Chinese Journal of Tissue Engineering Research ›› 2012, Vol. 16 ›› Issue (20): 3773-3778.doi: 10.3969/j.issn.1673-8225.2012.20.037

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Effect of mutant huntingtin on the expression levels and histone acetyltransferase activity of CREB-binding protein  

Cong Shu-yan, Zhang Wei, Wang Ya, Shao Hua, Feng Juan   

  1. Department of Neurology, Shengjing Hospital of China Medical University, Shenyang  110003, Liaoning Province, China
  • Received:2012-03-21 Revised:2012-04-12 Online:2012-05-13 Published:2012-05-13
  • Contact: Cong Shu-yan, Department of Neurology, Shengjing Hospital of China Medical University, Shenyang 110003, Liaoning Province, China congshuyan@hotmail.com
  • About author:Cong Shu-yan☆, Doctor, Associate professor, Department of Neurology, Shengjing Hospital of China Medical University, Shenyang 110003, Liaoning Province, China congshuyan@hotmail.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30971017*; the Doctoral Start-up Foundation of Liaoning Province, No. 20080526*

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Abstract:

BACKGROUND: Studies addressing the role of CREB-binding protein (CBP) in the pathogenesis of Huntington's disease are mainly concentrated in the inhibitory effect of aggregate formation on CBP.
OBJECTIVE: To investigate the effect of mutant huntingtin (htt) on the histone acetyltransferase (HAT) activity and expression levels of CBP in the cell model of Huntington’s disease.
METHODS: Neuronal phaeochromocytoma rat PC12 cells, stably inducible for GFP-tagged HD exon 1 with either 23 (normal) or 74 (expanded) glutamines (N-htt-Q23 or -Q74), driven by a doxycycline (dox)-dependent Tet-On promoter were used. N-htt expression was observed via immunofluorescence microscopy. The HAT activities of endogenous CBP were determined via a histone H4 acetylation assay with immunoprecipitated CBP. CBP protein levels were observed via Western blot. The effect of specific proteasome inhibitor on CBP protein level was investigated by adding MG-132. CBP mRNA levels were detected by Lightcycler PCR.
RESULTS AND CONCLUSION: Cells expressing N-htt-Q74 started to form visible aggregates 1 day after dox induction in a small percentage of cells (< 1%), while after 6 days aggregates were present in about 90% of cells. In contrast, N-htt-Q23 distributed evenly throughout the cells and did not form aggregates. Expression of soluble N-htt-Q74 already inhibited the HAT activity of CBP, the inhibitory effect was exacerbated by aggregate formation of N-htt-Q74, while N-htt-Q23 did not affect CBP HAT activity. Expression of soluble N-htt-Q74 already reduced CBP protein levels; the level of CBP was dramatically decreased after 6 days, while N-htt-Q23 did not affect the levels of CBP protein. Neither N-htt-Q74 nor -Q23 affected CBP mRNA levels. The proteasome inhibitor MG-132 partly restored CBP protein levels by affecting protein degradation in the cells expressing N-htt-Q74. The results indicated that decrement of CBP HAT activity and its protein level play important roles in the molecular pathogenesis HD. Aside from aggregated mutant N-htt, soluble mutant N-htt already represses CBP HAT activity and CBP level, providing theoretical evidence for administration of CBP and histone deacetylase inhibitor in the treatment of Huntington's disease in the early stages.

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