The STAT3 response of excitatory neurons to epileptogenic brain injury

摘要 摘要颞叶癫痫(TLE)是一种由分子级联病理改变介导的进行性疾病。 以及海马区神经回路重塑导致自发性癫痫易感性增加 和认知功能障碍。针对这些级联反应可以防止或逆转症状的进展，并具有 提供可行的疾病修正治疗的潜力，可以减少TLE患者的比例(&gt；30%) 对当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)通路最近被认为与TLE的发病机制有关。已知这条途径是 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WP1066可以极大地减少 匹罗卡品致痫持续状态动物模型的自发性反复发作次数 (Se)。虽然这暗示了JAK/STAT抑制剂作为疾病修改疗法的前景，但潜在的不利因素 全身性或全局性中枢神经系统通路抑制的影响限制了它们的使用。开发更具靶向性的治疗方法 将需要详细了解JAK/STAT在不同细胞类型中诱导的致痫反应。至 为此，我们开发了一种新的转基因株系，其中二聚体依赖的STAT3信号在功能上是 三苯氧胺敲除(FKO)诱导前脑兴奋性神经元(ENS)特异性Cre的表达 钙/钙调蛋白依赖蛋白激酶IIα(CamK2a)启动子。我们现在报告STAT3 KO in 兴奋性神经元(ENSTAT3fKO)显著降低癫痫的进展(SRS频率)。 海马区海人酸致小鼠TLE模型及对海人酸致小鼠记忆障碍的保护作用 根据情境恐惧条件反射进行评估。使用大量的海马区组织RNA测序数据，我们 进一步发现了IHKA模型的转录签名，该签名包含大量基因， 尤其是在突触可塑性和炎性基因网络中，它们在KA诱导的SE后下调 在野生型而不是eNSTAT3fKO小鼠中。在本应用程序中，我们将测试STAT3信号在 兴奋性神经元是通过选择性沉默调控基因而导致癫痫进展的关键驱动因素 突触可塑性和神经炎症。与使用多组学的开放发现和 定量分子成像(目标1和3)，结合电生理学和神经药理学 (目标2)，我们将阐明不同细胞类型内基因组对损伤(IHKA后24小时和4周)的反应 确定ENS中STAT3 KO在KA注射后抑制疾病进展的原因 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 从eNSTAT3KO对神经胶质细胞的影响看eNSTAT3信号与神经胶质细胞激活的关系 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 调控STAT3信号或其下游靶点是治疗干预的有前途的策略。

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.