Purinergic Signaling in Human Pluripotent Stem Cells Is Regulated by the Housekeeping Gene Encoding Hypoxanthine Guanine Phosphoribosyltransferase

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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

aDepartment 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

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

Lesch–Nyhan disease (LND) is an X-linked genetic disorder caused purine derivatives in modulating neurotransmitter release and by mutations of the hypoxanthine guanine phosphoribosyltrans- demonstrating the involvement of purinergic receptors in neu- ferase (HPRT) purine biosynthesis gene and characterized by aber- ronal growth and survival (9–13). A role of purinergic receptors rant purine metabolism, deficient basal ganglia dopamine levels, in neuronal differentiation has been reported as well (14, 15). dystonia, and severe neurobehavioral manifestations, including Recent studies also have established roles of nucleotides in compulsive self-injurious behavior. Although available evidence controlling several aspects of neurogenesis, neural differentia- has identified important roles for purinergic signaling in brain tion, and migration in the developing mammalian CNS through development, the mechanisms linking HPRT deficiency, purinergic interactions with two classes of purinergic P2 receptors, the pathways, and neural dysfunction of LND are poorly understood. ionotropic P2X receptors and the P2Y class of G protein-cou- In these studies aimed at characterizing purinergic signaling in pled receptors (GPCRs) (16–18). Other studies have shown that HPRT deficiency, we used a lentivirus vector stably expressing an HPRT fi ATP, acting via P2X and P2Y receptors, stimulates increased shRNA targeted to the gene to produce HPRT-de cient hu- proliferation of embryonic stem cells (19). Moreover, P2Y re- man CVB induced pluripotent stem cells and human HUES11 em-

ceptor activation in adult neural progenitor cells occurs at least BIOLOGY bryonic stem cells. Both CVB and HUES11 cells show >99% HPRT

