GABA(A) Receptor Subunit Regulation in Epileptogenesis

 描述(申请人提供)：脑源性神经营养因子(BDNF)是大脑中的一种关键信号分子，在功能上与基本的细胞过程有关，如与学习和记忆相关的过程。BDNF信号的改变被认为在包括癫痫在内的多种神经系统疾病的神经可塑性失调中起着关键作用，但这些重要大脑过程的复杂分子决定因素还不完全清楚。我们的实验室发现，BDNF部分地通过激活Janus Kinase(JAK)/信号转导和转录激活因子(STAT)信号通路来调节抑制。利用原代培养的神经元和啮齿动物模型，我们报道了脑源性神经营养因子诱导的JAK/STAT信号抑制癫痫持续状态和脑创伤后含有GABA受体的突触α1的表达，并且在损伤时抑制JAK/STAT可以减轻随后癫痫的严重程度。我们还有初步证据表明，BDNF诱导的JAK/STAT信号是通过激活细胞表面的TrkB，促进含有p75神经营养素受体的细胞内信号体(称为(I)p75NTRJ)内的JAK2自动磷酸化，导致STAT3的募集和激活而介导的。随后(I)p75NTRJ信号体进入细胞核可能改变多个靶基因的转录，包括可诱导的早期cAMP抑制物(ICER)，它结合并抑制SE后的α1 GABA基因。为了验证我们的假设，即BDNF诱导的JAK/STAT激活在脑损伤后受到失调调节，导致多个促进癫痫发生的基因的转录发生系统变化，我们将使用一种无偏见的转录切割方法，包括RNA-SEQ和带有pSTAT3、p75NTR、JAK2和ICER抗体的CHIP-SEQ，在一个临床前癫痫模型中，利用野生型和转基因小鼠，p75NTR或STAT3基因可诱导缺失。具体地说，我们将：1)确定(I)p75NTRJ在致痫脑损伤后改变其表达的靶基因，以及它们是否针对神经元或使用神经元选择性或全局p75NTR或STAT3缺失/抑制的一般细胞反应的一部分；2)机械测试目标1中确定的目标基因与用BDNF或海人藻酸盐治疗的单个神经元的基因组反应的关系；以及3)确定神经元选择性或全局移除p75NTR或STAT3在脑损伤后癫痫发生和认知共病中的功能后果。结合转录分析和小鼠遗传学在癫痫动物模型中，我们将扩大我们目前对脑损伤后GABAR基因调控的知识，以了解癫痫发生背后的基因组变化的复杂模式。这种理解对于癫痫修饰疗法的发展至关重要，这些修饰疗法针对的是广泛调节基因表达的细胞内途径，可能比孤立地针对单个候选基因的产物的修饰疗法更有效。

 DESCRIPTION (provided by applicant): Brain derived neurotrophic factor (BDNF), a critical signaling molecule in the brain, is functionally linked to essential cellular processes, such as those associated with learning and memory. Altered BDNF signaling is thought to play a crucial role in dysregulated neuroplasticity underlying multiple neurological diseases, including epilepsy, yet the complex molecular determinants of these important brain processes are not fully understood. Our laboratories discovered that BDNF modulates inhibition, in part, through activation of the Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling pathway. Employing primary cultured neurons and rodent models, we have reported that BDNF-induced JAK/STAT signaling represses the expression of synaptic α1 containing GABAA receptors (GABARs) following epileptogenic brain injuries, including status epilepticus (SE) and brain trauma (TBI), and that JAK/STAT inhibition at the time of injury can reduce the severity of subsequent epilepsy. We also have preliminary evidence that BDNF-induced JAK/STAT signaling is mediated by TrKB activation at the cell surface promoting JAK2 autophosphorylation within an intracellular signalsome containing p75 neurotrophin receptors (a complex referred to as (i)p75NTRJ), leading to STAT3 recruitment and activation. Subsequent transport of the (i)p75NTRJ signalsome into the nucleus may alter transcription of multiple target genes, including inducible early cAMP repressor (ICER) that binds to and represses the α1 GABAR gene after SE. To test our hypothesis that BDNF-induced JAK/STAT activation is dysregulated following brain injury causing a systematic change in the transcription of multiple genes that promote epileptogenesis, we will use an unbiased transcriptomic approach, including RNA-seq and ChIP-seq with antibodies to pSTAT3, p75NTR, JAK2, and ICER, in a preclinical epilepsy model utilizing wild-type and transgenic mice with inducible deletion of p75NTR or STAT3 genes. Specifically, we will: 1) Identify the target genes of (i)p75NTRJ that change their expression after epileptogenic brain injury and whether they are specific to neurons or part of a general cellular response using neuron selective or global p75NTR or STAT3 deletion/inhibition; 2) Mechanistically test the relationship of target genes identified in Aim 1 to the genomic response of individual neurons treated with BDNF or kainate; and 3) Determine the functional consequences of neuronal selective or global p75NTR or STAT3 removal on epileptogenesis and cognitive co-morbidities following brain injury. Combining transcriptomic analysis with mouse genetics in an animal model of epilepsy, we will expand our current knowledge of GABAR gene regulation after brain injury to appreciate the complex patterns of genomic changes that underlie epileptogenesis. Such understanding is essential for development of epilepsy modifying therapies targeting intracellular pathways that broadly regulate gene expression & may be more efficacious than those that target the products of single gene candidates in isolation.