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

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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. 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.