in part through effects on nucleotide-induced expression and on DEVELOPMENTAL knockdown and demonstrate markedly decreased expression of the purinergic P2Y1 receptor mRNA. In CVB cells, P2Y1 mRNA the function of transcription factors ERK1/2 and CREB phosphorylation (17). and protein down-regulation by HPRT knockdown is refractory to β activation by the P2Y1 receptor agonist ATP and shows aberrant P2Y receptors also affect the vital Wnt/ -catenin signaling purinergic signaling, as reflected by marked deficiency of the tran- pathway, which includes a major class of growth factors with scription factor pCREB and constitutive activation of the MAP potent effects on stem cells and developmental processes (20, kinases phospho-ERK1/2. Moreover, HPRT-knockdown CVB cells 21). Recent evidence shows that P2Y receptors mediate phos- β also demonstrate marked reduction of phosphorylated β-catenin. phorylation of glycogen synthase kinase (GSK-3 ), a key medi- These results indicate that the housekeeping gene HPRT regulates ator of Wnt signaling and nuclear transport of the transcription purinergic signaling in pluripotent human stem cells, and that this factor β-catenin (22). Furthermore, purinergic signaling has been regulation occurs at least partly through aberrant P2Y1-mediated reported to regulate GSK-3β and nuclear translocation of expression and signaling. We propose that such mechanisms may β-catenin in astrocytes (23). In addition, we recently reported play a role in the neuropathology of HPRT-deficiency LND and may evidence of major disruption of phosphorylated β-catenin point to potential molecular targets for modulation of this intrac- expression and impaired nuclear transport in HPRT-deficient table neurological phenotype. human fibroblasts (24). Our present study on the effect of HPRT deficiency on puri- neurodevelopment | neurotransmitters nergic signaling are based on the working hypothesis that alterations in purine pools resulting from defective salvage purine omplete genetic deficiency of the enzyme hypoxanthine biosynthesis might affect the function of purinergic receptors, Cguanine phosphoribosyltransferase (HPRT) causes Lesch– thereby leading to aberrant development of the CNS dopamine- Nyhan disease (LND), an intractable neurobehavioral disorder related pathways and aberrant dopaminergic neurogenesis. We characterized by hyperuricemia, cognitive impairment, dystonia have chosen to study mechanisms associated with that inter- with spasticity choreoathetosis and compulsive self-mutilation action in human induced pluripotent stem (iPS) and embryonic (1). The enzyme HPRT plays a major role in salvage purine stem (ES) cells because of the growing importance of such cells biosynthesis by catalyzing the conversion of hypoxanthine and in delineating the early molecular and cellular events in the guanine to the nucleotides inosine monophosphate (IMP) and defective neural development characteristic of LND and other guanosine monophosphate (1, 2). HPRT deficiency leads to the neurodegenerative and neurodevelopmental diseases (25). A hallmark biochemical defect in LND: greatly increased de novo fi purine synthesis, with resulting elevated levels of oxypurines and more detailed understanding of the effects of HPRT de ciency tissue accumulation of uric acid, gout, and life-threatening during embryogenesis is likely to clarify the developmental out- nephrolithiasis (3, 4). Although treatment with the xanthine oxi- comes of LND and identify potential new therapeutic targets. dase 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 Author contributions: L.M. designed research; L.M. and T.H.K. performed research; L.M., lasting improvement in neurological manifestations (5, 6). J.-E.K., A.M., and T.H.K. contributed new reagents/analytic tools; L.M., J.-E.K., A.M., T.H.K., PET scanning and postmortem studies of brains from patients and T.F. analyzed data; and L.M. and T.F. wrote the paper. with LND have demonstrated decreased levels of dopamine, The authors declare no conflict of interest. dopamine biosynthetic enzymes, and dopamine uptake in the This article is a PNAS Direct Submission. basal ganglia (7, 8). However, the mechanisms linking HPRT 1To whom correspondence should be addressed. E-mail: [email protected]. fi de ciency with the neurologic dysfunctions are little understood. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Recently, evidence has accumulated identifying key roles for 1073/pnas.1118067109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1118067109 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 Results expression is both qualitatively and quantitatively reduced in fi HPRT Knockdown in CVB iPS Cells. Expression of the shRNA tar- HPRT-de cient CVB cells. geted to HPRT profoundly reduces HPRT expression but does fi not produce major disturbances in the pluripotency of iPS cells. P2Y1 Expression in HPRT-De cient HUES11 Human ES Cells. To de- A termine whether aberrant P2Y1 expression is specific to CVB Fig. 1 shows mRNA levels of HPRT in CVB iPS cells trans- fi duced with the control anti-luciferase (shLux) and with the anti- iPS cells or is a more general consequence of HPRT de ciency in HPRT vector (shHPRT). HPRT mRNA expression was virtually pluripotent stem cells, we used real-time PCR to examine P2Y1 undetectable in this assay and was reduced by >99% in cells trans- expression in HPRT-knockdown human HUES11 ES cells. We found that in these cells, efficient (>95%) HPRT knockdown duced by shHPRT compared with control luciferase-knockdown A cells. Similarly, HPRT protein expression was markedly reduced with vector shHPRT measured by real-time PCR (Fig. 3 ), Western immunoblot analysis (Fig. 3B), and HPRT enzymatic in the HPRT-knockdown cells (Fig. 1B). Finally, TLC-based activity (Fig. 3C) leads to a significantly (∼60%) reduced ex- HPRT enzyme assays confirmed the virtual complete loss of pression of P2Y1 mRNA levels (Fig. 3D). Our results are con- HPRT enzymatic activity in HPRT-knockdown CVB cells, as in- sistent with changes seen in the HPRT-deficient CVB iPS cells. dicated by absence of IMP in the chromatography assay (Fig. 1C). The process of transduction of cells with the knockdown vectors P2Y1 Regulates CREB Expression and ERK1/2 Phosphorylation in CVB does not lead to major changes in the pluripotency state of the iPS Cells. Recent studies with murine adult neural progenitor cells cells, as measured by expression of the pluripotency markers β D E have shown that the P2Y1 receptor agonists ADP S and UTP Nanog and Oct-4 (Fig. 1 and ). stimulate the expression of phosphorylated CREB and ERK1/2, and that this stimulation is inhibited by the P2Y1 receptor an- Expression of Nucleotide Signaling Components. Transcription of tagonist MRS2179 (17). Because HPRT knockdown markedly nucleotide signaling pathway components was examined by down-regulates P2Y1 expression (Fig. 2 A and B), we examined quantitative PCR assays (Fig. 2). Total mRNA was extracted the effect of HPRT knockdown on phospho-CREB and phos- from undifferentiated CVB iPS cells transduced with the control pho-ERK1/2 expression in CVB cells. Our Western blot analyses shLux or the shHPRT vectors, and real-time PCR analyses were (Fig. 4A) demonstrate, as expected, that in control CVB cells the performed for the purinergic receptors P2Y1, P2Y2, P2X3, and P2Y1 agonist ATP up-regulates pCREB expression (lane b) adenosine A2a and the ectonucleotidase enzyme NTPase. Fig. compared with cells not exposed to ATP (lane a), and that the A 2 demonstrates that HPRT knockdown in CVB cells is ac- P2Y1 inhibitor MRS2179 abrogates that effect (lane c). In con- companied by a marked (∼90%) reduction of mRNA expression trast, HPRT-knockdown CVB cells show little or no detectable for the GPCR purinergic receptor P2Y1, a significant but less basal level pCREB expression (lane d) or induction of pCREB marked reduction of the ligand-gated receptor P2X3, and a sig- (lane e) after exposure to the P2Y1 agonist ATP. Paradoxically, nificant up-regulation of NTPase but no change in the expression treatment of the CVB knockdown cells with the P2Y1 inhibitor of P2Y2 or adenosine receptor A2a (Fig. 2C). Given that pre- MRS2179 leads to detectable expression of pCREB (lane f). vious studies have demonstrated an important role of P2Y1- mediated ATP signaling in neuronal migration (18), we used ERK1/2 Signaling. As shown in Fig. 4B, Western blot analysis of Western blot analysis to assess P2Y1 protein levels in control and CVB control HPRT-positive cells (lane a) demonstrates consti- HPRT-knockdown CVB cells. Fig. 2B shows that P2Y1 protein tutively 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.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1118067109 Mastrangelo et al. Downloaded by guest on October 1, 2021 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 BIOLOGY AMRNA were determined by real-time PCR. Experiments were performed in triplicate. Data are presented as mean ± SD. *P < 0.05. (B) Decreased P2Y1 DEVELOPMENTAL 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.

