title: Elucidating a normal function of huntingtin by functional and microarray analysis of huntingtin-null mouse embryonic fibroblasts.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Physiology, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA. Hua.Zhang@UTSouthwestern.edu
authors.forename: Hua
authors.initials: H
authors.lastname: Zhang
authors.forename: Sudipto
authors.initials: S
authors.lastname: Das
authors.forename: Quan-Zhen
authors.initials: QZ
authors.lastname: Li
authors.forename: Ioannis
authors.initials: I
authors.lastname: Dragatsis
authors.forename: Joyce
authors.initials: J
authors.lastname: Repa
authors.forename: Scott
authors.initials: S
authors.lastname: Zeitlin
authors.forename: György
authors.initials: G
authors.lastname: Hajnóczky
authors.forename: Ilya
authors.initials: I
authors.lastname: Bezprozvanny
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000235:genetics*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D000071058:Huntingtin Protein; D006816:Huntington Disease / Q000235:genetics; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000502:physiology*; D009687:Nuclear Proteins / Q000235:genetics* / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
chemical_list: C506812:Htt protein, mouse; D000071058:Huntingtin Protein; D053496:Inositol 1,4,5-Trisphosphate Receptors; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D015544:Inositol 1,4,5-Trisphosphate