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.

项目摘要 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， 阻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层中5-羟色胺轴突的延时图像。血清素轴突经历 安非他明治疗后出现大规模退行性变，并显示随后的缓慢，长距离 再生刺伤切断了新皮层中的5-羟色胺轴突， 切断的5-羟色胺轴突和从切断的末端重新生长，穿过刺裂区。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，但他们没有被安非他明治疗损坏，他们不能再生后刺伤横断。什么 是一种分子特化，它允许血清素轴突再生，而大脑中其他受损的轴突则会衰竭， 这样做？为了开始解决这个问题，我们从中缝背核收集了大量的5-羟色胺神经元 为了使用全基因组RNA测序（RNA-Seq）测量基因表达，这是 在安非他明或盐水治疗结束后1周进行。我们的初步数据显示 这些基因在安非他明诱发的轴突再生过程中在5-羟色胺神经元中显著上调。 在这里，我们建议完成和扩展这个屏幕作为第一步，以确定分子的要求， 血清素轴突再生目标1.哪些基因在5-羟色胺神经元中的差异表达 安非他明诱发的轴突在新皮层的再生初步数据显示， 在安非他明治疗结束后约1周， 与盐水处理的对照相比（使用p<0.05的错误发现率）。为了完成这个数据集，我们将 在t= 1天时使用安非他明和盐水处理的小鼠的时间过程来扩展这些RNA-Seq测量， 1周和18周。每组将有8只动物（4只雄性，4只雌性），我们将验证 感兴趣的是原位杂交和免疫组织化学。我们还将比较基因表达 5-羟色胺神经元对苯丙胺刺激的反应变化与多巴胺产生的变化 VTA的神经元。目标2.再生期间血清素神经元中哪些基因差异表达 新皮层刺伤后的5-羟色胺轴突与安非他明损伤相反， 仅产生局部的5-羟色胺和多巴胺轴突的轴突退化。刺伤会形成胶质瘢痕， 中风和其他形式的急性脑损伤。我们将比较中缝背核5-羟色胺神经元和腹侧被盖区 多巴胺神经元表达谱对刺伤和安非他明损伤的反应， 时间点。再生和非再生神经元中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5-羟色胺轴突的再生，并最终赋予 在大脑中的非肾上腺素能神经元上再生。