Epitranscriptomic regulation by m6A RNA methylation after stroke

许多功能基团共价连接到RNA上，以增强细胞转录组的功能。 特别是，甲基修饰的N6-甲基腺苷(M6A)被认为是 成人的大脑。由于其在mRNA处理中的不同和上下文特定的作用，m6A参与了这一进程 急性中枢神经系统损伤和慢性神经退行性疾病。 由于M6A在卒中后脑中的作用尚未得到评估，我们将检验“M6A高甲基化”的假说 是脑缺血后损伤的主要调节因子。 初步研究表明，短暂性局灶性脑缺血显著增加成人M6A甲基化 老鼠的大脑。我们还观察到，m6A写入器复合体没有改变，但大脑丰富的m6A橡皮擦FTO 在中风后的大脑中表达下调。初步研究表明，使用FTO AAV9治疗 载体增加了大脑FTO的表达，并防止了中风后大脑中m6A的高甲基化。这 表明FTO下调，而不是甲基化增加，是所观察到的持续的原因 M6A-中风后的甲基化。FTO AAV9还减少了继发性脑损伤，并改善了运动 局灶性脑缺血后两性小鼠的认知功能恢复。我们进一步观察到， 卒中后使用NADPH治疗可激活FTO并使m6A标记的RNA去甲基化。NADPH也受到保护 中风后的大脑。基于此，Aim 1将评估是否可以通过使用以下药物诱导FTO来保护中风后的大脑 NADPH遵循许多楼梯标准。 在缺血脑中被5倍高甲基化的122个RNA中，c-jun RNA的数量最多 19个真正的M6A基元。C-jun是细胞凋亡基因表达的中介物，FTO过表达 使c-jun基因去甲基化，并阻止其在中风后大脑中的翻译。在此基础上，Aim 2将进行测试 如果c-jun基因高-m6A甲基化介导中风后细胞凋亡和随之而来的脑损伤。 M6A阅读器YTHDF1的结合促进了m6A标记的转录本的翻译。初步数据显示 局灶性脑损伤诱导YTHDF1表达及m6A甲基化c-jun与YTHDF1结合增强 缺血症。我们进一步观察到，FTO的过度表达降低了m6A甲基化的c-jun的丰度 并降低了其与YTHDF1的结合。YTHDF1-/-小鼠未表现出c-jun蛋白的诱导表达 缺血症。这表明增加的m6A甲基化和YTHDF1结合都是翻译所必需的 C-Jun.基于此，Aim 3将测试m6A阅读器YTHDF1是否在脑缺血后损伤中发挥作用。 总体目标是了解中风后m6A甲基化的机制和治疗潜力。

Many functional groups attach covalently to RNAs to enhance the functionality of the cellular transcriptome. Particularly, the methyl group modification N6-methyladenosine (m6A) is regarded as the fifth nucleobase in the adult brain. With its diverse and context-specific roles in mRNA processing, m6A is implicated in the progression of acute CNS injuries and chronic neurodegenerative diseases. As the role of m6A in post-stroke brain is not yet evaluated, we will test the hypothesis “m6A hypermethylation is a major modulator of post-ischemic brain damage.” Preliminary studies showed that transient focal ischemia significantly increases the m6A methylation in adult mouse brain. We also observed that the m6A writer complex is unaltered, but the brain-enriched m6A eraser FTO was down-regulated in the post-stroke brain. Preliminary studies showed that treatment with an FTO AAV9 vector increased cerebral FTO expression and prevented m6A hypermethylation in the post-stroke brain. This indicates that FTO downregulation, rather than increased methylation, is responsible for the observed sustained m6A-hypermethylation after stroke. FTO AAV9 also decreased secondary brain damage, and improved motor and cognitive functional recovery in mice of both sexes subjected to focal ischemia. We further observed that post-stroke treatment with NADPH activates FTO and demethylates m6A-tagged RNAs. NADPH also protected post-stroke brain. Based on this, Aim 1 will evaluate if post-stroke brain can be protected by inducing FTO with NADPH following many STAIR criteria. Of all the 122 RNAs hypermethylated by >5-fold in the ischemic brain, c-Jun RNA harbors the highest number of 19 bonafide m6A motifs. c-Jun is a mediator of apoptotic gene expression, and FTO overexpression demethylated c-Jun mRNA and prevented its translation in the post-stroke brain. Based on this, Aim 2 will test if c-Jun hyper-m6A methylation mediates post-stroke apoptosis and the ensuing brain damage. Binding of m6A reader YTHDF1 promotes translation of m6A-tagged transcripts. Preliminary data showed induction of YTHDF1 expression and increased binding of m6A-methylated c-Jun to YTHDF1 following focal ischemia. We further observed that FTO overexpression decreased the abundance of m6A-methylated c-Jun and reduced its binding to YTHDF1. YTHDF1-/- mice didn’t show induction of c-Jun protein expression after ischemia. This indicates that both increased m6A methylation and YTHDF1 binding are essential for translation of c-Jun. Based on this, Aim 3 will test if m6A reader YTHDF1 plays a role in post-ischemic brain damage. The overall goal is to understand the mechanisms and therapeutic potential of m6A methylation after stroke.