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

应激诱导的 DNA 断裂对染色质结构、基因活性和神经元功能的影响

• 批准号: 10655982
• 负责人: Ram Madabhushi
• 金额: $ 79.98万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-05 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10655982
• 关键词: Affect; Agonist; Architecture; Area; Atrophic; Automobile Driving; Behavior; Behavioral; Behavioral Symptoms; Brain; Brain region; Brain-Derived Neurotrophic Factor; Breeding; Candidate Disease Gene; ChIP-seq; Chromatin; Chromatin Conformation Capture and Sequencing; Chromatin Loop; Chromatin Structure; Chronic; Chronic stress; Clinical Research; Cognition; Cognitive; Cognitive deficits; Complex; Confocal Microscopy; DLG4 gene; DNA; DNA Double Strand Break; Diazepam; ERG gene; Early Promoters; Enhancers; Enzymes; Epigenetic Process; Etoposide; Exposure to; FOSB gene; FRAP1 gene; Gene Expression; Genes; Genetic Transcription; Genome Mappings; Genotype; Heterochromatin; Hippocampus; Hour; Infusion procedures; Knowledge; Label; Length; Major Depressive Disorder; Maps; Mediating; Mental disorders; Molecular; Mus; NPAS4 gene; Neurobehavioral Manifestations; Neurobiology; Neurons; Pathway interactions; Pattern; Poison; Post-Traumatic Stress Disorders; Prefrontal Cortex; Prosencephalon; Proteins; Recurrence; Reporting; Rodent; Role; Site; Sorting; Stress; Stressful Event; Structure; Synapses; Synaptic plasticity; Synaptosomes; Testing; Topoisomerase; Work; candidate identification; chromatin remodeling; chromosome conformation capture; cohort; density; experience; experimental study; genome-wide; genomic locus; gephyrin; inhibitor; insight; lateral ventricle; nervous system disorder; novel; novel therapeutic intervention; osmotic minipump; preclinical study; promoter; receptor; transcriptome sequencing

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.

项目概要： 慢性应激导致皮质边缘神经元的分子适应和结构重塑 大脑区域，包括前额皮质 (PFC) 和海马体 (HPC)。这很重要，因为 PFC 和 HPC 集成在调节复杂行为和认知的大脑回路中。临床前和临床 研究表明，突触丢失以及 PFC 和 HPC 中连通性的降低会导致行为和行为障碍。 多种精神疾病的认知症状，例如创伤后应激障碍 (PTSD) 和重度抑郁症 抑郁症（MDD）。虽然之前的报告已经确定了候选基因和途径，但分子 导致基因活动模式和结构重塑持久应激变化的机制 神经元仍然未知。在初步研究中，将小鼠暴露于慢性不可预测压力（CUS）会引发 PFC 中应激激活神经元内 DNA 双链断裂 (DSB) 的形成。管理 GABAA 受体激动剂地西泮减少了应激激活神经元的数量和水平 DSBs，表明应激诱导的 DSBs 是由活动依赖性机制产生的。积累中 有证据表明，神经元活动会诱导拓扑异构酶，拓扑异构酶 II (Top2B) 生成 DSB 并促进介导经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 (ERG)，例如 Fos、Npas4、Egr1 和 Arc。这些结果表明 经验依赖的 DSB 形成可以调节应激诱导的基因活动模式和随后的 神经元的重塑。然而，PFC 和 HPC 神经元中应激诱导的 DSB 的位点尚未被绘制出来。 DSB 如何影响应激相关基因的活动模式尚未得到探索。 有趣的是，初步研究表明 Top2B 介导的 DSB 的反复和异位诱导 在培养的神经元中足以重现慢性应激诱导的各种基因表达谱 神经元活动反应基因，包括 ERG 和 Bdnf。初步染色体构象捕获 基于 (3C) 的实验（3C 和 4C-seq）进一步表明 DSB 通过改变 染色质拓扑结构。这些观察结果导致了这样的假设：慢性 DSB 形成过程中反复出现。 压力改变相关基因的染色质结构，从而稳定压力相关基因的活性 触发 PFC 和 HPC 神经元重塑和突触损失的模式。为了检验这个假设， 拟议的研究将绘制 CUS 诱导的 DSB 的全基因组位点，并利用 Top2b 的条件删除来 定义 DSB 如何影响 PFC 中转录以及神经元结构和功能的应激依赖性变化 和 HPC 投射神经元。此外，将采用基于 3C 的方法 (HiChIP) 来评估 DSB 如何 影响染色质结构的应激依赖性变化。这些努力共同将提供新颖的见解 研究驱动压力诱导的神经元适应的机制，并可能发现新的治疗策略 用于精神疾病，例如创伤后应激障碍 (PTSD) 和抑郁症 (MDD)。