A Dual Assay to Compare Protein Levels and Toxicity of Alpha-Synuclein Variants: Acute Expression of Wild-Type versus S129A

Parkinson’s disease (PD) affects over 12 million people worldwide and has the fastest-growing global impact. The pathological hallmark is the presence of Lewy bodies and Lewy neurites, which are intraneuronal lesions enriched in aggregated alpha-synuclein (αS) that is typically hyper-phosphorylated at serine 129. Therefore, lowering phosphoserine 129 (pS129) may be a viable therapeutic strategy to treat PD. However, pS129 has also been proposed to regulate synaptic transmission and αS degradation. In both cases, inhibiting pS129 could be detrimental. Here, we developed a sensitive assay in a human neuroblastoma model and utilized it to assess the relative expression levels and cytotoxicity of pS129 by comparing αS wild-type (WT) vs. S129A (pS129-deficient). We show that the S129A mutant does not affect the acute expression levels or toxicity of αS in a transient transfection paradigm. This provides new insight into the intricate interplay between αS, phosphorylation, toxicity, and degradation. Our assay provides a versatile platform for understanding disease-relevant mechanisms and opens novel avenues for the design of future therapeutic interventions in PD and other α-synucleinopathies.