The Regulatory Role of the Limk1/Cofilin Signaling Pathway in Spinal Motor Neuron

描述(申请人提供)：我们研究的长期目标是了解在发育过程中产生神经网络的机制，然后将这些知识应用于再生损伤或神经退行性疾病后丢失的神经回路。朝着这个目标迈出的重要一步是识别允许轴突导航到它们的突触目标的分子线索。然而，尽管许多将轴突定向投射到特定方向的外部因素都得到了很好的描述，但控制轴突的机制(S) 轴突生长的速度仍未确定。我们最近的研究揭示了这个问题；我们发现cofilin及其负调控因子Lim kinase1(LIMK1)控制着脊髓中一群后连合中间神经元的生长速度。因此，外在信号也指示轴突以特定的速度生长。这样的“时间”线索有可能控制解释方向信息的速度和/或时间，是确保轴突回路与发育中的胚胎的其余部分协调发展的重要机制。此外，这一发现提出了一种可能性，即在发育过程中控制轴突生长速度的信号通路可以被操纵，以在再生或神经保护的背景下加速轴突生长，从而加快人类患者神经回路再生的漫长过程。为了实现这一目标，我们将确定调节轴突生长速度的信号是否对在发育过程中建立脊髓运动神经回路以及在测试干细胞来源的MN功能的胚胎模型中是否重要。在这项提议的目标1中，我们将检验这一假设，即在发育过程中，cofilin和LIMK1激活状态之间的平衡控制着内源性运动轴突延伸的速度。我们将利用鸡胚胎体内电穿孔和小鼠功能丧失遗传学来提高发育中胚胎中cofilin的活性水平，并评估cofilin活性升高对 脊髓运动神经轴突延伸。在这项提议的目标2中，我们将检验这样一个假设，即在胚胎干细胞(ESC)来源的运动神经元(MN)中，提高cofilin水平会导致它们更快地产生功能运动神经回路。我们将使用慢病毒转染法在ESC来源的MN中内在地增加Cofilin的活性，然后评估运动轴突延展的速度以及它们在培养中形成功能神经回路的能力。胚胎干细胞来源的MN是一种很有前途的替代损伤或疾病后受损或丢失的MN的候选细胞。当与其他轴突再生疗法配对时，从胚胎干细胞来源的MNS本质上加速轴突延伸的能力可能会导致更有效的恢复时间。

DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the mechanisms that generate neuronal networks during development and then apply this knowledge to regenerating the neural circuitry lost after injury or neurodegenerative diseases. An important step towards this goal is to identify the molecular cues that permit axons to navigate towards their synaptic targets. However, although many of the extrinsic factors that orient axons to project in a particular direction are well-described, the mechanism(s) that control the rate of axon outgrowth remain unresolved. Our recent studies have shed light on this issue; we showed that cofilin, and its negative regulator Lim kinase 1 (Limk1), control the speed of growth for a population of dorsal commissural interneurons in the spinal cord. Thus, axons are also instructed by extrinsic signals to grow at a particular rate. Such "temporal" cues have the potential to control the rate and/or time at which directional information is interpreted and are a important mechanism that ensures that axonal circuits develop in concert with the rest of the developing embryo. Moreover, this finding raises the possibility that the signaling pathways that control the rate of axon growth during development could be manipulated to accelerate axonal outgrowth in a regenerative or neuroprotective context and thereby speed up the lengthy process of regrowing neural circuits in a human patient. To work towards this goal, we will determine whether the signals that regulate the rate of axon growth are important for the establishment of spinal motor circuits during development and in an embryonic model that tests the functionality of stem-cell derived MNs. In Aim 1 of this proposal we will test the hypothesis that the balance between the activation states of cofilin and Limk1 controls the rate of endogenous motor axon extension during development. We will utilize in ovo electroporation of chicken embryos and mouse loss-of-function genetics to increase the activity levels of cofilin in developing embryos and assess the effect of elevated cofilin activity on the rate and trajectory of spinal motor axon extension. In Aim 2 of this proposal we will test the hypothesis that elevating levels of cofilin in embryonic stem cell (ESC)-derived motor neurons (MN) results in their generating functional motor circuits more rapidly. We will use lentivirus transfection methods to intrinsically increase cofilin activity in ESC-derived MNs and then will assess the rate of motor axon extension as well as their ability to form functional neural circuits in culture. ESC-derived MNs are a promising candidate to replacing MNs that are damaged or lost after injury or disease. The ability to intrinsically accelerate axon extension from ESC-derived MNs may lead to more efficient recovery times when paired with other axon regeneration therapies.