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

项目摘要 虽然环状RNA（circRNA）在哺乳动物大脑中富集，但对其功能知之甚少。 可能与大脑功能和精神疾病有关在无偏circRNA分析和 在多个死后大脑队列的验证中，我们发现了circHomer 1，一个神经元富集的 circRNA在成年人的额叶皮层中大量表达，在成年人的前额叶皮层中显著减少。 精神疾病患者。我们的初步数据表明，circHomer 1可以抑制突触 通过竞争结合，在眶额皮质（OFC）内定位长Homer1b mRNA亚型 RNA结合蛋白（RBP）HuD，一种已知的神经元mRNA运输调节剂。此外，我们的飞行员 数据表明，circHomer 1可以影响突触能传递，恢复Homer 1b OFC中的水平可以挽救在circHomer 1敲低后观察到的认知灵活性的改变 （科威特第纳尔）。我们建议确定circHomer 1调节突触定位的机制， Homer1b（目的1），检测Homer1b在circHomer1介导的神经元功能改变中的作用， 认知灵活性（目标2），并确定新的人类神经元circHomer 1的上游调控因子 生物成因（Aim3）。我们的中心假设是，RBP结合在circHomer 1剪接点附近，以促进其 神经元内的生物发生，并且circHomer1可以隔离HuD与Homer1b的结合，从而抑制 Homer1b突触定位和破坏神经元活动和OFC功能。建议的理由 研究表明，阐明突触可塑性调节circRNA的未探索作用将揭示新的 与精神疾病相关的神经元功能紊乱的本质的分子机制的见解。 对于该提议，我们首先打算通过CRISPR介导的缺失circHomer 1基因来产生circHomer 1敲除（KO）小鼠。 反义内含子区所需的circHomer1回剪接，并检查对突触Homer1b的影响 表达，以及确定circHomer1和Homer1b之间是否存在HuD结合的竞争。 负责增加Homer1b突触定位作为circHomer1丢失的结果（Aim 1）。我们将 然后研究Homer1b水平改变对神经元活动和OFC功能的重要性（目的2）。最后， 在小鼠皮层神经元和iPSC中使用新型circRNA传感器方法以及基因组编辑， 我们将揭示神经元circHomer 1生物发生的上游调节因子（Aim 3）。的 以下三个具体目标将试图解决我们的假设：1）测试的假设，circHomer 1 通过竞争与OFC内的HuD结合来抑制Homer1b mRNA突触定位。2)测试 假设Homer1b OE足以损害OFC功能， circHomer 1缺失可以恢复神经元活性和OFC介导的认知灵活性。3)确定 circHomer 1生物发生在神经元内调节的机制。

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.