CYTOSKELETAL GENES DURING AXONAL REGENERATION

这个项目的长期目标是了解分子 哺乳动物神经元细胞骨架基因表达的调控机制 轴突损伤后，并确定 外周神经元和中枢神经元的损伤反应不同。大鼠 背根神经节(DRG)神经元在 特定微管蛋白mRNAs的稳态水平和快速下调 坐骨神经切断后神经细丝(NFm RNA)的表达。相比之下，中枢神经系统神经元 如视网膜神经节神经元(RGN)不能上调微管蛋白mRNA 损伤后NFmRNAs水平下降较慢。目前，它是 不知道这种差异是否是由于基因组的差异 转录水平的激活或与转录后的差异有关 影响mRNA稳定性的转录机制。的具体目标 这项提议是为了检验以下两个假设。第一个： 轴突切断对细胞骨架基因转录的不同影响 外周(DRG)和中枢(RGN)神经元。我们将对此进行测试 利用内含子特异性DNA探针进行原位杂交的假说 为了检测核因子-M的初级、未剪接的RNA转录本(“前-mRNAs”)， 背根神经节和视网膜神经节细胞核中的βIII和α1微管蛋白基因的组织学研究 切断术后不同时间取材。我们还将直接评估 正常人细胞核中细胞骨架mRNAs的转录水平 并采用核流出法切断背根神经节神经元。第二名： 外周和中枢神经元特异性细胞骨架mRNAs的稳定性 不同程度地受轴突切断的影响。我们将研究不同的 视网膜节切断后主要细胞骨架mRNAs的周转(半衰期) 在活体中使用3/H-尿苷脉冲追逐范例作为DRG神经元，并使用 一种转录-阻断范式，涉及微量注射 放线菌素D或DRB(5，6-dichloro-1-beta-ribofuranosylbenzimidazole)和 随着时间的推移监测随后的mRNAs丢失。最后，为了检查 蛋白质合成和多聚体缔合在细胞周转中的作用 在轴突切断的神经元中，我们将显微注射不同的mRNAs 翻译抑制剂(放线菌胺或嘌呤霉素)进入背根节或眼部 在轴突切开术之前或之后不久，并确定我们是否可以 预防神经切断引起的特定细胞骨架水平的变化 MRNAs。这项提议与健康相关的是，信息 将大大增强我们对分子的理解 哺乳动物神经元有效再生的基础，并可能 使分子策略的发展增强再生能力 在未来损伤后不能再生的神经元。

The long-term objectives of this project are to understand the molecular mechanisms regulating cytoskeletal gene expression in mammalian neurons after axonal injury, and to determine the molecular locus at which peripheral and central neurons differ in their injury response. Rat dorsal root ganglion (DRG) neurons exhibit robust increases in their steady-state levels of specific tubulin mRNAs and rapid downregulation of neurofilament (NF) mRNA levels after axotomy. In contrast, CNS neurons such as retinal ganglion neurons (RGN) fail to upregulate tubulin mRNA levels after injury and decrease NF mRNAs more slowly. Currently, it is not known whether such differences are due to disparities in genomic activation at the level of transcription or to differences in post- transcriptional mechanisms affecting mRNA stability. The specific aims of this proposal are to test the following 2 hypotheses. First: The transcription of cytoskeletal genes is differentially affected by axotomy in peripheral (DRG) and central (RGN) neurons. We will test this hypothesis by using intron-specific DNA probes for in situ hybridization to detect primary, unspliced RNA transcripts ("pre-mRNAs") of NF-M, betaIII and alpha1-tubulin genes in DRG and RGN nuclei in histological sections at various times after axotomy. We will also directly assess the transcription rate of cytoskeletal mRNAs in isolated nuclei from normal and axotomized DRG neurons using nuclear run-off methods. Second: The stability of specific cytoskeletal mRNAs in peripheral and central neurons is differentially affected by axotomy. We will examine differences in the turnover (half-life) of major cytoskeletal mRNAs in axotomized RGN as well as DRG neurons in vivo using a 3/H-uridine pulse-chase paradigm, and using a transcriptional-blockade paradigm involving microinjection of actinomycin D or DRB(5,6-dichloro-1-beta-ribofuranosylbenzimidazole) and monitoring the subsequent loss of mRNAs with time. Finally, to examine the role of protein synthesis and polysome-association in the turnover of cytoskeletal mRNAs in axotomized neurons, we will microinject different translation inhibitors (cycloheximide or puromycin) into the DRG or eye immediately before, or shortly after, axotomy and determine if we can prevent axotomy-induced changes in the levels of specific cytoskeletal mRNAs. The health-relatedness of this proposal is that the information gained will significantly enhance our understanding of the molecular underpinnings of effective regeneration in mammalian neurons, and may enable the development of molecular strategies to enhance regeneration in neurons which fail to regenerate following injury in the future.