Polo-like kinase 2 inhibition reduces serine-129 phosphorylation of physiological nuclear alpha-synuclein but not of the aggregated alpha-synuclein

Abstract

Accumulation of aggregated alpha-synuclein (α-syn) is believed to play a pivotal role in the pathophysiology of Parkinson’s disease (PD) and other synucleinopathies. As a key constituent of Lewy pathology, more than 90% of α-syn in Lewy bodies is phosphorylated at serine-129 (pS129) and hence, it is used extensively as a marker for α-syn pathology. However, the exact role of pS129 remains controversial and the kinase(s) responsible for the phosphorylation have yet to be determined. In this study, we investigated the effect of Polo-like kinase 2 (PLK2) inhibition on formation of pS129 using an ex vivo organotypic brain slice model of synucleinopathy. Our data demonstrated that PLK2 inhibition has no effect on α-syn aggregation, pS129 or inter-neuronal spreading of the aggregated α-syn seen in the organotypic slices. Instead, PLK2 inhibition reduced the soluble pS129 level in the nuclei. The same finding was replicated in an in vivo mouse model of templated α-syn aggregation and in human dopaminergic neurons, suggesting that PLK2 is more likely to be involved in S129-phosphorylation of the soluble physiological fraction of α-syn. We also demonstrated that reduction of nuclear pS129 following PLK2 inhibition for a short time before sample collection improves the signal-to-noise ratio when quantifying pS129 aggregate pathology.

Pathogenic Impact of α-Synuclein Phosphorylation and Its Kinases in α-Synucleinopathies

“…Furthermore, treatment with a designed highly selective inhibitor for PLK2, compound 37, reduced α-synuclein phosphorylation in rat brain [ 108 , 109 ]. However, the inhibitor had no effect on α-synuclein aggregation, p-Ser129, or inter-neuronal spreading of the aggregated α-synuclein in the S129A preformed fibril injection model [ 110 ]. These findings suggest that PLK2 could be a potential target for the development of novel therapies, although further optimization is still required at the current stage ( Figure 4 ).…”

“… Schematic pathways of α-synuclein phosphorylation by kinases in the neurodegeneration process and potential pharmacotherapeutic targets [ 56 , 104 , 106 , 107 , 108 , 109 , 110 ]. NO: nitric oxide; NOS: NO synthase; ROS: reactive oxygen species; L-NNA: N(omega)-nitro-L-arginine, a competitive inhibitor of nitric oxide synthase; GRK: G-protein-coupled receptor kinase; CKII: casein kinase II; PLK: polo-like kinase; PPX: pramipexole.…”

Polo-Like Kinase 2: From Principle to Practice

“…Phosphorylation at Ser129 also regulates the inhibition of TH by α-Syn; PLK2 reduces its ability to inhibit TH or activate PP2A by phosphorylation of α-Syn ( 126 ). PLK2 mainly phosphorylates soluble α-Syn ( 151 ); Inhibition of PLK2 triggers autophagic elimination of α-Syn ( 152 ). Oxidative stress might play a key role in PLK2 phosphorylation of α-Syn, and antioxidant NAC could completely block iron-induced up-regulation of PLK2, CK2 and p-Ser129 α-Syn ( 140 ); However, as PLK2 induces elevation of α-Syn in copper-treated SHSY5Y neuroblastoma cells, both PP2A level and oxidative status remains unchanged ( 153 ).…”

Neighbouring modifications interfere with the detection of phosphorylated alpha-synuclein at Serine 129: Revisiting the specificity of pS129 antibodies

“…Wakamatsu et al also reported that nuclear aSyn is preferentially phosphorylated compared to cytosolic aSyn 50 . However, most of these studies were based on immunohisto-/cytochemistry and relied on the use of a single pS129 antibody [50][51][52][53][54] (Figure S1). Furthermore, the cross-reactivity of pS129 antibodies towards nuclear proteins has not been systematically assessed.…”