Supplementary Material for: Induction of oxidative stress and alteration of synaptic gene expression in newborn hippocampal granule cells after developmental exposure to Aroclor 1254

Introduction: Hippocampal newborn neurons integrate into functional circuits where they play an important role in learning and memory. We previously showed that perinatal exposure to Aroclor 1254, a commercial mixture of polychlorinated biphenyls (PCBs) associated with alterations of cognitive function in children, disrupted the normal maturation of excitatory synapses in the dentate gyrus. We hypothesized that hippocampal immature neurons underlie some of the cognitive effects of PCBs. Methods: We used newly-generated neurons to examine the effects of PCBs in mice following maternal exposure. Newborn dentate granule cells were tagged with enhanced green fluorescent protein (EGFP) using a transgenic mouse line. The transcriptome of the newly generated granule cells was assessed using RNA sequencing. Results: Gestational and lactational exposure to 6 mg/kg/day of Aroclor 1254 disrupted the mRNA expression of 1308 genes in newborn granule cells. Genes involved in mitochondrial functions were highly enriched with 154 genes significantly increased in exposed compared to control mice. The upregulation of genes involved in oxidative phosphorylation was accompanied by signs of endoplasmic reticulum stress and an increase in lipid peroxidation, a marker of oxidative stress, in the subgranular zone of the dentate gyrus but not in mature granule cells in the granular zone. Aroclor 1254 exposure also disrupted the expression of synaptic genes. Using laser-captured subgranular and granular zones, this effect was restricted to the subgranular zone, where newborn neurons are located. Conclusion: Our data suggest that gene expression in newborn granule cells is disrupted by Aroclor 1254 and provide clues to the effects of endocrine disrupting chemicals on the brain.

引言：海马体新生神经元可整合入功能性神经环路，并在学习记忆过程中发挥重要作用。本课题组此前研究证实，围产期暴露于Aroclor 1254——一种与儿童认知功能改变相关的商用多氯联苯（polychlorinated biphenyls, PCBs）混合物——可破坏齿状回（dentate gyrus）兴奋性突触的正常成熟进程。本研究假设，海马体未成熟神经元是PCBs引发部分认知功能异常的潜在基础。
方法：本研究通过母鼠暴露构建染毒模型，探究PCBs对小鼠的影响。采用转基因小鼠品系，以增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）对新生齿状回颗粒细胞进行标记，并通过RNA测序（RNA sequencing）分析新生颗粒细胞的转录组。
结果：妊娠及哺乳期暴露于6 mg/kg/天剂量的Aroclor 1254，可使新生颗粒细胞内1308个基因的mRNA表达发生紊乱。线粒体功能相关基因显著富集，相较于对照组小鼠，暴露组有154个基因的表达量显著上调。氧化磷酸化相关基因的上调，伴随齿状回颗粒下层区（subgranular zone）出现内质网应激（endoplasmic reticulum stress）迹象，以及作为氧化应激（oxidative stress）标志物的脂质过氧化（lipid peroxidation）水平升高；而颗粒细胞层的成熟颗粒细胞未出现此类变化。Aroclor 1254暴露还可干扰突触相关基因的表达。通过激光捕获颗粒下层区与颗粒细胞层，本研究发现该效应仅局限于新生神经元所在的颗粒下层区。
结论：本研究数据表明，Aroclor 1254可干扰新生颗粒细胞的基因表达，同时为内分泌干扰化学物对大脑的作用机制提供了研究线索。