Zhang Hua, Das Sudipto, Li Quan-Zhen, Dragatsis Ioannis, Repa Joyce, Zeitlin Scott, Hajnóczky György, Bezprozvanny Ilya
Department of Physiology, UT Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
BMC Neurosci. 2008 Apr 15;9:38. doi: 10.1186/1471-2202-9-38.
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.
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.
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.
亨廷顿蛋白（Htt）中的聚谷氨酰胺扩增是亨廷顿舞蹈病（HD）的病因。Htt是一个必需基因，因为小鼠Htt基因同源物（Hdh）的缺失在小鼠胚胎期是致死性的。因此，除了阐明聚谷氨酰胺介导的病理学机制外，了解Htt蛋白在基础生物学和HD中的正常功能也很重要。
为了系统地探寻小鼠Htt的功能，我们利用了Hdh+/-和Hdh-floxed小鼠，构建了四个含有单拷贝Hdh基因的小鼠胚胎成纤维细胞（MEF）系（Hdh-HET）以及四个缺失Hdh基因的MEF系（Hdh-KO）。利用构建的细胞系，通过钙离子成像实验分析了Htt在钙离子（Ca2+）信号传导中的功能。我们发现，与Hdh-HET细胞相比，Hdh-KO细胞中由肌醇1,4,5-三磷酸受体（InsP3R）激活所产生的细胞质Ca2+尖峰以及随之而来的线粒体Ca2+信号受到了抑制。此外，在通透细胞实验中我们发现，Hdh-KO细胞中从内质网动员Ca2+的InsP3敏感性降低。这些结果表明，Htt在调节InsP3R介导的Ca2+信号传导中发挥重要作用。为了进一步评估Htt的功能，我们通过微阵列对构建的Hdh-HET和Hdh-KO细胞进行了全基因组转录谱分析。我们的结果显示，与Hdh-HET细胞相比，Hdh-KO细胞中有106个独特转录本下调超过两倍且p<0.05，173个独特转录本上调至少两倍且p<0.05。微阵列结果通过对一些受影响转录本的定量实时PCR得到了证实。从微阵列结果的注释中鉴定出了几条受Hdh基因缺失影响的信号通路。
对构建的Htt基因敲除MEF细胞的功能分析表明，Htt通过调节InsP3R对InsP3的敏感性在Ca2+信号传导中发挥直接作用。对Htt基因敲除细胞的全基因组转录谱分析产生了关于Htt在细胞中正常功能的新颖独特信息，这可能有助于我们对HD的理解和治疗。
