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

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 ß-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.

MicroRNA-mediated dysregulation of neural developmental genes in HPRT deficiency: clues for Lesch-Nyhan disease?

Mutations in the gene encoding the purine biosynthetic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause the intractable neurodevelopmental Lesch-Nyhan disease (LND) associated with aberrant development of brain dopamine pathways. In the current study, we have identified an increased expression of the microRNA miR181a in HPRT-deficient human dopaminergic SH-SY5Y neuroblastoma cells. Among the genes potentially regulated by miR181a are several known to be required for neural development, including Engrailed1 (En1), Engrailed2 (En2), Lmx1a and Brn2. We demonstrate that these genes are down-regulated in HPRT-deficient SH-SY5Y cells and that over-expression of miR181a significantly reduces endogenous expression of these genes and inhibits translation of luciferase plasmids bearing the En1/2 or Lmx1a 3'UTR miRNA-binding elements. Conversely, inhibition of miR181a increases the expression of these genes and enhances translation of luciferase constructs bearing the En1/2 and Lmx1a 3'UTR miRNA-binding sequences. We also demonstrate that key neurodevelopmental genes (e.g. Nurr1, Pitx3, Wnt1 and Mash1) known to be functional partners of Lmx1a and Brn2 are also markedly down-regulated in SH-SY5Y cells over-expressing miR181a and in HPRT-deficient cells. Our findings in SH-SY5Y cells demonstrate that HPRT deficiency is accompanied by dysregulation of some of the important pathways that regulate the development of dopaminergic neurons and dopamine pathways and that this defect is associated with and possibly due at least partly to aberrant expression of miR181a. Because aberrant expression of miR181a is not as apparent in HPRT-deficient LND fibroblasts, the relevance of the SH-SY5Y neuroblastoma cells to human disease remains to be proven. Nevertheless, we propose that these pleiotropic neurodevelopment effects of miR181a may play a role in the pathogenesis of LND.

Deficiency of the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase (HPRT) dysregulates neurogenesis.

Neuronal transcription factors play vital roles in the specification and development of neurons, including dopaminergic (DA) neurons. Mutations in the gene encoding the purine biosynthetic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause the resulting intractable and largely untreatable neurological impairment of Lesch-Nyhan disease (LND). The disorder is associated with a defect in basal ganglia DA pathways. The mechanisms connecting the purine metabolic defect and the central nervous system (CNS) phenotype are poorly understood but have been presumed to reflect a developmental defect of DA neurons. We have examined the effect of HPRT deficiency on the differentiation of neurons in the well-established human (NT2) embryonic carcinoma neurogenesis model. We have used a retrovirus expressing a small hairpin RNA (shRNA) to knock down HPRT gene expression and have examined the expression of a number of transcription factors essential for neuronal differentiation and marker genes involved in DA biosynthetic pathway. HPRT-deficient NT2 cells demonstrate aberrant expression of several transcription factors and DA markers. Although differentiated HPRT-deficient neurons also demonstrate a striking deficit in neurite outgrowth during differentiation, resulting neurons demonstrate wild-type electrophysiological properties. These results represent direct experimental evidence for aberrant neurogenesis in HPRT deficiency and suggest developmental roles for other housekeeping genes in neurodevelopmental disease.

A fractionation method to identify qauntitative changes in protein expression mediated by IGF-1 on the proteome of murine C2C12 myoblasts.

Although much is known about signal transduction downstream of insulin-like growth factor-1 (IGF-1), relatively little is known about the global changes in protein expression induced by this hormone. In this study, the acute effects of IGF-1 on the proteome of murine C2C12 cells were examined. Cells were treated with IGF-1 for up to 24 hours, lysed, and fractionated into cytosolic, nuclear, and insoluble portions. Proteins from the cytosolic fraction were further separated using a new batch ion-exchange chromatography method to reduce sample complexity, followed by two-dimensional (2D) electrophoresis, and identification of selected proteins by mass spectrometry. PDQuest software was utilized to identify and catalogue temporal changes in protein expression during IGF-1 stimulation. In response to IGF-1 stimulation, expression of 23 proteins increased at least three-fold and expression of 17 proteins decreased at least three-fold compared with control un-stimulated C2C12 cells. Changes in expression of selected proteins from each group, including Rho-GDI, cofillin, RAD50, enolase, IkappaB kinase b (IkappaBKb) and Hsp70 were confirmed by Western blotting. Additionally, the position of 136 'landmark' proteins whose expression levels and physicochemical properties did not change appreciably or consistently during IGF-1 treatment were mapped and identified. This characterization of large-scale changes in protein expression in response to growth factor stimulation of C2C12 cells will further help to establish a comprehensive understanding of the networks and pathways involved in the action of IGF-1.

Rethinking Human Enhancement as Collective Welfarism.

Human enhancement technologies are opening tremendous opportunities but also challenges to the core of what it means to be human. We argue that the goal of human enhancement should be to enhance quality of life and well-being not only of individuals but also of the communities they inhabit.

Insulin-like growth factor-1 coordinately induces the expression of fatty acid and cholesterol biosynthetic genes in murine C2C12 myoblasts.

