Identification and Characterisation of Tau Minimal Regions as Potential Pharmacological Targets Against Tau Spreading in Tauopathies

Narendran ANNADURAI

Doctoral thesis

Abstract:

Tau, a microtubule-associated protein present abundantly in neurons of the central nervous system, stabilises axonal microtubules, providing structural architecture to axons. Tau aggregation occurs in many neurodegenerative diseases collectively termed 'tauopathies', characterised by the intracellular deposits of fibrillar tau tangles. Tau seeding, the ability of prion-like tau to recruit and misfold inert tau to generate new seeds, is detected early in Alzheimer's disease (AD) human brains before the development of significant tau pathology. Circumstantial evidence shows that pathological 'tau seeds' spread across the brain through synaptically connected neurons, potentially contributing to AD pathogenesis. This work aimed to identify the minimal regions within the tau repeat domain that define seeding potency and the subsequent impact on intracellular native tau. R2 and R3 repeat domains formed seed-competent fibrils in the presence of heparin. Though R2 and R3 hold identical N-terminal hexapeptide and cysteine residue sequences, R3 but not R2 formed prion-like fibrils in the absence of heparin. Besides, cysteine to alanine substitution in R3 (C322A) abrogated its self-aggregation and seeding potency. R2 and R3 fibrils induced aggregation, pathological phosphorylation and oligomerisation of native tau in tau cell models expressing P301S or P301L tau. Protein fractions from biosensor cells seeded with R2 and R3 fibrils reseeded endogenous tau when introduced into a new set of biosensor cells. These findings suggest that under physiological conditions, while R3 may be the minimal region essential for pathological seed generation, R2 needs polyanionic cofactors to initiate pathogenic seeds. R2 and R3 fibrils induce template-induced misfolding and pathological hyperphosphorylation of intracellular inert tau, making intracellular tau seed-competent. Overall, the results of this study identify N-terminal hexapeptides and cysteine residue of R2 and R3 as druggable targets for early inhibition of prion-like seeds. Next, VQIVYK and cysteine of self-aggregating R3 were targeted with small molecules to abolish the prion-like activity of R3 aggregates. Antitumour drugs have been suggested to confer protection against neurodegeneration, supporting the idea of repurposing approved and experimental or investigational oncology drugs for AD therapy. Antitumour drugs that interacted with the N-terminal VQIVYK or the C-terminal region housing the cysteine residue (C322) prevented R3 dimerisation and abolish the generation of prion-like R3 seeds. Preformed R3 seeds capped with, or R3 aggregates formed in the presence of VQIVYK- or C322-targeting drugs have a reduced prion-like potency to cause aggregation of endogenous tau in tau biosensor cells. These findings indicate that VQIVYK- or C322-targeting drugs may act as prophylactic agents against tau seeding. Next, the mechanisms associated with clearing intracellular aggregates in biosensor cells seeded with R2 and R3 fibrils were studied. R2 and R3 seeds increased p62 levels in response to the build-up of intracellular tau aggregates. R2 and R3 seeds also reduced the autophagy marker indicated by the conversion of LC3-I to -II. These findings suggest a failure in autophagy initiation and autophagy flux in tau biosensor cells, causing a failure in the clearance of intracellular aggregates. Thus, the effect of autophagy induction on removing intracellular tau aggregates was studied. Autophagy inducers but not autophagy inhibitors reduced intracellular tau aggregates in biosensor cells seeded with R3 fibrils. Autophagy inducers also decreased triton-insoluble total and phosphorylated Ser262 tau. Altogether, evidence from this work supports the importance of targeting N-terminal hexapeptides or cysteines of R2 or R3 and enhancing cell clearance mechanisms for developing tau-targeted therapies.

Language used: English

Date on which the thesis was submitted / produced: 7. 2. 2023

Thesis defence

• Supervisor: Mgr. Viswanath Das, PhD.

Palacký University Olomouc

Faculty of Medicine and Dentistry

Doctoral programme / field:
Pediatrics / Pediatrics