with the P2Y1 agonist ATP (lane b). In contrast to the results Discussion obtained for pCREB, however, expression of pERK1/2 in ATP- Most concepts of pathogenesis in LND have centered on the treated cells is essentially unchanged in the presence of the disruption of normal purine metabolism resulting from impaired P2Y1 inhibitor MRS2179 (lane c). Strikingly, in contrast, HPRT- reutilization and excessive de novo purine biosynthesis and the knockdown cells show constitutive activation of pERK1/2 ex- putative neurodevelopmental and metabolic effects of the pression, even in the absence of the agonist ATP (lane d), and no resulting aberrant purine pools (3). The present study has iden- added effect of ATP (lanes e) or reduced expression in response tified an alternative potential mechanism for the dysregulation of to the P2Y1 inhibitor MRS2179 (lane f). These qualitative purine and purine nucleotide metabolism in HPRT deficiency, results were confirmed by quantitation of the Western blot involving severe disturbances in function of the purinergic re- analysis results shown in Fig. 4B. ceptor P2Y1 and its important role in neural development of the mammalian CNS. Recent studies have identified major roles Increased GSK-3β Phosphorylation in HPRT-Deficient CVB iPS Cells. for purines, purine nucleotides, and purinergic signaling in neu- Previous studies have identified a role for purinergic receptors in rogenesis and the development of neural pathways during em- GSK-3β phosphorylation and β-catenin nuclear translocation in bryonic CNS development (16–19). It is now recognized that in granule neurons (22). Accordingly, we used Western blot anal- cells of the developing mammalian CNS, binding of extracellular ysis to determine the levels of pGSK-3β in control and HPRT- nucleotides to purinergic receptors initiates protein kinase cas- knockdown CVB cells. Fig. 5A shows that pGSK-3β protein ex- cades that stimulate downstream signaling pathways, which in pression in HPRT-knockdown CVB cells (lane d) is up-regulated turn lead to cell proliferation, migration, differentiation, neurite compared with expression in control HPRT-positive CVB cells outgrowth, and synapse formation (10). (lane a). Moreover, CVB cells transduced with the control anti- Our demonstration of aberrant P2Y1 receptor expression luciferase vector show increased levels of pGSK-3β in response complements previous demonstrations of disturbed expression of to treatment with the purinergic receptor agonist ATP (lane b), other purines and related receptors, such as adenosine, dopa- whereas in contrast, HPRT-knockdown CVB cells are refractory mine, and serotonin receptors, in HPRT deficiency (27). Before to the inducing effects of ATP (lane e). In both control and the present study, there was no evidence suggesting that puri- HPRT-knockdown CVB cells, ATP-induced pGSK-3β expres- nergic receptors play a role in the HPRT-deficiency phenotype. sion is not affected by exposure of cells to the P2Y1 inhibitor Purinergic receptors are divided into two major families, P1 MRS2179 (lanes c and f). adenosine receptors and P2 receptors, which include ionotropic P2X and metabotropic P2Y receptors. To date, four subtypes Decreased β-Catenin and β-Catenin Phosphorylation in HPRT- of P1 receptors, seven subtypes of P2X receptors, and eight Deficient CVB iPS Cells. pGSK-3β is a key determinant of canoni- subtypes of P2Y receptors have been identified (11). The GPCR cal Wnt signaling through its role in regulating the phosphoryla- P2Y receptors are activated by a variety of nucleotides (e.g., tion and nuclear translocation of β-catenin (26). To characterize ATP, ADP, UTP, UDP, NAD+) or nucleotide sugars and in turn mechanisms associated with β-catenin signaling in HPRT-de- activate several cellular signaling pathways. Evidence has accu- ficient CVB cells, we used Western blot analysis to measure total mulated indicting the involvement of purinergic mechanisms in and phosphorylated β-catenin in control and HPRT-knockdown such pathological conditions as brain trauma and ischemia, CVB iPS cells. We found that HPRT-knockdown CVB cells neurodegenerative diseases, and neuropsychiatric disorders, in- have a marked reduction of total cellular β-catenin and a virtual cluding depression and schizophrenia (28, 29). Important puta- absence of pβ-catenin (Fig. 5B). tive developmental functions of purinergic receptors have been

