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

描述(申请人提供)：许多类型的侮辱或伤害会导致癫痫发作和神经元丧失，最终可能导致毁灭性和棘手的长期后果，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，特别是促神经生长因子在严重癫痫发作(癫痫持续状态；SE)后介导神经元凋亡的中心作用。神经营养因子最初的特征是对神经元的存活和分化作用，最初是作为前体合成的，被切割后释放C末端成熟形式，与Trk受体结合，促进神经元存活和突触作用。最近的研究表明，神经生长因子的前体形式(ProNGF)通过与p75和山梨素的受体复合体结合来启动神经元死亡，从而作为一种独特的配体。我们的实验室已经证明，在啮齿动物模型中，SE迅速诱导海马区的p75和proNGF。重要的是，功能阻断的抗ProNGF抗体的输注，或p75的基因缺失，挽救了SE后神经元的死亡，为防止SE后神经元丢失及其导致癫痫的发生提供了治疗机制和靶点。在这里，我们将这些合作研究扩展到利用在其内源启动子元件下表达HA表位标记的NGF或proNGF的新型敲入小鼠系。这些动物绕过了目前试剂灵敏度的限制，显著提高了我们检测内源性NGF的能力。因此，我们将能够确定在基础条件下和SE后神经元和神经胶质细胞中NGF/proNGF合成的细胞特异性和时间模式。利用proNGF小鼠，我们将定量评估SE诱导后增强的proNGF的效果，以评估过量的proNGF是否会导致神经细胞死亡加剧。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，并防止癫痫的发生。这些拮抗剂由FDA批准的药物组成，毒性最小，并有效地阻断proNGF诱导的培养神经元凋亡。确定有能力防止严重癫痫发作引起的神经元死亡的已批准药物的可能性具有巨大的临床应用潜力，无论是对癫痫患者群体，还是对那些因脑创伤或中风而癫痫发作的人。

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.