Elucidating a normal function of huntingtin by functional and microarray analysis of huntingtin-null mouse embryonic fibroblasts

Hua Zhang, Sudipto Das, Quan Zhen Li, Ioannis Dragatsis, Joyce Repa, Scott Zeitlin, György Hajnóczky, Ilya Bezprozvanny

Research output: Contribution to journalArticle

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Abstract

Background: The polyglutamine expansion in huntingtin (Htt) protein is a cause of Huntington's disease (HD). Htt is an essential gene as deletion of the mouse Htt gene homolog (Hdh) is embryonic lethal in mice. Therefore, in addition to elucidating the mechanisms responsible for polyQ-mediated pathology, it is also important to understand the normal function of Htt protein for both basic biology and for HD. Results: To systematically search for a mouse Htt function, we took advantage of the Hdh +/- and Hdh-floxed mice and generated four mouse embryonic fibroblast (MEF) cells lines which contain a single copy of the Hdh gene (Hdh-HET) and four MEF lines in which the Hdh gene was deleted (Hdh-KO). The function of Htt in calcium (Ca2+) signaling was analyzed in Ca2+ imaging experiments with generated cell lines. We found that the cytoplasmic Ca2+ spikes resulting from the activation of inositol 1,4,5-trisphosphate receptor (InsP3R) and the ensuing mitochondrial Ca2+ signals were suppressed in the Hdh-KO cells when compared to Hdh-HET cells. Furthermore, in experiments with permeabilized cells we found that the InsP3-sensitivity of Ca2+ mobilization from endoplasmic reticulum was reduced in Hdh-KO cells. These results indicated that Htt plays an important role in modulating InsP3R-mediated Ca2+ signaling. To further evaluate function of Htt, we performed genome-wide transcription profiling of generated Hdh-HET and Hdh-KO cells by microarray. Our results revealed that 106 unique transcripts were downregulated by more than two-fold with p < 0.05 and 173 unique transcripts were upregulated at least two-fold with p < 0.05 in Hdh-KO cells when compared to Hdh-HET cells. The microarray results were confirmed by quantitative real-time PCR for a number of affected transcripts. Several signaling pathways affected by Hdh gene deletion were identified from annotation of the microarray results. Conclusion: Functional analysis of generated Htt-null MEF cells revealed that Htt plays a direct role in Ca2+ signaling by modulating InsP3R sensitivity to InsP3. The genome-wide transcriptional profiling of Htt-null cells yielded novel and unique information about the normal function of Htt in cells, which may contribute to our understanding and treatment of HD.

Original languageEnglish (US)
Article number38
JournalBMC Neuroscience
Volume9
DOIs
StatePublished - Apr 15 2008

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Microarray Analysis
Fibroblasts
Huntington Disease
Gene Deletion
Genome
Genes
Inositol 1,4,5-Trisphosphate Receptors
Cell Line
Null Lymphocytes
Calcium Signaling
Essential Genes
Endoplasmic Reticulum
Real-Time Polymerase Chain Reaction
Down-Regulation
Pathology

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Cellular and Molecular Neuroscience

Cite this

Elucidating a normal function of huntingtin by functional and microarray analysis of huntingtin-null mouse embryonic fibroblasts. / Zhang, Hua; Das, Sudipto; Li, Quan Zhen; Dragatsis, Ioannis; Repa, Joyce; Zeitlin, Scott; Hajnóczky, György; Bezprozvanny, Ilya.

In: BMC Neuroscience, Vol. 9, 38, 15.04.2008.

Research output: Contribution to journalArticle

Zhang, Hua ; Das, Sudipto ; Li, Quan Zhen ; Dragatsis, Ioannis ; Repa, Joyce ; Zeitlin, Scott ; Hajnóczky, György ; Bezprozvanny, Ilya. / Elucidating a normal function of huntingtin by functional and microarray analysis of huntingtin-null mouse embryonic fibroblasts. In: BMC Neuroscience. 2008 ; Vol. 9.
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AU - Repa, Joyce

AU - Zeitlin, Scott

AU - Hajnóczky, György

AU - Bezprozvanny, Ilya

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