Transcriptional characterization of the Eif2b5[R191H] loss-of-function mouse model

The integrated stress response (ISR) is a conserved pathway in eukaryotic cells that is activated in response to multiple sources of cellular stress. Although acute activation of this pathway restores cellular homeostasis, intense or prolonged ISR activation perturbs cell function and may contribute to neurodegeneration. We have characterized an eIF2B loss of function (LOF) mouse model, carrying the homozygous Eif2b5[R191H] mutation that is homologous to the Vanishing White Matter Disease (VWMD)-causing human R195H variant. Characterization of previously reported transcript markers in animals 3-4 weeks of age confirmed ISR activation in the brain, but not in heterozygous or wild-type littermates.The robust elevation of ISR markers was further confirmed in bulk brain RNA sequencing analysis of a separate cohort of homozygous mutants and wild-type littermates. Known ATF4 targets feature prominently among the most strongly up-regulated genes in the eIF2B mutant compared to WT littermates. We generated homozygous Eif2b5[R191H] knock-in mice to create a model of Vanishing White Matter Disease (VWMD). Bulk tissue from the anterior right hemisphere from mutant (n=9) and wildtype (n=12) animals at 3-4 months of age was collected and bulk RNA-seq data was collected with the QuantSeq 3' mRNA-Seq protocol.整合应激反应（integrated stress response, ISR）是真核细胞中一条保守的信号通路，可被多种细胞应激源激活。尽管该通路的急性激活可恢复细胞稳态，但强烈或持续的ISR激活会扰乱细胞功能，并可能参与神经退行性变的发生发展。我们已对eIF2B功能丧失（loss of function, LOF）小鼠模型完成表征，该模型携带纯合子Eif2b5[R191H]突变，其与致脑白质消失症（Vanishing White Matter Disease, VWMD）的人类R195H变异体同源。对3~4周龄动物的已报道转录标志物进行检测，证实突变小鼠脑内存在ISR激活，而杂合子或野生型同窝仔鼠中未观察到此现象。对独立队列的纯合突变小鼠与野生型同窝仔鼠开展全脑组织批量RNA测序分析，进一步验证了ISR标志物的显著升高。与野生型同窝仔鼠相比，eIF2B突变小鼠中上调幅度最显著的基因中，已知的ATF4靶基因占比极高。我们构建了纯合子Eif2b5[R191H]敲入小鼠，以建立脑白质消失症（VWMD）的动物模型。我们收集了3~4月龄的突变型（n=9）与野生型（n=12）小鼠右侧前脑半球的组织样本，并采用QuantSeq 3'端mRNA测序实验方案获取了批量RNA-seq数据。