Universität zu Köln

Al Kabbani, Mohamed Aghyad ORCID: 0000-0001-6200-2448 (2024). Microtubule Integrity in Tauopathies: Exploring the Roles of TAU, Tubulin Tyrosine Ligase-Like Enzymes, and Tubulin Post-Translational Modifications. PhD thesis, Universität zu Köln.

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Abstract

Tauopathies, including Alzheimer’s disease (AD), represent a class of neurodegenerative disorders characterized by the pathological aggregation of the microtubule-associated protein TAU. Under physiological conditions, TAU stabilizes microtubules, which are essential for neuronal structure, intracellular transport, and synaptic function. However, in tauopathies, TAU detaches from microtubules, becomes hyperphosphorylated, missorted and aggregated, culminating in microtubule destabilization, synaptic dysfunction, and neuronal degeneration. Emerging evidence highlights the role of tubulin post-translational modifications (PTMs), such as polyglutamylation, in regulating microtubule stability. Polyglutamylation, mediated by Tubulin Tyrosine Ligase-Like (TTLL) enzymes, modulates interactions between microtubules, motor proteins, and severing enzymes. However, dysregulated polyglutamylation has been implicated in microtubule instability and neurodegeneration. This thesis hypothesizes that TTLL enzymes contribute to microtubule dysfunction in tauopathies and that targeting these enzymes can mitigate TAU-driven neurodegeneration. To address this hypothesis, the study utilized human induced pluripotent stem cell (iPSC)-derived cortical neurons (iNeurons) and pR5 mice, transgenic for P301L-TAU, as models of tauopathies. iNeurons were generated by differentiating iPSCs into cortical layer 2/3 glutamatergic neurons, and TAU pathology was induced via lentiviral expression of P301L-TAU or treatment with oligomeric amyloid-beta (oAβ). TAU missorting, microtubule PTMs, and synaptic integrity were assessed using immunocytochemistry, Western blotting, and fluorescence imaging. Lentiviral knockdown of TTLL enzymes was achieved using short hairpin RNA (shRNA) constructs, and the effects on microtubule stability and neuronal health were evaluated. To further characterize TTLL activity, recombinant TTLL enzymes were expressed in HEK293T cells to analyze their glutamylation profiles and their effects on microtubule stability, which was assessed via live-cell imaging of the microtubule plus end-binding protein EB3. Additionally, the efficacy of a novel TTLL inhibitor was tested for its ability to reverse pathological microtubule destabilization. The results demonstrated that P301L-TAU in pR5 mice pathologically missorted into the somatodendritic compartments, accompanied by reduced microtubule acetylation and elevated polyglutamylation in hippocampal neurons, correlating with microtubule fragmentation and impaired dendritic morphology in aged mice. However, P301L-TAU expression in iNeurons did not result in TAU missorting or aggregation, but only in elevated tubulin acetylation and polyglutamylation. On the other hand, oAβ treatment of iNeurons led to TAU missorting, combined with increased tubulin polyglutamylation, reduced acetylation, and synaptic loss, thus providing a suitable human tauopathy model. Knockdown of the major brain polyglutamylase TTLL1, and to a lesser extent TTLL4, in iNeurons significantly reduced tubulin polyglutamylation, mitigated TAU missorting, and partially protected synaptic integrity. Fluorescence resonance energy transfer (FRET)-based interaction studies in HEK293T cells revealed a direct interaction between TTLL1 and TAU, implicating TTLL1 as a mediator of TAU-driven microtubule dysfunction. Similarly, TTLL6, another polyglutamylase, was shown to destabilize microtubules in recombinant protein expression experiments in HEK293T cells. A novel TTLL inhibitor effectively blocked glutamylation activity across several TTLLs, reversed TTLL6-induced microtubule destabilization, and restored microtubule dynamics in HEK293T cells. These findings establish TTLL enzymes, particularly TTLL1, TTLL4, and TTLL6, as critical mediators of microtubule dysfunction in tauopathies, and contributors to the pathological cascade initiated by TAU missorting and aggregation. Targeting TTLLs offers a promising therapeutic strategy for tauopathies, with the potential to intervene early in disease progression by stabilizing microtubules and preserving neuronal health. The use of human iPSC-derived neurons in this research provided a physiologically relevant model to study TAU pathology and evaluate the therapeutic potential of TTLL inhibition. While this study demonstrates the feasibility of targeting TTLLs, future work will extend these findings to in vivo models and investigate the long-term efficacy and safety of TTLL-targeting gene therapy. In conclusion, this thesis highlights the interplay between TAU pathology and microtubule dynamics, uncovering TTLL enzymes as pivotal players in neurodegeneration. By shifting the focus from TAU aggregation to microtubule regulation, it identifies novel targets and therapeutic approaches that hold promise for mitigating the devastating effects of tauopathies such as AD.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Al Kabbani, Mohamed Aghyad mhd.al-kabbani@uk-koeln.de orcid.org/0000-0001-6200-2448 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-785328
Date:	2024
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Medicine > Humangenetik
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language TAU English Microtubules English Tubulin-tyrosine-ligase-like enzymes English
Date of oral exam:	6 February 2025
Referee:	Name Academic Title Zempel, Hans Dr. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/78532