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

Abstract

Aggregated α-synuclein (α-syn) accumulates in the neuronal Lewy body inclusions in Parkinson's disease and Lewy body dementia. Yet, under non-pathological conditions, monomeric α-syn is hypothesized to exist in an equilibrium between disordered cytosolic- and partially α-helical lipid-bound states: a feature presumably important in synaptic vesicle release machinery. The exact underlying role of α-syn in these processes, and the mechanisms regulating membrane-binding of α-syn remains poorly understood. Herein we demonstrate that PKR kinase can phosphorylate α-syn at several Ser/Thr residues located in the membrane-binding region that is essential for α-syn's vesicle-interactions. α-Syn phosphorylated by PKR or α-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-α-syn mutant displays reduced overall attachment to brain vesicles due to a decrease in vesicle-affinity of the last two thirds of α-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-α-syn inhibits α-syn oligomerization and completely abolishes nucleation, elongation and seeding of α-syn fibrillation in vitro and in cells, and prevents trans-synaptic spreading of aggregated α-syn pathology in organotypic hippocampal slice cultures. Overall, our findings demonstrate that normal and abnormal functions of α-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 α-syn aggregate-related diseases.

α-Synuclein and biological membranes: the danger of loving too much

“…Indeed the bound-unbound equilibrium can be altered by several factors such as the characteristics of the lipid bilayers, including curvature, charge, 51 levels of cholesterol, 63 packing defects and surface hydrophobicity, 41,44,[64][65][66] as well as the properties of aS, including mutations 57 and post-translational modifications. 67 The double-anchor mechanism clarifies a number of experimental observations where mutational variants affecting the membrane affinity of the two individual anchors impairs vesicle clustering, as observed in S. cerevisiae 18 or in aqueous solutions. 20 These investigations also identify a new link between functional and aberrant behaviour of membrane-bound aS.…”

“…αS oligomers were indeed shown to impair the SNARE formation by establishing aberrant interactions with the N-terminal region of synaptobrevin-2. 134 The concomitance of all PD familial variants in the membrane-binding region of αS also indicate alterations in the membrane binding, as demonstrated in vitro , 57,67,135 which may in turn affect the biological behaviour of the protein in the context of SV trafficking. 136,137 Finally, a link between dysfunction and aggregation has been proposed whereby the fibrils growth induces toxic effects by depleting αS monomers, leading to a loss of function of the protein.…”

“…4A ). This balance can be perturbed by external factors such as for example the lipid dyshomeostasis leading to an increase of GM content in the PM 79 as well as calcium bursts, 60 post-translational modifications 67 (particularly Ser 87, 87 Ser 129 88 and Tyr 39 82 ) or αS aggregation. These recent data thereby provide a link between functional and pathological properties of αS in the context of SV trafficking.…”

Structural basis of epitope recognition by anti-alpha synuclein antibodies MJFR14-6-4-2

“…Liposomes were prepared from DMPG and DMPC phospho-lipids as described previously [54]. Liposomes were mixed with α-synuclein in PBS (5 mg/ml liposomes, 0.5 mg/ml alpha-synuclein).…”

The Potential Regulation of A-to-I RNA Editing on Genes in Parkinson’s Disease

“…One regulator of α-synuclein, the highly conserved Argonaute 2 ( AGO2 ), is abnormally expressed in PD patients [ 12 ]. The inflammation-associated serine-threonine kinase ( EIF2AK2 ) regulates α-synuclein by directly phosphorylating the Ser129 residue, linking it to neurodegenerative disorders such as PD [ 13 , 14 ].…”