Objective/Rationale:
Phosphorylation of alpha-synuclein at serine 129 is characteristic of Parkinson's disease (PD) and related alpha-synulceinopathies. Unraveling the role of phosphorylation in modulating the physiological and pathogenic activities of alpha-syn requires identification of the kinases and phosphatases involved in regulating its phosphorylation in vivo. Studies from our laboratory demonstrate point to PLK2 and PLK3 as the primary PLKs responsible for alpha-syn phosphorylation. We plan to validate these findings in the pre-clinical model of PD.
Project Description:
In order to test the relationship between PLKs and alpha-syn phosphorylation and toxicity in vivo, vectors co-expressing PLK2 or PLK3 (or their kinase dead mutant) and alpha-syn under the control of two separate human synapsin promoters will be cloned in a pTR-UF20 backbone in order to 1) determine if the coexpression of PLK2 or PLK3 results in an increase in pS129 alpha-syn phosphorylation and 2) test the in vivo effects of increased alpha-syn phosphorylation. By comparing the results obtained from overexpressing both the wild type and kinase-dead mutants, we will be able to determine if the in vivo effects of the PLKs are linked to their kinase activity.
Next, we will create a second group of vectors to test for decreased S129 phosphorylation by employing an shRNA approach. These vectors will express shRNA (or mismatched control, scrPLK2/3) downstream of the H1 promoter targeting PLK2 or PLK3 while still overexpressing human alpha-syn. This approach will enable us to determine if the reducing/blocking the expression of PLK2 or PLK3 results in a significant decrease of pS129 ?-syn phosphorylation and evaluate the in vivo effects of decreased PLK expression and/or alpha-syn phosphorylation in the substantia nigra.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Together, our findings point to PLK2 and PLK3 as the primary PLKs responsible for alpha-syn phosphorylation. The efficiency with which these kinases phosphorylate alpha-syn provides unique opportunities to elucidate the molecular mechanisms and consequences of alpha-syn phosphorylation in vivo. These studies are essential for validating the potential of PLKs as therapeutic targets for treating PD and related synucleinopathies. The identification of the kinases and phosphatases involved in modulating its toxicity in vivo could present viable targets for small molecule-based therapeutic strategies for PD.
Anticipated Outcome:
A detailed molecular understanding of the functional consequences of phosphorylation could reveal new insight into the normal biology of alpha-syn and the mechanisms by which it contributes to neurodegeneration in PD and related disorders. More specifically, the proposed studies will allow us to answer the following questions:
- What is the relative contribution of the PLKs (PLK2 and/or PLK3) to alpha-synuclein phosphorylation and toxicity?
- Can we block alpha-synuclein aggregation and/or toxicity through modulation of its phosphorylation state(s) in vivo, i.e. by controlling the activity of PLK2 and/or PLK3?
Progress Report
These studies are aimed at determining if polo like kinase (PLK2 and PLK3) mediated phosphorylation of alpha-synuclein protects or enhances alpha-synuclein toxicity and if modulating PLK alters this effect. Thus far the investigators have generated PLK and PLK kinase-dead mutant viral vectors and are characterizing the effects in cell culture. Currently the vectors are being tested in pre-clinical models to determine if alpha-synuclein toxicity can be modulated in vivo.