Protein kinase R dependent phosphorylation of -synuclein regulates its membrane binding and aggregation

Aggregated alpha-synuclein (alpha-syn) accumulates in the neuronal Lewy body (LB) inclusions in Parkinson's disease (PD) and LB dementia. Yet, under nonpathological conditions, monomeric alpha-syn is hypothesized to exist in an equilibrium between disordered cytosolic- and partially alpha-helical lipid-bound states: a feature presumably important in synaptic vesicle release machinery. The exact underlying role of alpha-syn in these processes, and the mechanisms regulating membrane-binding of alpha-syn remains poorly understood. Herein we demonstrate that Protein kinase R (PKR) can phosphorylate alpha-syn at several Ser/Thr residues located in the membrane-binding region that is essential for alpha-syn's vesicle-interactions. alpha-Syn phosphorylated by PKR or alpha-syn isolated from PKR overexpressing cells, exhibit decreased binding to lipid membranes. Phosphorylation of Thr64 and Thr72 appears as the major contributor to this effect, as the phosphomimetic Thr64Glu/Thr72Glu-alpha-syn mutant displays reduced overall attachment to brain vesicles due to a decrease in vesicle-affinity of the last two thirds of alpha-syn's membrane binding region. This allows enhancement of the "double-anchor" vesicle-binding mechanism that tethers two vesicles and thus promote the clustering of presynaptic vesicles in vitro. Furthermore, phosphomimetic Thr64Glu/Thr72Glu-alpha-syn inhibits alpha-syn oligomerization and completely abolishes nucleation, elongation, and seeding of alpha-syn fibrillation in vitro and in cells, and prevents trans-synaptic spreading of aggregated alpha-syn pathology in organotypic hippocampal slice cultures. Overall, our findings demonstrate that normal and abnormal functions of alpha-syn, like membrane-binding, synaptic vesicle clustering and aggregation can be regulated by phosphorylation, e.g., via PKR. Mechanisms that could potentially be modulated for the benefit of patients suffering from alpha-syn aggregate-related diseases.