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)神经元内在生长能力差是导致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构成再生能力的内在神经生长机制。感觉神经元外周分支和中枢分支的不同再生能力表明，这两个分支的切断会导致神经元胞体产生不同的反应。尽管中枢神经系统环境恶劣，但接受外周突起切断(一种“条件性损伤”)的DRG神经元能够再生随后切断的中枢突起，这证明了促进CNS神经元生长状态和再生能力的可行性。这项建议的目的是利用细胞和分子方法来定义分子开关，该开关控制周围神经切断后DRG神经元的轴突生长。首先，一个候选的方法将被用来确定轴突切断后逆行运输的信号蛋白的磷酸化状态的变化，以及可能启动生长状态的刺激诱导的转录因子的活性状态的变化。其次，微阵列表达谱分析将被用作一种无偏见的方法，以确定在周围神经切断后DRG生长状态早期变化中重要的转录因子。最后，利用Sindbis病毒介导的过度表达分析和慢病毒介导的干扰RNA(RNAi)敲除，将在原代神经元培养实验中测试所确定的转录因子的功能意义。最后，有希望的转录因子将在在体脊髓损伤模型中用腺病毒介导的基因转移进行测试，以证明这些候选转录因子与脊髓损伤后轴突再生的相关性。申请者的长期目标是了解促进轴突再生的基本分子和细胞机制，这对于设计新的治疗中枢神经系统(中风、脊髓损伤、慢性进行性多发性硬化症)和周围神经病(PNS)的疾病至关重要，目前这些疾病继发于轴突损伤，预后较差。近期的目标是获得神经生物学中最先进的技术应用于临床相关问题的培训。候选人的职业发展计划、Tessier Lavigne博士和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.