Genomic Studies upon NeuroD6 overexpression in PC12 cells, in the presence or absence of an apoptotic stimulus

During neurogenesis, expression of the basic Helix-Loop-Helix NeuroD6/Nex1/MATH-2 transcription factor parallels neuronal differentiation, while maintaining the differentiated state in the mature nervous system. To further dissect NeuroD6 differentiation properties, we previously generated a NeuroD6-overexpressing stable PC12 cell line, PC12-ND6, which displays a neuronal phenotype characterized by spontaneous neuritogenesis, accelerated NGF-induced differentiation, and increased regenerative capacity. Furthermore, we reported that NeuroD6 promotes long-term neuronal survival upon oxidative stress triggered by serum deprivation. In this study, we identified the NeuroD6-mediated transcriptional regulatory pathways linking neuronal differentiation to survival, by conducting a genome-wide microarray analysis using PC12-ND6 cells and serum deprivation as a stress paradigm. Through a series of filtering steps and a gene-ontology analysis, we found that NeuroD6 promotes distinct but overlapping gene networks, consistent with the differentiation, regeneration, and survival properties of PC12-ND6 cells. Using a gene set enrichment analysis, we provide the first evidence of a compelling link between NeuroD6 and a set of heat shock proteins in the absence of stress, which may be instrumental to confer stress tolerance to PC12-ND6 cells. Immunocytochemistry results showed that HSP27 and HSP70 interact with cytoskeletal elements, consistent with their roles in neuritogenesis and preserving cellular integrity. HSP70 also colocalizes with mitochondria located in the soma, growing neurites and growth cones of PC12-ND6 cells prior to and upon stress stimulus, consistent with its neuroprotective functions. Collectively, our findings support the notion that NeuroD6 links neuronal differentiation to survival via the network of molecular chaperones and endows the cells with increased stress tolerance.在神经发生过程中，碱性螺旋-环-螺旋（basic Helix-Loop-Helix）转录因子NeuroD6/Nex1/MATH-2的表达与神经元分化进程同步，并在成熟神经系统中维持细胞的分化状态。为进一步解析NeuroD6的分化调控特性，我们此前构建了过表达NeuroD6的稳定PC12细胞系PC12-ND6，该细胞系呈现出以自发性神经突生成、加速的神经生长因子（Nerve Growth Factor, NGF）诱导分化以及增强的再生能力为特征的神经元表型。此外，我们曾报道NeuroD6可促进血清剥夺诱导的氧化应激环境下神经元的长期存活。本研究中，我们以PC12-ND6细胞系与血清剥夺应激模型开展全基因组微阵列分析，解析了NeuroD6介导的连接神经元分化与存活的转录调控通路。通过一系列筛选步骤与基因本体（gene ontology, GO）分析，我们发现NeuroD6可调控既相互独立又存在功能重叠的基因网络，这与PC12-ND6细胞的分化、再生及存活特性相符。借助基因集富集分析，我们首次证实：在无应激条件下，NeuroD6与一组热休克蛋白（heat shock proteins, HSP）存在显著关联，这可能为PC12-ND6细胞赋予应激耐受性提供关键助力。免疫细胞化学结果显示，HSP27与HSP70可与细胞骨架元件相互作用，这与其在神经突生成及维持细胞完整性中的功能一致。在应激刺激前后，HSP70还可与定位于PC12-ND6细胞胞体、生长神经突及生长锥中的线粒体共定位，这与其神经保护功能相符。综上，我们的研究结果支持以下观点：NeuroD6通过分子伴侣网络将神经元分化与存活联系起来，并使细胞获得更强的应激耐受能力。