Mastrangelo et al. PNAS Early Edition | 3of6 Downloaded by guest on October 1, 2021 been detected in rat brain between embryonic day 10 and post- natal 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 post- natal 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 in- creases 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), Fig. 3. Effect of HPRT knockdown on P2Y1 mRNA expression in human we have based much of our recent work and most of the present HUES11 ES cells. (A) HPRT mRNA levels were reduced by >99% in HPRT- study on the underlying working hypothesis that aberrant purine knockdown HUES11 cells. (B) Western blot analysis was performed to detect metabolism in LND plays a direct causative role in the neuro- expression of HPRT, and β-actin level was determined by stripping and logical phenotype of the disorder through disturbed purinergic reprobing the membrane. (C) Scintillation counting revealed an 88% re- signaling. In this study, we aimed to generate an in vitro human duction in HPRT enzyme activity. (D) Effect of HPRT knockdown on P2Y1 iPS and ES cell-based system in which to study the development mRNA expression. *P < 0.05. of dopaminergic pathways. To determine whether P2Y1 down- regulation by HPRT deficiency is specific for CVB iPS cells or is a more general feature of pluripotentiality in ES and iPS cells, inferred from in vivo studies showing transient and temporal we examined P2Y1 expression in HUES11 human ES cells (Fig. expression of defined purinergic receptor subtypes during de- 3). Studies of aberrations in gene expression in ES cells rather velopment (12, 30); for instance, P2Y1 purinergic receptors have 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.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1118067109 Mastrangelo et al. Downloaded by guest on October 1, 2021 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.

cells, ES cells are not complicated by the extensive epigenetic protein expression (37). CREB is expressed in many brain cells BIOLOGY

