Loss of Tubulin Tyrosine Ligase, a Novel Feature of Sporadic and Familial AD, is an Enhancer of Tau Phosphorylation

Background We have recently shown that loss of tubulin tyrosine ligase (TTL) and subsequent disruption of a‐tubulin retyrosination is a feature of AD and a promoter of synaptic loss by inhibiting microtubule entries into dendritic spines. TTL+/‐ mice have cognitive deficits, altered LTP, synapse loss and increased levels of detyrosinated and D2 tubulin, two tubulin modifications that accumulate on non‐dynamic microtubules. We also found that low levels of phospho‐tau correlated with higher levels of detyrosinated and D2 tubulin in pyramidal neurons residing in the anterior hippocampal formation of human AD brains, raising the question of whether improper microtubule longevity drives early stages of tau hyperphosphorylation in AD. Here, we demonstrate that loss of TTL enhances both AMPK activation and tau phosphorylation in cultured rat hippocampal and human cortical neurons, and present preliminary evidence on the mechanisms by which this regulation may occur and contribute to TTL‐loss dependent synaptic dysfunction. Method Human cortical neurons derived from iPSC lines in which the London familial APP mutation V717I was knocked into one allele of the IMR90 control using CRISPR/Cas9, were used as a cellular model of familial AD. Lentiviral infection with shRNA against TTL was used to silence TTL expression. Microtubule dynamics were measured by confocal time‐lapse microscopy in neurons expressing EB3‐EGFP, a protein that tracks the growing plus ends of microtubules. Immunoblotting was performed to measure both unmodified and modified levels of TLL, tubulins, AMPK subunits, total tau, p262 and AT8 phospho‐tau. Result In the APP neurons, we found that a reduction in TTL levels correlated with an increase in p262/AT8 phospho‐tau variants and phospho‐AMPK, the active state of one of the kinases that phosphorylates serine 262, a key residue in the binding of tau to microtubules. Knockdown of TTL was sufficient to reduce microtubule dynamics and increase both active AMPK levels and phospho‐tau. Conclusion Our results indicate that loss of TTL alone reduces dynamic microtubules, triggers tau kinase activation, and results in hyperphosphorylated tau. Together with our previously published data, these findings suggest that reduced levels of TTL act as one of the early drivers of tau hyperphosphorylation in AD.