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. α-Syn is a key constituent protein of Lewy pathology, and α-syn phosphorylated at serine-129 (pS129) constitutes more than 90% of α-syn in Lewy bodies and hence, it is used extensively as a pathological marker for the aggregated form of α-syn. However, the exact role of pS129 remains controversial as well as the kinase(s) responsible for the phosphorylation. In this study, we investigated the effect of Polo-like kinase 2 (PLK2) inhibition on formation of pS129 using 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 nuclear pS129 level confined in the nuclei. The same finding was replicated in an in-vivo mouse models of templated α-syn aggregation and human dopaminergic neurons, suggesting that PLK2 is more likely to be involved in S129 phosphorylation of soluble non-pathology related fraction of α-syn. We also demonstrated that reduction of nuclear pS129 but not the aggregates specific pS129 following PLK2 inhibition for a short time before sample collection improves the signal to noise ratio when quantifying pS129 aggregate pathology.

Optical pulse labeling studies reveal exogenous seeding slows α-synuclein clearance

“…1b). Recent studies have suggested a useful tool to distinguish between insoluble and soluble pSer129 α-syn is to reduce the levels of soluble pSer129 using the PLK1-3 inhibitor, BI2536 16 . We treated 9 DIV BSCs for 24 h until 10DIV with BI2536 and saw a reduction in soluble pSer129 to further characterize the α-syn properties at this stage (Fig.…”

“…BSCs were treated with 1 μM BI2536 (Aobious Inc, MA, USA), or dimethylsulfoxide (DMSO) control added to the media and 1 μL added on top of the slice, for 24 h from 9-10 DIV or 27-28DIV to reduce levels of soluble pSer129 α-syn and identify any insoluble pSer129 α-syn 16 .…”

Revisiting the specificity and ability of phospho-S129 antibodies to capture alpha-synuclein biochemical and pathological diversity

“…Wakamatsu et al also reported that nuclear aSyn is preferentially phosphorylated compared to cytosolic aSyn 51 . However, most of these studies were based on IHC/ICC and relied on the use of a single pS129 antibody [51][52][53][54][55] (Supplementary Fig. 1).…”

MED13 and glycolysis are conserved modifiers of α-synuclein-associated neurodegeneration

“…155 All the primary antibody dilution was 1:1000 except 1: 20,000 for alpha-tubulin. The primary antibodies used were anti-Skd (gift of Jessica Treissman, 1:1000), anti-Sima (Rb anti-Sima, gift of Pablo Wappner, 1:1000), anti-a-synuclein (Abcam, ab190376, 1:1000), anti-a-synuclein (clone 42, BD Biosciences, BD610787, 1:1000), anti-phospho-a-synuclein Serine 129 (clone D1R1R, Cell Signaling Technology, 23706), 156,157 anti-PGK (Merck, HPA073656, 1:1000), anti-PGI (Merck, HPA024305, 1:1000), anti-LDH (OAAB06120, Aviva Systems Biology, 1:1000) and anti-alpha-Tubulin (DSHB 12G10, 1:20000). 63,147 The images were acquired using ChemiDoc MP (Bio-Rad) and band intensity was analyzed using Image Lab (Bio-Rad), processed in Microsoft Excel, and plotted in GraphPad Prism.…”