modifications occurring during the generation of iPS cells by most and has been associated with cells involved in learning and DEVELOPMENTAL current methods of genetic reprogramming. In the HPRT knock- memory (38). From our results, we infer that HPRT deficiency down HUES11 cells, the same HPRT-knockdown vector used for may hamper the function of embryonic and developing CNS cells the CVB iPS cells produced a 99% reduction in HPRT gene ex- in creating an appropriate environment suitable for normal pression, markedly reduced HPRT protein expression and enzyme neurogenesis and neural pathway development, hallmarks of the activity, as well as marked reduction of P2Y1 mRNA expression, neuropathology of HPRT-deficiency LND. consistent with findings in CVB iPS cells (Fig. 3D). We recently reported dysregulated Wnt/β-catenin signaling in To test whether the HPRT down-regulation and resulting P2Y1 HPRT-deficient human fibroblasts (24) and presented further dysregulation were specific for the HPRT region targeted by the evidence for aberrant canonical Wnt-1 signaling in the HPRT- knockdown vector, we also examined the effect of commercially deficient iPS cells. The Wnt/β-catenin signaling pathway is available HPRT knockdown lentivirus vectors (MISSION lenti- a major pathway regulating the growth, differentiation, and fate viral transduction particles; Sigma-Aldrich) targeting five differ- of stem cells during embryogenesis (21). Moreover, Wnt/β-cat- ent regions of the HPRT mRNA in HUES11 cells. A mixture of enin signals are crucial for the expansion of neural progenitors those vectors produced a 60% HPRT knockdown compared with and neural differentiation, and Wnt3a has been shown to inhibit control expressing the anti-luciferase shLux vector as measured by stem cell pluripotency and promote neural induction (39, 40). quantitative PCR (Fig. S1A). The HPRT knockdown was ac- Activity of the canonical Wnt/β-catenin pathway depends on the companied by an ∼40% reduction in P2Y1 expression (Fig. S1B). amount, phosphorylation, and cellular distribution of the multi- Based on these results, we conclude that the effects of HPRT functional transcription factor β-catenin. Free cytosolic β-catenin knockdown on P2Y1 purinergic signaling is not an off-target levels are controlled through its recruitment to a degradative effect of the vector and is not specific to the CVB iPS cell line, complex that includes Axin protein, the tumor suppressor ade- but rather is a consequence of HPRT deficiency in both plurip- nomatous polyposis coli, and GSK-3β. Phosphorylation of otent human ES and iPS cells. β-catenin by GSK-3β promotes β-catenin degradation via the Interestingly, our results for CVB iPS cells are consistent with ubiquitin-proteasome pathway (26). Binding of Wnt ligands to a recognized role of P2Y receptors in adult neural progenitor their receptors activates the Wnt/β-catenin pathway through cells. Other investigators found that expression of the purinergic phosphorylation and resulting inhibition of GSK-3β, resulting in receptor P2Y1 occurs in the mouse embryonic ventricular zone stabilization and nuclear translocation of β-catenin to generate and subventricular zone at embryonic day 14–16, and that P2Y1 a transcriptional complex that includes phosphorylated CREB, knockdown plays a role in determining the correct migration of CREB-binding protein, or its protein homolog p300 and ulti- neural progenitor cells, presumably through ATP-mediated sig- mately to expression of downstream target genes (26, 41). Acti- naling (18). Furthermore, genetic knockdown of P2Y1 in vivo vation of ERK1/2 is known to induce phosphorylation of both has been shown to significantly impair the migration of progen- CREB and GSK-3β, thereby increasing the free β-catenin pro- itors to the cortical plate (18). tein pool (42). Inhibition of GSK-3β catalytic activity has been In the present study, we have demonstrated that P2Y1 recep- related to purinergic receptor function (22). Our present results tors in CVB iPS cells respond to the agonist ATP by enhanced suggest that alterations in pERK1/2 and pCREB levels might phosphorylation of CREB and ERK1/2 (17), but that HPRT- have an effect on β-catenin signaling pathways. deficient CVB cells show highly impaired CREB phosphorylation The present study indicates that HPRT may play a role in and constitutively activated ERK1/2 and insensitivity to activation determining aspects of neurogenesis and neurodevelopment by ATP. ERK protein kinases belong to the MAPK family, which during embryogenesis by regulating P2Y1 purinergic signaling. is known to respond to growth signals and regulate proliferation Our findings also support the potential usefulness of iPS and ES and differentiation (36). CREB is a target of intracellular sig- cells in modeling the developmental defects in a monogenic and naling pathways initiated by membrane receptors and functions yet highly complex neurodevelopmental disorder such as LND through modulation of calcium fluxes, cAMP levels, and MAPK and in establishing a vital role for the classical “housekeeping”

Mastrangelo et al. PNAS Early Edition | 5of6 Downloaded by guest on October 1, 2021 HPRT gene. Our results regarding the dysregulatory effects of human male karyotype and the ability to differentiate into cells of all three HPRT deficiency may provide insight into the normal mecha- primary germ layers (ectoderm, endoderm, and mesoderm). HUES11 hES cells nisms of neural pathway development and neurogenesis and the were provided by the University of California San Diego Human Stem Cell mechanisms underlying the neuropathology of LND, and may Core Facility. The studies with HUES11 cells were supported by nonfederal help identify new potential targets for therapy of the neurological grant funds and were approved by the University of California San Diego, phenotype of HPRT deficiency and related neurodevelopmental consistent with federal and state regulations under protocol UCSD E09-012. and neurodegenerative human diseases. 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 ACKNOWLEDGMENTS. We thank Zoë Vomberg and Megan Robinson (Uni- versity of California San Diego Human Stem Cell Core Facility) for their help- Young, University of California San Diego) were derived by established fi ful advice and technical assistance. We also thank Paul Insel for his assistance methods of transduction of donor normal primary dermal broblast cultures with manuscript preparation. The CVB iPS cells were a generous gift from with retroviral vectors encoding oct-4, sox-2, klf-4, c-myc, and +EGFP (43). Jessica Young (University of California San Diego). This work was supported Candidate iPS cells with ES cell-like morphology were found to have the by National Institutes of Health Grant R24DK082840 (to T.F. and L.M.) and molecular and cellular properties characteristic of iPS cells, including a normal the Lesch–Nyhan Disease Children’s Research Foundation.

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