Regulation of mammalian brain development by p21-activated kinase signaling pathways

标题：p21激活的激酶信号通路对哺乳动物大脑发育的调节*这项申请是我目前NSERC发现基金(RGPIN341498,2012-2017)的续订。*我研究计划的长期目标是了解支配大脑发育和功能的基本机制。为此，我们已经确定了一些对神经元的增殖、存活、迁移、形态形成和成熟至关重要的分子，这是一系列高度调控的过程，对正常的脑生长、神经回路的形成和功能至关重要。与这一应用特别相关的是，在我目前的NSERC发现拨款(2012-17年)的资助下，我们已经证明了缺乏p21激活的激酶(PAKs)的基因敲除(KO)小鼠在脑大小、皮质体积、神经前体细胞分裂和迁移方面都发生了变化。值得注意的是，这些KO小鼠在Lim-kinase1(LIMK1)和肌动蛋白结合蛋白cofilin的活性方面存在缺陷，这两个蛋白都是PAK的关键靶点，对肌动蛋白调节至关重要。此外，我们发现PAK可以对内源性大麻素(ECB)系统产生影响，ECB系统是胚胎大脑发育的各个方面的新兴参与者。这些结果导致了一种假设，即PAK通过协调肌动蛋白重组和ECB信号来调节皮质发育。为了解决这一假设，我们将使用多种技术，包括小鼠遗传学、免疫组织化学、成像、生化分析和电生理记录来实现三个特定的目标：*目的1.通过标记和跟踪不同PAK KO小鼠的神经前体细胞，确定PAK1和3在神经元增殖和迁移中的相对贡献。目的2.通过对神经元和突触形态的分析，确定PAK1和3在出生后早期神经元形态发生和突触成熟中的作用。目的3.通过研究LIMK1/cofilin和ECB信号的作用，阐明PAK1/3作用的分子机制。这一应用的结果将揭示PAK信号调节皮质和神经元发育的体内功能和潜在机制。这反过来将有助于我的长期目标，了解控制大脑发育和功能的基本过程。由于皮质畸形和帕金森病都与许多神经和精神障碍密切相关，这一建议也将为这些疾病的发病机制和治疗提供新的见解。********

Title: Regulation of mammalian brain development by p21-activated kinase signaling pathways******This application is a renewal of my current NSERC discovery grant (RGPIN341498, 2012-2017).******The long-term goal of my research program is to understand the fundamental mechanisms that govern brain development and function. To this end, we have identified a number of molecules that are important for neuronal proliferation, survival, migration, morphogenesis and maturation, a series of highly regulated processes critical for normal brain growth, neural circuit formation and function. With particular relevance to this application and with the funding of my current NSERC discovery grant (2012-17), we have demonstrated that knockout (KO) mice lacking the p21-activated kinases (PAKs), a family of protein kinases known to be important for cytoskeletal reorganization, are altered in brain size, cortical volume, neuronal progenitor cell division and migration. Significantly, these KO mice are defective in the activity of LIM-kinase 1 (LIMK1) and the actin-binding protein cofilin, both of which are key targets of PAKs important for actin regulation. Furthermore, we discovered that PAKs can exert effects on the endocannabinoid (eCB) system, an emerging player in various aspects of embryonic brain development. These results lead to a hypothesis that PAKs regulate cortical development via coordinating actin reorganization and eCB signaling. To address this hypothesis, we will use multiple techniques, including mouse genetics, immunohistochemistry, imaging, biochemical assays and electrophysiological recordings to achieve three specific aims: ******Aim 1. To determine the relative contribution of PAK1 and 3 in neuronal proliferation and migration by labeling and tracking neural progenitor cells in various PAK KO mice. ******Aim 2. To determine the role of PAK1 and 3 in early postnatal neuronal morphogenesis and synaptic maturation by analyzing neuronal and synaptic morphology. ******Aim 3. To elucidate the molecular mechanisms underlying PAK1/3 actions by investigating the role of LIMK1/cofilin and eCB signaling.******The results from this application will reveal the in vivo function and underlying mechanisms by which PAK signaling regulates cortical and neuronal development. This will in turn contribute to my long-term goal to understand the fundamental process governing brain development and function. Since both cortical malformations and PAKs are closely associated with many neurological and mental disorders, this proposal will also provide new insight into the pathogenesis and treatment of these diseases. ********