Mechanisms controlling distinct modes of adult axon growth

 描述（由申请人提供）：影响轴突生长作为在神经系统损伤、损伤或疾病的情况下促进恢复和减轻病理的手段是医疗保健和生物医学研究工作的主要目标。大量努力旨在诱导神经可塑性以增强轴突生长以建立功能适应性连接。然而，这些努力还必须防止而不是诱发适应不良的可塑性，这种平衡需要清楚地了解调节轴突生长的过程。阻碍理解神经可塑性的一个主要因素是，创伤性损伤的神经系统既包含直接损伤的轴突，也包含未损伤的轴突。该项目研究了关于成人神经系统中轴突生长的两种主要形式的机制的长期存在的问题和争议 - 受伤轴突的生长（再生）和未受伤轴突的生长（侧枝发芽 - CS）。我们的目标是客观地确定控制这些过程的内在分子机制的相似或不同程度。这样做将能够识别控制特定模式的基因组，因此可能有针对性地只影响该一种模式，或者可能在模式之间共享，因此有针对性地影响两种模式，并且可以识别出能够调节成人轴突可塑性的全新基因组。 轴突再生和CS在PNS中都相对稳健，并且可以清楚地区分受伤和未受伤的神经元。神经挤压提供了成功的轴突再生的模型。使用幸存的皮节模型（其中单个背根神经节的完整未受伤神经元通过去神经化其皮节边界的皮肤来诱导生长），我们生成了 CS 期间调节的基因的转录组谱。生物信息学分析表明，参与再生和 CS 的基因是高度不同的。使用基因删除转录因子（TF）的小鼠获得的初步数据似乎对每种轴突生长模式都具有特异性，支持了生长模式涉及单独的遗传程序的概念。目标 1 将使用模式特异性 TF 敲除的小鼠，通过轴突生长的行为和组织学评估，彻底检查基因缺失对不同轴突生长模式的影响。这将确定旗舰模式特定 TF 是否确实负责控制该单一模式。 我们有数据证明：1）特定模式转录因子的互斥表达；2）一种生长模式的“调节”似乎会影响另一种模式的功能执行。这是合理的，因为基因表达必须发生巨大的变化，并且可以延迟或阻止这种重大变化的适当协调。目标 2 将通过交替地将不同的模型应用于相同的神经元（即再生然后萌芽或萌芽然后再生）来测试这些模式相互负面影响并涉及相互排斥的遗传程序的假设。这将决定一种模式的执行如何影响另一种模式。 对于这两个目标，与初步数据一致的实验结果将有力地支持这样的概念：确实存在两种不同的生长模式，每种模式都有不同的遗传控制。与初步数据相反的结果可能包括：1）对某些神经群体的影响，但对其他神经群体没有影响，这表明可能仍然存在具有不同遗传控制的不同模式，但这些模式可能不是基于损伤状态，而是基于细胞类型，和/或2）明显的模式特异性遗传控制系统充当促进者，但不是必需的（即，在它们不存在的情况下，过程无论如何都会发生，但要慢得多），这表明不一定存在两种完全不同的神经群体。模式。所有结果都将有助于解决有关成人神经系统轴突生长的概念框架和特定分子控制。

 DESCRIPTION (provided by applicant): Affecting axonal growth as a means to enhance recovery and alleviate pathology in conditions of nervous system injury, insult, or disease is a major goal for the healthcare and biomedical research endeavors. Significant effort is directed at inducing neural plasticity to enhance axonal growth to establish functionally- adaptive connections. However, these efforts must also prevent, and not induce, maladaptive plasticity, a balance which requires a clear understanding of the processes regulating axon growth. A major factor confounding efforts to understand neural plasticity is that the traumatically-injured nervou system contains both directly-injured axons and the NON-injured axons. This project examines the long-standing question and controversy regarding the mechanisms of the two major forms of axon growth in the adult nervous system - growth of injured axons (Regeneration) and that of non-injured axons (Collateral Sprouting - CS). We aim to objectively determine the degree to which the intrinsic molecular mechanisms controlling these processes are similar or different. Doing so will enable identification of sets of genes which control a specific mode and may thus be targeted to affect just that one mode, or may be shared between modes and thus targeted to affect both, and could identify an entire new set of genes capable of regulating adult axonal plasticity. Axonal Regeneration and CS are both relatively robust in the PNS, and the injured and non-injured neurons can be clearly separated. Nerve crush provides a model of successful axon regeneration. Using the spared dermatome model (where intact non-injured neurons of a single dorsal root ganglion are induced to grow by denervating the skin bordering their dermatome) we have generated a transcriptomic profile of genes regulated during CS. Bioinformatic analyses indicate that the genes involved in regeneration and CS are highly distinct. Preliminary data using mice with genetic deletion of transcription factors (TFs) that appear to be specific for each axon growth mode supports the concept that the growth modes involve separate genetic programs. Aim 1 will use mice with mode-specific-TF knockout to thoroughly examine the impact of the gene- deletions on the different modes of axon growth using behavioral and histological assessments of axon growth. This will determine if the flagship mode-specific-TFs are indeed responsible for controlling that single mode. We have data demonstrating 1) a mutually-exclusive expression of the mode-specific TFs and 2) that "conditioning" with one mode of growth appears to influence the functional execution of the other mode. This is rational considering that there must be a large change in which genes are expressed, and proper orchestration of such a significant change could be delayed or prevented. Aim 2 will test the hypothesis that the modes negatively-influence each other and involve mutually-exclusive genetic programs by alternately applying the different models to the same neurons (i.e., regeneration-then-sprouting or sprouting-then-regeneration). This will determine how execution of one mode influences the other. For both Aims, experimental outcomes in accord with preliminary data would strongly support the concept that there are indeed two different growth modes, each with distinct genetic control. Outcomes contrary to preliminary data could include 1) effects on some neural populations but not others, which would suggest that there may still be distinct modes with distinct genetic control, but that these modes may be based not on injury- status, but on cell-type, and/or 2) that the apparent mode-specific genetic control systems act as facilitators but are not necessary (i.e., in their absence the processes occur anyway, but much more slowly), which would suggest that there are not necessarily two fully-distinct modes. All outcomes will serve to address both the conceptual framework and the specific molecular control regarding axon growth in the adult nervous system.