The STAT3 response of excitatory neurons to epileptogenic brain injury

兴奋性神经元对致癫痫性脑损伤的 STAT3 反应

• 批准号: 10119388
• 负责人: Amy R. Brooks-Kayal
• 金额: $ 57.86万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-15 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10119388
• 关键词: Affect; Anabolism; Animals; Antibodies; Apoptosis; Astrocytes; Behavioral; Brain; Brain Injuries; Ca(2+)-Calmodulin Dependent Protein Kinase; Cell Nucleus; Cells; Ceramides; ChIP-seq; Complex; Contralateral; Data Set; Development; Disease; Electroencephalography; Epilepsy; Epileptogenesis; Frequencies; Future; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genome; Hippocampus (Brain); Hour; Hyperactive behavior; Immunity; Individual; Inflammation; Injections; Interneurons; Intervention; Intractable Epilepsy; Ion Channel Gating; Ipsilateral; Janus kinase; Laboratories; Lead; Ligands; Long-Term Depression; Measures; Membrane; Memory; Memory impairment; Metabolism; Microglia; Modeling; Molecular; Molecular Profiling; Monitor; Mus; Neurons; Pathway interactions; Patients; Pharmacology; Pilocarpine; Population; Predisposition; Process; Prosencephalon; Reporting; Role; STAT protein; STAT3 gene; Seizures; Severities; Signal Transduction; Site; Sphingolipids; Status Epilepticus; Structure; Symptoms; Synaptic plasticity; Tamoxifen; Technology; Temporal Lobe Epilepsy; Testing; Time; Transcriptional Regulation; Transgenic Organisms; Up-Regulation; Video Recording; brain tissue; calmodulin-dependent protein kinase II; cell type; conditioned fear; early detection biomarkers; excitatory neuron; granule cell; hippocampal pyramidal neuron; in vivo; inhibitor/antagonist; kainate; knock-down; metabolome; metabolomics; mouse model; neural circuit; neuroprotection; promoter; receptor binding; relating to nervous system; response; trafficking; transcriptome; transcriptome sequencing; transcriptomics; voltage

ABSTRACT Temporal lobe epilepsy (TLE) develops after a period of ongoing molecular cascades and neural circuit remodeling in the hippocampus resulting in increased susceptibility to spontaneous seizures. Targeting these cascades in TLE patients could reverse their symptoms and have the potential to provide a viable disease- modifying treatment, especially for the large portion of over 30% of TLE patients who do not respond to any available treatments. In recent years, the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway has been implicated in temporal lobe epilepsy (TLE). The JAK/STAT pathway is known to be involved in inflammation and immunity, and only more recently has been shown to be associated with neuronal functions such as synaptic plasticity. Our laboratories previously showed that a JAK inhibitor, WP1066, could greatly reduce the number of spontaneous seizures that animals went on to develop over time in the pilocarpine model of status epilepticus (SE). We have continued to investigate the mechanism of JAK/STAT- induced epileptogenic responses through the use of a new transgenic line we developed where STAT3 knockdown (KD) can be controlled by tamoxifen-induced CRE expression specifically in forebrain excitatory neurons via the Calcium/Calmodulin Dependent Protein Kinase II alpha (CamK2a) promoter. We now report that this knockdown of STAT3 (nSTAT3KD) markedly reduces spontaneous seizure frequency in the intrahippocampal kainate model (IHKA) and “rescues” mice from KA-induced memory deficits as measured by Contextual Fear Conditioning. Recently, using deep RNA-sequencing we also discovered transcriptomic signatures 24 hours after SE that occur in response to IHKA injections (ipsilateral and contralateral to the injection site) and are reversed by nSTAT3KD, especially for those genes important in sphingolipid metabolism: a regulator of neuronal structure, and the trafficking, stability, and function of multiple membrane bound receptors, including ligand- and voltage-gated ion channels. These findings, taken together with our preliminary IHKA metabolome, brings us to propose the following unique hypothesis that there is a JAKx/STAT3 pathway in excitatory forebrain neurons that becomes activated in response to prolonged seizures and that identifying the cells most susceptible to STAT3 signaling during the epileptogenic process will provide a window on basic circuitry that underlies memory formation, and most importantly, the brain's susceptibility to epilepsy development. To test this hypothesis, we have three Aims using state of the art molecular technologies (metabolomic profiling, single nuclei RNA sequencing, and chromatin immunoprecipitation sequencing) to interrogate the molecular signature of the hippocampus (24 h, 2 wk, and 4 wk after IHKA SE) . The emerging transcriptome for STAT3 in the context of epilepsy suggests that it may be useful for identifying potential epileptogenic gene networks that were previously unknown, selecting early-detection biomarkers that inform seizure susceptibility, as well as choosing new targets for the future treatment of intractable epilepsies.

摘要 颞叶癫痫（TLE）是在一段时间的持续分子级联和神经回路后发生的 海马的重塑导致对自发性癫痫发作的易感性增加。靶向这些 TLE患者的级联反应可以逆转他们的症状，并有可能提供一个可行的疾病- 修改治疗，特别是对于大部分超过30%的TLE患者，他们对任何治疗都没有反应。 可用的治疗。近年来，Janus激酶/信号转导和转录激活因子 (JAK/STAT）通路与颞叶癫痫（TLE）有关。已知JAK/STAT途径 参与炎症和免疫，只是最近才被证明与 神经元功能，如突触可塑性。我们的实验室先前表明JAK抑制剂WP 1066， 可以大大减少动物随着时间的推移自发癫痫发作的次数， 癫痫持续状态（SE）的匹罗卡品模型。我们继续研究JAK/STAT的机制， 通过使用我们开发的一种新的转基因株系诱导癫痫反应， 敲低（KD）可以通过他莫昔芬诱导的CRE表达控制，特异性地在前脑兴奋性 神经元通过钙/钙调蛋白依赖性蛋白激酶II α（CamK 2a）启动子。我们现在报告， 这种STAT 3的敲低（nSTAT 3 KD）显著降低了癫痫患者的自发发作频率。 海马内红藻氨酸盐模型（IHKA）和“拯救”小鼠免于KA诱导的记忆缺陷，如通过 情境恐惧条件反射。最近，使用深度RNA测序，我们还发现了转录组学 SE后24小时出现的响应于IHKA注射（注射的同侧和对侧）的特征 位点），并被nSTAT 3 KD逆转，特别是对于那些在鞘脂代谢中重要的基因： 神经元结构的调节剂，以及多种膜结合受体的运输、稳定性和功能， 包括配体和电压门控离子通道。这些发现，连同我们的初步IHKA 代谢组学，使我们提出以下独特的假设，即在代谢组中存在JAKx/STAT 3途径。 兴奋性前脑神经元，在长时间癫痫发作时被激活， 在癫痫发生过程中对STAT 3信号最敏感的细胞将提供一个基本的 记忆形成的基础电路，最重要的是，大脑对癫痫的易感性 发展为了验证这一假设，我们有三个目标，使用最先进的分子技术 （代谢组学分析、单核RNA测序和染色质免疫沉淀测序）， 询问海马的分子特征（在IHKA SE后24小时、2周和4周）。新兴 在癫痫背景下STAT 3的转录组表明，它可能有助于识别潜在的 癫痫基因网络是以前未知的，选择早期检测生物标志物， 癫痫发作的敏感性，以及选择新的目标，为未来的治疗顽固性癫痫。