CYTOSKELETAL GENES DURING AXONAL REGENERATION

轴突再生过程中的细胞骨架基因

• 批准号: 2264211
• 负责人: Monica Oblinger
• 金额: $ 23.43万
• 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 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2264211
• 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 调节哺乳动物神经元细胞骨架基因表达的机制 轴突损伤后，并确定其分子位点 周围神经元和中枢神经元的损伤反应不同。 鼠 背根神经节（DRG）神经元表现出其 特定微管蛋白 mRNA 的稳态水平和快速下调 轴索切除术后神经丝 (NF) mRNA 水平。 相比之下，中枢神经系统神经元 例如视网膜神经节神经元 (RGN) 无法上调微管蛋白 mRNA 损伤后的水平并更缓慢地减少 NF mRNA。 目前，它是 不知道这种差异是否是由于基因组差异造成的 转录水平的激活或后转录水平的差异 影响 mRNA 稳定性的转录机制。 具体目标 该提案旨在检验以下两个假设。 第一： 轴索切断术对细胞骨架基因的转录有不同的影响 外周（DRG）和中枢（RGN）神经元。 我们将测试这个 通过使用内含子特异性 DNA 探针进行原位杂交的假设 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 betaIII 和 alpha1-微管蛋白基因 轴切术后不同时间的切片。 我们还将直接评估 正常分离细胞核中细胞骨架 mRNA 的转录率 并使用核径流方法对 DRG 神经元进行轴切。 第二： 外周和中枢神经元中特定细胞骨架 mRNA 的稳定性 轴索切断术的影响不同。 我们将研究以下方面的差异 轴突 RGN 中主要细胞骨架 mRNA 的周转（半衰期） 使用 3/H-尿苷脉冲追踪范例作为体内 DRG 神经元，并使用 涉及显微注射的转录阻断范例 放线菌素 D 或 DRB（5,6-二氯-1-β-呋喃核糖基苯并咪唑）和 随着时间的推移监测随后的 mRNA 损失。 最后，要检查 蛋白质合成和多核糖体缔合在周转中的作用 轴突神经元中的细胞骨架 mRNA，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 在轴突切除术之前或之后不久，确定我们是否可以 防止轴切术引起的特定细胞骨架水平的变化 mRNA。 该提案与健康的相关性在于信息 所获得的成果将显着增强我们对分子的理解 哺乳动物神经元有效再生的基础，并且可能 促进分子策略的发展以增强再生 未来受伤后无法再生的神经元。

项目成果

Cytotypic differences in the protein composition of the axonally transported cytoskeleton in mammalian neurons.

哺乳动物神经元中轴突运输的细胞骨架的蛋白质组成的细胞型差异。

• DOI: 10.1523/jneurosci.07-02-00453.1987
• 发表时间: 1987
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Oblinger,MM;Brady,ST;McQuarrie,IG;Lasek,RJ
• 通讯作者: Lasek,RJ

A comparison of peripheral and central axotomy effects on neurofilament and tubulin gene expression in rat dorsal root ganglion neurons.

外周和中央轴索切断术对大鼠背根神经节神经元神经丝和微管蛋白基因表达的影响的比较。

• DOI: 10.1523/jneurosci.10-07-02215.1990
• 发表时间: 1990
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Wong,J;Oblinger,MM
• 通讯作者: Oblinger,MM

Comparison of changes in beta-tubulin and NF gene expression in rat DRG neurons under regeneration-permissive and regeneration-prohibitive conditions.

再生允许和禁止再生条件下大鼠 DRG 神经元 β-微管蛋白和 NF 基因表达变化的比较。

• DOI: 10.1016/0006-8993(94)91238-6
• 发表时间: 1994
• 期刊: Brain research
• 影响因子: 2.9
• 作者: Jiang,YQ;Pickett,J;Oblinger,MM
• 通讯作者: Oblinger,MM

Coordinate regulation of tubulin and microtubule associated protein genes during development of hamster brain.

仓鼠大脑发育过程中微管蛋白和微管相关蛋白基因的协调调节。

• DOI: 10.1016/0165-3806(94)90212-7
• 发表时间: 1994
• 期刊: Brain research. Developmental brain research
• 作者: Oblinger,MM;Kost,SA
• 通讯作者: Kost,SA

Biochemical composition and dynamics of the axonal cytoskeleton in the corticospinal system of the adult hamster.

成年仓鼠皮质脊髓系统轴突细胞骨架的生化组成和动力学。

• DOI: 10.1007/bf01001353
• 发表时间: 1988
• 期刊: Metabolic brain disease
• 影响因子: 3.6
• 作者: Oblinger,MM