Mechanisms controlling distinct modes of adult axon growth

 描述(由申请人提供)：在神经系统损伤、侮辱或疾病的情况下，影响轴突生长作为促进恢复和减轻病理的一种手段是医疗保健和生物医学研究工作的主要目标。在诱导神经可塑性以促进轴突生长以建立功能适应性连接方面做出了重大努力。然而，这些努力也必须防止而不是诱导适应不良的可塑性，这种平衡需要清楚地了解调节轴突生长的过程。一个主要的因素混淆了理解神经可塑性的努力，那就是创伤损伤的神经系统既包含直接损伤的轴突，也包含非损伤的轴突。本研究旨在探讨成人神经系统轴突生长的两种主要形式--损伤轴突生长(再生)和非损伤轴突生长(侧枝萌发-CS)的机制。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一组基因，因此可能被定位为仅影响一种模式，或者可能在两种模式之间共享并因此被定位为影响两种模式，并可能识别能够调节成人轴突可塑性的一整套新基因。三叉神经节的轴突再生和CS都比较健壮，损伤神经元和非损伤神经元可以清晰地区分开来。神经挤压提供了一个成功再生轴突的模型。使用备用皮肤刀模型(在该模型中，单个背根神经节中未受损伤的完整神经元通过去神经支配其皮肤刀边缘的皮肤来诱导生长)，我们已经生成了CS过程中调节的基因的转录图谱。生物信息学分析表明，参与再生和CS的基因是高度不同的。使用转录因子(TF)基因缺失的小鼠的初步数据支持这一概念，即生长模式涉及不同的遗传程序。目的1将使用具有模式特异性Tf基因敲除的小鼠，通过对轴突生长的行为和组织学评估，彻底检查基因缺失对轴突生长的不同模式的影响。这将确定旗舰模式特定的TF是否真的负责控制该单一模式。我们有数据表明，1)特定于模式的TF的互斥表达，以及2)一种生长模式的“限制”似乎影响另一种模式的功能执行。考虑到必须有一个表达基因的大变化，而这种重大变化的适当协调可能会被推迟或阻止，这是合理的。目的2将通过交替地将不同的模式应用于相同的神经元(即，再生-然后萌发或萌芽-然后再生)来检验这两种模式相互负面影响并涉及互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一个概念，即确实存在两种不同的增长模式，每种模式都有不同的基因控制。与初步数据相反的结果可能包括1)对一些神经群体的影响，但不包括其他，这表明可能仍然存在具有不同基因控制的不同模式，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)表面模式特有的基因控制系统起到促进器的作用，但不是必需的(即，在没有它们的情况下，过程无论如何都会发生，但速度要慢得多)，这表明不一定存在两种完全不同的模式。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问题。

 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.