BACKGROUND: We present evidence that a major aspect of the mechanism of acute signal transduction regulation by insulin-like growth factor-1 (IGF-1) in cultured murine myoblasts is associated with a broad perturbation of many components of cholesterol and fatty acid biosynthetic pathways. RESULTS: We have used microarray transcriptional analysis to examine the acute effects of IGF-1 on global patterns of gene expression in C2C12 myoblasts and have identified approximately 157 genes that are up-regulated and 75 genes down-regulated from 2- to 6-fold after treatment with IGF-1. Of the up-regulated genes, 19 genes are associated with cholesterol biosynthesis and 5 genes specify aspects of fatty acid biosynthesis. In addition 10 recognized transcription factors are significantly induced by IGF-1 at 1 hour. CONCLUSION: The SREBPs, important regulators of fatty acid and cholesterol biosynthesis, operate via a post-transcriptional route and no significant transcriptional induction was observed in the 4 hr of IGF-1 treatment. Since there are no prior reports of significant and coordinated perturbations of fatty acid and cholesterol biosynthetic pathways with IGF-1 in muscle cells, these findings provide a substantive expansion of our understanding of IGF-1 action and the signal transduction pathways mediated by it, its variants and insulin.

Characterization of the gene delivery properties of baculoviral-based virosomal vectors.

The current study reports the production of baculoviral-virosomal vectors consisting of lipoplexes and of the viral glycoprotein (GP64) of baculovirus Autographa californica multiple nucleopolyhdrovirus (AcMNPV). This study demonstrates that such complexes have an increased transfection capability in a number of cells, including undifferentiated H9 human embryonic stem H9hES cells compared to lipoplexes alone. The GP64-mediated enhancement of gene transfer of lipoplexes is inhibited by the addition of anti-GP64 neutralizing antibody and by a modified GP64 protein, but is however less potent than vesicular stomatitis virus glycoprotein (VSV-G)-mediated enhancement of gene transfer of lipoplexes. This difference may be explained in part by the dissimilarity in the fusogenic properties of their respective viral glycoprotein.

Next Generation "Omics" Approaches in the "Fight" against Blood Doping.

Despite being prohibited by the World Anti-Doping Agency (WADA), blood manipulations such as the use of recombinant human erythropoietin and blood transfusions are a well-known method used by athletes to enhance performance. Direct detection of illicit blood manipulation has been partially successful due to the short detection window of the substances/methods, sample collection timing, and the use of sophisticated masking strategies. In response, WADA introduced the athlete biological passport (ABP) in 2009, which is an individualised longitudinal monitoring approach that tests primarily haematologic biomarkers of doping in order to identify atypical variability in response(s) in athletes, highlighting a potential doping violation. Although the implementation of the ABP has been an encouraging step forward in the quest for clean/drug-free sport, this detection method has some limitations. To reduce the risk of being detected by the ABP method, athletes are now resorting to microdoses of prohibited blood boosting substances to prevent abnormal fluctuations in haematologic biomarkers, thereby reducing the sensitivity of the ABP detection method. Recent studies from numerous laboratories, including our own, have confirmed the potential of transcriptomic microarrays, which can reveal distinct changes in gene expression after blood manipulations, to enhance the ABP. There is, therefore, an urgent need to intensify research efforts that involve transcriptomics and other state-of-the-art molecular methods, collectively known as "omics", e.g., proteomics (proteins) and metabolomics (metabolites), in order to identify new and even more robust molecular signatures of blood manipulation that can be used in combination with the ABP and, intriguingly, even as a stand-alone test.

Alzheimer's disease shares gene expression aberrations with purinergic dysregulation of HPRT deficiency (Lesch-Nyhan disease).

Transcriptomic studies of murine D3 embryonic stem (ES) cells deficient in the purinergic biosynthetic function hypoxanthine guanine phosphoribosyltransferase (HPRT) and undergoing dopaminergic neuronal differentiation has demonstrated a marked shift from neuronal to glial gene expression and aberrant expression of multiple genes also known to be aberrantly expressed in Alzheimer's and other CNS disorders. Such genetic dysregulations may indicate some shared pathogenic metabolic mechanisms in diverse CNS diseases.

Discrepant effects of culture conditions on survival and function of dopaminergic neurons.

Primary midbrain cultures are valuable for probing the function of dopaminergic neurons and for elucidating the factors that cause their dysfunction and degeneration. To allow more effective control of the culture environment, we have characterized the survival, differentiation, and trophic factor response of dopaminergic neurons in the absence of serum. Combinations of media and supplements markedly affected all three indices measured. Combinations that produced maximal dopaminergic neuron survival are different from those that result in maximal differentiation and trophic factor response. Furthermore, antioxidant treatment was effective with only one medium/supplement combination, indicating that these neurons were not degenerating as a result of oxidative stress in the majority of culture conditions used in this study. These results demonstrate that dopaminergic neurons can be grown in serum-free conditions but that the choice of culture conditions has a marked influence on cell survival and function.

Oxidative stress and dopamine deficiency in a genetic mouse model of Lesch-Nyhan disease.

Lesch-Nyhan disease, a neurogenetic disorder caused by congenital deficiency of the purine salvage enzyme hypoxanthine guanine phosphoribosyl transferase, is associated with a prominent loss of striatal dopamine. The current studies address the hypothesis that oxidant stress causes damage or dysfunction of nigrostriatal dopamine neurons in a knockout mouse model of the disease, by assessing several markers of oxidative damage and free radical scavenging systems. Some of these measures provided evidence for an increase in oxidative stress in the mutant mice (aconitase activity, oxidized glutathione, and lipid peroxides), but others did not (superoxide dismutase, protein thiol content, carbonyl protein content, total glutathione, glutathione peroxidase, catalase, and thiobarbituric reducing substances). Immunolocalization of heme-oxygenase 1 provided no evidence for oxidative stress restricted to specific elements of the striatum or midbrain in the mutants. Striatal dopamine systems of the mutant mice were more vulnerable to a challenge with the neurotoxin 6-hydroxydopamine, but they were not protected by cross-breeding the mutants with transgenic mice over-expressing superoxide dismutase. Overall, these data provide evidence for increased oxidative stress, but the failure to protect the knockout mice by over-expressing SOD1 argues that oxidative stress is not the sole process responsible for the loss of striatal dopamine.