Dr. Rosenberry and his laboratory colleagues study three components from human brain that may play roles in Alzheimer's disease (AD). One component, β-amyloid (Aβ), is composed of 40- and 42-residue Aβ peptides. The brains of AD patients contain large numbers of Aβ amyloid fibrils in the form of senile plaques. It now appears likely that Aβ peptides and fibrillar Aβ aggregates play an important role in the development of AD, and therapeutic strategies that prevent aggregate formation are attractive. Since cellular interfaces affect aggregation rates and aggregate structures, we are investigating the ways in which several interfaces, including polar-nonpolar interfaces and anionic micellar surfaces, alter aggregation in vitro. Interfaces in general promote aggregation by bringing Aβ peptides into closer proximity, but some interfaces also selectively stabilize Aβ42 aggregates called oligomers. We propose that inhibitors of Aβ aggregation or promoters of Aβ disaggregation in vitro will reduce the amyloid burden in AD patients. To test this proposal, we are investigating selected monoclonal antibodies and peptides that prevent or reverse the assembly or growth of Aβ aggregates. We are determining the affinities of potent blockers with both monomers and aggregates and the size and structure of the inhibited complexes. Potent blocking agents also will be examined in a transgenic mouse model of AD (Tg2576). These mice exhibit increased levels of Aβ40 and Aβ42, dystrophic neurites, Aβ deposits in the amygdala, hippocampus, and cortex, and marked deficits in synaptic transmission and memory. We will determine whether the blocking agents slow or reverse the appearance of these pathological markers in the mice.
Another way of preventing the formation of toxic Aβ aggregates is to block the protease cleavages that give rise to the 40- and 42-residue Aβ peptides. The protease that generates the N-terminus of these peptides in brain tissue is called BACE. We have constructed a high-level expression system for the production of quantities of secreted recombinant BACE sufficient for its detailed structural and functional analysis. Through collaboration with another laboratory that specializes in X-ray crystallography, we will determine the three-dimensional structure of BACE and its complexes with inhibitors. We are also analyzing the cleavage sequence specificity and enzymatic mechanism of BACE using various peptide substrates as well as site specific mutants of recombinant BACE.
A third component of interest is acetylcholinesterase (AChE), an enzyme that controls communication between nerve cells by the neurotransmitter acetylcholine. This communication is disrupted by the death of nerve cells in AD patients, and inhibitors of AChE are approved as drugs to elevate acetylcholine and aid neuronal function in these patients. AChE is also important in another context: It is i nactivated by toxic agents like organophosphates, and this can lead to neuromuscular paralysis and death. Individuals risk exposure to organophosphates in certain pesticides and in chemical warfare agents, and we are pursuing new therapeutic strategies to protect against organophosphate poisoning. The active site gorge of AChE contains two sites of ligand binding, an acylation site near the base of the gorge and a peripheral site at the mouth of the gorge some 1.5 - 2.0 nm from the acylation site. This peripheral site is an attractive target for the design of new drugs that might selectively protect against organophosphate reaction at the acylation site, and we have clarified its role in AChE function. We first showed that small ligands which bind selectively to the peripheral site inhibit AChE by slowing the rates at which substrates enter and exit the acylation site. We then found that substrates like acetylthiocholine itself transiently bind to the peripheral site to accelerate their entry into the acylation site. We also observed conformational interaction between the peripheral and acylation sites. These studies have led us embark on the design of new cyclic peripheral site ligands that may block the access of organophosphates but not acetylcholine to the acylation site, thereby offering protection against organophosphate toxicity.
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