Role of CLASP in neuronal morphogenesis during development

描述（由申请人提供）：神经元形态发生是一个高度调节的过程，最终取决于神经元细胞骨架响应细胞外信号的重塑。以往对神经元细胞骨架的研究主要集中在细胞外信号对肌动蛋白丝的调控上。很少有研究已经做了调查如何神经元微管（MT）的调节。MT的调节涉及脑发育的每个步骤，例如神经前体细胞的不对称细胞分裂、神经元迁移、轴突生长和引导、树突发育和突触发生。许多MT调节蛋白在发育过程中的突变通常与精神疾病有关，这强调了MT调节在正常大脑发育中的重要性。因此，我们的长期目标是了解如何管理神经元形态发生的细胞外信号转导到MT重组神经元，这是必要的适当的轴突和树突的发展。我们的初步研究揭示了一种新的机制，MT加上末端跟踪蛋白，CLASPs，调节哺乳动物轴突和树突的生长。我们发现，与其他只跟踪MT正端的+TIP不同，CLASP显示与神经元中MT（或MT晶格）的正端或沿着侧的双重结合。在功能上，我们表明，这种独特的双MT CLASPs的结合行为，使他们能够差异调节MT组织和轴突生长在不同的神经元。在再生的感觉神经元中，CLASP主要与MT+末端结合并起支持轴突快速生长的作用。相比之下，CLASP在发育中的皮质神经元中显示出沿着MT侧边的结合增加，并起到限制轴突生长的作用。除了限制轴突生长，我们还提供了CLASPs功能支持皮质神经元树突发育的证据。有趣的是，轴突导向因子Slit也起到排斥轴突的作用，同时促进树突生长。由于CLASP已被放置在Slit的下游，以介导果蝇的轴突排斥，我们假设CLASP，其独特的双重MT结合特性，可能是Slit-Robo信号转导的收敛目标，以调节哺乳动物轴突和树突的发育。因此，本研究的总体目标是阐明CLASP在皮质发育过程中响应细胞外信号调节神经元形态发生中的作用。为了验证这一假设，我们将1）阐明CLASP调节MT以控制轴突生长和树突发育的分子机制，2）使用体外轴突导向测定和细胞培养来确定CLASP在Slit介导的轴突排斥和树突生长中的作用，以及3）确定CLASP在皮质神经元轴突生长/导向中的体内作用，以及使用子宫内电穿孔在皮质发育期间的树突发育。这项研究将揭示新的分子机制，细胞外线索调节MT控制神经元形态发生。公共卫生相关性：神经元形态发生，包括轴突生长、导向和树突生长，是发育过程中神经回路形成或损伤后神经修复的关键分子事件。发育过程中的这些错误被认为会导致许多神经发育障碍。因此，我们提出的研究不仅有助于我们了解神经回路在发育过程中是如何形成的，而且还为如何促进损伤后的神经再生提供了有价值的信息。

DESCRIPTION (provided by applicant): Neuronal morphogenesis is a highly regulated process that ultimately depends on the remodeling of neuronal cytoskeleton in response to extracellular cues. Most previous studies of neuronal cytoskeleton focus on the regulation of actin filaments by extracellular cues. Very few studies have been done to investigate how neuronal microtubules (MTs) are regulated. Regulation of MTs is involved in every step of brain development, such as asymmetrical cell division of neural precursor cells, neuronal migration, axon growth and guidance, dendrite development, and synaptogenesis. Mutation of many MT regulating proteins during development is often associated with mental illnesses, underscoring the importance of MT regulation in normal brain development. Thus, our long-term goal is to understand how extracellular signals governing neuronal morphogenesis are transduced into MT reorganization in neurons that is necessary for proper axonal and dendritic development. Our preliminary study has revealed a novel mechanism by which MT plus end tracking proteins, CLASPs, regulate mammalian axonal and dendritic growth. We found that, unlike other +TIPs that only track MT plus ends, CLASPs display dual bindings to either the plus ends or along the sides of MTs (or MT lattices) in neurons. Functionally, we show that this unique dual MT binding behavior of CLASPs allows them to differentially regulate MT organization and axon growth in different neurons. In regenerating sensory neurons, CLASP mainly bind to MT plus ends and function to support fast axon growth. In contrast, CLASP in developing cortical neurons show increased binding along the side of MTs and act to restrict axon growth. In addition to restricting axon growth, we also provide evidence that CLASPs function to support the development of cortical neuron dendrites. Interestingly, axon guidance cue Slit also functions to repel axons and in the meantime promote dendritic growth. Because CLASP has been placed downstream of Slit to mediate axon repulsion in Drosophila, we hypothesize that CLASPs, with their unique dual MT binding property, may be converging targets of Slit-Robo signaling to regulate mammalian axonal and dendritic development. Thus, the overall goal of this study is to elucidate the role of CLASP in regulation of neuronal morphogenesis in response to extracellular cues during cortical development. To test this hypothesis, we will 1) elucidate the molecular mechanism by which CLASPs regulate MTs to control axon growth and dendritic development, 2) determine the roles of CLASPs in Slitmediated axon repulsion and dendritic growth using in vitro axon guidance assay and cell cultures, and 3) determine the in vivo roles of CLASPs in cortical neuron axon growth/guidance, and dendritic development during cortical development using in utero electroporation. This study will reveal novel molecular mechanisms by which extracellular cues regulate MTs to control neuronal morphogenesis. PUBLIC HEALTH RELEVANCE: Neuronal morphogenesis, including axon growth, guidance, and dendrite growth are key molecular events underlying the formation of the neural circuit during development, or neural repair after injuries. Mistakes in these processes during development are believed to cause many neurodevelopmental disorders. Therefore, our proposed study will not only help us understand how neural circuits form during development, but also provide valuable information of how to promote neural regeneration following injuries.