Live‐cell imaging of internalised oligomeric tau reveals differences between neuronal cell types

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AbstractBackgroundIt has recently been established that pathological forms of aggregated tau spread from affected to recipient cells in the brain, facilitating disease progression. Here we have applied live‐cell imaging to visualise internalised tau in recipient neuronal cells.MethodRecombinant human 2N4R tau was aggregated using heparin and dithiothreitol, sonicated, and labelled with the fluorescent dye, Cy5. Differentiated SH‐SY5Y human neuroblastoma cells (dSH‐SY5Y), mouse primary cortical neurons (PCNs) and human induced pluripotent stem cell‐derived neurons (iPSCs) were exposed to Cy5‐tau for up to 7 days. Confocal microscopy was used to visualise internalised Cy5‐tau in live‐cells and paraformaldehyde‐fixed cells. BODIPY 493/503 and Lysotracker dyes were used to label neutral lipids and lysosomes, respectively.ResultLive‐cell imaging showed rapid (within five minutes) internalisation of Cy5‐tau by all cell types, appearing as small speckles (<0.1µm diameter), with larger masses (>0.5 µm diameter) forming over time. In contrast, only small speckles of Cy5‐tau were detectable inside fixed cells. The size of Cy5‐tau accumulations in PCNs, but not in dSH‐SY5Y or iPSCs, increased further over time, reaching up to 6 µm diameter after 7 days. Lysotracker labelling of live cells indicated significant colocalisation with Cy5‐tau in all three cell types. BODIPY also partially colocalised with both Lysotracker and Cy5‐tau in PCN and iPSCs, but not in dSH. Furthermore, some large (>5 µm diameter) Cy5‐tau spheres in PCNs were Lysotracker‐negative, indicating that incorporation of Cy5‐tau may deacidify lysosomes.ConclusionLive‐cell imaging of neuronal cells has revealed previously unreported internalised tau structures that are not detectable in fixed cells. The sizes of tau spheres that form inside cells exposed to oligomeric tau differ between neuronal cell types, which is a consideration for selecting the most appropriate disease model. The colocalisation between Cy5‐tau and specific organelle labelling indicated tau intracellular trafficking pathway.