Establishing a neuron-astrocyte co-culture system to model non-cell autonomous mechanisms of neurotoxicity and synaptotoxicity in the context of Alzheimer’s disease

Abstract

Aim of the work: Early alterations in AD remain ill-defined and are likely associated with alterations at the level of dendritic spines as well as neuroinflammatory processes. So far, there is a lack of simple, yet validated in vitro models to study cellular and molecular events that may be relevant in early AD. The goal of this thesis was to establish and optimize an in vitro platform of primary mouse neurons following a bottom-up approach to model the process of neuronal synaptic degeneration in the context of AD. Based on a relatively well-described protocol originally published by Banker et al., our assay offers a facile and reproducible protocol, where primary neurons are co-cultured with primary astrocytes providing paracrine support, enabling proper in vitro differentiation of neurons (74, 75). Importantly, this model overcomes some of the difficulties of complex in vivo studies as it provides separate access to two main cell types of the CNS, while it still retains some complexity allowing experimental manipulations in a dish. Accordingly, we aimed to characterize our system by modeling astrocyte-mediated neuronal changes by addressing the following key aspects: 1) Characterizing acutely dissected CNS tissue and resulting primary CNS cultures in regard to survival and cell-specific marker expression of astrocytes and neurons the co-culture assay. 2) Tracking neuronal growth and differentiation in co-cultures, thus determining the optimal time window for experimental manipulations. 3) Comparison of co-cultured neurons with neurons in monocultures in terms of neuronal health status and morphometric indices. 4) Probing the impact of experimental manipulation - including Aβ42-related effects and effects of LPS - on both astrocytes and neurons to model non-cell autonomous effects on neurotoxicity and synaptotoxicity.