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

描述（由申请人提供）：我们研究的长期目标是了解在发育过程中产生神经元网络的机制，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。实现这一目标的一个重要步骤是确定允许轴突向其突触靶点导航的分子线索。然而，尽管许多定向轴突以在特定方向投射的外在因素被很好地描述，但是控制轴突投射的机制仍然不清楚。 轴突长出的速度仍然没有解决。我们最近的研究揭示了这个问题;我们表明，cofilin及其负调节因子Lim激酶1（Limk 1）控制脊髓背连合中间神经元群体的生长速度。因此，轴突也被外在信号指示以特定的速率生长。这种“时间”线索有可能控制方向信息被解释的速率和/或时间，并且是确保轴突回路与发育中的胚胎的其余部分一起发育的重要机制。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率的信号通路可以被操纵，以加速再生或神经保护背景下的轴突生长，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率的信号是否对发育期间和测试干细胞衍生MN功能的胚胎模型中脊髓运动回路的建立很重要。在本提案的目的1中，我们将检验以下假设：在发育过程中，cofilin和Limk 1的激活状态之间的平衡控制内源性运动轴突延伸的速率。我们将利用鸡胚胎的卵内电穿孔和小鼠功能丧失遗传学来增加发育中胚胎中cofilin的活性水平，并评估升高的cofilin活性对胚胎发育的速率和轨迹的影响。 脊髓运动轴突延伸。在本提案的目标2中，我们将检验以下假设：升高胚胎干细胞（ESC）衍生的运动神经元（MN）中的cofilin水平导致其更快地产生功能性运动回路。我们将使用慢病毒转染方法来内在地增加ESC衍生的MN中的cofilin活性，然后将评估运动轴突延伸的速率以及它们在培养物中形成功能性神经回路的能力。胚胎干细胞来源的骨髓细胞是一个有前途的候选人，以取代受损或丢失后的损伤或疾病的骨髓细胞。当与其他轴突再生疗法配对时，从ESC衍生的MN内在地加速轴突延伸的能力可能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.