Effects of T217 Phosphorylation on Tau Localization and Microtubule Binding

Abstract

AbstractBackgroundBiomarker‐based early detection of Alzheimer’s disease (AD) is necessary for developing therapies that counter AD symptom onset. Promising biomarkers include tau phosphorylated at T181, T217 or T231, levels of which can be measured in cerebrospinal fluid or plasma. Little is known about functional consequences of tau phosphorylation at these sites, though, especially for taupT217. We thus sought to identify where taupT217 is localized in the brains of human AD patients and AD model mice, and in cultured mouse neurons, and, in a more functional context, to determine if phosphorylation at T217 affects tau binding to microtubules.MethodBrain tissue from human AD patients and AD model mice, and cultured mouse neurons were analyzed by western blotting and immunofluorescence microscopy to compare the intracellular localization of taupT217, taupT181, taupT231 (AT180 epitope) and taupS396,pS404 (PHF1 epitope). EGFP‐tau (2N4R) was expressed in CV‐1 African green monkey kidney fibroblasts as wild type (WT), T217A non‐phosphorylatable, and T217E pseudo‐phosphorylated tau versions of the fluorescent fusion protein. Fluorescence recovery after photobleaching (FRAP) microscopy was used to assay turnover rates of the EGFP‐tau fusion proteins on microtubules, a surrogate measurement of affinity.ResultIn human AD brains and AD model mice, taupT217 was abundant in the somatodendritic compartment and co‐localized with neurofibrillary tangles. In cultured neurons, taupT217 was concentrated in somatodendritic puncta that were closely associated with dendritic spines. Both in vivo and in cultured neurons taupT217 co‐localized only partially with taupT181 and taupT231. Exposure of cultured neurons to extracellular oligomers of tau, but not amyloid‐β, increased intracellular taupT217. In CV‐1 cells, EGFP‐tauT217E recovered from photobleaching faster than EGFP‐tauT217A or EGFP‐tauT217.ConclusionIntracellular taupT217 is enriched at locations that normally lack much tau and is increased by extracellular tau oligomers. TaupT217 is enriched near dendritic spines, suggesting a possible role for T217 phosphorylation in altering synaptic activity. The faster fluorescence recovery of EGFP‐tauT217E compared to EGFP‐tauT217A or EGFP‐tauT217 implies that T217 phosphorylation reduces tau’s affinity for microtubules.