Targeting TTLL1 Alleviates Aβ-Induced Microtubule Disruption and TAU Pathology in Human iPSC-Derived Cortical Neurons

Background: Microtubules play a crucial role in neuronal structure and function, with their stability and dynamics regulated by posttranslational modifications (PTMs) such as polyglutamylation. In Alzheimer's disease (AD), the microtubule-associated protein TAU becomes mislocalized into the somatodendritic compartment ('TAU missorting'), dissociates from microtubules, aggregates into neurofibrillary tangles, and contributes to microtubule destabilization and neuronal death. Objectives and Methods: Here, we investigated the role of tubulin tyrosine ligase-like proteins (TTLLs) in TAU missorting and microtubule dysregulation using human-induced pluripotent stem cell (hiPSC)-derived cortical neurons treated with oligomeric amyloid-beta (oAβ) to replicate AD-like conditions. TTLL1, TTLL4, and TTLL6 were selectively knocked down (KD) to assess their impact on TAU missorting and microtubule stability. Fluorescence resonance energy transfer (FRET) microscopy was used to examine proximities between TAU and TTLL proteins. Results: We observed TAU missorting, increased tubulin polyglutamylation, decreased tubulin acetylation associated with microtubule destabilization, and synaptic declustering in oAβ-treated neurons. TTLL1 KD significantly reduced TAU missorting, tubulin polyglutamylation, and synaptic disintegration, while TTLL4 KD showed moderate effects, and TTLL6 KD restored microtubule acetylation. Importantly, TTLL KD did not impair neuritic networks, dendritic complexity, or neuronal activity. FRET microscopy in HEK293T cells revealed a close molecular proximity between TAU and TTLL1 consistent with a potential direct or complex-mediated association, but not with other TTLLs, suggesting a direct role of TTLL1 in TAU-mediated toxicity. Conclusions: Our findings identify TTLL1 as a promising therapeutic target for limiting TAU-associated cytoskeletal pathology in AD. These results support further development of pharmacological or genetic strategies targeting TTLL1 as a disease-modifying approach for AD and related tauopathies.