Impact of small molecules and peptides on tau microtubule interaction and tau aggregation in primary peripheral neurons and models of Alzheimer’s and Parkinson’s disease
[EN] Tau is a member of the microtubule associated proteins family, it is abundant in neurons of both central and peripheral nervous systems of vertebrates. It is estimated that 80% of tau is bound to microtubule (MT) at every time point. Tau is a neuron-specific protein, and involved in promoting M...
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| Formato: | tesis doctoral |
| Fecha de publicación: | 2023 |
| País: | España |
| Recursos: | Consejo Superior de Investigaciones Científicas (CSIC) |
| Repositorio: | DIGITAL.CSIC. Repositorio Institucional del CSIC |
| OAI Identifier: | oai:digital.csic.es:10261/355207 |
| Acesso em linha: | http://hdl.handle.net/10261/355207 |
| Access Level: | acceso abierto |
| Palavra-chave: | Tubulina Microtubulos Alzheimer Taxol Epotilona http://metadata.un.org/sdg/3 Ensure healthy lives and promote well-being for all at all ages |
| Resumo: | [EN] Tau is a member of the microtubule associated proteins family, it is abundant in neurons of both central and peripheral nervous systems of vertebrates. It is estimated that 80% of tau is bound to microtubule (MT) at every time point. Tau is a neuron-specific protein, and involved in promoting MT polymerization and providing support to the MT network. Tau hyperphosphorylation can lead to the formation of neurofibrillary tangles that are observed in several neurodegenerative diseases collectively called tauopathies. The most common tauopathy is Alzheimer’s disease at which there is a change of tau’s function that leads to disturbances in the MT network and ultimately to neuron death. In this thesis, I tested a panel of small molecules with respect to tau-MT interaction and tau aggregation, and their possible utility to treat neurodegenerative diseases. Firstly, I tested four MT stabilizers that could act as tau biomimetic in situations where tau changed its activity that led to MT network dynamic alterations. These small compounds were: Epothilone-D, Zampanolide, Pelophen-B and Paclitaxel. To do so: (i) I studied their effect in cells on the MT cytoskeleton from the biological and the structural biological aspects employing two in vitro models (differentiated PC12 neuronal-like cells and primary mouse dorsal root ganglia (DRG) neurons) transfected or infected to express either human full-length tau (tau-441wt) or an aggregation-prone mutant (tau-ΔK280). Both proteins were tagged with a photoactivatable green fluorescent protein to monitor tau-MT interaction in living cells using fluorescence decay after photoactivation followed by mathematical modelling of the decay curves. (ii) Knowing the structural distortion introduced by MT stabilizers in clinical use, I studied the effect of both tau and MT stabilizing molecules on MT structure using X-ray fiber diffraction. (iii) I characterized the potential of the MT stabilizers to protect a human neuroblastoma cell line, SH-SY5Y, against okadaic acid-induced hyperphosphorylation. Finally, (iv) I studied the potential of these compounds to reach the central nervous system by conducting an in vitro blood brain barrier penetration (BBB) assay. Secondly, I evaluated a novel polypharmacological small compound, PHOX15, which had an anti-aggregation potential. To do so, I determined the diffusion (unbound soluble fraction) of tau-441wt and tau-ΔK280 in DRG neurons. The aggregation-prone tau-ΔK280 showed a significantly increased effective diffusion compared to tau-441wt indicating reduced interaction with MT due to aggregates formation. The results indicated that Epothilone-D, Zampanolide and Paclitaxel increased the interaction of tau with MTs, at non-toxic concentrations, in both the PC12 model neurons and mouse DRG. On the other hand, Pelophen-B did not alter neuronal MT-tau-441wt interaction, and showed a neuroprotective effect on hyperphosphorylated SH-SY5Y cells. Additionally, it could permeate the central nervous system according to the in vitro BBB assay. Finally, I found that PHOX15 was able to restore the tau-ΔK280-MT interaction to a physiological level similar to this measured for the tau-441wt control one. In summary, this study indicates that it is possible to employ small molecules to modulate the tau-tubulin interaction in cell lines and primary culture neuronal models, and to target tau aggregation-like conditions. This thesis provides an approach to the characterization of candidate compounds targeting tauopathies. |
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