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）神经元的内在生长能力差是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是阐明再生能力基础上的内在神经元生长机制的理想选择。感觉神经元外周分支和中枢分支的再生能力不同，这意味着对这两个分支的切开在神经元细胞体中会产生不同的反应。促进中枢神经系统神经元生长状态和再生能力的可行性通过惊人的观察得到证明，尽管中枢神经系统环境恶劣，但经历外周突轴切（一种“条件反射损伤”）的DRG神经元能够再生其随后被轴切的中枢神经过程。本研究的目的是利用细胞和分子方法来定义外周轴突切开术后DRG神经元中控制轴突生长的分子开关。首先，一种候选方法将用于确定轴切后逆行转运信号蛋白磷酸化状态的变化，以及可能启动生长状态的刺激诱导转录因子活性状态的变化。其次，微阵列表达谱分析将作为一种无偏倚的方法来识别在外周轴切开术后DRG生长状态早期变化中重要的转录因子。最后，鉴定的转录因子的功能意义将在初级神经元培养实验中进行测试，使用Sindbis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。最后，有希望的转录因子将在体内脊髓损伤模型中进行测试，使用腺病毒介导的基因转移来证明这些候选因子与脊髓损伤后轴突再生的相关性。申请人的长期目标是了解促进轴突再生的基本分子和细胞机制，这对于设计针对CNS（中风、脊髓损伤、慢性进行性多发性硬化症）和PNS（周围神经病变）疾病的新疗法至关重要，这些疾病目前继发于轴突损伤，预后不良。当前的目标是获得在神经生物学最先进的技术应用到临床相关问题的培训。候选人的职业发展规划，博士的指导；Tessier Lavigne和Mobley的临床培训计划，以及斯坦福大学神经内科的承诺，将使这一目标得以实现。

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.