Periodic Reporting for period 2 - InterTAU (Integrative structural biology of pathological tau protein, an appealing therapeutic target for Alzheimer´s disease modifying drugs)
Periodo di rendicontazione: 2023-07-01 al 2025-06-30
Neurodegenerative diseases represent a large burden to healthcare systems worldwide. The common denominator of neurodegeneration is the presence of misfolded and aggregated forms of specific proteins, which are suspected to be the culprits of disease progression. Neuronal protein tau is found aggregated in the most severe neurodegenerative tauopathies including Alzheimer's disease (AD). In the project we aimed to monitor conformation changes within the Tau protein variants that lead to its pathological forms characteristic in AD.
The detailed characterization of conformational changes of Tau protein towards its pathological forms will provide the basis for novel diagnostic and therapeutic strategies for AD and other tauopathies.
There are two overall project objectives:
i) To delineate pathways underlying pathologic tau polymerization, taking advantage of synergy created by the multidisciplinary InterTau network
ii) To establish a collaborative structure-characterizing platform based on existing and newly created tools to identify innovative interventional and diagnostics targets in the tau assembly cascade
After fruitful collaboration between project partners over more than five years of project duration, reflected by 12 publications, we contributed to better description of tau conformational and chemical changes leading to its aggregation and uncovered novel properties of nervous system underlying tau pathology propagation/healing.
The detailed characterization of conformational changes of Tau protein towards its pathological forms will provide the basis for novel diagnostic and therapeutic strategies for AD and other tauopathies.
There are two overall project objectives:
i) To delineate pathways underlying pathologic tau polymerization, taking advantage of synergy created by the multidisciplinary InterTau network
ii) To establish a collaborative structure-characterizing platform based on existing and newly created tools to identify innovative interventional and diagnostics targets in the tau assembly cascade
After fruitful collaboration between project partners over more than five years of project duration, reflected by 12 publications, we contributed to better description of tau conformational and chemical changes leading to its aggregation and uncovered novel properties of nervous system underlying tau pathology propagation/healing.
Within the synergic multidisciplinary workflow of InterTau consortium, we focused on the structural investigation of the Tau protein in its monomeric and fibrillary form, with consequences on the molecular pathways in organism. In particular, the impact of different phosphorylations and truncations was studied. We have optimized the production of sufficient amounts of recombinant Tau, its fragments, and their different phosphorylated variants in high purity. It included also the production of isotopically labelled Tau variants. Subsequently, the Tau samples including its phosphorylated variants and homologous Map2c were analysed by solution NMR spectroscopy where we managed to obtain almost complete assignment by applying the advanced nonuniformly sampled methods. This NMR assignment was subsequently used for structural characterization and elucidation of the binding epitopes towards the selected binding proteins, e.g. 14-3-3 proteins in dimeric and monomer states (Crha et al., Int J Biol Macromol. 2024, Jansen et al., FEBS J. 2025).
14-3-3 proteins were chosen as highly relevant partner proteins as they are also present in neurofibrilay tangles in AD. In terms of efficient monitoring of oligomeric changes in 14-3-3s as well as their complexation with phosphorylated proteins, we developer efficient 19F NMR assay (Náplavová et al., Int J Biol Macromol. 2025). 14-3-3 binding dynamics was also characterized by in silico simulation methods, which allowed us to describe a putative allosteric site on the outer surface of molecule regulating 14-3-3 activity (Barrera et al., Protein Sci. 2025). Efficient collaboration with US expert project partner explored methodology for high-efficiency trifluoromethyl-methionine incorporation into the proteins (Zhu et al., Angew Chem Int Ed Engl. 2025).
In terms of Tau fibrils, the partial solid-state NMR assignment was obtained that allowed us to characterize the differences in the Tau fibrilization and their formation initiated by seeding particles from truncated Tau. For these studies, we developed a method for obtaining disease-like Tau aggregates in vitro, suitable for future drug development purposes. The results of such collaborative research were summarized in the form of an original paper and a review with the common leitmotif – exploitation of solid-state NMR in tauopathies (Kitoka et al., Angew Chem Int Ed Engl. 2024, Kitoka et al., Front Mol Biosci. 2021)
The impact of different external factors, such as buffer conditions, on Tau fibrils formation has been monitored by atomic force microscopy and cryo-electron microscopy. These results are currently summarized within another collaborative manuscript. As majority of produced biomaterials are stored in frozen state, we examined this process and revealed the need to consider not only the choice of excipients but also their concentrations and freezing conditions when designing stable formulations for freeze-drying or frozen storage (Veselý et al., Int J Pharm. 2025)
Using animal models, we showed a little effect of chronic inflammation on the tau phosphorylation and disease progression (Basheer et al., Front Aging Neurosci 2024). These findings offer new insights into the relationship between neuroinflammation and tau pathology, challenging the prevailing view that chronic inflammation uniformly exacerbates tau pathology in neurodegenerative diseases. Another animal study within the project described how the specific attenuation of extracellular matrix can result in enhanced cross talk between synapses, spillover of transmitters, formation of new synaptic contacts, and thus increased synaptic plasticity, with consequences for brain clearance and therapeutics delivery (Syková et al. J. Neurosci. 2025)
Broader neuroscience research community was approached by two review publications in AD related fields (Cehlar et al., Int. J. Mol. Sci.2024 Basheer et al., Acta Neuropathol Commun 2024).
