Imaging flow cytometry reveals LPS-induced changes to intracellular intensity and distribution of α-synuclein in a TLR4-dependent manner in STC-1 cells

[Display omitted] •Gastrointestinal α-synuclein aggregation and the microbiome are linked to Parkinson’s disease.•α-synuclein in enteroendocrine cells of the gut epithelium is understudied.•The effect of microbial lipopolysaccharide on STC-1 cells was assessed using confocal microscopy and imaging flow cytometry.•Imaging flow cytometry showed subtle changes to intracellular α-synuclein localisation.•Complementary use of these techniques is encouraged for further studies of α-synuclein function in the gut. Parkinson’s disease is a chronic neurodegenerative disorder, where pathological protein aggregates largely composed of phosphorylated α-synuclein are implicated in disease pathogenesis and progression. Emerging evidence suggests that the interaction between pro-inflammatory microbial factors and the gut epithelium contributes to α-synuclein aggregation in the enteric nervous system. However, the cellular sources and mechanisms for α-synuclein pathology in the gut are still unclear. The STC-1 cell line, which models an enteroendocrine population capable of communicating with the gut microbiota, immune and nervous systems, was treated with a TLR4 inhibitor (TAK-242) prior to microbial lipopolysaccharide (LPS) exposure to investigate the role of TLR4 signalling in α-synuclein alterations. Antibodies targeting the full-length protein (α-synuclein) and the Serine-129 phosphorylated form (pS129) were used. Complex, multi-parametric image analysis was conducted through confocal microscopy (with Zen 3.8 analysis) and imaging flow cytometry (with IDEAS® analysis). Confocal microscopy revealed heterogenous distribution of α-synuclein and pS129 in STC-1 cells, with prominent pS129 staining along cytoplasmic processes. Imaging flow cytometry further quantified the relationship between various α-synuclein morphometric features. Thereafter, imaging flow cytometry demonstrated a dose-specific effect of LPS, where the low (8 μg/mL), but not high dose (32 μg/mL), significantly altered measures related to α-synuclein intensity, distribution, and localisation. Pre-treatment with a TLR4 inhibitor TAK-242 alleviated some of these significant alterations. This study demonstrates that LPS-TLR4 signalling alters the intracellular localisation of α-synuclein in enteroendocrine cells in vitro and showcases the utility of combining imaging flow cytometry to investigate subtle protein changes that may not be apparent through confocal microscopy alone. Further investigation is requir