The impact of stress-induced DNA breaks on chromatin structure, gene activity, and neuron function

项目总结： 慢性应激引起皮质边缘神经元的分子适应和结构重塑 大脑区域，包括前额叶皮质(PFC)和海马体(HPC)。这一点很重要，因为PFC 和HPC整合在大脑回路中，调节复杂的行为和认知。临床前和临床 研究表明，PFC和HPC的突触丢失和连接性降低有助于行为和 几种精神障碍的认知症状，如创伤后应激障碍(PTSD)和 抑郁障碍(MDD)。虽然以前的报告已经确定了候选基因和途径，但分子 导致持续应激诱导的基因活性模式改变和结构重塑的机制 神经元仍然是未知的。在初步研究中，将小鼠暴露在慢性不可预测的应激(CUS)中会引发 前额叶核内应激激活神经元DNA双链断裂的形成。管理 GABAA受体激动剂地西潘减少了应激激活神经元的数量和水平 这表明应激诱导的双链断裂是由活性依赖机制产生的。积累 有证据表明，神经元活动诱导拓扑异构酶(Top2B)产生 DSB并促进介导体验驱动突触的重要基因子集的转录 变化，包括早期反应基因(ERGs)，如Fos、Npas4、Egr1和Arc。这些结果表明 依赖经验的DSB的形成可以调节压力诱导的基因活动模式和随后的 神经元的重塑。然而，应激诱导的PFC和HPC神经元中DSB的位置还没有被定位 DSB如何影响与压力相关的基因活动模式还没有被探索过。 有趣的是，初步研究表明，Top2B介导的DSB的反复和异位诱导 足以概括慢性应激诱导的各种基因表达谱 神经元活性反应基因，包括ERGs和BDNF。初步捕获染色体构象 基于(3C)的实验(3C和4C-SEQ)进一步表明，DSB通过改变基因活性模式来调节基因活动模式 染色质拓扑。这些观察结果导致了一种假设，即慢性阻塞性肺疾病期间复发的DSB的形成 压力改变相关基因的染色质结构，进而稳定压力相关基因的活性。 触发PFC和HPC神经元重塑和突触丢失的模式。为了检验这一假设， 拟议的研究将绘制CUS诱导的DSB的全基因组位置图，并利用Top2b的条件删除来 明确DSB如何影响PFC中应激依赖的转录和神经元结构和功能的变化 和HPC投射神经元。此外，还将采用基于3C的方法(HiChIP)来评估DSB 影响染色质结构的应激依赖变化。总而言之，这些努力将提供新的见解 研究应激诱导神经元适应的机制，并可能发现新的治疗策略 用于精神障碍，如创伤后应激障碍和MDD。

PROJECT SUMMARY: Chronic stress causes molecular adaptations and structural remodeling of neurons within corticolimbic brain areas, including the prefrontal cortex (PFC) and hippocampus (HPC). This is important because the PFC and HPC are integrated in brain circuits that regulate complex behaviors and cognition. Preclinical and clinical studies indicate that synapse loss and reduced connectivity in the PFC and HPC contribute to behavioral and cognitive symptoms in several psychiatric disorders, such as post-traumatic stress disorder (PTSD) and major depressive disorder (MDD). While previous reports have identified candidate genes and pathways, the molecular mechanisms that cause lasting stress-induced changes in gene activity patterns and structural remodeling in neurons remain unknown. In preliminary studies, exposing mice to chronic unpredictable stress (CUS) triggered the formation of DNA double strand breaks (DSBs) within stress-activated neurons in the PFC. Administration of the GABAA receptor agonist, diazepam, diminished both the number of stress-activated neurons and the levels of DSBs, suggesting that stress-induced DSBs are generated by activity-dependent mechanisms. Accumulating evidence indicates that neuronal activity induces the topoisomerase, topoisomerase II (Top2B) to generate DSBs and promote the transcription of an important subset of genes that mediate experience-driven synaptic changes, including early response genes (ERGs), such as Fos, Npas4, Egr1, and Arc. These results suggest that experience-dependent DSB formation could regulate stress-induced gene activity patterns and subsequent remodeling of neurons. Yet the sites of stress-induced DSBs in PFC and HPC neurons have not been mapped and how DSBs affect stress-related gene activity patterns has not been explored. Interestingly, preliminary studies revealed that recurrent and ectopic induction of Top2B-mediated DSBs in cultured neurons is sufficient to recapitulate chronic stress-induced gene expression profiles for various neuronal activity-responsive genes, including ERGs and Bdnf. Preliminary chromosome conformation capture (3C)-based experiments (3C and 4C-seq) further suggest that DSBs regulate gene activity patterns by altering chromatin topology. These observations have led to the hypothesis that recurrent DSB formation during chronic stress alters chromatin architecture at associated genes, which in turn, stabilizes stress-related gene activity patterns that trigger neuronal remodeling and synapse loss in the PFC and HPC. To test this hypothesis, the proposed studies will map genome-wide sites of CUS-induced DSBs and utilize conditional deletion of Top2b to define how DSBs affect stress-dependent changes in transcription and neuronal structure and function in PFC and HPC projection neurons. Additionally, 3C-based methods (HiChIP) will be employed to assess how DSBs affect stress-dependent changes to chromatin architecture. Together, these efforts will provide novel insights into the mechanisms driving stress-induced neuronal adaptations, and may uncover new therapeutic strategies for psychiatric disorders, such as PTSD and MDD.