Altered Expression of Ion Channels in Alzheimer s disease

In Alzheimer s disease there is a build-up of protein in the brain that is thought to cause the death of nerve cells. We have found that this protein, named amyloid beta protein (Abeta), causes changes in the electrical activity of nerve cells when in a form that is not usually associated with the disease. In fact, we suspect that this may be a normal function of the protein. We believe that during the disease process Abeta] may lose its ability to alter electrical activity and this leads to death of nerve cells. This idea needs to be tested and to do this we will measure the effects of normal Abeta and abnormal Abeta on the electrical activity of individual nerve cells. In separate experiments we will also be able to track changes in levels of proteins involved in causing electrical activity in nerve cells (ion channels), and view their position within the nerve cells. This will give us more detailed information on the changes that occur and their consequences during the disease.
Because we believe that changes in protein will result in nerve cell death we will also look at how ion channel activity changes in nerve cells that are just beginning to enter the cell death death process. This will allow us to directly measure differences between the electrical activity of nerve cells that are healthy and those that are not. The information gained from these experiments will tell us how important changes in electrical activity in the brain of Alzheimer s disease patients are in causing the death of the nerve cells. This is vitally important since, unlike almost all other cell types, nerve cells do not divide and replace themselves. If we can prevent the cell death caused by Abeta we may therefore be able to slow or halt the progression of Alzheimer s disease.

Recent evidence from a number of sources implicates a role for ion channels in Alzheimer s disease. We have evidence suggesting that the main constituent of senile plaques, amyloid beta protein (Abeta), has a profound effect on ion channel expression in central neurones. In this proposal we hypothesise that Abeta acts as a physiological mediator of ion channel expression and loss of this function by aggregation/oligomerisation in Alzheimer s disease results in neurotoxicty. In order to test this idea we will measure ion channel currents (primarily K+ and Ca2+) and action potential parameters (amplitude, duration, afterhyperpolarisation, firing rates) in mouse hippocampal slices that have been treated for varying lengths of time with different forms of Abeta that are implicated in either AD or in normal function (monomeric, oligomeric and aggregated Abeta). Experiments will require application of Abeta peptides prepared from cells engineered to secrete Abeta (7PA2 cells) to slices (acutely pepared or organotypic) for times varying from 1-48 hours. The effects of inhibiting or increasing Abeta production in slices using pharmacological means (inhibitors of beta- and gamma-secretase and neprilysin) and shRNAi technology will be measured in voltage clamp and current clamp experiments in order to determine the effects of altering endogenous Abeta production on ion channel expression. To compliment patch-clamp study of functional ion channel expression we will use western blot to measure levels of ion channel subunits and immunohistochemistry to localise changes in subcellular distribution of ion channels.

The mechanism by which Abeta alters ion channel activity will be investigated and we will concentrate primarily on the signaling pathway that gives rise to inhibition of long term potentiation in slices. The relevance of changes in ion channel expression to cell death in Alzheimer s disease will be investigated by imaging neurons in the earliest stages of apoptosis with annexin 5. This will allow these neurons to be patched and ion channel currents measured. These experiments will be carried out in slices treated with Abeta or in slices or acutely dissociated neurones prepared from a triple transgenic mouse model of Alzheimer s disease.

These data will provide information on some of the basic mechanisms involved in the neurodegeneration associated with the Alzheimer s disease process. Furthermore, they will allow us to define the role of ion channels in that process and identify the ionic currents that may be involved in cell death or neuroprotective responses.