A kinase pathway required for cortical neuron polarization

描述(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，研究已经涉及到几个编码激酶、磷酸酶、小GTP酶、微管相关蛋白和支架蛋白的基因在神经元极性的建立中。这些研究大多使用经典的海马神经元分离培养模型，它们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活体神经元极化中发挥主要作用，以及(Ii)了解细胞外信号如何极化细胞内的反应，从而导致轴突和树突的指定。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LKB1(也称为PAR4或STK11)是活体皮质神经元极化所必需的(Barnes等人)。单元格按下)。使用有条件的皮质特异性基因敲除，我们发现LKB1是体内神经元极化过程中轴突启动所必需的，但对神经元迁移或树突形成并不是必需的。我们的初步结果表明，LKB1的极化活性既需要与其必要的辅助激活剂-假性激酶Strad1相互作用，也需要通过蛋白激酶A(PKA)或p90核糖体S6激酶(P90RSK)对丝氨酸431(S431)进行磷酸化，这两种已知的激酶介导了细胞外信号控制轴突形态发生的一些作用。一旦激活，LKB1就会磷酸化SAD-A/B激酶，而SAD-A/B激酶是LKB1指定神经元极性所必需的，部分原因是它们能够磷酸化微管相关蛋白(MAP)，如Tau。综上所述，我们的初步结果使我们假设，这一新的多激酶途径是体内轴突指定所必需的，它将细胞外信号与神经元极化背后的细胞内反应联系起来。我们建议通过以下方法检验这一假设：(1)确定LKB1是否在整个发育中的CNS和PNS中用于指定神经元的极性，(目的2)确定先前被证明激活PKA和p90RSK的细胞外信号如BDNF或WNT7a是否可以诱导丝氨酸431的磷酸化并控制轴突指定，(目的3)测试这些细胞外信号、TrkB和Frz3的受体是否对于皮质轴突指定是必需的。Frz3特别令人感兴趣，因为Frz3基因敲除胚胎在轴突形成方面显示出强烈的缺陷，这一表型几乎与我们的条件性LKB1基因敲除无关。这一提议将为在活体中指定神经元极性的分子机制提供新的见解。项目叙述现代神经科学中的一个主要问题集中在神经元如何在发育过程中建立联系。我们确定了一个基因(LKB1)，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调节该基因功能的因素。最近的证据表明，LKB1的中断可能参与阿尔茨海默病等肌萎缩侧索硬化症。因此，我们的工作将为了解这种破坏性神经退行性病变背后的分子机制提供重要而新颖的线索。

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.