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

描述（由申请人提供）：许多类型的侮辱或伤害导致癫痫发作和神经元丧失，最终可能导致毁灭性和难以处理的长期后果，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proNGF在严重癫痫发作（癫痫持续状态；SE）后介导神经元凋亡中的核心作用。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，最初作为前体合成，被裂解以释放与Trk受体结合的c端成熟形式，以促进神经元存活和突触作用。最近的研究表明，NGF的前体形式（proNGF）作为一种独特的配体，通过结合p75和sortilin受体复合物来启动神经元死亡。我们的实验室已经证明，在啮齿动物模型中，SE可以快速诱导海马中的p75和proNGF。重要的是，输注功能阻断抗prongf抗体或p75基因缺失可挽救SE后的神经元死亡，为预防SE后神经元丢失及其后果癫痫发生提供了一种治疗机制和靶点。在这里，我们扩展了这些合作研究，利用表达ha表位标记的NGF或内源性启动子元件下的proNGF的新型敲入小鼠系。这些动物绕过了目前试剂敏感性的限制，并显着提高了我们检测内源性NGF的能力。因此，我们将能够在基础条件下和SE后确定神经元和胶质细胞中NGF/proNGF合成的细胞特异性和时间模式。使用proNGF小鼠，我们将定量评估SE诱导后proNGF增强的效果，以评估过量的proNGF是否会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的proNGF对癫痫发展的影响。最后，也是最重要的，我们将研究在高通量药物筛选中发现的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阻断prongf诱导的培养神经元凋亡。确定有能力防止严重癫痫发作导致的神经元死亡的已批准药物的可能性，对于癫痫患者群体以及因脑外伤或中风而遭受癫痫发作的患者具有重大的临床应用潜力。

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.