The small peptide beta-amyloid, AB, has been implicated in the neurodegenerative process in Alzheimer's disease. The applicant has played a major role in defining AB toxicity and has now obtained data showing that it causes cultured neurons to die by an apoptotic pathway exhibiting membrane blabbing, chromatin condensation and DNA fragmentation. Classically apoptotic or programmed cell death was defined morphologically by initial surface blabbing, chromatin condensation and nuclear shrinkage, followed by nuclear fragmentation into nuclear bodies and polyribosome dispersal while limited ER remains and mitochondria remain normal. Only late in the secondary necrosis stage does the plasma membrane disintegrate. In contrast, necrotic cell death is characterized by early dilation of the ER and mitochondria and membrane disruption. The proposal is to define the sequence of cellular and molecular events initiated by the response to AB focussing on the induction of immediate early genes, IEGs, (c-fos and c-jun and later c- myc and junB) and the role of calcium and other second messengers in inducing fos and jun and DNA fragmentation. The applicant has preliminary data that AB induces the IEGs, c-fos and c-jun. In other cell systems, antisense inhibition of fos and jun has inhibited apoptosis.
Aim 1 proposes to define the detailed time course of c-jun and c-fos induction in response to AB.
Aim 2 proposes to use antisense oligos to block this induction and determine whether cell death is prevented. In other systems where apoptosis has been shown to result in fos and jun induction, the time course parallels that of DNA fragmentation.
Aim 4 is to determine the time course for DNA fragmentation and see if the time course is the same for c-fos and c-jun induction. It is also proposed to determine whether anti-sense treatment as in aim 2 protects against DNA fragmentation. C-myc is another IEG shown to be involved in apoptosis by antisense and expression studies and there are a number of others including bcl-2, bcl- x, ced-3 and ced-9.
Aim 3 is directed at determining changes in other IEGs, focussing on c-myc and junB in response to AB. Based on work in other apoptotic systems and other labs working on AB toxicity as well as their own data, the applicants hypothesize that AB apoptosis is mediated by a transient rise in intracellular calcium, followed by a delayed rise which precedes DNA fragmentation.
In aim 5, the applicant seeks to better define the role of calcium as a mediator of the AB response using calcium imaging, histochemical assessment of DNA fragmentation using in situ end labelling of free 3'-OH groups on fragments and light microscopy.
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