Study of PAK3 in epilepsy-associated defects in synaptic plasticity

项目摘要/摘要 美国有300多万人患有癫痫。除了无端的癫痫发作， 认知功能减退和记忆障碍是与癫痫有关的常见并发症，但我们的 这方面的知识是有限的。我们的初步工作表明，癫痫与泛素E3相关 连接酶Nedd4-2通过促进肌动蛋白结合蛋白的磷酸化来介导肌动蛋白聚合 在长时程突触增强(LTP)的诱导过程中的cofilin。LTP描述了 突触的效率，对认知和记忆的形成至关重要。为了识别泛素化 Nedd4-2的底物负责cofilin的磷酸化，我们鉴定了一个正在研究中的IDG合格的PAK3 激酶，作为Nedd4-2的潜在底物，在LTP过程中促进cofilin的磷酸化。这位飞行员 项目的制定是为了测试我们的假设，即LTP的诱导抑制Nedd4-2以稳定PAK3和 随后诱导cofilin磷酸化和肌动蛋白聚合。在目标1中，我们将确定PAK3如何 Nedd4-2泛素化以及Nedd4-2的癫痫相关突变如何破坏这种泛素化。 我们还将确定LTP的诱导是否抑制Nedd4-2，使PAK3在LTP期间稳定。 在目标2中，我们将使用最近开发的光遗传学方法来快速稳定Nedd4-2中的PAK3 LTP诱导过程中的条件性基因敲除(CKO)神经元，意在恢复cofilin 磷酸化和肌动蛋白聚合，最终恢复LTP。我们希望我们的项目能进一步推动我们的 了解IDG合格蛋白PAK3在突触可塑性中的作用，有助于解释癫痫患者的认知能力下降。

PROJECT SUMMARY/ABSTRACT Epilepsy affects more than 3 million people in the United States. In addition to unprovoked seizures, cognitive decline and memory impairment are common comorbidities associated with epilepsy, but our knowledge in this area is limited. Our preliminary work suggests that an epilepsy associated ubiquitin E3 ligase Nedd4-2 mediates actin polymerization through promoting phosphorylation of an actin binding protein cofilin during the induction of long-term synaptic potentiation (LTP). LTP describes long‐lasting increments of synaptic efficiency and is crucial for cognition and memory formation. In an effort to identify ubiquitination substrates of Nedd4-2 responsible for cofilin phosphorylation, we identified PAK3, an IDG-eligible understudied kinase, as a potential substrate of Nedd4-2 that contributes to cofilin phosphorylation during LTP. This pilot project is formulated to test our hypothesis that induction of LTP de-represses Nedd4-2 to stabilize PAK3 and subsequently induce cofilin phosphorylation and actin polymerization. In Aim 1, we will determine how PAK3 is ubiquitinated by Nedd4-2 and how this ubiquitination is disrupted by epilepsy associated mutations in Nedd4-2. We will also determine whether induction of LTP de-represses Nedd4-2 to allow PAK3 stabilization during LTP. In Aim 2, we will employ a recently developed optogenetic approach to rapidly stabilize PAK3 in Nedd4-2 conditional knockout (cKO) neurons during the induction of LTP, with the intention to restore cofilin phosphorylation and actin polymerization, and ultimately to restore LTP. We expect our project to further our knowledge of an IDG-eligible protein PAK3 in synaptic plasticity and help explain cognitive decline in epilepsy.