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

Lasse Reimer; Hjalte Gram; Nanna Møller Jensen; Cristine Betzer; Li Yang; Lorrain Jin; Min Shi; Driss Boudeffa; Giuliana Fusco; Alfonso De Simone; Deniz Kirik; Hilal A Lashuel; Jing Zhang; Poul Henning Jensen

doi:10.1093/pnasnexus/pgac259

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

PNAS Nexus. 2022 Nov 16;1(5):pgac259. doi: 10.1093/pnasnexus/pgac259. eCollection 2022 Nov.

Authors

Lasse Reimer^{1

2}, Hjalte Gram^{1

2}, Nanna Møller Jensen^{1

2}, Cristine Betzer^{1

2}, Li Yang³, Lorrain Jin³, Min Shi³, Driss Boudeffa⁴, Giuliana Fusco⁵, Alfonso De Simone⁶, Deniz Kirik⁷, Hilal A Lashuel⁴, Jing Zhang^{3

8}, Poul Henning Jensen^{1

2}

Affiliations

¹ Danish Research Institute of Translational Neuroscience - DANDRITE, Aarhus University, 8000 Aarhus C, Denmark.
² Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
³ Department of Pathology, University of Washington School of Medicine, Seattle WA 98195, USA.
⁴ Laboratory of Molecular and Chemical Biology of Neurodegeneration, School of Life Sciences Brain Mind Institute, Station 19, 1015 Lausanne, Switzerland.
⁵ Centre for Misfolding Diseases,Department of Chemistry, University of Cambridge, CB2 1EW, UK.
⁶ Department of Pharmacy, University of Naples, 80131, Naples, Italy.
⁷ Brain Repair and Imaging in Neural Systems, Department of Experimental Medical Science, Lund University, 22184 Lund, Sweden.
⁸ Department of Pathology, Zhejiang University School of Medicine and the First Affiliated Hospital, 310003 Hangzhou, China.

Abstract

Aggregated α-synuclein (α-syn) accumulates in the neuronal Lewy body (LB) inclusions in Parkinson's disease (PD) and LB dementia. Yet, under nonpathological 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 Protein kinase R (PKR) 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.