RNA-Sequencing and proteomics approaches reveal multi-cellular deficits in the cortex of Rett syndrome mice

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the transcriptional regulator MeCP2. RTT is characterized by having apparently normal development until 6-18 months, when a progressive decline in motor and language functions begins and breathing abnormalities and seizures present. Despite intense research, the molecular targets of MeCP2 and their contribution to the disease are unknown. Here we present the first comprehensive and comparative transcriptomic and proteomic analysis in a RTT mouse model. Examining whole cortex tissue in symptomatic males (Mecp2Jae/y) and wild-type littermates, we have identified 391 genes and 465 proteins considered to be significantly altered. We observed an overall poor correlation between global gene and protein expression (Pearson correlation 0.12), yet 35 hits were common to both data sets, with 12 hits not described elsewhere. These 35 hits indicate disrupted cellular metabolism, calcium signaling, protein stability, DNA binding and cytoskeletal cell structure in the RTT cortex. Pathway analysis in both data sets identified biological pathways ubiquitous to multiple cell types as well as cell type specific pathways, underscoring the contributions of multiple central nervous system (CNS) cell populations to the disease pathogenesis. These findings prompted us to compare identified 'hits' to a publicly available database containing CNS cell type specific gene expression. This indicated approximately 32% of differentially expressed (DE) genes and 16% proteins were highly enriched in unique CNS cell types, while the remaining DE genes and proteins were ubiquitously expressed and not ascribable to any unique cell population. Our comparative transcriptome and proteome analysis in the cortex of RTT mice supports previous works indicating widespread CNS dysfunction. Wild-type (WT) males were bred with heterozygous Mecp2tm1.1Jae/+ (Jaenisch) female mice. The whole cortex of mutant male mice (Mecp2Jae/y) along with WT littermates were collected after postnatal day 60 (P60+). An n of 4 biological replicates per genotype were used, with WT animals serving as controls. For RNA-Sequencing, 2 technical replicates were run per biological replicate.雷特综合征（Rett syndrome, RTT）是一种由转录调节因子MeCP2突变引发的X连锁神经发育障碍。该疾病的典型特征为患者在6至18月龄前发育看似正常，随后运动与语言功能逐渐衰退，并伴随呼吸异常与癫痫发作。尽管学界已开展大量深入研究，但MeCP2的分子靶点及其在疾病发生中的作用机制仍未明确。 本研究首次针对雷特综合征小鼠模型开展全面的比较转录组学与蛋白质组学分析。研究人员对出现症状的雄性模型小鼠（Mecp2Jae/y）及其野生型同窝幼鼠的全皮层组织进行检测，共鉴定出391个显著差异表达基因及465个显著差异表达蛋白质。 本研究观察到全局基因与蛋白质表达之间整体相关性较差（皮尔逊相关系数为0.12），但两组数据集共有35个重合靶点，其中12个为此前未被报道的新发现。这35个重合靶点提示，雷特综合征小鼠皮层中存在细胞代谢、钙信号通路、蛋白质稳定性、DNA结合以及细胞骨架结构的紊乱。 对两组数据集的通路分析不仅鉴定出多种细胞类型共有的生物学通路，还发现了细胞类型特异性通路，这凸显了中枢神经系统（central nervous system, CNS）多种细胞群在疾病发病机制中的贡献。基于上述发现，研究团队将鉴定出的靶点与公开的中枢神经系统细胞类型特异性基因表达数据库进行了比对。结果显示，约32%的差异表达（differentially expressed, DE）基因与16%的蛋白质在特定中枢神经系统细胞类型中高度富集，而剩余的差异表达基因与蛋白质则呈普遍表达状态，无法归因于某一独特细胞群。 本研究在雷特综合征小鼠皮层中开展的比较转录组学与蛋白质组学分析，印证了此前研究提出的中枢神经系统广泛功能异常的结论。 实验中，野生型（wild-type, WT）雄性小鼠与杂合子Mecp2tm1.1Jae/+（Jaenisch）雌性小鼠进行繁育。在出生后第60天（postnatal day 60, P60+）后，采集突变型雄性小鼠（Mecp2Jae/y）及其野生型同窝幼鼠的全皮层组织。每组基因型设置4个生物学重复，以野生型动物作为对照。对于RNA测序（RNA-Sequencing），每个生物学重复设置2个技术重复。