Investigating in vivo the principles governing the spread of tau phosphorylation and amyloid-beta excitotoxicity

Abstract

Alzheimers disease (AD) is a devastating neurodegenerative disease which presents clinically with progressive loss of memory, executive function and cognitive abilities.Traditionally AD is diagnosed post-mortem by presence of two hallmark lesions, extracellular plaques comprised of amyloid-beta (Aβ) and intracellular neurofibrillary tangles (NFTs) containing hyperphosphorylated microtubule-associated protein tau.The current available therapies for the treatment of AD only provide mild symptomatic relief and cannot halt the ongoing disease progression. Therefore, the research presented focuses on the mechanisms underlying disease propagation specifically that of hyperphosphorylated tau pathology, and understanding potential disease modifying therapeutics targeting known mechanisms, specifically TatNR2b9c peptide induced neuroprotection against Aβ induced excitotoxicity.Current approaches to investigate tau propagation have utilised tau transgenic recipient mice, mutant tagged tau constructs and whole brain lysates to establish tau seeding and spreading in vitro. To circumvent caveats of this technique and progress our understanding of tau seeding events we proposed a two-pronged approach.Firstly, we created an endogenous model of tau hyperphosphorylation by inhibiting tau dephosphorylation with the unilateral injection of the inhibitor okadaic acid. By keeping the concentration and volume low we were able to show by ELISA that the spread of the inhibitor could be restricted, and this single exposure was sufficient to induce tau hyperphosphorylation, increases in insolubilty and thioflavin-S NFT-Like immunoreactivity at both the injection site and anatomically distinct non-exposed brain regions. Secondly, we injected exosome and large extracellular vesicle fractions derived from tau transgenic mice into human tau expressing mice and compared there seeding ability to brain lysate. Under our experiment parameters we did not observe any NFT positivity and could not recreate previous seeding using brain lysates. Interestingly, both brain lysates and exosomes increased AT8 phosphorylation and oligomeric tau deposition in the hippocampus, whereas large extracellular vesicles did not.To address whether the Tat-NR2b9c peptide can provide long-term protection against Aβ induced excitotoxicity we treated mutant amyloid precursor protein 3 transgenic mice at both young and old age and investigated amyloid levels and NMDA receptor subunit expression in synaptic and extrasynaptic zones.Unfortunately, lack of a robust behavioural deficit in our aged APP23 cohort prevented us from performing a therapeutic intervention study. However, biochemical analysis of aged Tat-NR2b9c revealed a similar effect on synaptic redistribution of NMDA receptor subunits as that observed in young treated mice, indicating that the peptide may be a viable therapeutic prospect for late stage intervention in AD.This work demonstrates that mechanisms underlying tau spreading may also be studied by using endogenous mouse tau models. An avenue whic...