INVESTIGATING PRESYNAPTIC DISRUPTION IN ORGANOTYPIC HIPPOCAMPAL SLICE CULTURES FROM HU-APP TRANSGENIC MICE

Abstract

Loss of presynaptic proteins has been reported as an early and clinically-relevant alteration in the brains of patients with Alzheimer's disease. Understanding the mechanisms by which synapses are disrupted requires a model system that represents all major CNS cell types, is amenable to repeated analysis, and can be readily manipulated. Long-term organotypic hippocampal slice cultures (OHSCs) from neonatal amyloid mice offer an excellent compromise between in vivo and primary culture studies, largely retaining the cellular composition and neuronal architecture of the in vivo hippocampus, but with the in vitro advantages of accessibility to live imaging, sampling and intervention. OHSCs are made using tissue from P6-P9 TgCRND8 mice and maintained on membrane inserts for up to 2 months. Pathology development was closely monitored using Aβ ELISA, western blotting for synaptic proteins, and immunofluorescence for Aβ, neurons and glia. The mechanisms behind presynaptic disruption were probed via pharmacological manipulation of Aβ production and genetic knockout of endogenous mouse tau. Aβ1–40 and Aβ1–42 rise progressively in transgenic slice culture medium and stabilise when regular feeding balances continued production. In contrast, intra-axonal Aβ continues to accumulate in close correlation with a specific decline in presynaptic proteins. There are no fibrillar plaques under these conditions indicating that plaques are not needed for synaptic disruption. Beta secretase inhibitor treatment completely abolished the accumulation of Aβ1–42 in the medium but surprisingly this did not rescue synaptophysin levels, even when continually present from before synaptophysin declines. This raises the question of whether BACE1-independent APP products underlie some synaptic defects and provides an effective experimental system to test this. Interestingly, genetic deletion of tau does rescue the loss of synaptophysin, opening lines of investigation for potential therapeutics. Organotypic brain slices from TgCRND8 mice represent an important new system for understanding mechanisms of presynaptic disruption induced by the presence of mutant huAPP. The pathology observed in these cultures will allow for rapid assessment of disease modifying compounds in a system amenable to manipulation and observation. Future work will involve examination of the mechanisms resulting in synaptophysin depletion, particularly in relation to the involvement of tau and alternative APP processing products.