Is a specific chemical modification of amyloid beta-peptides important in Alzheimer's disease?
Detection of cross-linked amyloid-beta oligomers in Alzheimer's disease by mass spectrometry
This study seeks to determine some of the mechanisms by which amyloid beta aggregates and ultimately how these aggregates work to assemble in amyloid plaques. Using a systematic approach, Dr. Rosenberry's team is working on developing ways of studying these aggregates that exist at extremely low concentrations in the brain.
A hallmark of Alzheimer's disease (AD) is the presence of deposits called amyloid plaques in the brain. These plaques are mostly composed of a peptide called amyloid-beta. Recent research indicates that soluble clusters of Abeta, called oligomers, are perhaps the primary cause of AD. We are trying to determine the chemical structure of these oligomers and, in particular, whether the structure contains a feature called a cross-link. This is a challenging goal, as the amount of these oligomers in the brain is too low for conventional chemical analyses. To our knowledge, no other researchers have tried to address this question in a technically rigorous manner. Our project could have significant therapeutic benefits. Diagnostic tests could be designed to specifically quantify cross-linked oligomers in cerebrospinal fluid and correlate their levels with cognitive deficits in elderly individuals. Other tests of the neurotoxicity of these oligomers could be conducted by chemical synthesis of their precise structures and evaluation their toxicity in cellular and intact animal assays. Such synthetic peptides could even be investigated as possible vaccines. However, before we can determine whether cross-linked oligomers are enriched in individuals with AD, we must first develop the analytical techniques required for sensitive oligomer detection. This developmental effort is best directed at synthetic Abeta oligomers that are prepared and chemically cross-linked in our laboratory. Our grant proposal has three aims. In Aim 1 we will prepare stable oligomers of Abeta by chemical cross-linking in vitro and isolate individual oligomers. In Aim 2 we will evaluate and then establish procedures to detect covalently cross-linked Abeta oligomers and identify sites of cross-linking by two mass spectrometry techniques. In Aim 3 we will apply our optimized techniques to determine the structure and amounts of Abeta oligomers that can be isolated from AD brain tissue.
The Alzheimer's disease (AD) field has placed emphasis on the hypothesis that small, soluble oligomers of amyloid beta (Aβ) peptides, particularly Aβ42, are key toxic components that initiate the disease pathology. Peptides are small units of proteins and oligomers are created by chemically "bunching together" many single peptide units. In this case, scientists believe that units of Aβ42 peptides are clumping together to cause the toxicity. However, no laboratory has yet been able to isolate from cells an amount of endogenous (or naturally-found) Aβ oligomers at levels sufficient for determination of the structure of this toxin. Once the Aβ42 oligomer structure is known, then how this toxic initiator leads to disease can be discovered and methods to counteract these effects can be designed.
We are attempting to reconstitute toxic Aβ oligomers from synthetic Aβ42. However, the synthetic Aβ42 oligomers we have produced differ from the endogenous Aβ oligomers in two important respects: They are less stable; and they do not segregate into discrete oligomers by chromatographic procedures (methods that separate and measure objects of different sizes). Our working hypothesis to account for this difference is the following: Endogenous oligomers initially associate non-covalently (a tentative, easily-broken association) at cellular anionic interfaces and then become covalently cross-linked (a chemically solid association) by unknown cellular agents at these interfaces. We are developing techniques to detect synthetic Aβ42 that is covalently cross-linked and then use this knowledge to examine whether endogenous Aβ oligomers have been chemically cross-linked. We have made progress in producing and characterizing synthetic Aβ42 oligomers on detergent and phospholipid micelles that mimic the natural cells' membranes, and we have examined the covalent cross-linking of various forms of Aβ42. We used mass spectrometry studies (a procedure that measures the sizes of protein fragments) to determine that the cross-linking of Aβ42 (by a natural cell protein called transglutaminase) is largely specific for positions Q15 to K28 in the Aβ42 peptide. Finally, we evaluated procedures for isolating endogenous Aβ42 oligomers. Defining these structures could allow the development of diagnostic tests for AD that would quantify these oligomers in cerebrospinal fluid and correlate their levels with cognitive deficits in elderly individuals.
First published on: June 10, 2008
Last modified on: April 12, 2011