Modulating ProNGF-induced cell death in Epilepsy: strategies for neuroprotection

描述（由申请人提供）：许多类型的损伤或损伤会导致癫痫发作和神经元丢失，最终可能导致毁灭性和难以治愈的长期后果，包括认知障碍、慢性癫痫和残疾。我们的实验室已经确定了神经营养因子，特别是神经生长因子原在介导神经细胞凋亡后严重癫痫（癫痫持续状态; SE）的核心作用。神经营养因子最初以其对神经元的存活和分化作用为特征，最初合成为前体，其被切割以释放C-末端成熟形式，其结合Trk受体以促进神经元存活和突触作用。最近的研究表明，前体形式的神经生长因子（proNGF）作为一个独特的配体结合p75和分拣蛋白的受体复合物，启动神经元死亡。我们的实验室已经证明，SE在啮齿动物模型中快速诱导海马中的p75和proNGF。重要的是，功能阻断性抗proNGF抗体的输注或p75的基因缺失挽救了SE后的神经元死亡，为预防SE后的神经元损失及其后果癫痫发生提供了治疗机制和靶点。在这里，我们扩展这些合作研究，利用新的敲入小鼠品系，表达HA-表位标记的神经生长因子，或其内源性启动子元件下的proNGF。这些动物规避了目前试剂灵敏度的限制，并显着提高了我们检测内源性NGF的能力。因此，我们将能够确定细胞特异性和时间模式的神经生长因子/神经生长因子原合成神经元和神经胶质细胞在基础条件下，SE后。使用proNGF小鼠，我们将定量评估SE诱导后增强的proNGF的作用，以评估过量的proNGF是否导致神经元细胞死亡加剧。我们还将使用视频脑电图来评估过量proNGF对癫痫发展的后果。最后，也是最重要的，我们将研究在高通量药物筛选中确定的proNGF拮抗剂是否可以预防或减轻SE后的神经元死亡，并预防癫痫发作。这些拮抗剂由FDA批准的药物组成，具有最小的毒性，并有效地阻断proNGF诱导的培养神经元凋亡。鉴定有能力预防严重癫痫发作引起的神经元死亡的批准药物的可能性对于癫痫患者群体和由于脑创伤或中风而遭受癫痫发作的那些患者两者的临床应用具有显著的潜力。

DESCRIPTION (provided by applicant): Many types of insults or injury lead to seizures and neuronal loss, which ultimately can lead to devastating and intractable long-term consequences, including cognitive impairment, chronic epilepsy and disability. Our labs have identified a central role of neurotrophins, particularly proNGF in mediating neuronal apoptosis following severe seizures (status epilepticus; SE). Neurotrophins, initially characterized for their survival and differentiative actions on neurons, are initially synthesized as precursors that are cleaved to release C-terminal mature forms that bind to Trk receptors to promote neuronal survival, and synaptic actions. Recent studies suggest that the precursor form of NGF (proNGF) acts as a distinct ligand by binding to a receptor complex of p75 and sortilin to initiate neuronal death. Our labs have demonstrated that SE rapidly induces p75 and proNGF in the hippocampus, in rodent models. Importantly, infusion of function blocking anti-proNGF antibodies, or genetic deletion of p75, rescues neuronal death following SE, providing a mechanism and target for therapy to prevent neuronal loss after SE, and its consequence, epileptogenesis. Here we extend these collaborative studies to utilize novel knock-in mouse lines that expressed HA-epitope tagged NGF, or proNGF under its endogenous promoter elements. These animals circumvent the current limitations in sensitivity of reagents, and markedly enhance our ability to detect endogenous NGF. Thus we will be able to identify cell specific and temporal patterns of NGF/proNGF synthesis in neurons and glia under basal conditions and after SE. Using the proNGF mouse, we will quantitatively evaluate the effects of augmented proNGF following SE induction, to assess whether excess proNGF leads to exacerbated neuronal cell death. We will also use video-EEG to assess the consequence of excess proNGF on the development of epilepsy. Finally, and most important, we will investigate whether antagonists of proNGF, identified in a high through-put drug screen, can prevent or attenuate neuronal death following SE and prevent the onset of epilepsy. These antagonists consist of FDA-approved drugs that have minimal toxicity and efficiently block proNGF-induced apoptosis in cultured neurons. The possibility of identifying approved drugs that have the capacity to prevent neuronal death from severe seizures has significant potential for clinical application, both for epileptic patient populations, and for those suffering seizures as a consequence of brain trauma or stroke.