Optimization of α‐Synuclein and Tau Detection by Immunoblot in Enteroendocrine Cell Lines

BACKGROUND: Enteroendocrine cells (EECs) are dispersed along the intestinal mucosa and transduce luminal stimuli into hormonal signals. EECs exhibit neuron-like features and express both α-synuclein and tau, two proteins pathologically and genetically linked to Parkinson disease (PD). These observations support the hypothesis that EECs may be involved in disease development in the "body-first" PD subtype. Cellular models represent invaluable tools for studying the role of α-synuclein and tau in PD pathogenesis. However, the sensitivity and specificity of commercial antibodies for detecting α-synuclein and tau in EEC cell lines remain unclear. METHODS: We tested by immunoblot a panel of commercial total-α-synuclein and total-tau antibodies on protein lysates from three EEC cell lines: GLUTag, NCI-H716, and STC-1. Pharmacological and biochemical manipulations were applied to assess the specificity of antibodies against phosphorylated α-synuclein and tau. KEY RESULTS: Five antibodies detected total α-synuclein in NCI-H716 lysates, whereas the antibodies D1M9X and Tau12 detected total tau in GLUTag and NCI-H716 lysates, respectively. α-synuclein and tau protein levels were comparable between naïve and differentiated NCI-H716 cells. Four phospho-specific antibodies revealed phospho-α-synuclein S129 in NCI-H716. Of the nine phospho-tau antibodies tested, six recognized tau phosphorylated at specific epitopes (T181, S199, T231, S356, S396, and S404) in GLUTag cells. CONCLUSIONS AND INFERENCES: Our results indicate that NCI-H716 cells represent an ideal EEC model for studying α-synuclein expression and phosphorylation, whereas GLUTag cells are preferable for investigating tau protein biology. This work provides a comprehensive antibody toolbox to dissect the physiological and pathological role of α-synuclein and tau in EECs.