31 October 2007. Since they were discovered to be a major toxic species, Aβ oligomers have haunted many a research lab. Their presence is difficult to detect in vivo and their full range of toxicities unclear. In the October 22 Journal of Neurochemistry online, researchers report small advances on both fronts. A research team led by Lee-Way Jin, University of California, Davis, report that Congo red and thioflavin T analogs can detect Aβ oligomers in living animals, while researchers at the University of South Carolina, Columbia, report how oligomers may facilitate transmigration of monocytes across the blood-brain barrier by priming endothelial cells.
A way to quantify Aβ oligomers in the living brain is widely regarded as a key research priority. An oligomer-specific imaging agent might help identify individuals at early stages of AD, provide a pathological correlate of cognitive decline independent of amyloid fibrils, and offer a readout on therapeutic effects. To date, the only means of reliably measuring oligomers is by using oligomer-specific antibodies, which are of little use for brain imaging.
Jin and colleagues adopted a different approach. They tested if Aβ oligomers can be detected with Congo red (CR) and its derivative BSB ([trans, trans]-1-bromo-2,5-bis-[3-hydroxycarbonyl-4-hydroxy]styrylbenzene), or thioflavin T (ThT) and its analogs PIB and IBOX ([2’-4’-dimethylaminophenyl]-6-iodobenzoxazole). The scientists found that all compounds bound to synthetic Aβ oligomers, with PIB, IBOX and ThT having the highest affinities, and CR and BSB having the highest total binding. The results suggest that Aβ oligomers contain some high-affinity sites for ThT derivatives, and a greater number of low-affinity sites for CR and BSB.
Can these compounds detect soluble Aβ oligomers (AβO) in vivo? Because of the diffuse nature of oligomers and their small size, “compounds with substantially higher AβO-binding affinities need to be developed for use as probes,” the authors write. These CR and ThT derivatives could serve as a starting point, as first author Izumi Maezawa and colleagues found that the ligands do bind oligomers extracted from AD brain tissue. The ligands also stained sections from 3xTg-AD mice, which develop intraneuronal Aβ oligomers between 4 and 6 months of age. In those sections, the punctate staining pattern obtained using BSB colocalized with binding of the A11 oligomer antibody (see ARF related news story). PIB and IBOX gave similar staining. The authors further report that these three ligands bound to inclusions in MC65 neuroblastoma cells lines, which they use as a model of intraneuronal accumulation of Aβ40 oligomers. Finally, to test if the ligands could serve as an in-vivo tracer, the authors stereotactically injected BSB into the brain of 7-month-old 3xTg mice, where it was rapidly taken up by hippocampal neurons and bound intraneuronal inclusions, as judged by subsequent postmortem analysis with A11.
All told, the findings suggest that CR and ThT derivatives could eventually make useful probes if their binding affinities for oligomers could be increased. The authors suggest that affinities similar to those used for imaging neurotransmitter receptors (dissociation constants of less than 1nM) would be in order.
The second oligomer advance comes courtesy of Francisco Gonzalez-Velazquez and Melissa Moss at the University of South Carolina. These investigators tackled the question of Aβ’s role in the vasculature. Though it is well established that Aβ can trigger responses in endothelial cells that may contribute to trans-endothelial migration of immune cells and neuroinflammation (see ARF related news story), the species of Aβ responsible for this remains to be identified.
Toward this goal, the authors tested Aβ for effects on human brain microvascular endothelial cell (HBMVEC) monolayers. They found that an unpurified mixture of Aβ comprising monomers, oligomers, and fibrils made THP-1 monocytes more adhesive to the monolayers by twofold, and that twice as many THP-1 cells migrated through the monolayers. Neither isolated Aβ monomers nor fibrils were able to repeat this effect, but isolated soluble aggregates did.
How might Aβ oligomers stimulate this effect? One possibility is that they simply kill off endothelial cells, effectively punching a hole through which monocytes can squeeze. But the authors found that the increased adhesion and transmigration through the HBMVEC monolayers occurred in the absence of endothelial cell death. “We therefore speculate that Aβ stimulates cell signaling pathways responsible for endothelial activation via its interaction with cell surface receptors,” they write. While the identity of the precise form of Aβ responsible for this is not known, the authors did find that endothelial adhesion correlated with oligomer size—smaller aggregates of 30-40 nm radius worked best, while those over 100 nm had little effect.—Tom Fagan.
Maezawa I, Hong H-S, Liu R, Wu C-Y, Cheng RH, Kung M-P, Kung HF, Lam KS, Oddo S, LaFerla FM, Jin L-W. Congo red and thioflavin-T analogs detect Abeta oligomers. J Neurochem. 2007 Oct 22 online. Abstract
Gonzalez-Velasquez, FJ, Moss MA. Soluble aggregates of the amyloid-beta protein activate endothelial monolayers for adhesion and subsequent transmigration of monocyte cells. J Neurochem. 2007 Oct 22 online. Abstract