Dynamic reversibility of α‐synuclein serine‐129 phosphorylation is impaired in synucleinopathy models.

α‐Synuclein phosphorylation at serine‐129 (pS129) is a widely used surrogate marker of pathology in Parkinson's disease and other synucleinopathies. However, we recently demonstrated that phosphorylation of S129 is also a physiological activator of synaptic transmission. In a feed‐forward fashion, neuronal activity triggers reversible pS129. Here, we show that Parkinson's disease‐linked missense mutations in SNCA impact activity‐dependent pS129. Under basal conditions, cytosol‐enriched A30P, H50Q, and G51D mutant forms of α‐synuclein exhibit reduced pS129 levels in rat primary cortical neurons. A53T pS129 levels are similar to wild‐type, and E46K pS129 levels are higher. A30P and E46K mutants show impaired reversibility of pS129 after stimulation. For the engineered profoundly membrane‐associated α‐synuclein mutant "3K" (E35K + E46K + E61K), de‐phosphorylation was virtually absent after blocking stimulation, implying that reversible pS129 is severely compromised. Importantly, pS129 excess resulting from proteasome inhibition is also associated with reduced reversibility by neuronal inhibition, kinase inhibition, or phosphatase activation. Our findings suggest that perturbed pS129 dynamics are probably a shared characteristic of pathology‐associated α‐synuclein, with possible implications for synucleinopathy treatment and diagnosis. Synopsis: Perturbed phospho‐S129 dynamics are a shared characteristic of various models of α‐synuclein dyshomeostasis, with possible implications for synucleinopathy treatment and diagnosis.α‐Synuclein solubility and pS129 accumulation are inversely proportional.α‐Synuclein dyshomeostasis impairs the reversibility of polo‐like‐kinase 2 (Plk2)‐ and protein phosphatase 2A (PP2A)‐modulated activity‐dependent pS129.Impaired pS129 reversibility is likely to be an early pathological change that precedes α‐synuclein aggregation. [ABSTRACT FROM AUTHOR]