Gene expression underlying serotonin axon regrowth in the adult mammalian brain

成年哺乳动物大脑中血清素轴突再生的基因表达

• 批准号: 9765426
• 负责人: Susan M. Dymecki
• 金额: $ 20.74万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9765426
• 关键词: Acute Brain Injuries; Address; Adult; Aftercare; Amphetamines; Animals; Astrocytes; Axon; Bacterial Artificial Chromosomes; Biological Models; Brain; Caliber; Cells; Chemicals; Cicatrix; Cognition Disorders; Cytosine; Data; Data Set; Development; Dioxygenases; Dopamine; Dorsal; Dose; Dynein ATPase; Enzymes; Expression Profiling; Failure; Female; Fiber; Gene Expression; Gene Expression Profile; Genes; Harvest; Histones; Homeostasis; Image; Immediate-Early Genes; Immunohistochemistry; Impaired cognition; In Situ Hybridization; Inflammation; Injury; JUNB gene; Label; Left; Lesion; Measurement; Measures; Molecular; Mood Disorders; Motor; Mus; Myelin; Natural regeneration; Neocortex; Neurons; Neurotransmitters; Paralysed; Pathway interactions; Peripheral; Population; Proteins; Recovery; Recovery of Function; Research Personnel; Retrograde Degeneration; Rodent; Running; Saline; Sampling; Scanning; Screening Result; Serotonin; Signal Transduction; Site; Somatosensory Cortex; Spinal; Stroke; Suggestion; TPH2; Time; Tissue-Specific Gene Expression; Tissues; Transgenic Organisms; Transmembrane Domain; Traumatic Brain Injury; Ventral Tegmental Area; axon growth; axon injury; density; differential expression; dopaminergic neuron; dorsal raphe nucleus; experimental study; genome-wide; immunoreactivity; in vivo; injured; interest; male; neocortical; novel therapeutics; planar cell polarity; receptor; response; serotonin transporter; therapy development; transcriptome sequencing; two-photon

Project Summary It is widely believed that damaged axons in the adult mammalian brain have little capacity to regrow, thereby impeding functional recovery after injury. Serotonin neurons appear to be a notable exception. We have produced time-lapse images of serotonin axons in the neocortex of the adult mouse. Serotonin axons undergo massive retrograde degeneration following amphetamine treatment and show subsequent slow, long-distance regrowth. A stab injury that transects serotonin axons running in the neocortex is followed by local regression of cut serotonin axons and by regrowth from cut ends that projects across the stab rift zone. By contrast, dopamine fibers originating in neurons of the ventral tegmental area (VTA) are also small-diameter unmyelinated axons, yet they are not damaged by amphetamine treatment and they fail to regrow after stab transection. What are the molecular specializations that allow serotonin axons to regrow while other injured axons in the brain fail to do so? To begin to address this question we have harvested pools of serotonin neurons from the dorsal raphe of adult mice in order to measure gene expression using genome-wide RNA sequencing (RNA-Seq). This was done 1 week after the end of either amphetamine or saline treatment. Our preliminary data have revealed a set of genes that are significantly upregulated in serotonin neurons during amphetamine-evoked axon regrowth. Here, we propose to complete and extend this screen as a first step to define the molecular requirements for serotonin axon regrowth. Aim 1. What genes are differentially expressed in serotonin neurons during amphetamine-evoked regrowth of their axons in the neocortex? Our preliminary data reveal ~50 upregulated and ~60 downregulated genes measured ~1 week after the end of amphetamine treatment as compared with saline-treated-controls (using a false-discovery rate of p<.05). To complete this data set, we shall extend these RNA-Seq measurements using a time course of amphetamine and saline-treated mice at t= 1 day, 1 week and 18 weeks. There will be 8 animals per group (4 male, 4 female), and we shall validate genes of interest with in situ hybridization and immunohistochemistry. We shall also compare the gene expression changes in response to amphetamine challenge in serotonin neurons with those changes produced in dopamine neurons of the VTA. Aim 2. What genes are differentially expressed in serotonin neurons during regrowth of serotonin axons following a neocortical stab lesion? In contrast to amphetamine lesions, stab lesions produce only local axonal regression of serotonin and dopamine axons. Stab lesions form a glial scar, as occurs in stroke and other forms of acute brain injury. We shall compare dorsal raphe serotonin neuron and VTA dopamine neuron expression profiles in response to stab and amphetamine injury using the same post-injury time points. The results of these two complementary injuries in regenerating and non-regenerating neurons will inform later manipulative experiments to modulate serotonin axon regrowth and, ultimately, confer the capacity to regrow on non-serotonergic neurons in the brain.

项目概要 人们普遍认为，成年哺乳动物大脑中受损的轴突几乎没有再生能力，因此 妨碍受伤后的功能恢复。血清素神经元似乎是一个值得注意的例外。我们有 制作了成年小鼠新皮质中血清素轴突的延时图像。血清素轴突经历 安非他明治疗后出现大规模逆行性变性，并随后表现出缓慢、长距离的退化 再生。刺伤横断在新皮质中运行的血清素轴突，随后出现局部退化 被切割的血清素轴突，以及从穿过刺裂裂区的切割端重新生长。相比之下， 起源于腹侧被盖区 (VTA) 神经元的多巴胺纤维也是小直径无髓鞘的 轴突，但它们不会因安非他明治疗而受损，并且在刺伤横断后无法再生。什么 是允许血清素轴突重新生长而大脑中其他受损轴突失效的分子特化 这样做吗？为了开始解决这个问题，我们从中缝背侧收集了血清素神经元池 使用全基因组 RNA 测序 (RNA-Seq) 测量成年小鼠的基因表达。这是 安非他明或盐水治疗结束后 1 周进行。我们的初步数据揭示了一组 在苯丙胺诱发的轴突再生过程中，血清素神经元中显着上调的基因。 在这里，我们建议完成并扩展此屏幕，作为定义分子要求的第一步 血清素轴突再生。目标 1. 哪些基因在血清素神经元中差异表达 安非他命引起新皮质轴突的再生？我们的初步数据显示~50 安非他明治疗结束后约 1 周测量到上调和约 60 个下调基因，如下所示 与生理盐水处理的对照相比（使用 p<.05 的错误发现率）。为了完成这个数据集，我们将 使用安非他明和盐水处理的小鼠在 t = 1 天的时间过程来扩展这些 RNA-Seq 测量， 1周和18周。每组有 8 只动物（4 只雄性，4 只雌性），我们将验证 对原位杂交和免疫组织化学感兴趣。我们还将比较基因表达 血清素神经元对安非他明刺激的反应发生变化以及多巴胺产生的变化 VTA 的神经元。目标 2. 血清素神经元再生过程中哪些基因差异表达 新皮质刺伤后血清素轴突的变化？与安非他明损伤相反，刺伤损伤 仅产生血清素和多巴胺轴突的局部轴突回归。刺伤损伤形成神经胶质疤痕，如发生的那样 中风和其他形式的急性脑损伤。我们将比较中缝背侧血清素神经元和 VTA 使用相同的损伤后对刺伤和安非他明损伤作出反应的多巴胺神经元表达谱 时间点。再生和非再生神经元的这两种互补损伤的结果将 为后来的操纵实验提供信息，以调节血清素轴突的再生，并最终赋予能力 在大脑中的非血清素能神经元上重新生长。