Alpha-synuclein overexpression triggers divergent cellular responses and post-translational modifications in SH-SY5Y and ReNcell VM models

Alpha-synuclein (-syn) overexpression models are widely used to unravel the molecular mechanisms of Parkinsons disease (PD), particularly in light of the dose-dependent transition between its physiological and toxic roles. However, existing systems rely on inducible expression, lack robust dose stratification and comparative cellular contexts. Here, we developed and characterized a panel of stable neuronal cell lines in two human cellular models (SH-SY5Y neuroblastoma cells and ReNcell VM neural progenitors) overexpressing GFP-tagged wild-type (WT) or A53T mutant -syn at low and high overexpression levels. Utilizing this framework, we demonstrated that A53T consistently induces cytotoxicity, oxidative stress and mitochondrial dysfunction in both cell types. In contrast, WT -syn had divergent effects depending on the cellular context. In SH-SY5Y cells, it enhanced mitochondrial function and viability, whereas in ReNcell VM cells, the same protein triggered mitochondrial impairment and elevated oxidative stress. This opposing metabolic response was reflected in increased respiratory activity in SH-SY5Y cells and a marked decline across WT -syn overexpressing ReNcell VM. Importantly, post-translational modification (PTM) landscape of overexpressed WT -syn varied dramatically by cell type. ReNcell VM cells exhibited more robust modifications signatures, even in the absence of overt aggregation, which highlights a cell-type-specific PTM landscape that may contribute to differential vulnerability. Our findings underscore a complex interplay between -syn dosage, mutational status, cellular environment, and PTM profiles highlighting that neuronal vulnerability in PD is context-dependent. This work establishes a modular in vitro platform for dissecting -syn pathology and testing targeted therapeutic strategies grounded in cell-type specificity.