Gorecki Anastazja M, Anyaegbu Chidozie C, Fitzgerald Melinda, Fuller Kathryn A, Anderton Ryan S
School of Health Sciences, University of Notre Dame Australia, Fremantle, WA, Australia; Curtin Health Innovation Research Institute, Curtin University, Ralph and Patricia Sarich Neuroscience Research Institute, Nedlands, WA, Australia; Perron Institute for Neurological and Translational Science, Ralph and Patricia Sarich Neuroscience Research Institute, Nedlands, WA, Australia; School of Biological Sciences, University of Western Australia, Crawley, WA, Australia.
Curtin Health Innovation Research Institute, Curtin University, Ralph and Patricia Sarich Neuroscience Research Institute, Nedlands, WA, Australia; Perron Institute for Neurological and Translational Science, Ralph and Patricia Sarich Neuroscience Research Institute, Nedlands, WA, Australia.
Methods. 2025 Feb;234:93-111. doi: 10.1016/j.ymeth.2024.10.009. Epub 2024 Oct 30.
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 required to understand the apparent dose-dependent effects of LPS on α-synuclein in the gut epithelium in healthy states as well as conditions such as Parkinson's disease.
帕金森病是一种慢性神经退行性疾病，其中主要由磷酸化α-突触核蛋白组成的病理性蛋白质聚集体与疾病的发病机制和进展有关。新出现的证据表明，促炎微生物因子与肠道上皮之间的相互作用促成了肠神经系统中α-突触核蛋白的聚集。然而，肠道中α-突触核蛋白病理的细胞来源和机制仍不清楚。
STC-1细胞系模拟能够与肠道微生物群、免疫系统和神经系统进行通讯的肠内分泌细胞群，在暴露于微生物脂多糖（LPS）之前用TLR4抑制剂（TAK-242）处理，以研究TLR4信号传导在α-突触核蛋白改变中的作用。使用靶向全长蛋白（α-突触核蛋白）和丝氨酸-129磷酸化形式（pS129）的抗体。通过共聚焦显微镜（使用Zen 3.8分析）和成像流式细胞术（使用IDEAS®分析）进行复杂的多参数图像分析。
共聚焦显微镜显示STC-1细胞中α-突触核蛋白和pS129的异质分布，沿细胞质突起有明显的pS129染色。成像流式细胞术进一步量化了各种α-突触核蛋白形态特征之间的关系。此后，成像流式细胞术显示了LPS的剂量特异性效应，其中低剂量（8μg/mL）而非高剂量（32μg/mL）显著改变了与α-突触核蛋白强度、分布和定位相关的指标。用TLR4抑制剂TAK-242预处理减轻了其中一些显著改变。
本研究表明，LPS-TLR4信号传导在体外改变了肠内分泌细胞中α-突触核蛋白的细胞内定位，并展示了结合成像流式细胞术来研究单独通过共聚焦显微镜可能不明显的细微蛋白质变化的实用性。需要进一步研究以了解LPS在健康状态以及帕金森病等疾病条件下对肠道上皮中α-突触核蛋白的明显剂量依赖性效应。
