| Abstract No.: | C-C3116 |
| Country: | Canada |
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| Title: | THE ROLE OF CHOLINE ACETYLTRANSFERASE IN CHOLINERGIC NEURON FUNCTION AND AMYLOID PRECURSOR PROTEIN PROCESSING |
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| Authors/Affiliations: | 1 Maureen Kinghorn; 1 Maureen Kinghorn*; 1 Sandy Gill; 1 Ewa Jaworski; 1 R Jane Rylett;
1 Robarts Research Institute, London, ON, Canada
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| Content: | Choline acetyltransferase (ChAT) is the enzyme that synthesizes acetylcholine from acetyl Coenzyme-A and choline in cholinergic presynaptic nerve terminals. There is increasing evidence that ChAT is involved in the link between cholinergic neurons and toxic beta-amyloid (A-beta) peptides characteristic of Alzheimer’s Disease (AD) pathology. Previous research in our lab has shown that there are changes in several genes involved in amyloid precursor protein (APP) processing in cells stably expressing either 69- or 82-kDa ChAT.
Objectives
These studies examine the functional outcomes of ChAT expression in terms of its effect on APP processing and A-beta production. Furthermore, the specific role of several proteins translated from the genes found to have altered expression profiles in the presence of ChAT will be examined.
Methods
RT-PCR – RT-PCR will be done using primers for APP, ADAM10, PAR4, BACE2, and PEN2 - genes which have previously been found to be affected in IMR32 neuroblastoma cells expressing ChAT. RNA from cells transiently transfected with 69- and 82-kDa ChAT cDNA will be used to see if there are changes in mRNA production as compared with wild type cells.
ELISA – In order to address the functional aspect of ChAT expression in cells, an ELISA is being performed to assess A-beta1-40 and A-beta1-42 production in cells expressing ChAT Using media surrounding cells expressing wt, 69- and 82-kDa ChAT, A-beta1-40 and Abeta1-42 production will be measured as compared to total A-beta content in the cells.
Western Blot Analysis – For protein expression studies, cell lysates were collected and analyzed by Western blot using antibodies for PAR4, BACE2, and ADAM10. Expression of protein was quantified from immunoblots after being normalized to the actin loading control.
Results
Previous data in the lab and preliminary data from this experiment have shown changes in ADAM10, PAR4, BACE2 and PEN2 that are consistent with those changes seen from previous microarray data. Similarly, previous ELISAs performed have shown that A-beta1-40 and A-beta1-42 production has a tendency to decrease in cells expressing ChAT, although the results have not yet been confirmed as significant. There is a decreasing trend in the expression of ADAM on immunoblots of cell lysate containing ChAT as compared with wt. No significance has been found at this time, but further experiments will be done to substantiate this trend.
Conclusions
These experiments show that ChAT plays an important role in cholinergic neuron function as well as playing a potential role in the processing of APP. Future studies will look at mutant forms of ChAT which cannot enter the nucleus of the cells, in order to examine whether APP processing is affected when ChAT does not have access to the nucleus. As it is already known that ChAT is specifically decreased in cases of AD, ascertaining the pathway affected as a result of that change would provide valuable information for better understanding the disease, its underlying pathophysiology and its mode of progression
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