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of the SNCA gene could be found in an extensive investigation of primate brain specimens (M.G. Schlossmacher, unpublished data).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,...  Read more

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of the SNCA gene could be found in an extensive investigation of primate brain specimens (M.G. Schlossmacher, unpublished data).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:
El-Agnaf, O. M. A., Salem, S. A., Paleologou, K. E., Cooper, L. J., Fullwood ,N. J., Gibson, M. J., Curran, M. D., Court, J. A., Mann, D. M. A., Ikeda, S. I., Cookson, M. R., Hardy, J., and Allsop, D. (2003a). α-Synuclein implicated in Parkinson’s disease is present in extracellular biological fluids, including human plasma. FASEB J 17, 1315-1317. El-Agnaf, O. M. A., Walsh, D. M., and Allsop, D. (2003b). Soluble oligomers for the diagnosis of neurodegenerative diseases. Lancet (Neurol.); 2, 461-462. El-Agnaf, O. M. A., Paleologou, K. E., Greer, B., Abogrein, A. M., King, J. E., Salem, S. A., Fullwood, N. J., Benson, F. E., Hewitt, R., Ford, K. J., Martin, F. L., Harriott, P., Cookson, M. R. and Allsop, D. (2004). A strategy for designing inhibitors of α-synuclein aggregation and toxicity as a novel treatment for Parkinson's disease and related disorders. FASEB J. 18, 315-317. El-Agnaf, O. M. A., Salem, S. A., Paleologou, K. E., Curran, M.D., Gibson, M. J., Court, J. A., Michael, S. G. and Allsop, D. (2005). Detection of oligomeric forms of α-synuclein protein in cerebrospinal fluid and plasma as a potential biomarker for Parkinson’s disease. FASEB J. (In press). Lee, H. J., Patel, S. And Lee, S. J. (2005). Intravesicular localization and exocytosis of alpha-synuclein and its aggregates. J Neurosci; 25:6016-24.

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