Investigation of a Tau aggregation inhibitor peptide and peptide-conjugated liposomes as a potential treatment for Alzheimer’s Disease
Creators: name: Reich, Niklas
Publisher: name: Lancaster University
Year: 2023

description: Although amyloid beta (Aβ)-targeting antibodies have recently been approved to reduce cognitive decline in Alzheimer’s disease (AD), the most common form of dementia, the utility of these drugs is questionable due to dangerous side effects. As a therapeutic alternative to Aβ, inhibiting the pathological aggregation of Tau has gained traction. Our group has recently developed a Tau aggregation inhibitor peptide, RI-AG03, to complement our Aβ aggregation inhibitor peptide RI-OR2. My aim in this thesis was to investigate the protective effects of RI-AG03 in Tauopathy cell models and 1N4R Tau (P301S)-transgenic PS19 mice. This included the use of doxycycline-inducible and full-length (2N4R) Tau (P301L)-expressing SH-SY5Y cells, termed Clone 4, and Tau RD (V337M/P301L)-EYFP-expressing HEK293 cells, termed Clone 9, to test the peptide. Moreover, the cellular uptake mechanisms and subcellular distributions of polyarginine (polyR)- or transactivator of transcription (TAT)- cell penetrating peptide-linked variants of RI-AG03, unconjugated liposomes and RI-AG03-conjugated liposomes were studied. RI-AG03-polyR treatment increased total Tau levels in Tauopathy cell and animal models. In Clone 9 cells, RI-AG03-polyR weakly co-localised with monomeric and oligomeric, but not larger, Tau RD-EYFP inclusions. Curiously, both RI-AG03-polyR and a scrambled control peptide reduced the proportions of soluble Tau in Clone 9 cells, while image analysis showed that Cy5-RI-AG03-polyR treatment increased the number of aggregates, suggesting a pro-aggregatory effect. Despite this, RI-AG03-polyR or RI-AG03-TAT improved cell viability and reduced membrane rupture in Clone 9 cells (this effect was even more pronounced when the peptides were conjugated to liposomes), whilst a scrambled control peptide had no such effects. Cellular trafficking studies revealed that only the uptake of RI-AG03-polyR/TAT-conjugated liposomes, whose internalisation was 3-fold higher than that of unconjugated liposomes, was significant in SH-SY5Y cells. Uptake occurred mainly via direct membrane penetration and macropinocytosis, in agreement with fair or moderate co-localisation with lysosomes and macropinosomes. Strikingly, conjugating RI-AG03-polyR to liposomes avoided peptide entrapment in cell organelles. Due to using too young animals in a preliminary in vivo study, the potential effects of RI-AG03-polyR on cerebral Tau pathology and glial inflammation in PS19 mice could not be characterised. However, immunoblotting showed that RI-AG03-polyR administration doubled baseline postsynaptic density protein 95 (PSD-95) levels in the brains of both wild-type and PS19 mice, whilst both RI-AG03-polyR and its scrambled version increased cerebral pro-apoptotic caspase 3 activity in vivo. Finally, RI-AG03-polyR-treated control animals showed two-fold increased p62 levels, but no changes in the autophagy markers Beclin 1 and LC3. Overall, my findings suggest that RI-AG03 and RI-AG03-conjugated liposomes protect from Tau propagation and toxicity in non-neuronal cells. However, given that the data suggests negative effects of the dosing regimen used in the in vivo pilot studies, substantial further studies are necessary to determine whether the peptides might represent an effective AD therapy in pre-clinical models.