Peptide-based inhibitors of tau aggregation as a potential therapeutic for Alzheimer's disease

Abstract

Tauopathies are a group of neurodegenerative diseases characterized by the deposition of misfolded and aggregated Tau protein in patients’ brains. Alzheimer's disease (AD), the most common tauopathy and leading cause of dementia, affects over 55 million people worldwide, with prevalence projected to double every two years. Despite this enormous socioeconomic burden, there is still limited availability of disease-modifying therapies for AD. In 2022, our group developed RI-AG03, a peptide-based inhibitor targeting Tau aggregation-a key pathological hallmark of AD and demonstrated its inhibitory effect against truncated Tau aggregation in vitro. However, the necessity of assessing Tau aggregation inhibitors using physiologically relevant models is highlighted by observations that certain compounds, such as D-tlkivw, inhibit truncated Tau aggregation but fail against aggregation of 2N4R Tau. In this project, using heparin-induced aggregation of physiologically relevant 2N4R Tau, I confirmed that RI-AG03 inhibits this aggregation in a dose-dependent manner with an IC50 of 4.64 μM, comparable to its activity against truncated TauΔ1-250. The inhibition mechanism of RI-AG03 under the same condition was also evaluated in this project. The previous study employing heparin-induced aggregation of TauΔ1-250 indicated that RI-AG03 may inhibit Tau aggregation by redirecting the canonical aggregation pathway and promoting the formation of off-pathway species. The inhibition mechanism assessment performed in this project using heparin-induced aggregation of 2N4R Tau supported this hypothesis. The result of this assessment revealed that an off-pathway Tau species, RITA2N4R, was formed at the endpoint of RI-AG03 redirected, heparin-induced aggregation of 2N4R Tau. This species exhibited a morphology resembling nascent canonical 2N4R Tau filaments, but with much reduced cross β-sheet contents. Moreover, differential centrifugation and propagation competence test performed in this project also demonstrated that RITA2N4R has similar density but significantly reduced competence to seed new aggregation compared to canonical 2N4R Tau filaments. In this project, the in vitro inhibitory effect of RI-AG03 was also assessed using 0N3R PAD12, a pseudo phosphorylated 3R Tau construct that recapitulates the effects of Tau hyperphosphorylation. Notably, 0N3R PAD12 spontaneously forms filaments adopting AD fold in the absence of artificial inducers such as heparin, allowing the assessment to be performed under more tauopathy relevant conditions. RI-AG03 exhibited a significant inhibitory effect in this assessment, further confirming its potential as a therapeutic candidate. The previous study also confirmed that RI-AG03 alleviates Tau toxicity in vivo, as shown by extended lifespan and rescued eye degeneration in Drosophila models expressing 2N4R Tau pan-neuronally or specifically in the eyes. In this project, I provided direct evidence linking this attenuation of Tau toxicity to the inhibition of Tau aggregation. When RI-AG03 was administered to Drosophila models expressing either 2N4R Tau or the pseudo phosphorylated Tau construct 2N4R E14, which recapitulates the effects of Tau hyperphosphorylation, a significant inhibition or a clear trend toward inhibition of aggregation was observed in the two models, respectively.