Unraveling the biogenesis and molecular mechanisms of a neuronal-enriched circRNA altered in psychiatric disease

项目总结 尽管环状RNA(CircRNAs)在哺乳动物的大脑中含量丰富，但人们对它们的了解很少 可能与大脑功能和精神疾病有关。在无偏见的CircRNA分析和 在多个死后大脑队列中的验证中，我们发现了一种富含神经元的CircHmer 1 CircRNA在成人额叶皮质中大量表达，而在成年大鼠的前额叶皮质中表达显著减少 患有精神病的受试者。我们的初步数据表明，CircHmer 1可以抑制突触 通过竞争结合，在眶前叶皮质(OFC)内定位长Hmer 1b mRNA异构体 与RNA结合蛋白(RBP)HUD有关，HUD是已知的神经元mRNA运输的调节因子。另外，我们的飞行员 有数据表明，CircHmer 1可以影响谷氨酸能突触传递，恢复Hmer 1b OFC中的水平可以挽救CircHmer 1基因敲除后观察到的认知灵活性的变化 (KD)。我们建议确定CircHmer 1调节突触定位的机制。 Hmer 1b(目标1)，以研究Hmer 1b在Hmer 1介导的神经元功能和细胞周期改变中的作用 认知灵活性(目标2)，并确定人类神经元环路Hmer 1的新的上游调节因子 生物发生(Aim3)。我们的中心假设是，限制性商业惯例结合在CircHmer 1剪接接头附近，以促进其 神经元内的生物发生和CircHmer 1可以隔离HUD与Hmer 1b的结合，从而抑制 Hmer 1b突触定位和扰乱神经元活动和OFC功能。建议的理由是 研究表明，阐明突触可塑性调节CircRNAs的未知作用将发现新的 与精神疾病相关的神经功能障碍的本质的分子力学洞察。 对于这一提议，我们首先打算通过CRISPR介导的缺失来产生CircHmer 1基因敲除(KO)小鼠 反义内含子区域和检测对突触Hmer 1b的影响 表达，以及确定CircHmer 1和Hmer 1b之间对HUD结合的竞争是否 负责由于CircHmer 1的丢失而导致Hmer 1b突触定位的增加(目标1)。我们会 然后研究Hmer 1b水平改变对神经元活动和OFC功能的重要性(目标2)。最后， 利用一种新的CircRNA传感器方法，结合基因组编辑，在小鼠皮质神经元和IPSC中- 衍生神经元培养，我们将揭示神经元CircHmer 1生物发生的上游调节因子(目标3)。这个 以下三个具体目标将尝试解决我们的假设：1)测试CircHmer 1的假设 通过与OFC内的HUD竞争结合，抑制Hmer 1b基因的突触定位。2)测试 假设Hmer 1b OE足以损害OFC功能，并将其表达正常化如下 CircHmer 1缺失可以恢复神经元活动和OFC介导的认知灵活性。3)确定 CircHmer 1生物发生在神经元内的调节机制。

PROJECT SUMMARY Although circular RNAs (circRNAs) are enriched in the mammalian brain, very little is known about their potential involvement in brain function and psychiatric disease. Following unbiased circRNA profiling and validation in multiple postmortem brain cohorts, we have uncovered that circHomer1, a neuronal-enriched circRNA abundantly expressed in the adult frontal cortex, is significantly reduced in the prefrontal cortex of subjects with psychiatric disease. Our preliminary data suggest that circHomer1 can inhibit the synaptic localization of the long Homer1b mRNA isoform within the orbitofrontal cortex (OFC), by competing for binding to the RNA-binding protein (RBP) HuD, a known regulator of neuronal mRNA trafficking. Moreover, our pilot data suggest that circHomer1 can influence glutamatergic synaptic transmission and that restoring Homer1b levels in the OFC can rescue the alterations in cognitive flexibility observed following circHomer1 knockdown (KD). We propose to determine the mechanisms by which circHomer1 regulates the synaptic localization of Homer1b (Aim 1), to examine the role of Homer1b in circHomer1-mediated alterations in neuronal function and cognitive flexibility (Aim 2), and to identify novel upstream regulators of human neuronal circHomer1 biogenesis (Aim3). Our central hypothesis is that RBPs bind near the circHomer1 splice junction to promote its biogenesis within neurons and that circHomer1 can sequester HuD from binding to Homer1b, thereby inhibiting Homer1b synaptic localization and disrupting neuronal activity and OFC function. The rationale of the proposed research is that elucidating the unexplored role of synaptic plasticity-regulating circRNAs will uncover novel molecular mechanistic insights into the nature of neuronal function disturbances related to psychiatric disease. For this proposal we first intend to generate circHomer1 knockout (KO) mice via CRISPR-mediated deletion of antisense intronic regions required for circHomer1 backsplicing and examine the impact on synaptic Homer1b expression, as well as determine whether competition for HuD binding between circHomer1 and Homer1b is responsible for the increased Homer1b synaptic localization as a result of loss of circHomer1 (Aim 1). We will then examine the importance of altered Homer1b levels for neuronal activity and OFC function (Aim 2). Finally, using a novel circRNA sensor approach together with genome editing in mouse cortical neurons and iPSC- derived neuronal cultures we will uncover upstream regulators of neuronal circHomer1 biogenesis (Aim 3). The following three specific aims will attempt to address our hypotheses: 1) Test the hypothesis that circHomer1 inhibits Homer1b mRNA synaptic localization via competing for binding to HuD within the OFC. 2) Test the hypothesis that Homer1b OE is sufficient to impair OFC function and that normalizing its expression following circHomer1 loss can restore neuronal activity and OFC-mediated cognitive flexibility. 3) Determine the mechanism by which circHomer1 biogenesis is regulated within neurons.