Aggregation inhibits alpha-synuclein dephosphorylation resulting in the observation of pathological enrichment

Abstract

AbstractAlpha-synuclein is an intrinsically disordered protein that aggregates in the brain in several neurodegenerative diseases collectively called synucleinopathies. Phosphorylation of alpha-synuclein (αsyn) at serine 129 (PSER129) was considered rare in the healthy human brain but is enriched in pathological αsyn aggregates and is used as a specific marker for disease inclusions. However, recent observations challenge this assumption by demonstrating that PSER129 results from neuronal activity and can be readily detected in the non-diseased mammalian brain. Here, we tested experimental conditions under which two distinct PSER129 pools, endogenous-PSER129 and aggregated-PSER129, could be detected and differentiated in the brain. Results showed that in the mouse brain, perfusion fixation conditions greatly influenced detection of endogenous-PSER129, with endogenous-PSER129 being nearly undetectable after delayed perfusion fixation (30min and 1hr post-mortem interval). Exposure to anesthetics (e.g., ketamine and xylazine) before perfusion did not significantly influence endogenous-PSER129 detection or levels. In situ, non-specific phosphatase (i.e., calf alkaline phosphatase, CIAP) selectively dephosphorylated endogenous-PSER129 while preformed fibril-seeded aggregates and genuine disease aggregates (Lewy pathology and Papp–Lantos bodies in Parkinson’s disease and multiple systems atrophy brain, respectively) were resistant to CIAP-mediated dephosphorylation. Phosphatase resistance of aggregates was abolished by sample denaturation. We conclude that in the mammalian brain, there is an abundant steady state of endogenous-PSER129, and PSER129 association with pathology is, at least partially, due to dephosphorylation resistance of αsyn aggregates. Our findings have implications for the mechanism of PSER129-accumulation in the synucleinopathy brain.