CYTOSKELETAL GENES DURING AXONAL REGENERATION

轴突再生过程中的细胞骨架基因

• 批准号: 2264209
• 负责人: Monica Oblinger
• 金额: $ 21.87万
• 依托单位: ROSALIND FRANKLIN UNIV OF MEDICINE & SCI
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2264209
• 关键词: axon reaction; cycloheximide; cytoskeletal proteins; cytoskeleton; denervation; gene expression; genetic regulation; genetic transcription; in situ hybridization; injury; laboratory rat; messenger RNA; nervous system regeneration; neurofilament proteins; northern blottings; nuclear runoff assay; nucleic acid probes; polymerase chain reaction; protein biosynthesis; puromycin; retinal ganglion; spinal ganglion; tubulin

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.

该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，并确定分子位点， 外周和中枢神经元的损伤反应不同。 大鼠 背根神经节（DRG）神经元在其活动中表现出强烈的增加。 特异性微管蛋白mRNA的稳态水平和 神经丝蛋白（NF）mRNA水平。 相比之下，CNS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NF mRNA水平降低更慢。 目前正在 尚不清楚这种差异是否是由于基因组差异造成的。 在转录水平上的激活或转录后的差异， 影响mRNA稳定性的转录机制。 的具体目标 本提案旨在检验以下两个假设。 First：The 细胞骨架基因的转录受到轴突切断的不同影响 在外周（DRG）和中枢（RGN）神经元中。 我们将测试这个 内含子特异性DNA探针原位杂交假说 为了检测NF-M的初级未剪接RNA转录物（“前mRNA”）， 组织学中DRG和RGN核中的β III和α 1-微管蛋白基因 切片后的不同时间轴切。 我们还将直接评估 正常人分离细胞核中细胞骨架mRNA的转录率 和轴突切断DRG神经元使用核径流方法。 第二：The 外周和中枢神经元中特异性细胞骨架mRNA的稳定性 受到轴突切断术的不同影响。 我们将研究不同的 轴突切断的RGN中主要细胞骨架mRNA的周转（半衰期）也 作为DRG神经元在体内使用3/H-尿苷脉冲追踪范例，并使用 一种转录阻断范式，涉及显微注射 放线菌素D或DRB（5，6-二氯-1-β-呋喃核糖基苯并咪唑），和 监测mRNA随时间的后续损失。 最后，检查 蛋白质合成和多核糖体联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 翻译抑制剂（放线菌酮或嘌呤霉素）进入DRG或眼睛 在轴突切断术之前或之后不久， 防止轴突切断术诱导的特定细胞骨架水平的变化 mRNA。 这项建议的健康相关性在于， 将大大增强我们对分子生物学的理解。 哺乳动物神经元有效再生的基础， 使分子策略的发展，以提高再生， 这些神经元在未来受伤后无法再生。