One of the major pathological findings in Alzbeimer's disease is the degeneration of cholinergic neurons located in the basal forebrain. The long term goal of this laboratory is to understand the mechanisms that act to maintain the expression of appropriate functions by these cell most studies of cholinergic neurons have used the activity of chorine acetyltransferase (ChAT), the biosynthetic enzyme for the neurotransmitter acetylcholine, as a measure of the integrity of their cholinergic properties. Yet, relatively little is known about the human enzyme. Preliminary experiments using exons derived from the human ChAT gene have demonstrated that two species of ChAT mRNA are expressed in both adult human nucleus basalis and human cholinergic neuroblastoma cells. One of these RNA species is far more abundant than the other in human nucleus basalis and the corresponding mRNA is induced by nerve growth factor (NGF) in cultures of rat embryo medial septal neurons. Furthermore, the two ChAT mRNAs differ within their protein coding sequences, suggesting that human cholinergic neurons express two isoforms of the ChAT protein.
The specific aims of this proposal are (1) to completely characterize each form of human ChAT mRNA and (2) to identify and characterize the corresponding protein isoforms. Using exons from the complete human ChAT gene, both species of human ChAT mRNA will be mapped by nuclease protection and Northern blotting procedures in order to determine the extent to which they differ in their protein coding domains and in their untranslated sequences which may suggest possible mechanisms through which the splicing of nuclear ChAT RNA is regulated. The sequences of the corresponding proteins will be predicted from these data and used to design synthetic peptides for the production of ChAT isoformspecific monoclonal antibodies. These antibodies will be used to purify each of the ChAT proteins and to identify differences, such as stability, subcellular location, or kinetic properties, between the two proteins which could have significant consequences for cholinergic neuron function. The identification of two human ChAT proteins would finally resolve a longstanding controversy concerning the presence of multiple ChAT isoforms in the vertebrate central nervous system. More importantly, the identification of CHAT isoforms with different properties would force a re-evaluation of the pathology of cholinergic neurons in Alzheimer's disease and, perhaps, of the therapeutic regimens directed at these neurons.
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