Circadian clock-mediated neuronal gene expression in Aß toxicity and excitotoxicity

In recent decades, transcriptome analysis has been widely used to understand human disease pathogenesis and identify therapeutic targets and biomarkers. Accumulated reports in patients with neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, have also provided evidence of dysregulated gene expression related to neuropathogenesis. However, obtaining samples from neurodegenerative patients is more challenging than other human diseases due to low accessibility. Also, brain tissues and cerebrospinal fluid are composed of multiple cell types, so they are unsuitable for obtaining neural cell-type-specific gene expression profiles. Thus, we here report gene expression profiles in primary neuronal cultures exposed to Aß toxicity and glutamate excitotoxicity to understand pathological gene expression in neurons. By RNA-sequencing analysis, we compare transcriptomes and find that two groups of genes show similar expression patterns in Aß toxicity excitotoxicity—they are either up- or down-regulated in both conditions. Genes in the two groups are related to synaptic function and cell signaling, which are well-known biological functions altered in Aß toxicity and excitotoxicity. Interestingly, the analysis reveals a possibility that circadian clock (molecular oscillator generating daily rhythms)-related genes are dysregulated in both conditions. We confirm the reduced circadian transcription factor Bmal1 levels in Aß toxicity and glutamate excitotoxicity. RNA-sequencing analysis in Bmal1-deleted neurons shows potential relationships between BMAL1 and synaptic functions. Thus, this transcriptome study provides evidence of the potential roles of the circadian clock in neuropathogenesis. Overall design: Transcriptomes in two neuropathological conditions, Aß toxicity and glutamate excitotoxicity, were examined. Total RNAs were extracted in primary neuronal cultures and subjected to RNA-sequencing analysis to understand neuron-specific gene expression profiles. Then, similar experiments were performed in neurons cultured from the circadian transcription factor Bmal1-deleted mice. We compared transcriptomes between the samples to understand their relationships.近数十年来，转录组（transcriptome）分析已被广泛应用于解析人类疾病的发病机制，并助力治疗靶点与生物标志物的发掘。针对阿尔茨海默病、帕金森病等神经退行性疾病患者的大量研究报道，也已证实存在与神经发病机制相关的基因表达失调现象。然而，由于样本可及性较低，从神经退行性疾病患者体内获取样本的难度远高于其他人类疾病。此外，脑组织与脑脊液由多种细胞类型构成，无法用于获取神经细胞类型特异性的基因表达谱。为此，本研究报道了暴露于β淀粉样蛋白（Aβ）毒性与谷氨酸兴奋性毒性（glutamate excitotoxicity）的原代神经元培养物的基因表达谱，以解析神经元中的病理性基因表达特征。通过RNA测序（RNA-sequencing）分析，本研究对转录组进行了比较，发现两组基因在Aβ毒性与谷氨酸兴奋性毒性条件下呈现相似的表达模式：即在两种处理条件下均呈现上调或下调趋势。这两组基因均与突触功能及细胞信号转导相关，而这两类生物学过程正是已知的Aβ毒性与兴奋性毒性中发生改变的核心环节。有趣的是，本分析还揭示了一种可能性：即昼夜节律钟（circadian clock，调控每日节律的分子振荡器）相关基因在两种处理条件下均存在表达失调。本研究证实，在Aβ毒性与谷氨酸兴奋性毒性条件下，昼夜节律转录因子BMAL1的表达水平均出现下调。对Bmal1基因敲除神经元开展的RNA测序分析，揭示了BMAL1与突触功能之间潜在的调控关联。因此，本项转录组研究为昼夜节律钟在神经发病机制中的潜在作用提供了实验依据。实验设计：本研究对两种神经病理条件（Aβ毒性与谷氨酸兴奋性毒性）下的转录组进行了检测。从原代神经元培养物中提取总RNA，随后进行RNA测序分析，以获取神经元特异性的基因表达谱。随后，本研究以昼夜节律转录因子Bmal1基因敲除小鼠来源的神经元为材料，开展了相同的实验。通过比对各组样本的转录组数据，解析其间的分子关联。