Sharma Dharmendra, Kim Min Soo, D'Mello Santosh R
Department of Biological Sciences, Southern Methodist University, Dallas, TX 75272, USA.
Quantitative Biomedical Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Exp Biol Med (Maywood). 2015 Feb;240(2):242-51. doi: 10.1177/1535370214551688. Epub 2014 Sep 25.
The molecular mechanisms underlying neuronal death are poorly understood. One of the most widely used models to study neuronal death are cultured cerebellar granule neurons (CGNs) which undergo apoptosis when switched from a medium containing depolarizing levels of potassium (HK) to a medium with low non-depolarizing levels of potassium (LK). Previously, other labs have used DNA microarray analysis to characterize gene expression changes in LK-treated CGNs. However, microarray analysis is only capable of measuring the status of known transcripts, and expression of low-abundance mRNAs is often not detected by the hybridization-based approach. We have used RNA-sequencing to conduct a more detailed and comprehensive analysis of gene expression changes in CGNs induced to die by LK treatment. RNA-seq investigates the status of both known transcripts as well as exploring new ones and is substantially more sensitive than the microarray approach. We have found that the expression of 4334 genes is significantly altered in LK-treated CGNs with 2199 being up-regulated while 2135 are down-regulated. Genes functioning in cell death and survival regulation, cell growth and proliferation and molecular transport were most affected by LK treatment. Further, a large number of genes involved in nervous system development and function were also deregulated. Analysis of signaling pathways that were affected in LK-induced death included but were not limited to mitochondrial dysfunction and oxidative phosphorylation, consistent with a number of studies showing perturbations of these pathways in neurodegenerative disorders. Thus, our study identifies a large number of new genes that are affected during the process of neuronal death. While a majority of these changes may reflect consequences of the induction of neuronal death, many of the genes that we have identified are likely to be critical and potentially novel mediators of neuronal death, including death associated with neurodegenerative disease.
神经元死亡背后的分子机制仍知之甚少。研究神经元死亡最常用的模型之一是培养的小脑颗粒神经元（CGNs），当从含有去极化水平钾离子的培养基（HK）转换到含有低非去极化水平钾离子的培养基（LK）时，这些神经元会发生凋亡。此前，其他实验室已使用DNA微阵列分析来表征LK处理的CGNs中的基因表达变化。然而，微阵列分析仅能测量已知转录本的状态，基于杂交的方法通常无法检测到低丰度mRNA的表达。我们使用RNA测序对LK处理诱导死亡的CGNs中的基因表达变化进行了更详细和全面的分析。RNA测序不仅研究已知转录本的状态，还探索新的转录本，并且比微阵列方法灵敏得多。我们发现，在LK处理的CGNs中，4334个基因的表达发生了显著变化，其中2199个上调，2135个下调。在细胞死亡和存活调节、细胞生长和增殖以及分子运输中起作用的基因受LK处理的影响最大。此外，大量参与神经系统发育和功能的基因也失调了。对LK诱导死亡中受影响的信号通路的分析包括但不限于线粒体功能障碍和氧化磷酸化，这与许多研究表明这些通路在神经退行性疾病中受到干扰一致。因此，我们的研究鉴定出了大量在神经元死亡过程中受影响的新基因。虽然这些变化中的大多数可能反映了神经元死亡诱导的后果，但我们鉴定出的许多基因可能是神经元死亡的关键且潜在的新介质，包括与神经退行性疾病相关的死亡。
