4 June 2009. In Alzheimer disease research, well over a decade of intense research into fluid biomarker candidates has reached a point where a so-called “pathological signature” of amyloid-β and tau proteins is beginning to emerge from the 59-center Alzheimer’s Disease Neuroimaging Study (Shaw et al., 2009). This signature validates on a larger scale a number of earlier studies that had shown essentially the same thing (Fagan et al., 2007; Fagan et al., 2006; Li et al., 2007; Hansson et al., 2006; Welge et al., 2009). Coming, as it did, with ups and downs along the way, this search for a fluid test is guiding researchers who are working to develop similar markers for α-synuclein and progranulin, two major proteins involved in many of the overlapping forms of dementia at issue in earlier parts of this news series. The 9th International Conference AD/PD, held last March in the Czech capital city of Prague, as well as an immediately preceding workshop on dementia with Lewy bodies (DLB) and Parkinson disease dementia (PDD) in Germany, showcased a rapidly growing field of groups who are racing to broaden the field. Here is a selection.

First, α-synuclein (for progranulin, see Part 7). The three investigators who started fluid-based markers on this intraneuronal protein are Michael Schlossmacher, now at the University of Ottawa, Canada, Brit Mollenhauer, now at Paracelsus-Elena-Klinik in Kassel, who worked in Schlossmacher’s former lab at Brigham and Women’s Hospital in Boston, and Omar El-Agnaf, now at United Arab Emirates University in Al Ain, UAE. All three collaborated extensively, first to build ELISA assays and to show that these assays can quantify α-synuclein in normal human cerebrospinal fluid (CSF), then to show that the CSF concentration of this protein normally declines with age and declines even further with Parkinson disease (Tokuda et al., 2006). A first cross-sectional study compared CSF α-synuclein concentration in various patient groups, i.e., AD, DLB, PD, multiple system atrophy (MSA), and Creutzfeldt-Jakob disease (CJD) with controls. These studies found the lowest levels in PD and MSA, whereas AD and controls had similar and higher levels, and DLB lay in between. In CJD, α-synuclein was curiously elevated, perhaps because the rapid cell death in this condition dumps this protein into the CSF so that it serves as a marker of degeneration in this situation, much as tau is viewed in AD (Mollenhauer et al., 2008).

However, the same difficulty that has dogged CSF measurements of Aβ since the beginning (e.g., Seubert et al., 1992) quickly caught up with α-synuclein, too. Its concentration varies greatly from person to person, creating enough overlap that the test in its original form is unable to distinguish which group a given person falls into. While as a group, the values of people with PD always cluster at the bottom, any given person with PD might have a value higher than a control or an AD patient. Moreover, other research groups, using their own, different ELISAs, have been unable so far to replicate Mollenhauer and colleagues’ result, calling into question whether α-synuclein can serve as a robust diagnostic marker to distinguish between overlapping diseases (Ohrfelt et al., 2009; Spies et al. 2009; Noguchi-Shinohara et al., 2009). In Prague, debate centered on the different assays and antibodies different groups are using to measure α-synuclein. Schlossmacher noted that his and collaborators’ ELISA is extensively validated. Other scientists agreed that before a final word can be spoken, more tests in additional patient cohorts, independent replication, a comparison of methods, and exchange of antibodies are needed.

“Right now, many groups are trying to measure CSF α-synuclein and are having a hard time seeing good separation between the groups. We, too, see a very narrow range of values,” said James Galvin of Washington University, St. Louis, Missouri. “But that’s no reason to be discouraged. It may just take more standardization of the steps and the right tools to get it down.”

In Kassel, Mollenhauer presented new data on total CSF α-synuclein measured in a separate cross-sectional cohort of clinically diagnosed patients. Again, PD and MSA lay at the bottom, AD and controls at the top, DLB in between. The overlap remained extensive, though expressing α-synuclein concentration relative to total protein teased the groups apart somewhat. In Prague, Mollenhauer’s poster of a separate series of 41 autopsy-confirmed cases showed that their CSF measurement matched the working diagnosis they had received during life.

