In vivo modulation of polo-like kinases supports a key role for PLK2 in Ser129 α-synuclein phosphorylation in mouse brain

Abstract

α-Synuclein is the major component of Lewy bodies. α-Synuclein phosphorylated at Ser 129 (Phospho-α-Syn) is the most common synuclein modification observed in Parkinson's disease pathology and transgenic animal models. Polo-like kinase 2 (PLK2) was previously proposed as an important kinase in α-synuclein phosphorylation at Ser129. To better understand the role of PLK2 in α-synuclein phosphorylation in vivo, we further evaluated the effect of PLK2 genetic knockdown and pharmacological inhibition on Phospho-α-Syn levels in different brain regions of PLK2 knockout (KO), heterozygous (Het) and wild-type (WT) mice. Whereas PLK2 knockdown had no effect on Total-α-synuclein brain levels, it resulted in a gene-dosage dependent, albeit incomplete, reduction of endogenous Phospho-α-Syn levels in all brain regions investigated. No compensatory induction of other α-synuclein kinases (PLK3, casein kinase-2, G-protein-coupled receptor kinase 5 (GRK5) and GRK6) was observed at the mRNA level in the PLK2 KO mouse brain. To determine whether increased activity of another PLK family member is responsible for the residual Phospho-α-Syn levels in the PLK2 KO mouse brain, the pan-PLK inhibitor BI 2536 was tested in PLK2 KO mice. Whereas BI 2536 reduced Phospho-α-Syn levels in WT mice, it did not further reduce the residual endogenous Phospho-α-Syn levels in PLK2 KO and Het mice, suggesting that a kinase other than PLK1-3 accounts for the remaining PLK inhibitor-resistant pool in the mouse brain. Moreover, PLK3 KO in mice had no effect on both Total- and Phospho-α-Syn brain levels. These results support a significant role for a PLK kinase in phosphorylating α-synuclein at Ser129 in the brain, and suggest that PLK2 is responsible for this activity under physiological conditions.

Keywords: 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; ANOVA; BI 2536; CEB; CHAPS; CSNK2a2; Ce; Co; EDTA; EGTA; ELISA; G-protein-coupled receptor kinase; GRK; GusB; HRP; Het; Hi; IV; IgG; IgM; KO; Ob; PBS; PCR; PD; PLK; PLK2; Parkinson’s disease; Phospho-α-Syn; Po; SDS; SNCA; St; TBST; TH; Total-α-Syn; Tris-buffered saline with Tween; VM; WT; alpha prime subunit of casein kinase II; analysis of variance; brain; cell extraction buffer; cerebellum; cerebral cortex; enzyme-linked immunosorbent assay; ethylene glycol tetraacetic acid; ethylenediaminetetraacetic acid; heterozygous; hippocampus; horseradish peroxidase; immunoglobulin G; immunoglobulin M; intravenous; knockout; olfactory bulb; phosphate-buffered saline; phosphorylated α-Synuclein; polo-like kinase; polymerase chain reaction; pons-medulla; qRT-PCR; quantitative real-time reverse-transcription PCR; sodium dodecyl sulfate; striatum; synuclein; thalamus and hypothalamus; total α-synuclein; ventral midbrain; wild-type; α-synuclein mRNA; β-glucuronidase.