Final Report Summary - PLKS IN PD (Elucidating the Role of Phosphorylation by Polo-like kinases in Modulating Alpha-Synuclein Aggregation and Toxicity in Parkinson's disease and Related Disorders)
The objective of this proposal is to elucidate the role of phosphorylation in modulating alpha-synuclein (a-syn) aggregation and toxicity in Parkinson's disease (PD) and other related disorders. This was done using cellular and animal models of synucleopathies. We focused on the implication of Polo-like kinases (Plks), a family of serine/threionin kinases, in the phosphorylation of a-syn. We sought to understand the molecular mechanism by which the phosphorylation, by this family of kinases, modulates a-syn aggregation and neurotoxicity in vitro and in vivo. More specifically, we focused on the following specific aims:
Aim one: To characterise the in vitro phosphorylation of members of the synuclein family (a, b, g) by the Polo-like kinases (Plk1, Plk2, Plk3 and Plk4), using a combination of biochemical, mass spectrometry and cell culture approaches.
Aim two: To elucidate the structural basis of Plks-syn interactions in vitro.
Aim three: To determine the relationship between a-syn phosphorylation by Plks and a-syn aggregation and/or neurotoxicity in vivo. Towards this goal we will take two different approaches based on:
a) Performing comparative studies to determine if there is a correlation between the localisation and expression levels of Plks with that of a-syn and S129 phosphorylation in synucleinopathy diseased brains.
b) AAV-mediated Co-expression of a-syn (WT and mutants) and Plks directly in the substantia nigra of rat models of PD.
Aim one: Our data showed that only PLK1, two and three, but not PLK4, are able to phosphorylate a-syn in vitro. Moreover, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis demonstrated that a-syn phosphorylation occured at a single residue, Ser129. Furthermore, we demonstrated, for the first time, that PLK2 and PLK3 are also able to phosphorylate beta-syn at the residue Ser118. We confirmed theses data in cell culture and our data demonstrate that a-syn is quantitatively phosphorylated by PLK1, two and three in vitro and in cell culture based assays.
Aim two: To study the protein-protein interaction of a-syn and PLK2, we used a biochemical approach based on co-immunoprecipitation of both proteins. We showed that, after cross-linking in cells we could immunopreciptate PLK2 with a-syn. This observation demonstrates that a protein-protein interaction exists between PLK2 and a-syn in vivo. Moreover, we showed the Polo-box domain, a domain implicated in protein-protein interactions between PLKs and their substrates, is not required for a-syn phosphorylation. This data demonstrates that PLK2 kinase domain alone is sufficient to phosphorylate a-syn.
Aim three: To determine the relevance of Plks interactions and phosphorylation in the normal biology and pathogenesis of the a-syn, we performed a comparative study on the expression levels and co-localisatoin of Plks and a-syn in normal and diseased brains. Using antibodies against Plks and phosphoryalated a-syn, we showed that the levels of PLK2 and PLK3 were significantly increased in synucleinopathies disesased brains, including Alzheirme's disease, Lewy Body Disease and Multiple Sytem Atrophy cases. The presence and levels of Plks, and WT and phosphorylated forms of a-syn were determined in cytosolic, membrane and nuclear fractions. These studies allowed us to determine if the levels of Plks in each fraction correlates with the levels of phosphorylated syn and/or specific form of synucleiopathies. Moreover, we showed also a significant increase of PLK2 and PLK3 in transgenic mice model of synucleinopathies. Interstingly, PLK2 and three co-localise with phosphorylated a-syn in hippocampus, cortex and midbrain. Together, these observations demonstrate that PLK2 and PLK3 levels are increased in synucleinopathies and co-localise with phosphoryated a-syn at ser129.
Our effort now is focused in overexpressing a-syn and PLK2 in a genetic rat model of PD and studying the cellular and behavioral effect of a-syn phosphorylation in vivo.
Aim one: To characterise the in vitro phosphorylation of members of the synuclein family (a, b, g) by the Polo-like kinases (Plk1, Plk2, Plk3 and Plk4), using a combination of biochemical, mass spectrometry and cell culture approaches.
Aim two: To elucidate the structural basis of Plks-syn interactions in vitro.
Aim three: To determine the relationship between a-syn phosphorylation by Plks and a-syn aggregation and/or neurotoxicity in vivo. Towards this goal we will take two different approaches based on:
a) Performing comparative studies to determine if there is a correlation between the localisation and expression levels of Plks with that of a-syn and S129 phosphorylation in synucleinopathy diseased brains.
b) AAV-mediated Co-expression of a-syn (WT and mutants) and Plks directly in the substantia nigra of rat models of PD.
Aim one: Our data showed that only PLK1, two and three, but not PLK4, are able to phosphorylate a-syn in vitro. Moreover, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) analysis demonstrated that a-syn phosphorylation occured at a single residue, Ser129. Furthermore, we demonstrated, for the first time, that PLK2 and PLK3 are also able to phosphorylate beta-syn at the residue Ser118. We confirmed theses data in cell culture and our data demonstrate that a-syn is quantitatively phosphorylated by PLK1, two and three in vitro and in cell culture based assays.
Aim two: To study the protein-protein interaction of a-syn and PLK2, we used a biochemical approach based on co-immunoprecipitation of both proteins. We showed that, after cross-linking in cells we could immunopreciptate PLK2 with a-syn. This observation demonstrates that a protein-protein interaction exists between PLK2 and a-syn in vivo. Moreover, we showed the Polo-box domain, a domain implicated in protein-protein interactions between PLKs and their substrates, is not required for a-syn phosphorylation. This data demonstrates that PLK2 kinase domain alone is sufficient to phosphorylate a-syn.
Aim three: To determine the relevance of Plks interactions and phosphorylation in the normal biology and pathogenesis of the a-syn, we performed a comparative study on the expression levels and co-localisatoin of Plks and a-syn in normal and diseased brains. Using antibodies against Plks and phosphoryalated a-syn, we showed that the levels of PLK2 and PLK3 were significantly increased in synucleinopathies disesased brains, including Alzheirme's disease, Lewy Body Disease and Multiple Sytem Atrophy cases. The presence and levels of Plks, and WT and phosphorylated forms of a-syn were determined in cytosolic, membrane and nuclear fractions. These studies allowed us to determine if the levels of Plks in each fraction correlates with the levels of phosphorylated syn and/or specific form of synucleiopathies. Moreover, we showed also a significant increase of PLK2 and PLK3 in transgenic mice model of synucleinopathies. Interstingly, PLK2 and three co-localise with phosphorylated a-syn in hippocampus, cortex and midbrain. Together, these observations demonstrate that PLK2 and PLK3 levels are increased in synucleinopathies and co-localise with phosphoryated a-syn at ser129.
Our effort now is focused in overexpressing a-syn and PLK2 in a genetic rat model of PD and studying the cellular and behavioral effect of a-syn phosphorylation in vivo.