Quantifying the Acute Toxicity of Alpha-Synuclein Membrane-Accumulation in a Cell-Based Assay

Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects over 12 million people worldwide. A central pathological feature is the accumulation of aggregated alpha-synuclein (αS) in Lewy bodies and Lewy neurites. Engineered αS variants such as 3K (E35K+E46K+E61K) and KLK (KTKEGV→KLKEGV in 6 repeats) have been shown to enhance membrane binding and aggregation propensity, contributing to cellular toxicity. To further investigate the impact of 3K and KLK on αS biology, we developed a sensitive assay in a human neuroblastoma model to assess expression levels and cytotoxicity. Relative to wild-type αS, the 3K mutant exhibited reduced steady-state expression and increased toxicity, consistent with prior reports. In contrast, the KLK mutant showed no marked change in protein expression but induced significantly higher toxicity, more than the 3K variant. Furthermore, stearoyl-CoA desaturase (SCD) inhibition partially rescued the toxicity of 3K and KLK αS, with 3K showing greater restoration of cell growth than KLK, validating the sensitivity of our live-cell assay to detect pharmacological responses. These findings underscore the utility of our assay in dissecting disease-relevant mechanisms and highlight the potential of engineered αS variants to model pathogenic features of PD. This platform offers a versatile tool for evaluating therapeutic strategies targeting αS aggregation and toxicity in PD and related synucleinopathies.