The Intrinsic Neuronal Growth State and CNS Regeneration

描述（由申请人提供）：成人中枢神经系统（CNS）神经元固有生长能力较差是大脑和脊髓轴突再生失败的主要原因。背根神经节（DRG）中的初级感觉神经元非常适合于阐明再生能力背后的内在神经元生长机制。感觉神经元的外周和中央分支的不同再生能力意味着两个分支的轴突切断导致神经元细胞体中的不同反应。增强CNS神经元的生长状态和再生能力的可行性通过以下惊人的观察来证明：尽管CNS环境恶劣，但经历外周突轴突切断术（“条件性损伤”）的DRG神经元能够再生其随后轴突切断的中枢突。本研究的目的是利用细胞和分子的方法来确定在已经经历外周轴突切断的DRG神经元中控制轴突生长的分子开关。首先，候选人的方法将被用来确定两个变化后，轴突损伤后的逆行运输的信号蛋白的磷酸化状态，并确定刺激诱导的转录因子的活性状态的变化，可能会启动的生长状态。其次，微阵列表达谱分析将被用作一种无偏的方法，以确定转录因子，是重要的外周轴突切断后的DRG生长状态的早期变化。最后，将使用辛德毕斯病毒介导的过表达分析和慢病毒介导的干扰RNA（RNAi）敲低递送在原代神经元培养测定中测试所鉴定的转录因子的功能意义。最后，有前途的转录因子将在体内脊髓损伤模型中使用腺病毒介导的基因转移进行测试，以证明这些候选物与脊髓损伤后轴突再生的相关性。申请人的长期目标是理解促进轴突再生的基本分子和细胞机制，这对于设计用于CNS（中风、脊髓损伤、慢性进行性多发性硬化）和PNS（周围神经病）中的疾病的新疗法是至关重要的，这些疾病目前具有继发于轴突损伤的不良预后。近期目标是获得在神经生物学中应用最先进技术解决临床相关问题的培训。候选人的职业发展计划，Tessier Lavigne和Mobley博士的指导，临床培训计划以及斯坦福大学神经病学系的承诺将允许实现这一目标。

DESCRIPTION (provided by applicant): The poor intrinsic growth capacity of adult central nervous system (CNS) neurons is a major contributor to failed axon regeneration in the brain and spinal cord. Primary sensory neurons in the dorsal root ganglia (DRG) are ideally suited to elucidate the intrinsic neuronal growth mechanisms that underlie regenerative ability. The differential regenerative capacity of the peripheral and central branches of sensory neurons implies that axotomies of the two branches result in different responses in the neuronal cell body. The feasibility of boosting the growth state and regenerative ability of CNS neurons is demonstrated by the striking observation that DRG neurons that have undergone peripheral process axotomy (a "conditioning lesion") are able to regenerate their subsequently axotomized central process despite a hostile CNS environment. The goal of this proposal is to utilize cellular and molecular approaches to define the molecular switch that controls axon growth in DRG neurons that have undergone peripheral axotomy. First, a candidate approach will be used to identify both changes in the phosphorlyation state of retrogradely transported signaling proteins after axotomy and identify changes in the activity state of stimulus-induced transcription factors that may initiate the growth state. Second, microarray expression profile analysis will be used as an unbiased approach to identify transcription factors that are important in early changes in the DRG growth state after peripheral axotomy. Finally, the functional significance of the transcription factors identified will be tested in a primary neuronal culture assay using Sindbis virus-mediated over-expression analysis and Lentivirus-mediated delivery of interfering RNA (RNAi) knockdown. Finally, promising transcription factors will be tested in an in-vivo spinal cord lesion model using adenovirus-mediated gene transfer to demonstrate the relevance of these candidates to axonal regeneration after spinal cord injury. The long term goal of the applicant is to understand basic molecular and cellular mechanisms that promote axon regeneration, which is critical to devising new therapies for diseases in both the CNS (stroke, spinal cord injury, chronic progressive multiple sclerosis) and PNS (peripheral neuropathies) that currently carry poor prognosis secondary to axonal damage. The immediate goal is to obtain training in the application of state-of-the-art techniques in neurobiology to clinically relevant problems. The candidate's career development plan, guidance from Drs. Tessier Lavigne and Mobley, clinical training plan, and commitment from the Stanford Department of Neurology, will allow the realization of this goal.