A kinase pathway required for cortical neuron polarization

描述（由申请人提供）：轴突和树突的极化是神经元在大脑中整合和传递信息的能力的基础。在过去的几年里，研究已经牵连的几个基因编码激酶，磷酸酶，小GTP酶，微管相关蛋白，和支架蛋白在建立神经元极性。这些研究大多采用经典的分离海马神经元培养模型，其与体内脑发育的相关性尚不确定。目前该领域最紧迫的挑战是（i）确定这些蛋白质是否在体内神经元极化中起主要作用，以及（ii）了解细胞外信号如何影响细胞内反应，从而导致轴突和树突特化。使用候选方法，我们最近获得了初步结果，证明丝氨酸/苏氨酸激酶LKB 1（也称为Par 4或STK 11）是体内皮层神经元极化所需的（巴恩斯等人，Cell -出版中）。使用条件性皮质特异性敲除，我们发现LKB 1是体内神经元极化过程中轴突起始所必需的，但对神经元迁移或树突形成不是必需的。我们的初步研究结果表明，LKB 1的极化活性需要与其必要的共激活剂，假激酶Strad 1和丝氨酸431（S431）的磷酸化蛋白激酶A（PKA）或p90核糖体S6激酶（p90 RSK），已知介导的一些细胞外线索控制轴突形态发生的影响的两种激酶。一旦被激活，LKB 1磷酸化SAD-A/B激酶，这是LKB 1部分通过磷酸化微管相关蛋白（MAP）如Tau的能力来指定神经元极性的能力所必需的。两者合计，我们的初步结果使我们假设，这种新的多激酶途径是必需的轴突规格在体内连接细胞外信号的细胞内反应神经元极化。我们建议通过以下方式来检验这一假设：（目的1）确定LKB 1是否是整个发育中的CNS和PNS所必需的，以用于神经元极性的特化，（目的2）确定细胞外线索如BDNF或Wnt 7a先前显示激活PKA和p90 RSK是否可以诱导丝氨酸431的磷酸化并控制轴突特化，（目的3）测试这些细胞外线索的受体，TrkB和Frz 3是皮质轴突规范所必需的。Frz 3是特别感兴趣的，因为Frz 3基因敲除胚胎在轴突形成中显示出强烈的缺陷，这是一种与我们的条件性LKB 1基因敲除几乎相同的表型。这一建议将提供新的见解的分子机制，指定在体内神经元极性。项目叙述在现代神经科学的一个主要问题集中在神经元如何建立连接在发展过程中。我们确定了一个基因（LKB 1），它是大脑皮层轴突形成的基础，我们建议研究在发育过程中是什么调节这个基因的功能。最近的证据表明，LKB 1的破坏可能参与tau蛋白病，如阿尔茨海默病。因此，我们的工作将提供重要的和新的线索，这种破坏性的神经退行性病变的分子机制。

DESCRIPTION (provided by applicant): The polarization of axon and dendrites underlies the ability of neurons to integrate and transmit information in the brain. In the past few years, studies have implicated several genes encoding kinases, phosphatases, small GTPases, microtubule-associated proteins, and scaffolding proteins in the establishment of neuronal polarity. Most of these studies used the classic model of dissociated hippocampal neuron culture and their relevance to brain development in vivo is uncertain. The most pressing challenges in the field are now (i) determining if these proteins play major roles in neuronal polarization in vivo and (ii) understanding how extracellular cues polarize intracellular responses leading to axon and dendrite specification. Using a candidate approach, we recently obtained preliminary results demonstrating that the serine/threonine kinase LKB1 (also known as Par4 or STK11) is required for polarization of cortical neurons in vivo (Barnes et al. Cell - in press). Using a conditional cortex-specific knockout, we found that LKB1 is specifically required for axon initiation during neuronal polarization in vivo but is not essential for neuronal migration or dendrite formation. Our preliminary results suggest that the polarizing activity of LKB1 requires both interaction with its necessary co-activator, the pseudo-kinase Strad1 and phosphorylation on Serine 431 (S431) by protein kinase A (PKA) or p90 Ribosomal S6 Kinase (p90RSK), two kinases known to mediate some of the effects of extracellular cues controlling axon morphogenesis. Once activated, LKB1 phosphorylates SAD-A/B kinases which are required for the ability of LKB1 to specify neuronal polarity in part through their ability to phosphorylate microtubule associated proteins (MAPs) such as Tau. Taken together, our preliminary results lead us to hypothesize that this novel multi-kinase pathway is required for axon specification in vivo by linking extracellular signals to the intracellular responses underlying neuronal polarization. We propose to test this hypothesis by (Aim 1) determining if LKB1 is required throughout the developing CNS and PNS for the specification of neuronal polarity, (Aim 2) determining if extracellular cues such as BDNF or Wnt7a previously shown to activate PKA and p90RSK can induce phosphorylation of Serine 431 and control axon specification, (Aim 3) testing if receptors for these extracellular cues, TrkB and Frz3 are required for cortical axon specification. Frz3 is of particular interest since Frz3 knockout embryos show a strong defect in axon formation, a phenotype almost indentical to our conditional LKB1 knockout. This proposal will provide novel insights into the molecular mechanisms specifying neuronal polarity in vivo.Project narrative One major question in modern neuroscience focuses on how neurons establish connections during development. We identified a gene (LKB1) that underlies the formation of axons in the cerebral cortex and we propose to study what regulate the function of this gene during development. Recent evidence suggested that LKB1 disruption could participate to tauopathies such as Alzheimer's disease. Therefore our work will provide important and novel clues regarding the molecular mechanisms underlying this devastating neurodegenerative pathology.