The results of InterTAU were disseminated also on numerous scientific conferences involving multidisciplinary audience from clinical and experimental neuroscientists to biochemists, biotechnologists, biophysicists and structural biologists. The public was targeted by open days at project partners with emphasis on the future generation of young students. The main exploitation route consists in identified molecular and conformational targets on tau protein, which may contribute to the design of novel therapeutic and diagnostic tools for tauopathies.
14-3-3 proteins were chosen as highly relevant partner proteins as they are also present in neurofibrilay tangles in AD. In terms of efficient monitoring of oligomeric changes in 14-3-3s as well as their complexation with phosphorylated proteins, we developer efficient 19F NMR assay (Náplavová et al., Int J Biol Macromol. 2025). 14-3-3 binding dynamics was also characterized by in silico simulation methods, which allowed us to describe a putative allosteric site on the outer surface of molecule regulating 14-3-3 activity (Barrera et al., Protein Sci. 2025). Efficient collaboration with US expert project partner explored methodology for high-efficiency trifluoromethyl-methionine incorporation into the proteins (Zhu et al., Angew Chem Int Ed Engl. 2025).
In terms of Tau fibrils, the partial solid-state NMR assignment was obtained that allowed us to characterize the differences in the Tau fibrilization and their formation initiated by seeding particles from truncated Tau. For these studies, we developed a method for obtaining disease-like Tau aggregates in vitro, suitable for future drug development purposes. The results of such collaborative research were summarized in the form of an original paper and a review with the common leitmotif – exploitation of solid-state NMR in tauopathies (Kitoka et al., Angew Chem Int Ed Engl. 2024, Kitoka et al., Front Mol Biosci. 2021)
The impact of different external factors, such as buffer conditions, on Tau fibrils formation has been monitored by atomic force microscopy and cryo-electron microscopy. These results are currently summarized within another collaborative manuscript. As majority of produced biomaterials are stored in frozen state, we examined this process and revealed the need to consider not only the choice of excipients but also their concentrations and freezing conditions when designing stable formulations for freeze-drying or frozen storage (Veselý et al., Int J Pharm. 2025)
Using animal models, we showed a little effect of chronic inflammation on the tau phosphorylation and disease progression (Basheer et al., Front Aging Neurosci 2024). These findings offer new insights into the relationship between neuroinflammation and tau pathology, challenging the prevailing view that chronic inflammation uniformly exacerbates tau pathology in neurodegenerative diseases. Another animal study within the project described how the specific attenuation of extracellular matrix can result in enhanced cross talk between synapses, spillover of transmitters, formation of new synaptic contacts, and thus increased synaptic plasticity, with consequences for brain clearance and therapeutics delivery (Syková et al. J. Neurosci. 2025)
Broader neuroscience research community was approached by two review publications in AD related fields (Cehlar et al., Int. J. Mol. Sci.2024 Basheer et al., Acta Neuropathol Commun 2024).
The results of InterTAU were disseminated also on numerous scientific conferences involving multidisciplinary audience from clinical and experimental neuroscientists to biochemists, biotechnologists, biophysicists and structural biologists. The public was targeted by open days at project partners with emphasis on the future generation of young students. The main exploitation route consists in identified molecular and conformational targets on tau protein, which may contribute to the design of novel therapeutic and diagnostic tools for tauopathies.
The formed InterTau consortium significantly strengthens the collaboration between the involved institutions that include both academic institutions as well as biotech companies. It allows the intense flow of know-how between them and also allows the students and young scientists to experience the biotech environment and consider their future carrier in the commercial sector. Summer school organized within the InterTau project also allowed very valuable experience for the participating students.
The established collaborations within the Rise mobility InterTau project initiated another successful consortium project Horizont Europa: Excellence Hubs: HORIZON-WIDERA-2022-ACCESS-04 (ID: 101087124, period: 2023-2026): Alzheimer's Disease Diagnostics Innovation and Translation to Clinical Practice in Central Europe that involves 4 institutions from the InterTau project (academic: MU, INSAS; industrial: BioVendor, Geneton). Jozef Hritz is also main coordinator in this new ADDIT-CE project and greatly benefitted from the obtained experiences by coordinating the InterTau project. ADDIT-CE project also involves Czech and Slovak Alzheimer’s Societies that will communicate the outcomes of both projects ADDIT-CE and InterTau to the general public.
The established collaborations within the Rise mobility InterTau project initiated another successful consortium project Horizont Europa: Excellence Hubs: HORIZON-WIDERA-2022-ACCESS-04 (ID: 101087124, period: 2023-2026): Alzheimer's Disease Diagnostics Innovation and Translation to Clinical Practice in Central Europe that involves 4 institutions from the InterTau project (academic: MU, INSAS; industrial: BioVendor, Geneton). Jozef Hritz is also main coordinator in this new ADDIT-CE project and greatly benefitted from the obtained experiences by coordinating the InterTau project. ADDIT-CE project also involves Czech and Slovak Alzheimer’s Societies that will communicate the outcomes of both projects ADDIT-CE and InterTau to the general public.