Substantia nigra from different ages of Rat and iron-challenged SH-SY5Y transcriptome profile. Substantia nigra from different ages of Rat and iron-challenged SH-SY5Y transcriptome profile

Iron is known to be gradually accumulated at the substantia nigra region of the midbrain as getting old, considered as one of the main risk factors of Parkinson’s disease and other neurodegenerative diseases in humans. Heavy metal ions like iron could induce reactive oxygen species, making toxic stress inside the cell, but how the neurons at the substantia nigra are protected from this age-related iron overload has not been fully elucidated. Here, using the rat brains of different ages, we examined the cellular responses of the region of substantia nigra against the age-related iron accumulation. By analyzing the transcriptome of the region we observed the iron deposition, we identified the elevation of stress response genes in the older animals. To determine the genes related to iron response independent of neurodevelopment also elevated as getting old, we challenged a similar amount of irons to the neuroblastoma cell line SH-SY5Y and analyzed their transcriptomic responses. The pathway related to ER stress by protein folding is significantly upregulated among various stress responses altered by iron overload both at the rat brain and the cell. Our result provides a detailed cellular response of neuronal cells against the iron overload causing age-related neurodegenerative diseases. Overall design : Total RNA was extracted from Substantia nigra in each lobe of 4 male 15-month-old, 4 male 6-month-old, 5 female 6-month-old, and 5 female 6-week-old rat. SH-SY5Y was treated with media mixed with 1mM or 2mM ferrous iron for 4 passages with non-treated control. Triplicate samples were prepared in each concentration.

已知随着机体衰老，铁会逐渐在中脑的黑质（substantia nigra）区域蓄积，这被认为是人类罹患帕金森病及其他神经退行性疾病的主要危险因素之一。铁等重金属离子可诱导活性氧（reactive oxygen species）产生，进而引发细胞内毒性应激，但中脑黑质神经元如何免受这种年龄相关性铁过载的影响，目前尚未完全阐明。
本研究采用不同年龄段的大鼠脑组织，探究了黑质区域针对年龄相关性铁蓄积的细胞应答反应。通过对该区域的转录组（transcriptome）进行分析，我们在检测到铁沉积的同时，发现老年动物体内的应激反应基因表达显著上调。
为鉴定出不依赖神经发育且随衰老表达上调的铁应答相关基因，我们将等量亚铁离子处理神经母细胞瘤细胞系SH-SY5Y，并分析其转录组应答变化。在大鼠脑组织与细胞的铁过载所致的多种应激反应中，蛋白质折叠相关的内质网（endoplasmic reticulum，ER）应激通路均显著上调。
本研究结果阐明了神经元细胞针对可引发年龄相关性神经退行性疾病的铁过载的详细细胞应答机制。
实验整体设计：从4只15月龄雄性、4只6月龄雄性、5只6月龄雌性以及5只6周龄雌性大鼠的各脑叶黑质中提取总RNA。将神经母细胞瘤细胞系SH-SY5Y置于添加了1mM或2mM亚铁离子的培养基中传代培养4代，并设置未处理组作为对照。每个浓度均设置三份重复样本。