METHOD FOR INVESTIGATING CHROMOSOMES IN HUMAN NEURONS

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Nondividing human neurons have not been accessible to analysis by conventional cytogenetic techniques which rely upon the cycling of a cell into mitosis in order to visualize the chromosomes. Premature chromosome condensation (PCC) methods, based on cell fusion between mitotic and nonmitotic cell populations, do not require such cycling. The PCC technique will be developed and standardized on human neural tissue to permit one to see, for the first time, the chromosomes of nontransformed human neurons. Once standardized, the PCC-neuron technique will provide a valuable tool for the examination of chromosomal changes in neurons isolated from brains of individuals with dementia of the Alzheimer type. The nature and extent of such changes could yield important information on etiological mechanisms of this disorder. Pure populations of neurons from frozen autopsy, unfrozen autopsy, and biopsy specimens will be isolated. Populations of mitotic CHO and HeLa cells will be prepared. The neurons will be fused with the mitotic cells using either UV-inactivated Sendai virus or polyethylene glycol as a fusogen. The viral technique is done in suspension, the polyethylene glycol technique, in monolayer. At first, both techniques will be employed, using both HeLa and CHO mitotic cells. Evaluation of fusion and PCC outcomes from preliminary trials will determine the method and cell type of choice for all future experiments. The prematurely condensed chromosomes of the neurons will be banded and analyzed for chromosome number, chromosome breakage, and chromosome morphology. The three different sources of tissue will be compared for any differences in the efficiency of fusion, PCC induction, or for differences in chromosomal analyses. After the PCC-neuron technique is standardized on normal tissue, samples from temporal lobes from 10 familial Alzheimer cases and 10 control cases, matched for age, sex, and post-mortem time will be analyzed. Only individuals aged less than 65 years will be included. Chromosomal analysis will consist of the examination of numerical and structural changes.