Transcription profiling of mouse brain tissue from HtrA2 knockouts

Cellular stress responses can be activated following functional defects in organelles such as mitochondria and the endoplasmic reticulum. Mitochondrial dysfunction caused by loss of the serine protease HtrA2 leads to a progressive movement disorder in mice and has been linked to parkinsonian neurodegeneration in humans. Here we demonstrate that loss of HtrA2 results in transcriptional up-regulation of nuclear genes characteristic of the integrated stress response, including the transcription factor CHOP, selectively in the brain. We also show that loss of HtrA2 results in the accumulation of unfolded proteins in the mitochondria, defective mitochondrial respiration and enhanced production of reactive oxygen species that contribute to the induction of CHOP expression and to neuronal cell death. CHOP expression is also significantly increased in Parkinson's disease patients' brain tissue. We therefore propose that this brain-specific transcriptional response to stress may be important in the advance of neurodegenerative diseases. Experiment Overall Design: This experiment was set out to identify genes that are differentially expressed in brain tissue from the cortex of HtrA2 knockout (KO) mice compared to wild type (WT) littermates. We chose a cortical region since there is no evidence of neuronal loss in this region enabling us to compare mRNA transcripts in identical cellular populations. Brains of littermate WT and HtrA2 KO mice were dissected to obtain cortex tissue at post-natal day 29 (P29). RNA was isolated and samples were processed for hybridisation (6 samples in total, 3 replicates for each genotype).细胞器（如线粒体、内质网）功能异常可激活细胞应激反应。丝氨酸蛋白酶HtrA2缺失所致的线粒体功能障碍，可引发小鼠进行性运动障碍，并与人类帕金森样神经退行性变相关。本研究证实，HtrA2缺失可在大脑中选择性诱导整合应激反应特征性核基因的转录上调，其中包括转录因子CHOP。本研究还发现，HtrA2缺失会导致线粒体内未折叠蛋白蓄积、线粒体呼吸功能缺陷以及活性氧簇（reactive oxygen species）生成增加，而这些变化可诱导CHOP表达并引发神经元细胞死亡。帕金森病患者脑组织中CHOP的表达水平同样显著升高。因此，我们提出这种脑组织特异性应激转录应答可能在神经退行性疾病的进展中发挥重要作用。 实验整体设计：本实验旨在筛选HtrA2基因敲除（KO）小鼠大脑皮层组织相较于野生型（WT）同窝对照小鼠，存在差异表达的基因。我们选择大脑皮层作为研究区域，是因为该区域尚无神经元丢失的报道，可确保我们在相同细胞群体中比较信使RNA（mRNA）转录本的表达水平。分别采集同窝出生的WT和HtrA2 KO小鼠在出生后第29天（P29）的大脑，解剖分离获取皮层组织。提取RNA后，对样本进行杂交检测（总共6个样本，每个基因型设置3个生物学重复）。