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 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.

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  1. α-Synuclein (α-syn) is mainly expressed by neuronal cells, and is generally considered to exist as a cytoplasmic protein. However, our recent studies have shown that neuronal cells in culture constitutively secrete α-syn into the culture medium, and that α-syn is normally present in CSF and peripheral plasma (El-Agnaf et al., 2003a). Our findings suggest that cells normally secrete α-syn into their surrounding media, both in vitro and in vivo. Future cell biological research will have to address the mechanism by which α-syn protein can be found in the extracellular space under physiological conditions (El-Agnaf et al., 2003a; Lee et al., 2005), since no alternative splice variant has been published to date that would direct the nascent α-syn protein into and through the secretary pathway, and no such transcript of the SNCA gene could be found in an extensive investigation of primate brain specimens (M.G. Schlossmacher, unpublished data). We have suggested that the detection of extracellular α-syn and/or its modified forms in body fluids, particularly in peripheral plasma, offers new opportunities for the development of diagnostic tests for PD and related diseases (El-Agnaf et al., 2003a). The mounting evidence for soluble oligomers being the pathogenetic species that drive neurodegeneration and neuronal cell death, led us to hypothesize that the detection of soluble oligomers of α-syn in biological fluids could have potential use as a biomarker for PD and related diseases (El-Agnaf et al., 2003b). Therefore, we developed a simple and novel ELISA method that specifically recognizes only oligomeric species of α-syn. We employed this method to probe for oligomeric forms of α-syn in human CSF and in plasma. Interestingly, based on our preliminary results, the ELISA was able to detect α-syn oligomers in post mortem CSF from some PD and DLB patients, but, in contrast, only a very low signal was obtained from all control samples tested. These data suggested a higher amount of α-syn oligomer production in PD patients either in vivo or during post-mortem autolysis (El-Agnaf et al., 2005). These promising results led us to carry out a more extensive study on the more accessible peripheral blood plasma. We found that there was a highly statistically significant difference between PD samples and controls, with most of the PD samples giving high signals, whereas only a few control samples gave a high signal (El-Agnaf et al., 2005). If α-syn oligomerization occurs before the death of nigral neurons in PD, then our ELISA could potentially provide a diagnostic tool for the detection of oligomers in the early stages of the disease. This could lead to earlier detection and neuroprotective treatment intervention for high-risk subjects in the future. More extensive clinical studies will be required to confirm and extend our results and to validate the ELISA as a potential diagnostic test for disease state. As a matter of fact, in collaboration with M.G. Schlossmacher we recently began enrollment of subjects in a prospective, case-control study of 300 persons to be conducted over two years to monitor their oligomeric and total α-syn load in peripheral plasma, this study is funded by Michael J. Fox Foundation. Furthermore, it will be interesting to determine if there is any correlation between the detection of α-syn oligomers and the severity and/or the stage of the disease and/or the rate of its progression. Studies performed on blood samples from familial PD cases will be useful to further validate the ELISA as an early diagnostic method. We also recognize that any medication including dopaminergic replacement therapy, taken by previously diagnosed PD patients could influence the ELISA results. Our novel ELISA can also be used for high-throughput screening for modulators of α-syn oligomerization as potential novel drugs for PD and related disorders during preclinical validation studies, prior to their evaluation in rodent or nonhuman primate models of PD (El-Agnaf et al., 2004). Moreover, the principle of our ELISA could be applied for the development of similar sensitive diagnostic tests for the presence of other forms of oligomeric protein aggregates, such as those found in AD (Aβ and tau) and the TSEs (PrP).

    References:

    . Alpha-synuclein implicated in Parkinson's disease is present in extracellular biological fluids, including human plasma. FASEB J. 2003 Oct;17(13):1945-7. PubMed.

    . A strategy for designing inhibitors of alpha-synuclein aggregation and toxicity as a novel treatment for Parkinson's disease and related disorders. FASEB J. 2004 Aug;18(11):1315-7. PubMed.

    . Detection of oligomeric forms of alpha-synuclein protein in human plasma as a potential biomarker for Parkinson's disease. FASEB J. 2006 Mar;20(3):419-25. PubMed.

    . Intravesicular localization and exocytosis of alpha-synuclein and its aggregates. J Neurosci. 2005 Jun 22;25(25):6016-24. PubMed.

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References

News Citations

  1. Sorrento: ADNI Imagines the Future of AD Imaging
  2. Synuclein and Parkinson's—It's All in the Dose

Other Citations

  1. ARF related Live Discussion

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