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, 8 (10), e74967
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The Housekeeping Gene Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Regulates Multiple Developmental and Metabolic Pathways of Murine Embryonic Stem Cell Neuronal Differentiation

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The Housekeeping Gene Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Regulates Multiple Developmental and Metabolic Pathways of Murine Embryonic Stem Cell Neuronal Differentiation

Tae Hyuk Kang et al. PLoS One.

Abstract

The mechanisms by which mutations of the purinergic housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) cause the severe neurodevelopmental Lesch Nyhan Disease (LND) are poorly understood. The best recognized neural consequences of HPRT deficiency are defective basal ganglia expression of the neurotransmitter dopamine (DA) and aberrant DA neuronal function. We have reported that HPRT deficiency leads to dysregulated expression of multiple DA-related developmental functions and cellular signaling defects in a variety of HPRT-deficient cells, including human induced pluripotent stem (iPS) cells. We now describe results of gene expression studies during neuronal differentiation of HPRT-deficient murine ESD3 embryonic stem cells and report that HPRT knockdown causes a marked switch from neuronal to glial gene expression and dysregulates expression of Sox2 and its regulator, genes vital for stem cell pluripotency and for the neuronal/glial cell fate decision. In addition, HPRT deficiency dysregulates many cellular functions controlling cell cycle and proliferation mechanisms, RNA metabolism, DNA replication and repair, replication stress, lysosome function, membrane trafficking, signaling pathway for platelet activation (SPPA) multiple neurotransmission systems and sphingolipid, sulfur and glycan metabolism. We propose that the neural aberrations of HPRT deficiency result from combinatorial effects of these multi-system metabolic errors. Since some of these aberrations are also found in forms of Alzheimer's and Huntington's disease, we predict that some of these systems defects play similar neuropathogenic roles in diverse neurodevelopmental and neurodegenerative diseases in common and may therefore provide new experimental opportunities for clarifying pathogenesis and for devising new potential therapeutic targets in developmental and genetic disease.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HPRT knockdown in murine ESD3 cells.
(A) Reduction of HPRT mRNA expression as assayed by qPCR; (B) reduction of immunoreactive HPRT protein as determined by Western blot analysis; (C) reduction of HPRT enzymatic activity as measured by absent detectable IMP production as detected by autoradiography of a thin layer chromatography of cell lysate (6).
Figure 2
Figure 2. Neuron-like cells generated from WT control and HPRT-knockdown ESD3 cells.
Figure 3
Figure 3. Regulation of pluripotency markers at the embryonic stem cell, SNM and neuronal (DA) stages of neuronal differentiation of WT control and HPRT-knockdown ESD3 cells, as measured by qPCR analysis.
(A) Expression of Oct-4, showing expected down regulation; (B) expression of Nanog during differentiation; (C) expression of Sox2 during differentiation. Control cells show the expected down regulation at the SNM and DA stages but HPRT-knockdown cells show up-regulated expression during both SNM and DA stages of differentiation; (D) expression of the neuronal marker β-III tubulin showing the expected up-regulation in control cells, but reduced expression during differentiation of HPRT knockdown cells; (E) up-regulation of the dopaminergic neuronal marker DAT in control cells and HPRT-knockdown cells.
Figure 4
Figure 4. Immunohistochemical detection of the neuronal marker β-III tubulin and tyrosine hydroxylase (TH) in ESD3 cells at the final 14-day neuronal stage of SNM differentiation.
(A) β-III tubulin in WT control cells; (B) TH expression in control WT cells; (C) merged β-III tubulin and TH expression in WT control cells; (D) β-III tubulin in HPRT-knockdown cells; (E) TH in HPRT-knockdown cells, and (F) merged β-III tubulin and TH expression in HPRT-knockdown cells.
Figure 5
Figure 5. FACS analysis of control WT (A) and HPRT-knockdown (B) cells expressing TH.
The cell types demonstrate indistinguishable high percentages of TH-positive cells.
Figure 6
Figure 6. Western blot analysis for glial markers MBP and Olig2 of control WT cells and HPRT-knockdown ESD3 cells at the final neuronal stage of differentiation.
Both markers demonstrate a significant increase of expression in the HPRT-knockdown cells.
Figure 7
Figure 7. Mean shift and polarization of canonical pathways.
Gene expression of 802 canonical pathways was tested for (A) shift in mean expression and (B) polarization, each test aggregating paired differences between wild-type (WT) and HPRT-knockdown (KD) at each time point during the differentiation. Rows are significant pathways, organized by source database and with the top three WT and KD genes provided underneath the pathway name. Columns are the module response over time, with red/blue indicating over-expression in WT/KD cells and diameter indicating the fraction of genes in the pathway with differential expression. The mean-shift test statistic is the mean log-ratio of WT to KD expression aggregated over time points for genes within a pathway; the polarization test statistic is the variance of gene log-ratios within a pathway. For both tests, the null distribution is obtained by permutation of sample labels. Thresholds are two false discoveries (FDR  =  0.22) for mean shift and one false discovery (FDR  =  0.042) for polarization.

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