The overall objective of this project is to obtain cells which can be used for transplantation to replace lost neurons in Alzheimer's and other neurodegenerative diseases involving cholinergic neurons. Two general approaches will be investigated; one involves transfecting genes of the key cholinergic proteins into cells capable of acquiring a neuronal phenotype, while the other involves the use of promoter elements from the ChAT gene to select out cholinergic neurons from fetal neuronal cell cultures containing cholinergic precursor cells. The project involves 4 specific aims. The first involves the use of cell transfection techniques to introduce the three major components of the cholinergic cell, the choline transporter, choline acetyltransferase, and the acetylcholine transporter into cells capable of acquiring a neuronal phenotype. These include NT-2 cells, RN33B cells, and embryonic neuronal precursor cells. Constructs containing the cholinergic specific genomic sequences will be used to express drug resistant genes in embryonic cholinergic neuronal precursor cells. Growth in the presence of the drug will be used to select for the cholinergic cells from this population. The second specific aim involves characterization of the transfected or isolated cells in terms of cholinergic function. This will focus on the ability of these cells to synthesize and store acetylcholine in synaptic vesicles, and to release acetylcholine upon depolarization. The ability of these cells to establish functional interactions with host neurons will initially be examined in vitro by co-culturing these cells with fetal brain tissue from various brain regions and looking for synaptic contacts. Thirdly, the transfected or isolated cells will be tested for their ability to survive intracerebral implantation in rats and to continue to synthesize, store, and secrete acetylcholine. Lastly, we will determine the ability of the implanted cells to ameliorate specific behavioral deficits in animal models of basal forebrain degeneration. It is anticipated that these studies will provide the basis for the further development of neuronal cells which can be used in gene replacement therapy for Alzheimer's and related diseases.
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