Abstract
Serine 129 (S129) phosphorylation of α-synuclein (αSyn) is a central feature of Lewy body (LB) disease pathology. Although the neighboring tyrosine residues Y125, Y133, and Y136 are also phosphorylation sites, little is known regarding potential roles of phosphorylation cross-talk between these sites and its involvement in the pathogenesis of LB disease. Here, we found that αSyn aggregates are predominantly phosphorylated at Y136 in the Lewy body dementia brain, which is mediated by unexpected kinase activity of Casein kinase 2 (CK2). Aggregate formation with S129 and Y136 phosphorylation of recombinant αSyn (r-αSyn) were induced by CK2 but abolished by replacement of S129 with alanine (S129A) in vitro. Mutation of Y136 to alanine (Y136A) promoted aggregate formation and S129 phosphorylation of r-αSyn by CK2 in vitro. Introduction of Y136A r-αSyn oligomers into cultured cells exhibited increased levels of aggregates with S129 phosphorylation compared to wild-type r-αSyn oligomers. In addition, aggregate formation with S129 phosphorylation induced by introduction of wild-type r-αSyn oligomers was significantly attenuated by CK2 inhibition, which resulted in an unexpected increase in Y136 phosphorylation in cultured cells. Our findings suggest the involvement of CK2-related αSyn Y136 phosphorylation in the pathogenesis of LB disease and its potential as a therapeutic target.
Highlights
Lewy body (LB) diseases, including Lewy body dementia (LBD) and Parkinson’s disease (PD), are progressive diseases characterized by extensive accumulation of intracellular proteinaceous inclusions composed mainly of aggregated α-synuclein in the brain called LB
Insoluble aggregates of αSyn are predominantly phosphorylated on Y136 as well as serine 129 (S129) within the C‐terminal region in the LBD brain Brain lysates from LBD patients were analyzed by SDSPAGE followed by immunoblotting with anti-αSyn antibody, and all exhibited a band at approximately 20 kDa with another band just below 20 kDa likely corresponding to full-length and cleaved αSyn, respectively (Fig. 1b)
Consistent with our previous report [29], these results suggest that the detergent-insoluble multimer of αSyn highly phosphorylated at S129 with molecular weight > 250 kDa is present in the diffuse neocortical LBD (DN-LBD) brain
Summary
Lewy body (LB) diseases, including Lewy body dementia (LBD) and Parkinson’s disease (PD), are progressive diseases characterized by extensive accumulation of intracellular proteinaceous inclusions composed mainly of aggregated α-synuclein (αSyn) in the brain called LB. Earlier in vitro and vivo studies yielded contrasting results regarding the significance of S129 phosphorylation (pS129) for LB formation, showing facilitatory [10, 32], inhibitory [6, 24], or no effect [17, 30] of phosphorylation on αSyn aggregation. ΑSyn appears to be phosphorylated at Y125 in the human brain, but the role of pY125 in αSyn aggregate formation has not been fully elucidated. Immunoblotting analysis indicated the presence of phosphorylated Y133 (pY133) at similar levels in LBD, PD, and control brains [9], suggesting that pY133 may not be crucial for LB pathology. There have been few studies regarding the presence of phosphorylated Y136 (pY136) in the human brain and its physiological roles and implications in the pathogenesis of LB disease
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