Novel PD Tools: Generation of Posttranslationally Modified Alpha-synuclein

Objective/Rationale:             
There is increasing evidence linking specific posttranslational modification (folding, cutting, etc. of proteins before maturation) and alpha-synuclein aggregation and lewy body (abnormal protein aggregates in nerve cells) formation. Yet little is still known concerning the effect of these modifications on the physiopathologic properties of alpha-synuclein and the relative contribution of each modification in the pathogenesis of Parkinson’s disease (PD).  For many of these modifications, the enzymes involved remain unknown and/or achieving site-specific modification in vitro or in vivo has not been possible. This knowledge gap has significantly limited our ability to quantitatively assess the levels of these modifications in vivo, and the relationship between these modifications and disease progression.

Project Description:             
The primary objective of this project is to support The Michael J. Fox Foundation’s effort, in collaboration with Proteos Inc. and Biomer Technology, to transfer the technology developed by our group to introduce single or multiple disease-associated, site-specific modifications in alpha-synuclein. This effort will scale up the production of three disease-associated modified phosphorylated (pS129 and pY125) and nitrated (nY125) forms of alpha-synuclein. We will carry out detailed studies to validate and characterize these modified proteins. In addition, we will develop novel non-hydrolyzable analogues of phosphorylated alpha-synuclein to facilitate studies to elucidate the role of these modifications in the pathogenesis of PD. Finally, we will work with the Foundation to make all these reagents available to researchers for the development of assays, for biomarker research, and as standards for cellular and in vivo studies.

Relevance to Diagnosis/Treatment of Parkinson’s Disease:                     
The availability of these proteins is crucial for advancing current research programs aimed at 1) the characterization of post-translational modifications in humans and in pre-clinical models of PD; 2) biomarkers research, target identification and drug discovery research; 3) development of approaches for detection and quantitative assessment of these modifications in vivo and in biological fluids (e.g. ELISA or LUminx-based). Indeed, the lack of such tools has been a major obstacle for advancing these efforts and enabling quantitative understanding of the relationship between specific post-translational modifications and PD progression and pathology.

Anticipated Outcome:          
The availability of these reagents is crucial to unraveling the roles of post-translational modifications in regulating the structure, aggregation and toxicity of alpha-synuclein in PD. In addition, these research tools will enable us and others in the field to address key mechanistic questions that cannot be addressed using existing tools, including:

1. What percentage of alpha-synuclein is modified in vivo and is there a correlation between the level of modified alpha-synuclein and disease progression?
2. Do alpha-synuclein modifications occur before and/or after alpha-synuclein aggregation and lewy body formation?
3. Is there cross-talk between the different post-translational modifications in alpha-synuclein?
4. What are the natural enzymes involved in regulating each of these modification?
5. What is the effect of each modification on the stability, degradation and functional properties of alpha-synuclein?
6. How do these disease-associated modifications alter alpha-synuclein interactions with other proteins and ligands?