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. 2011 Jan 28;6(1):e16572.
doi: 10.1371/journal.pone.0016572.

HPRT deficiency coordinately dysregulates canonical Wnt and presenilin-1 signaling: a neuro-developmental regulatory role for a housekeeping gene?

Tae Hyuk Kang  1 , Ghiabe-Henri Guibinga, H A Jinnah, Theodore Friedmann
Affiliations

HPRT deficiency coordinately dysregulates canonical Wnt and presenilin-1 signaling: a neuro-developmental regulatory role for a housekeeping gene?

Tae Hyuk Kang et al. PLoS One. .

Erratum in

  • PLoS One. 2011;6(3). doi: 10.1371/annotation/893f09fd-ab24-4119-93f4-44a03a3342f9. Jinnah, H A [added]

Abstract

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.

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Conflict of interest statement

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

Figures

Figure 1
Figure 1. Western blot analysis of total and phosphorylated β-catenin in wild type and HPRT knockdown cells.
Undifferentiated control cells transduced with a control luciferase vector (lane 1), undifferentiated cells transduced with the HPRT knockdown vector (2), luciferase-transduced cells at 12 days of differentiation (3) and HPRT-knockdown differentiated cells at 12 days of differentiation (4). Bottom panels represent GAPDH and lamin loading controls.
Figure 2
Figure 2. Quantitative PCR analysis of the neural transcription factors En1 and Lmx1a.
In both the undifferentiated basal state and after differentiation, there is marked reduction of expression of both transcription factors in the knockdown cells (filled columns). Error bars (Mean + SEM).
Figure 3
Figure 3. Western blot analysis of PS-1 in wild type and HPRT-knockdown SH-SY5Y cells.
Control SH-SY5Y cells were transduced by a retrovirus vector encoding luciferase (Lux-ND) or the anti-HPRT shRNA (sh2-ND) before and after 12 days of differentiation with retinoic acid (Lux-D-12 and sh2-D-12). The 55 kDa full-length PS-1 (FL PS1) and the 34 kDa NTF are present in approximately equal amounts in the undifferentiated control and knockdown cells. After 12 days of differentiation, FL and NTF of PS-1 are markedly reduced. However, at both time, the 23 kDa form of the CTF is markedly reduced in knockdown cells.
Figure 4
Figure 4. Complexed β-catenin and PS-1 in wild type and HPRT-knockdown SH-SY5Y cells.
SH-SY5Y cell proteins were immunoprecipitated by antibody to PS-1 (upper panel) and to β-catenin (lower panel) and reacted with β-catenin or PS-1 antibodies. Un-differentiated control cells transduced by luciferase vector (lane 1), undifferentiated HPRT-knockdown cells (lane 2), 12-day differentiated control cells (lane 3) and 12-day HPRT-knockdown cells (lane 4).
Figure 5
Figure 5. Western blot analysis of PS-1 in fibroblasts from LND patients.
WT cells demonstrate very low levels of PS-1 processing to the N- and C-terminal fragment (NTF and CTF, respectively). In both LND cells, processing to both the NTF and CTF proteolytic products is markedly increased compared with that seen in HPRT-positive control WT human fibroblasts.
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