3 August 2009. It’s not just about dimers, dodecamers, and protofibrils anymore. With its wide range of presentations on the topic of small aggregates of Aβ (and α-synuclein, for that matter), the International Conference on Alzheimer’s Disease made it plainly obvious that the interest in these tiny culprits has branched out beyond the initial debates about whether they even exist and which forms are important. Held 11-16 July, ICAD showed that, in particular, the twin challenges of detecting small aggregates in human body fluids and of detoxifying them (see ARF companion story) are drawing new labs and new techniques to the field. Here is a summary of some selected examples; as always, the Alzforum welcomes reader comments on similar approaches.
Several groups are working on detecting oligomers of Aβ in body fluids. Some of those are developing methods other than ELISA, which in the past has been the staple of Aβ measurement. To quantify Aβ as a clinical biomarker, Xmap multiplex technology is increasingly replacing conventional ELISAs, which have long been dogged by great variability between labs. But to pinpoint oligomeric Aβ specifically, here’s something completely different.
At ICAD, Susanne Aileen Funke, working with Dieter Willbold at the Forschungszentrum Jülich, near Düsseldorf, Germany, presented a method that detects only Aβ aggregates, not its monomers. What’s more, it does so with a sensitivity down to a single aggregate, Funke said. In clinical AD research, it’s by now widely accepted that CSF Aβ42 levels plummet before a person develops AD. This biomarker has entered diagnostic practice in some centers, and has even become an inclusion criterion in a clinical trial of a new γ-secretase inhibitor. This concerns the Aβ monomer, however. Many scientists believe its drop-off reflects amyloid deposition in the brain, whereas others suspect that it might reflect a corresponding increase of aggregated Aβ in CSF (Englund et al., 2009). This implies that other forms of Aβ could be found in CSF besides the monomer if only scientists had a robust search method.
This is where Funke comes in. She is applying to Aβ detection a method called Surface-FIDA (short for fluorescence-intensity-distribution-analysis), which was originally developed for prion aggregate detection by fluorescence correlation spectroscopy (FCS). With this method, Funke immobilizes the aggregates contained in a few microliters of CSF onto a glass surface coated with an anti-Aβ capture antibody. Then she decorates them with two separate monoclonal antibodies, and lets a confocal microscope beam scan the surface to count the resulting fluorescence bursts. The trick is that only aggregates attract enough antibodies to give rise to a fluorescent burst; the monomers that are also present in the CSF sample do not. To preclude more than one antibody binding to monomer, the two different decoration antibodies target overlapping epitopes on Aβ.
“We can detect a single aggregate. That can never be done by ELISA. With ELISA, you detect a summarized signal of all aggregates in one well,” said Funke. In her talk, Funke showed three ways of validating that monomeric Aβ does not emit fluorescence bursts. She cross-correlated the signals from each decoration antibody, showed a linear dose response correlation, and eliminated the fluorescence bursts from an aggregate-spiked Aβ sample by adding the denaturing agent SDS.
In a first attempt to test the raw assay on clinical samples, CSF from three AD patients indeed appeared to contain more aggregates than control CSF (Funke et al., 2007). Since then, Funke and Willbold have improved the assay. It was also adapted to confocal imaging with laser scanning microscopes, which are more abundant than FCS devices. Moreover, this new version of the assay is able to determine the composition of each aggregate (e.g., Aβ40 and Aβ42) and robustly measures in the picogram range, Funke showed.
How does FIDA compare to ELISA? There is no publication on this yet, but Funke said that in a side-by-side experiment of the same clinical CSF samples, the ELISA test showed a decrease of Aβ42 in AD whereas FIDA showed an increase in oligomers/aggregates. In a separate ICAD presentation, Seong An at Kyungwon University, working with SangYun Kim at Seoul National University, both in the Republic of Korea, reported a similar approach using multiple antibodies whose epitopes overlap in order to distinguish oligomers from monomers. This group does not use FIDA, but they, too, reported seeing an increase of Aβ aggregates in CSF of AD patients. This group is actively investigating Aβ oligomer detection in blood.
In terms of moving the FIDA system toward clinical application, Funke said the next steps are to optimize the assay for CSF and for blood. Once that’s done, her team needs to test it on larger numbers of well-characterized clinical samples. She also wants to check which kinds of aggregates occur in CSF and blood of healthy people and patients—are they made of Aβ40, 42, or perhaps the pyroglutamated Aβ3-42? The scientists plan to see if the size, number, or composition of such aggregates changes with age and as people approach disease, and whether this test can predict that a person will develop AD. For a detailed description of Surface-FIDA and a publication list, see the institute’s website.
Funke adapted this detection method to Alzheimer disease after learning it initially from Eva Birkmann at the same institute (see institute website). Birkmann had developed Surface-FIDA for the detection of single prion particles. Her laboratory is presently adapting it to do so in CSF and plasma of cattle and sheep, as well as in the human prion disease Creutzfeld-Jakob disease. Most of currently available tests for the diagnosis of prion diseases in animals exploit the proteinase K resistance of PrPSc. In an e-mail to ARF, Birkmann wrote that her lab has not yet compared that established test and Surface-FIDA side by side. The hope is that, eventually, Surface-FIDA might provide a more accurate tool for human and veterinary diagnosis.
Regarding new collaborations, Birkmann recently teamed up with Brit Mollenhauer at the Paracelsus-Elena Klinic in Kassel, who has co-developed an ELISA to quantify monomeric α-synuclein in CSF of patients with PD and DLB. Their goal is to see if Surface-FIDA can detect oligomeric α-synuclein in this fluid, too. That such oligomers are there seems likely not only by analogy to AD. Prior hints exist as well. For one, Omar el-Agnaf at United Arab Emirates University in Al Ain, UAE, has reported seeing them with an ELISA both in brain extracts and CSF of PD (see ARF related Prague conference story). For another, Walter Schulz-Schaeffer at the University of Goettingen at ICAD presented an update to his previously published PET blot method (Kramer et al., 2007). This simple method reveals an overwhelming abundance of small α-synuclein aggregates in brain sections of people who had Parkinson’s or DLB, whereas neighboring brain sections stained conventionally may reveal at best a few scattered Lewy bodies or Lewy neurites. Other clinically relevant brain areas contain the small aggregates but no Lewy pathology at all. According to Schulz-Schaeffer, small α-synuclein aggregates are the species to watch. If you can see them.—Gabrielle Strobel.