2 December 2005. Biomarkers are the buzz of the field these days, and the hum was heard inside the beltway at the 35th Annual Conference of the Society for Neuroscience held in the US capital earlier this month. While the Alzforum has followed developments in AD biomarkers closely (see, e.g., ARF related news story and ARF related Live Discussion), we also keep our noses to the ground for likely portents of other neurodegenerative diseases, particularly Parkinson disease (PD). The Washington meeting featured updates on the search for PD biomarkers, and in these early days, α-synuclein seems to be a frontrunner.
Michael Schlossmacher, Brigham and Women's Hospital, Boston, reported that his lab's efforts to develop antibody-based diagnostics to measure both total and oligomeric-only forms of the protein in the peripheral plasma are bearing fruit (Program No. 13.6). An ELISA (enzyme-linked immunosorbent assay), which uses a sandwich combination of a monoclonal antibody and an affinity purified polyclonal antibody to detect total synuclein, gave good specificity and sensitivity, Schlossmacher reported, while a test designed by his collaborator, Omar el-Agnaf, United Arab Emirates University, Dubai, to specifically detect oligomeric forms does not recognize the monomer (el-Agnaf et al., FASEB J, 2005, in press). In blood from synuclein-null mice "spiked" with α-synuclein, the ELISA can detect over 90 percent of added protein.
This group has begun preliminary tests to evaluate the antibody-based diagnostic and to determine if blood α-synuclein can be validated as a test for PD, multiple system atrophy, dementia with Lewy bodies, or other synucleinopathies. In blinded tests during the development phase, the researchers found that total α-synuclein levels were much higher in two of eight samples received from their collaborator at the NIH, David Miller. These samples turned out to be from the same PD patient, who had a synuclein triplication (see ARF related news story). In a pilot trial, Schlossmacher detected that in samples from 14 living subjects from their Movement Disorder Clinic, there was no statistical difference between total plasma synuclein in six sporadic PD patients and six normal and neurological controls, but levels of synuclein were twofold higher in the two other synucleinopathy patients tested, one with multiple system atrophy and the other suffering from dementia with Lewy bodies. Schlossmacher emphasized that these sample numbers are small and that further testing is required.
However, he also revealed that the parallel assay for oligomeric α-synuclein did detect differences between sporadic PD patients and controls. In all six sporadic PD patients, blood oligomeric α-synuclein was statistically elevated (P <0.001), suggesting that the oligomeric-to-total α-synuclein ratio might be the important number. To validate α-synuclein as a biomarker, a longer trial with much larger sample numbers is needed, suggested Schlossmacher. With the financial support of a grant from the Michael J. Fox Foundation to search for PD biomarkers, Schlossmacher and his team, together with Clemens Scherzer, also at Brigham and Women's Hospital, and his group, will analyze the blood synuclein protein levels and peripheral blood mRNA profiles, respectively, in 300 subjects over 2 years.
How α-synuclein, a cytosolic protein, makes its way into the blood is presently unclear. In related work, Valerie Cullen from the same group used the same ELISA test to show that when the gene for α-synuclein was transfected into dopaminergic neural cells in culture, the protein was readily detected in the culture medium. Cullen was unable to detect another cytosolic protein, lactate dehydrogenase (LDH), in the same culture medium. Cullen is currently investigating if lipid binding to α-synuclein, or phosphorylation of it, are relevant to its release into the cell medium.
Continuing the theme of phosphorylation, David Miller, from the National Institute on Aging in Bethesda, Maryland, reported that phosphorylated α-synuclein might serve as a biomarker. Miller and coworkers, including NIA colleagues Mark Cookson and Andrew Singleton, as well as Tamie Chilcote at Elan Pharmaceuticals in South San Francisco, tested both the soluble and insoluble (SDS-solubilized) extracts of brain tissue samples for total (Syn-1 antibody) and serine 129 phosphorylated (Elan’s #11A5 antibody) synuclein. Miller reported that while comparably small amounts of phospho-α-synuclein were found in the soluble fractions of both controls and triplication cases, the amount of phosphorylated protein in the SDS-solubilized fraction was much greater in the extracts obtained from PD tissue than control. The finding suggests that, like tau, synuclein is phosphorylated prior to its aggregation. Miller also reported that he detected phospho-synuclein in blood samples from two patients who have synuclein triplication, and indicated that further studies on blood phospho-synuclein as a possible biomarker are under way.
There may be other molecules that might predict or confirm a diagnosis. Clemens Scherzer reported a proteomics-based approach to find them. Scherzer and colleagues used gene microarrays to get transcriptional profiles of blood samples from PD patients and normal controls. Correcting for blood type and looking for differential expression between normal and PD samples, Scherzer reported that he narrowed the field down from 22,000 potential transcripts to eight that were statistically significant. He reported that using expression values of these eight transcripts allowed him to generate a risk marker score that strongly identified PD patients from a set of 66 subjects that included healthy and disease controls.—Tom Fagan.