On balance, then, the early days of α-synuclein biomarker research have made clear that this protein can be directly measured in the CSF (and peripheral blood; see ARF related news story), and, least with one assay, trends downward from controls to DLB and PD, Schlossmacher said. But besides technical collaboration to streamline protocols, much more scientific work remains to be done, he added. Challenges include understanding where exactly the CSF α-synuclein comes from (the brain, the periphery, the choroid plexus could all contribute), what different species of α-synuclein occur in CSF (truncated, full-length, or modified), and which one of those best indicates disease. To see how these species change in the same person over time, Schlossmacher’s and Mollenhauer’s groups have begun longitudinal studies.

Galvin foresees a future where academic centers interested in earlier-stage clinical trials use a CSF assay for α-synuclein to distinguish preclinical AD from preclinical DLB. An α-synuclein imaging ligand is not on the horizon (see Part 5 of this series), but amyloid imaging is available and it shows a large fraction of non-demented elderly people who have brain amyloid and may turn out to be presymptomatic for either AD or DLB. Most DLB cases share Aβ and α-synuclein pathology; hence, an α-synuclein fluid assay could conceivably flag amyloid-positive people who are at high risk for future DLB, much like combining amyloid imaging with CSF Aβ/tau measurement is predicting who will develop AD symptoms. Other groups are drilling deeper with Aβ biochemistry, measuring some of its truncated and oxidized forms to distinguish between AD and DLB (Bibl et al., 2006; Bibl et al., 2007).

“In our studies, we already have a number of people who are PIB positive and are not demented, but when you look at them with some of the biomarkers we are developing, some of these people are clustering with the DLB group. The idea is to be able to diagnose preclinical DLB,” Galvin said. The Kassel meeting ended with the designation of a working group to hammer out a research path toward that goal, Mollenhauer wrote to ARF.

For his part, El-Agnaf has focused on measuring oligomers of α-synuclein, initially in plasma (El-Agnaf et al., 2006) and more recently in brain extracts. In Prague, he showed the results of a study looking for such oligomers in lysates prepared from postmortem brains of people who had suffered from DLB. As measured by a sandwich ELISA El-Agnaf developed with a commercial antibody that recognizes α-synuclein aggregates but not monomers, these brains contained far higher concentrations of α-synuclein oligomers than control or AD brains. The data showed less overlap between the groups, but no clear separation, either (Paleologou et al., 2009). Since then, the researchers used their ELISA on CSF samples and again found high levels. A final cohort of 60 samples from people with PD and controls displayed, again, a group difference but also a spread of the individual data points and overlap between the groups. Calculating the ratio of oligomeric α-synuclein to total α-synuclein improved the separation, El-Agnaf noted. “This is the first time we have been able to detect soluble oligomers from CSF in humans,” El-Agnaf said in Prague, and here, too, the work of replication and broadening the effort is only just beginning.

Meanwhile, research underpinning the rationale for going after oligomeric α-synuclein in body fluids is advancing in parallel. Here, too, Prague offered some news. For example, in the last talk of the AD/PD conference, Kostas Vekrellis of the Biomedical Research Foundation Academy of Athens, Greece, reported that secreted α-synuclein oligomers are up to no good. Even though α-synuclein is primarily a cytosolic protein, scientists know that cultured cells can release it. Cells also can take up external α-synuclein, usually at their peril as they tend to die soon after, Vekrellis said.

Vekrellis investigated this apparent toxicity with lines of human neuroblastoma cells that can be induced to express wild-type α-synuclein. Soluble monomeric and oligomeric α-synuclein showed up in the conditioned medium from these cells. Using liquid chromatography-mass spectrometry proteomics and electron microscopy, Vekrellis and colleagues showed that the cells actively export α-synuclein via an exosome pathway that itself depends on intracellular calcium. These cells sustained no harm from the α-synuclein. But their conditioned medium, when squirted onto primary rat cortical neurons, killed those cells. A high-molecular-weight fraction of α-synuclein species proved toxic. Medium depleted of α-synuclein, or medium subjected to oligomer-busting compounds such as scylloinositol did not, pointing to oligomers as the active component. This new data invoke parallels with amyloid-β oligomers, which have been shown to impair synaptic activity and to damage cells, and whose sensitivity to scylloinositol has led to a Phase 2 trial. For more on oligomers in mixed disease, see Part 4 of this series.—Gabrielle Strobel.

This is Part 6 of a nine-part series. See also Part 1, Part 2, Part 3, Part 4, Part 5, Part 7, Part 8, Part 9.