27 August 2003. Neuritic plaques are classic, obvious markers of Alzheimer’s disease, but it is a puzzle why their density is a poor predictor of disease symptoms. Recently, scientists have come to suspect that soluble oligomeric, not fibrillary, Aβ, may be the culprit in synaptic degeneration in AD. In the August 18 online PNAS, another piece of evidence implicating oligomers appeared in the latest edition of PNAS. Part of this paper was previously presented at the Society for Neuroscience meeting (see ARF related news story). Using an antibody specific for Aβ oligomers, Yuesong Gong and colleagues in William Klein’s group at Northwestern University, Chicago uncovered their presence in regions of AD brain that typically suffer the most degeneration, and also showed that they cluster on neuronal surfaces, binding with ligand-like specificity.
First, Gong et al. raised synthetic antibodies against Aβ oligomers and tested for immunoreactivity in tissue samples taken from the frontal cortex of AD and age-matched control brains. Consistent with results from other groups (see ARF related news story), AD cortex reacted robustly to the antibody, whereas control cortex showed little response. In some individual comparisons, AD tissue had as much as 70 times higher reactivity to oligomers than did control tissue.
Next, the authors visually examined the binding pattern of oligomers on cultured neurons, looking at cultured rat hippocampal neurons incubated with synthetic Aβ oligomers (or ADDLs), AD brain extract, or control tissue extract. Under the immunofluorescent microscope, distinct clusters of binding sites lit up on the surfaces of neurons treated with synthetic ADDL or AD brain extract. The authors note that the punctate distribution of binding sites is reminiscent of clusters of membrane rafts or synaptic terminals.
Finally, ligand overlay assays suggest that oligomers could perhaps act as ligands for neural membrane proteins. The authors tested synthetic oligomers with rat membrane proteins, and found that oligomers bound with high affinity to a small number of proteins in hippocampal and cortical, but not cerebellar, membranes. In a separate test, different concentrations of ADDL were applied to cortical and cerebellar cultures, revealing that ADDL at micromolar doses was selectively toxic to cortical neurons, as evidenced by a decrease in the level of MTT reduction. The authors suggest that the difference in oligomer binding seen in the different brain areas reflects differences in their intrinsic vulnerability to oligomer toxicity.
For hippocampal and cortical membrane preparations, synthetic ADDL binding was observed to proteins around 140 and 260 and, less so, 100 kDA. Binding in the p140 and p260 bands increased when the membrane protein mixture was enriched with rafts, suggesting that the binding sites may be concentrated in these membrane domains.
Human AD brain-derived extract yielded slightly different binding patterns from those of synthetic oligomers. The same three bands (p100, p140, and p260) showed up in the overlay pattern, but in this case, binding in p100 was the most robust. In contrast, the antibody did not bind detectably to control brain extract of any region.
P140 and p260 binding was also present in assays of synthetic oligomer and human membrane protein preparations. However, binding in both bands was significantly lower in AD brain than in control brain, which the authors say is consistent with reduction or degeneration of cells containing oligomer binding proteins in AD.
The authors suggest that, taken together, oligomers act as ligands to a small number of membrane proteins located in cortical and hippocampal neurons, and that these proteins may be disrupted or reduced in AD. It is not clear from this finding that the site of oligomer binding is actually in the synapses or in the rafts, although it’s been shown that oligomers have detrimental effects on LTP, thought to underlie synaptic plasticity (see ARF related news story). Nonetheless, the authors say, the finding of elevated levels of oligomers and their high binding affinity in brain areas most vulnerable to degeneration in AD further builds the case for focusing on oligomers in therapeutic strategies.-I-han Chou.
I-han Chou is a science writer based in Japan.
Gong Y, Chang L, Viola KL, Lacor PN, Lambert MP, Finch CE, Krafft GA, Klein WL. Alzheimer's disease-affected brain: Presence of oligomeric A(beta) ligands (ADDLs) suggests a molecular basis for reversible memory loss. Proc Natl Acad Sci U S A. 2003 Aug 18. Abstract