Al Kabbani, M. A. | bioRxiv Science

bioRxiv2024

Expression of recombinant human glutamylating TTLLs in human cells leads to differential tubulin glutamylation patterns, with only TTLL6 disrupting microtubule dynamics

Polyglutamylation, a post-translational modification (PTM) catalyzed by a subset of Tubulin Tyrosine Ligase-Like (TTLL) family enzymes, regulates microtubule dynamics through its influence on interactions with microtubule-associated, motor, and severing proteins, and has recently also been implicated in genetic and neurodegenerative diseases. In this study, we characterized the glutamylation activity of various glutamylating TTLLs in human embryonic kidney 293T (HEK293T) cells, revealing distinct patterns of mono- and polyglutamylation among TTLL family members, with TTLL4 and TTLL11 exhibiting the strongest chain initiation and elongation activities, respectively. We found that TTLL6 expression uniquely decreased microtubule stability, with live-cell imaging of end-binding protein (EB3) showing a TTLL6-induced decrease in microtubule stability. To explore therapeutic modulation of TTLL activity, we tested LDC10, a novel TTLL inhibitor, which successfully blocked glutamylation across all TTLLs investigated in this study, while also reversing the microtubule-destabilizing effects of TTLL6. These findings identify a potential pathogenic role of TTLL6 in microtubule dynamics and highlight LDC10 as a promising pharmacological tool to counteract TTLL-induced microtubule destabilization.

cell biology↗

bioRxiv2024

Knockdown of TTLL1 reduces Aβ-induced TAU pathology in human iPSC-derived cortical neurons

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 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. 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 (oA{beta}) to replicate AD-like conditions. TTLL1, TTLL4, 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 interactions between TAU and TTLL proteins. We observed TAU missorting, increased tubulin polyglutamylation, decreased microtubule stability, and synaptic declustering in oA{beta}-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 revealed a potential interaction between TAU and TTLL1, but not other TTLLs, suggesting a direct role of TTLL1 in TAU-mediated toxicity. Our findings indicate that targeting TTLL1, either alone or in combination with other TTLLs, may be a promising therapeutic strategy to counteract microtubule and synaptic dysfunction in AD and related neurodegenerative disorders.

neuroscience↗