT-deficient cell lines (33). Other studies have demonstrated species-dependent alterations in the activity of membrane NTPase, the enzyme involved in the rapid metabolism of ATP, in HPRT-deficient human and murine cells (34). 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 (8, 35), we have based much of our recent work and most of the present study on the underlying working hypothesis that aberrant purine metabolism in LND plays a direct causative role in the neurological phenotype of the disorder through disturbed purinergic signaling. In this study, we aimed to generate an in vitro human iPS and ES cell-based system in which to study the development of dopaminergic pathways. To determine whether P2Y1 downregulation by HPRT deficiency is specific for CVB iPS cells or is a more general feature of pluripotentiality in ES and iPS cells, we examined P2Y1 expression in HUES11 human ES cells (Fig. 3). Studies of aberrations in gene expression in ES cells rather than iPS cells can be highly informative, because, unlike iPS

Fig. 4. P2Y1 mediates pCREB (A) and pERK1/2 (B) expression in CVB iPS cells. Stimulation of cells with 300 μM ATP was performed for 5 min in CTLs (lane b) and HPRT-knockdown cells (lane e). The P2Y1 receptor antagonist MRS2179 (100 μM) was added to growth media of control (lane c) and HPRT-knockdown (lane f) CVB iPS colonies for 30 min, after which cells were supplemented with 300 μM ATP for another 5 min. GAPDH was used as a loading control. Data are presented as mean ± SD. Lane a, CTL; lane b, CTL + ATP; lane c, CTL + MRS2179 + ATP; lane d, shHPRT; lane e, shHPRT + ATP; lane f, shHPRT + MRS2179 + ATP. Treatments: ATP, 300 μM for 5 min; MRS2179, 100 μM for 30 min. *P < 0.05.

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cells, ES cells are not complicated by the extensive epigenetic modifications occurring during the generation of iPS cells by most current methods of genetic reprogramming. In the HPRT knockdown HUES11 cells, the same HPRT-knockdown vector used for the CVB iPS cells produced a 99% reduction in HPRT gene expression, markedly reduced HPRT protein expression and enzyme activity, as well as marked reduction of P2Y1 mRNA expression, consistent with findings in CVB iPS cells (Fig. 3D). To test whether the HPRT down-regulation and resulting P2Y1 dysregulation were specific for the HPRT region targeted by the knockdown vector, we also examined the effect of commercially available HPRT knockdown lentivirus vectors (MISSION lentiviral transduction particles; Sigma-Aldrich) targeting five different regions of the HPRT mRNA in HUES11 cells. A mixture of those vectors produced a 60% HPRT knockdown compared with control expressing the anti-luciferase shLux vector as measured by quantitative PCR (Fig. S1A). The HPRT knockdown was accompanied by an ∼40% reduction in P2Y1 expression (Fig. S1B). Based on these results, we conclude that the effects of HPRT knockdown on P2Y1 purinergic signaling is not an off-target effect of the vector and is not specific to the CVB iPS cell line, but rather is a consequence of HPRT deficiency in both pluripotent human ES and iPS cells. Interestingly, our results for CVB iPS cells are consistent with a recognized role of P2Y receptors in adult neural progenitor cells. Other investigators found that expression of the purinergic receptor P2Y1 occurs in the mouse embryonic ventricular zone and subventricular zone at embryonic day 14–16, and that P2Y1 knockdown plays a role in determining the correct migration of neural progenitor cells, presumably through ATP-mediated signaling (18). Furthermore, genetic knockdown of P2Y1 in vivo has been shown to significantly impair the migration of progenitors to the cortical plate (18). In the present study, we have demonstrated that P2Y1 receptors in CVB iPS cells respond to the agonist ATP by enhanced phosphorylation of CREB and ERK1/2 (17), but that HPRTdeficient CVB cells show highly impaired CREB phosphorylation and constitutively activated ERK1/2 and insensitivity to activation by ATP. ERK protein kinases belong to the MAPK family, which is known to respond to growth signals and regulate proliferation and differentiation (36). CREB is a target of intracellular signaling pathways initiated by membrane receptors and functions through modulation of calcium fluxes, cAMP levels, and MAPK Mastrangelo et al.

