NEURONAL TURNOVER IN ADULT BRAIN USING AMS FOR RETROSPECTIVE BIRTH DATING CELLS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。已知神经发生发生在成年动物大脑的特定区域，但成年人类大脑中神经发生的程度和可比性更难确定，到目前为止基本上是未知的。用于细胞测年的传统方法提供的信息有限，或者不适合人类使用。因此，目前还没有可用的方法来研究人类的细胞周转。我们建议开发一种细胞追溯出生年龄的方法。我们感兴趣的是使用核弹脉冲碳14(C14)测年作为一种测量成人大脑中特定细胞群体的近似年龄的方法。该方法建立在确定同位素C14在基因组DNA中的比例的基础上。C14的测量将使用高灵敏度的加速器质谱仪(AMS)进行。细胞终末分化后，不会再分裂。由于最后一次细胞分裂代表细胞合成DNA的最后时间点，其染色体DNA将反映细胞出生的年龄。传统上，C14相对于其他碳物种的缓慢衰减使其具有多年的时间分辨率，然而，由于20世纪50年代末和60年代初的核试验，大气中C14的水平急剧上升。自那以后，这一水平以指数方式下降，使人们能够在几年的范围内解决C14的差异。因为DNA的C14含量反映了它合成的时间，所以确定染色体DNA的C14含量将使我们能够追溯出生日期细胞，从而建立细胞周转。细胞周转的信息对于理解基本的生物过程至关重要。除了对正常的细胞周转感兴趣外，许多疾病被认为是在新细胞的生成过程中受到影响。有关疾病状态下细胞周转的信息可能为疾病的病理过程提供新的见解，并可能提出新的治疗策略。使用AMS从马脑DNA中测定C14的初步实验已经产生了令人鼓舞的结果。下一步是看看AMS从马的血液、大脑和牙齿中提取的DNA中测定C14的准确性。然后将使用FACS分析来分离特定的细胞群体(这允许分离特定的细胞群体，例如，可以使用神经元特异性标记，如Neun、微管蛋白或Thy1)来分选神经元。一旦这项技术建立起来，我们的目标是转移到人类材料上，并研究特定人类病理中的细胞周转。获得人类身体材料的道德许可已经得到批准。项目更新：我们使用这一策略来确定成人大脑皮层中细胞的年龄，并表明尽管非神经细胞是交换的，但枕叶神经元与个体一样老，支持了出生后神经发生不在这一区域发生的观点。追溯出生年代是一种普遍适用的策略，可以用来测量生理和病理条件下男性的细胞周转。年内发表了两篇论文，目前正在审查第三篇论文。《细胞》杂志的封面上刊登了神经细胞测年的照片。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Neurogenesis is known to occur in specific regions of the adult animal brain, but the extent and comparability of neurogenesis in the adult human brain is much harder to determine, and to date largely unknown. Traditional methods used for dating cells are limited in the information they provide, or are not appropriate for human use. Thus, currently there is no method available to study cellular turnover in man. We propose to develop a method for the retrospective birth dating of cells. We are interested in using bomb pulse carbon-14 (C14) dating as a method for measuring the approximate age of specific populations of cells in the adult human brain. This method is based on establishing the proportion of the isotope C14 in genomic DNA. C14 measurements will be made using the highly sensitive accelerator mass spectrometer (AMS). After a cell has terminally differentiated it does not divide again. Since the last cell division represents the last time point when the cell synthesized DNA, its chromosomal DNA will reflect the age when the cell was born. Traditionally, the slow decay of C14 relative to other carbon species has given it a temporal resolution of many years, however due to nuclear tests in the late 1950s and early 1960s, the level of C14 in the atmosphere has increased dramatically. This level has since dropped off in an exponential fashion, allowing one to resolve C14 differences in the range of years. Because DNA has a C14 content reflective of the time when it was synthesized, establishing the C14 content of chromosomal DNA will enable us to retrospectively birth date cells, and thus establish cellular turnover. Crucial to the understanding of basic biological processes, is information about cellular turnover. As well as having an interest in normal cellular turnover, many diseases are thought to be affected in their generation of new cells. Information about cellular turnover in disease states may provide novel insights into the pathological processes of the disease, and possibly suggest new therapeutic strategies. Preliminary experiments using AMS to date C14 from horse brain DNA have yielded encouraging results. The next step is to look at how accurately AMS dates C14 from DNA extracted from horse blood, brain and teeth. Particular populations of cells will then be isolated using FACS analysis (which allows specific cell populations to be isolated e.g. one can sort for neurons using neuronal specific markers such as NeuN, ¿ tubulin or Thy1). Once this technique has been established we aim to move onto human material, and study cellular turnover in specific human pathologies. Ethical permission to obtain postmortem human material has been granted. Project update: We used this strategy to determine the age of cells in the cortex of the adult human brain, and showed that whereas non-neuronal cells are exchanged, occipital neurons are as old as the individual, supporting the view that postnatal neurogenesis does not take place in this region. Retrospective birth dating is a generally applicable strategy that can be used to measure cell turnover in man under physiological and pathological conditions. Two papers were published during the year and a third is currently under review. The neuronal cell dating work was featured on the cover of Cell.