The Role of m6A-RNA Methylation in Memory Formation and Recall and Its Modulation and Influence on Long-Term Outcomes as a Consequence of Early Life Lead Exposure

转录调控是神经系统正常发育和功能的核心。失调症 转录和转录后调控通路与多种神经发育相关 疾病和障碍，包括发育性铅(铅)暴露，这往往会导致显著和 持续的认知和行为缺陷。我们之前曾报道过铅引起的甲基化改变。 河马基因启动子区域及铅对组蛋白翻译后修饰的影响 铅致认知功能障碍动物的海马区和内侧前额叶皮质 性别、铅暴露量和发育窗口期作为影响因素。虽然之前的工作已经 侧重于铅对DNA相关机制的影响，这是一种额外的分子转录调控 在这方面还没有研究的机制是RNA修饰。来自我们实验室支持的初步数据 假设通过N6-甲基腺苷(m6A/m)直接RNA甲基化可能是一个重要的机制 导致发育铅暴露后转录组的功能改变。直接修改 RNA似乎提供了对RNA功能的额外一层控制，用于微调转录 对环境的反应，对正常的可塑性和记忆功能至关重要。考虑到消极的一面 铅暴露对神经可塑性和记忆的影响，我们认为有一个潜在的重要 M6A/m在铅中毒相关基因特异性反应转录后调控中的作用 可塑性/记忆力受损。我们的假设是，这个丰富的表位转录标记，m6A/m，不仅 在正常情况下在调节记忆过程中起着重要作用，但在早期被改变 铅暴露对铅的转录和行为反应有不利影响。我们将对此进行测试 以下目标的假设：AIM1：检查转录组范围的m6A/m谱在多大程度上 男性和女性的mPFC和Hipp CA1受发育性铅暴露的影响，并与 伴随着记忆缺陷的表现。假设：将有全脑区域不同的转录组 M6A/m的富集谱(由m6A-eCLIP(增强型紫外光交联和免疫沉淀)检测)， 特别是与记忆/可塑性相关的基因和途径有关，这些基因和途径在 铅暴露，记忆受损的动物；AIM2：检测m6a/m修饰在 通过调节m6A/m水平对铅暴露动物mPFC和CA1的影响初步数据显示 铅暴露记忆障碍大鼠m6A水平降低和FTO表达增加，我们将探索 病毒介导靶向敲除FTO对铅性记忆障碍的潜在疗效 MPFC和CA1的m6A/m水平和记忆/可塑性相关基因的表达。我们假设，通过 提高m6A/m水平，至少可以部分改善铅中毒所致的记忆障碍。

Regulation of transcription is central to proper nervous system development and functioning. Dysregulation of transcriptional and post-transcriptional regulatory pathways are associated with various neurodevelopmental diseases and disorders including developmental lead (Pb) exposure, which often results in significant and persistent cognitive and behavioral deficits. We have previously reported Pb-induced methylation changes at gene promoter regions and effects of Pb on various post-translational histone modifications (PTHMs) in hippo- campus (HIPP) and medial prefrontal cortex (mPFC) in animals with Pb-induced cognitive dysfunction, with sex, amount of Pb exposure, and developmental window of exposure as effect modifiers. While prior work has focused on effects of Pb on DNA-related mechanisms, an additional molecular transcriptional control mechanism that has not been studied in this regard is RNA modification. Preliminary data from our lab support the hypothesis that direct RNA methylation via N6-methyladenosine (m6A/m), may be an important mechanism contributing to the functionally altered transcriptome after developmental Pb exposure. Direct modifications of RNA appear to provide an additional layer of control over RNA function for the fine-tuning of transcriptomic responses to the environment, critical for normal plasticity and memory function. Considering the negative impact that Pb exposure has on neural plasticity and memory, we suggest that there is a potentially important role of m6A/m in post-transcriptional regulation of gene-specific responses associated with Pb-induced plasticity/ memory impairments. Our hypothesis is that this abundant epitranscriptomic mark, m6A/m, not only plays an important role in modulating memory processes under normal circumstances but is altered by early Pb exposure with an adverse influence on transcriptional and behavioral responses to Pb. We will test this hypothesis in the following aims: Aim1: Examine the extent to which transcriptome-wide m6A/m profiles in mPFC and HIPP CA1 and are altered by developmental Pb exposure in males and females and associated with expression of memory deficits. Hypothesis: There will be brain region-distinct transcriptome-wide enrichment profiles of m6A/m (detected by m6A-eCLIP (enhanced UV-crosslinking and immunoprecipitation), particularly associated with memory/plasticity-related genes and pathways, that are aberrantly modulated in Pb-exposed, memory-impaired animals; Aim2: Examine the functional significance of m6A/m modification in mPFC and CA1 in Pb-exposed animals through manipulation of m6A/m levels. As preliminary data indicate decreased m6A levels and increased FTO expression in Pb-exposed rats with memory deficits, we will explore the potential efficacy of viral-mediated targeted knockdown of FTO on Pb-induced memory impairments and m6A/m levels and memory/plasticity-related gene expression in mPFC and CA1. We hypothesize that by increasing m6A/m levels we will at least partially ameliorate the Pb-induced memory impairment.