protein expression (37). CREB is expressed in many brain cells and has been associated with cells involved in learning and memory (38). From our results, we infer that HPRT deficiency may hamper the function of embryonic and developing CNS cells in creating an appropriate environment suitable for normal neurogenesis and neural pathway development, hallmarks of the neuropathology of HPRT-deficiency LND. We recently reported dysregulated Wnt/β-catenin signaling in HPRT-deficient human fibroblasts (24) and presented further evidence for aberrant canonical Wnt-1 signaling in the HPRTdeficient iPS cells. The Wnt/β-catenin signaling pathway is a major pathway regulating the growth, differentiation, and fate of stem cells during embryogenesis (21). Moreover, Wnt/β-catenin signals are crucial for the expansion of neural progenitors and neural differentiation, and Wnt3a has been shown to inhibit stem cell pluripotency and promote neural induction (39, 40). Activity of the canonical Wnt/β-catenin pathway depends on the amount, phosphorylation, and cellular distribution of the multifunctional transcription factor β-catenin. Free cytosolic β-catenin levels are controlled through its recruitment to a degradative complex that includes Axin protein, the tumor suppressor adenomatous polyposis coli, and GSK-3β. Phosphorylation of β-catenin by GSK-3β promotes β-catenin degradation via the ubiquitin-proteasome pathway (26). Binding of Wnt ligands to their receptors activates the Wnt/β-catenin pathway through phosphorylation and resulting inhibition of GSK-3β, resulting in stabilization and nuclear translocation of β-catenin to generate a transcriptional complex that includes phosphorylated CREB, CREB-binding protein, or its protein homolog p300 and ultimately to expression of downstream target genes (26, 41). Activation of ERK1/2 is known to induce phosphorylation of both CREB and GSK-3β, thereby increasing the free β-catenin protein pool (42). Inhibition of GSK-3β catalytic activity has been related to purinergic receptor function (22). Our present results suggest that alterations in pERK1/2 and pCREB levels might have an effect on β-catenin signaling pathways. The present study indicates that HPRT may play a role in determining aspects of neurogenesis and neurodevelopment during embryogenesis by regulating P2Y1 purinergic signaling. Our findings also support the potential usefulness of iPS and ES cells in modeling the developmental defects in a monogenic and yet highly complex neurodevelopmental disorder such as LND and in establishing a vital role for the classical “housekeeping” PNAS | February 28, 2012 | vol. 109 | no. 9 | 3381

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Fig. 5. Effect of HPRT knockdown on pGSK-3β, β-catenin, and phosphorylated β-catenin in CVB iPS cells. (A) Increased expression of pGSK-3β in CVB iPS CTL cells stimulated with ATP (300 μM; 5 min) (lane b) and increased pGSK-3β in HPRT-knockdown CVB iPS cells (lane d) compared with CTL (lane a). Lane a, CTL; lane b, CTL + ATP; lane c, CTL + MRS2179 + ATP; lane d, shHPRT; lane e, shHPRT + ATP; lane f, shHPRT + MRS2179 + ATP. Treatments: ATP, 300 μM for 5 min; MRS2179, 100 μM for 30 min. (B) Decreased expression of β-catenin and undetectable levels of phosphorylated β-catenin in HPRT-deficient CVB iPS cells. Data are presented as mean ± SD. *P < 0.05.

HPRT gene. Our results regarding the dysregulatory effects of HPRT deficiency may provide insight into the normal mechanisms of neural pathway development and neurogenesis and the mechanisms underlying the neuropathology of LND, and may help identify new potential targets for therapy of the neurological phenotype of HPRT deficiency and related neurodevelopmental and neurodegenerative human diseases.

human male karyotype and the ability to differentiate into cells of all three primary germ layers (ectoderm, endoderm, and mesoderm). HUES11 hES cells were provided by the University of California San Diego Human Stem Cell Core Facility. The studies with HUES11 cells were supported by nonfederal grant funds and were approved by the University of California San Diego, consistent with federal and state regulations under protocol UCSD E09-012. For the details regarding the methods and data analyses see SI Materials and Methods.

Materials and Methods CVB iPS and HUES11 ES Cells. Human CVB iPS cells (a generous gift from Jessica Young, University of California San Diego) were derived by established methods of transduction of donor normal primary dermal fibroblast cultures with retroviral vectors encoding oct-4, sox-2, klf-4, c-myc, and +EGFP (43). Candidate iPS cells with ES cell-like morphology were found to have the molecular and cellular properties characteristic of iPS cells, including a normal

ACKNOWLEDGMENTS. We thank Zoë Vomberg and Megan Robinson (University of California San Diego Human Stem Cell Core Facility) for their helpful advice and technical assistance. We also thank Paul Insel for his assistance with manuscript preparation. The CVB iPS cells were a generous gift from Jessica Young (University of California San Diego). This work was supported by National Institutes of Health Grant R24DK082840 (to T.F. and L.M.) and the Lesch–Nyhan Disease Children’s Research Foundation.

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