Chow N, Cox C, Callahan L M, Weimer J M, Guo L, Coleman P D
Department of Neurobiology and Anatomy, University of Rochester, Rochester, NY 14642, USA.
Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9620-5. doi: 10.1073/pnas.95.16.9620.
Many changes have been described in the brains of Alzheimer's disease (AD) patients, including loss of neurons and formation of senile plaques and neurofibrillary tangles. The molecular mechanisms underlying these pathologies are unclear. Northern blot, dot-blot, and reverse transcription-coupled PCR analyses have demonstrated altered expression levels of multiple messages in AD brain. Because not all cells are equally affected by the disease, these methods obviously cannot study the changes in relation to disease states of individual cells. We address this problem by using antisense RNA profiling of single cells. We present expression profiles of single neurons at early and late stages of AD and describe statistical tools for data analysis. With multivariate canonical analysis, we were able to distinguish the disease state on the basis of altered expression of multiple messages. To validate this approach, we compared results obtained by this approach with results obtained by in situ hybridization analysis. When the neurofilament medium subunit was used as a marker, our results from an antisense RNA profiling revealed no change in neurofilament medium subunit expression between early- and late-stage AD, consistent with findings obtained with in situ hybridization. However, our results obtained by either analysis at the single-cell level differed from the reported decrease in AD neocortex obtained by Northern blot analysis [Kittur, S., Hoh, J., Endo, H., Tourtellotte, W., Weeks, B. S., Markesbery, W. & Adler, W. (1994) J. Geriatr. Psychiatry Neurol. 7, 153-158]. Thus, the strategy of using the single-cell antisense RNA approach to identify altered gene expression in postmortem AD brain, followed by detailed in situ hybridization studies for genes of interest, is valuable in the study of the molecular mechanisms underlying AD neuropathology.
阿尔茨海默病（AD）患者大脑中出现了许多变化，包括神经元丧失以及老年斑和神经原纤维缠结的形成。这些病变背后的分子机制尚不清楚。Northern印迹法、斑点印迹法和逆转录偶联PCR分析表明，AD大脑中多种信息的表达水平发生了改变。由于并非所有细胞都受到该疾病的同等影响，这些方法显然无法研究单个细胞疾病状态相关的变化。我们通过单细胞反义RNA分析来解决这个问题。我们展示了AD早期和晚期单个神经元的表达谱，并描述了数据分析的统计工具。通过多变量典型分析，我们能够根据多种信息表达的改变来区分疾病状态。为了验证这种方法，我们将这种方法获得的结果与原位杂交分析获得的结果进行了比较。当使用神经丝中型亚基作为标志物时，我们反义RNA分析的结果显示AD早期和晚期之间神经丝中型亚基表达没有变化，这与原位杂交的结果一致。然而，我们在单细胞水平上通过任何一种分析获得的结果与Northern印迹分析报道的AD新皮质中的下降情况不同[Kittur, S., Hoh, J., Endo, H., Tourtellotte, W., Weeks, B. S., Markesbery, W. & Adler, W. (1994) J. Geriatr. Psychiatry Neurol. 7, 153 - 158]。因此，使用单细胞反义RNA方法来识别死后AD大脑中基因表达的改变，随后对感兴趣的基因进行详细的原位杂交研究的策略，在研究AD神经病理学潜在分子机制方面是有价值的。
