The STAT3 response of excitatory neurons to epileptogenic brain injury

摘要 颞叶癫痫（Temporallobeepilepsy，TLE）是一种由分子级联病理改变介导的进行性疾病 海马神经回路重塑导致对自发性癫痫发作的易感性增加 和认知功能障碍靶向这些级联反应可以预防或逆转症状进展， 提供可行的疾病改善治疗的潜力，可以减少TLE患者的比例（> 30%） 对目前的药物治疗没有反应。Janus激酶/信号转导和转录激活因子 (JAK/STAT）通路参与了TLE的发病机制。这条途径被认为是 参与炎症和免疫，并对神经元功能如突触可塑性和 突触发生我们的实验室先前表明，STAT3抑制剂WP 1066可以大大降低 匹罗卡品诱导的癫痫持续状态动物模型中的自发性复发性癫痫发作（SRS）次数 （SE）.虽然这表明JAK/STAT抑制剂作为疾病修饰疗法的前景，但潜在的不良反应可能会增加。 全身或全身CNS途径抑制作用限制了它们的应用。开发更有针对性的治疗方法 需要详细了解JAK/STAT在不同细胞类型中诱导的致癫痫反应。到 为此，我们已经开发了一种新的转基因系，其中二聚体依赖性STAT3信号传导在功能上是 敲除（fKO）的三苯氧胺诱导的Cre表达，特别是在前脑兴奋性神经元（eNs）通过 钙/钙调蛋白依赖性蛋白激酶II α（CamK2a）启动子。我们现在报告，STAT3 KO在 兴奋性神经元（eNSTAT3fKO）显著降低癫痫的进展（SRS频率）， 海马内红藻氨酸（IHKA）TLE模型，并保护小鼠免受红藻氨酸（KA）诱导的记忆缺陷 根据情境恐惧条件反射评估。使用大量海马组织RNA测序的数据，我们 进一步发现了包含大量基因的用于IHKA模型的转录组学标记， 特别是在KA诱导SE后下调的突触可塑性和炎症基因网络中， 在野生型而非eNSTAT3fKO小鼠中。在本申请中，我们将测试STAT3信号转导在细胞内表达的假设。 兴奋性神经元是癫痫进展的关键驱动力，通过选择性沉默基因， 突触可塑性和神经炎症。通过集成使用多组学的开放式发现， 定量分子成像（目标1和3），结合电生理学和神经药理学 (Aim 2），我们将阐明基因组对不同细胞类型损伤（IHKA后24小时和4周）的反应 并确定为什么STAT3 KO在eNs中抑制KA注射后的疾病进展， eNSTAT3表达缺失对兴奋性和抑制性神经元的间接影响。我们还将确定 通过检测eNSTAT3KO对胶质细胞的影响，研究eNSTAT3信号传导与胶质细胞活化之间的关系。 小胶质细胞和星形胶质细胞的转录组和炎症标志物。我们的结果将确定细胞类型特异性 STAT3信号传导或其下游靶点的调节是有希望的治疗干预策略。

Abstract Temporal lobe epilepsy (TLE) is a progressive disorder mediated by pathological changes in molecular cascades and neural circuit remodeling in the hippocampus resulting in increased susceptibility to spontaneous seizures and cognitive dysfunction. Targeting these cascades could prevent or reverse symptom progression and has the potential to provide viable disease-modifying treatments that could reduce the portion of TLE patients (>30%) not responsive to current medical therapies. The Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway has recently been implicated in the pathogenesis of TLE. This pathway is known to be involved in inflammation and immunity, and to be critical for neuronal functions such as synaptic plasticity and synaptogenesis. Our laboratories previously showed that a STAT3 inhibitor, WP1066, could greatly reduce the number of spontaneous recurrent seizures (SRS) in an animal model of pilocarpine-induced status epilepticus (SE). While this suggests promise for JAK/STAT inhibitors as disease-modifying therapies, the potential adverse effects of systemic or global CNS pathway inhibition limits their use. Development of more targeted therapeutics will require a detailed understanding of JAK/STAT-induced epileptogenic responses in different cell types. To this end, we have developed a new transgenic line where dimer-dependent STAT3 signaling is functionally knocked out (fKO) by tamoxifen-induced Cre expression specifically in forebrain excitatory neurons (eNs) via the Calcium/Calmodulin Dependent Protein Kinase II alpha (CamK2a) promoter. We now report that STAT3 KO in excitatory neurons (eNSTAT3fKO) markedly reduces the progression of epilepsy (SRS frequency) in the intrahippocampal kainate (IHKA) TLE model and protects mice from kainic acid (KA)-induced memory deficits as assessed by Contextual Fear Conditioning. Using data from bulk hippocampal tissue RNA-sequencing, we further discovered a transcriptomic signature for the IHKA model that contains a substantial number of genes, particularly in synaptic plasticity and inflammatory gene networks, that are down-regulated after KA-induced SE in wild-type but not eNSTAT3fKO mice. In this application, we will test the hypothesis that STAT3 signaling in excitatory neurons is a key driver of epilepsy progression via the selective silencing of genes that regulate synaptic plasticity and neuroinflammation. With an integration of open discovery using multiomics and quantitative molecular imaging (Aims 1 and 3), in combination with electrophysiology and neuropharmacology (Aim 2), we will elucidate the genome’s response to injury (24 h and 4 wks after IHKA) within different cell types and determine why STAT3 KO in eNs inhibits disease progression after KA injection by identifying direct and indirect effects of loss of eNSTAT3 expression on both excitatory and inhibitory neurons. We will also determine the relationship between eNSTAT3 signaling and glial activation by examining effects of eNSTAT3KO on the glial transcriptome and inflammatory markers of microglia and astrocytes. Our results will ascertain if cell-type specific modulation of STAT3 signaling or its downstream targets are promising strategies for therapeutic intervention.