Peptide Biomarkers for Parkinson’s Disease
This proposal is designed to meet several major challenges of current biomarker research, specifically: 1) significant variations associated with antibody-based protein assays, 2) low sensitivity and specificity of blood based markers, and 3) detection of PD at early stages. To address the problems of antibody-based assays, the strategy used in this proposal will focus on the development of targeted mass spectrometry-based techniques, such as selected reaction monitoring (SRM), to validate peptide markers derived from proteins either replicated in previous proteomics profiling, or known to be critical to PD pathogenesis, e.g., α-synuclein, parkin and LRRK2 from cerebrospinal fluid and plasma-derived exosomes.
Objective, reliable, and reproducible biomarkers are clearly needed to assist with accurate diagnosis of Parkinson disease (PD), especially at early stages, as well as for facilitating differential diagnosis and disease monitoring. Targeted mass spectrometry-based techniques, such as selected reaction monitoring (SRM), will be used to identify unique peptide markers derived from proteins either showing promise in previous proteomics profiling, or known to be critical to PD pathogenesis, e.g., α-synuclein, parkin and LRRK2, in human cerebrospinal fluid (CSF). To facilitate discovery and validation of blood based biomarkers, a specific population of central nervous system derived plasma exosomes, the cargo-carrying microvesicles recognized recently to transport biomolecules among different cells or organ systems, will be isolated before SRM analysis. The unique peptide markers will be tested in several large, well-established cohorts, e.g., Udall Centers affiliated with the University of Washington and University of Pennsylvania and PPMI (Parkinson Progression Marker Initiative), with cross-sectional and longitudinal samples collected, along with extensive clinical characterization. Finally, to improve early diagnosis, we will make use of two cohorts consisting of subjects at elevated risk for PD (i.e., asymptomatic subjects with LRRK2 mutations or anosmia/hyposmia), with the goal of discovering biomarkers capable of identifying subjects with early or premotor PD. The studies designed for this project, if successful, have the potential to result in a panel(s of biomarkers that are robust, with less variation than can currently be achieved, and in a body fluid that is readily accessible in a regular clinical setting. Markers for early diagnosis and progression of PD are critical in understanding how to arrest or slow PD progression.
Goals of this project:
1. SRM assay development and optimization for the following protein/peptide biomarker candidates in CSF: α-synuclein (α-syn total and phosphorylated or other post-translationally modified peptide species), tau (total and phosphorylated), DJ-1, LRRK2, PINK1, parkin, amyloid beta peptide 1-42 (Aβ42). Flt3 ligand, fractalkine and neurofilament light chain
2. Expansion of CSF protein/peptide discovery/replication by mass spectrometry through use of additional CSF samples from U Washington/U Pennsylvania Udall Center and PDBP (baseline) cohorts. The 5 protein/peptide CSF biomarker panel (SPP1, LRP1, CSF1R, VEPHA4, and TIMP1) will be further tested and validated
3. Replicate a panel of five glycopeptides (derived from PRNP, HSPG2, MEGF8, NCAM1, and ICAM1) in an independent cohort of blood samples from UW/UPenn and the PDBP at baseline
4. SRM assay optimization for plasma exosomal alpha-synuclein measurements
5. Proteomic screening for plasma exosomal proteins/peptides other than alpha-syn and SRM assay development for the identified peptides
6. Replication and validation of PD progression and early PD biomarker candidates in longitudinal CSF samples from PDBP and PPMI cohorts.