Tau, is an intrinsically disordered protein crucial for stabilizing and polymerizing microtubules (MTs). However, in Alzheimer's disease (AD), Tau undergoes hyperphosphorylation, leading to its misfolding and detachment from MTs, forming droplets termed liquid-liquid phase separation (LLPS), believed to be a precursor to Tau aggregates. Despite being a biomarker for AD, the role of AT8 phosphorylation (S202, T205, and S208) in Tau spread and toxicity remains unclear. To address this issue, we generated Tau variants with different AT8 phosphorylation states: PTau 2A-, 208- (lacking AT8 phosphorylation), PTau 2A+, 208+ (with AT8 phosphorylation), PTau 2A+, S208- (lacking S208 phosphorylation), and PTau 2A-, S208+ (with S208 phosphorylation). These variants were subjected to LLPS and aggregation assays by incubating them with heparin and used to challenge HEK-293T cells and primary neuronal cultures expressing either wild-type or P301L Tau mutant sensitive to Tau seeding. Our aim is to explore the effects of Tau variants on microtubule organization, Tau propagation, neurotoxicity, and neural physiology using confocal and super-resolution microscopy. Preliminary findings show intact Tau-Tubulin interaction in non treated WT or P301L Tau-expressing HEK cells using a Tau-Tau split Venus BiFC assay. However, heparin induced Tau fibrils addition induces intracellular Tau inclusions, particularly in P301L Tau. This was also evidenced by increased sarkosyl insoluble P301L Tau aggregates following fibril treatment using WB analysis. Our future work involves swiftly assessing the variants seeding activity using high-content screening methods. We aim to demonstrate an increased seeding activity and disrupted Tau-Tubulin interaction upon AT8 phoshphorylation, focusing on the S208 residue.