Objective/Rationale:
Diagnosis of Parkinson’s disease would be facilitated by the discovery of objective biomarkers for identifying it at early stages and differentiating it from other related disorders. Cerebrospinal fluid (CSF) level of alpha-synuclein, a key component of Lewy bodies, is among the most promising candidates to date, but does not correlate with disease severity, or differentiate between parkinsonian disorders. Based on the recent discovery that monitoring the level of a modified form of alpha-synuclein, ps129, improves performance of this biomarker, we will examine other modified forms in CSF and develop assays to measure them, laying the foundation for studies to determine whether they may serve as improved biomarkers for Parkinson’s disease.
Project Description:
We will first determine whether two modified forms of alpha-synuclein, ps87 and py125 are present in human CSF, by isolating them using specifically targeted antibodies, and identifying them by mass spectrometry. We will then develop and optimize antibody-based Luminex assays for each candidate. Finally, using the optimized assays, we will examine the circadian variation of the CSF levels of each candidate, including ps129, for which an assay has already been developed.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Currently, diagnosis of Parkinson’s disease, particularly at early stages, is complicated by its non-specific symptoms. Discovery of biomarkers that are sensitive and specific for Parkinson’s disease would allow earlier and more precise diagnosis, which would improve the potential to begin treatments at the earliest stages, when neuron-sparing therapies would be most useful, and to improve studies of new treatments by identifying appropriate patient cohorts.
Anticipated Outcome:
By the end of this study, we expect to have optimized assays for modified forms of alpha-synuclein in human CSF, as well as an understanding of how the levels of these biomarker candidates vary over the course of a day. These tools will then be available for large studies to determine their usefulness for diagnosing Parkinson’s disease.
Final Outcome
We identified both phosphorylated alpha-synuclein species (pS87 and pY125) in cerebrospinal fluid, using mass spectrometry. Our current technique is able to reliably identify the phosphorylated peptides in small samples, requiring <100 ul of starting material. These peptides are currently being validated using labeled standards to confirm the identity of the detected peptides. We have developed assays capable of detecting the proteins in CSF, and carried out a variety of experiments establishing their specificity for the phosphorylated proteins. However, sensitivity and accuracy of these assays are not yet fully validated. We are investigating adopting alternative assay techniques to improve the results of these assays.