Primary cortical cell tri-culture to study effects of amyloid-beta on microglia function and neuroinflammation

Introduction: Etiology and progression of sporadic Alzheimer’s Disease (AD) have been studied for decades, but underlying mechanisms of the disease are still not fully revealed. One generally-accepted mechanism is that soluble Aβ oligomers throughout the brain play a role in neuroinflammation, synapse loss, and neuronal death. However, the underpinning processes of cellular uptake of Aβ and its interplay with neuroinflammation remain elusive. One prominent hypothesis of Aβ accumulation in the brain is the dysfunctional microglia with impaired clearance of Aβ and altered motility, both of which are detrimental to neuronal viability and function. Studying these complex factors directly in vivo, where numerous confounding signals exist, is extremely challenging. There is, therefore, a need for an in vitro model that more accurately captures the in vivo phenomena in the context of AD.

Materials and

Methods: Tri-cultures of neurons, astrocytes, and microglia were established using protocols previously described (Goshi and Seker et al., 2020). On day in vitro 7 (DIV 7) both neuron-astrocyte co- and tri-cultures were challenged with fluorescently-labeled Aβ (FITC-Aβ) particles for 48-96 hours. The cells were then stained for βIII tubulin and Iba1, and epifluorescence images were analyzed to extract number of FITC-Aβ particles. Additionally, cytokine profile of conditioned culture media was analyzed. To study the microglial motility in response to Aβ, microglia was isolated from the tri-culture to form a mono-culture and FITC-Aβ was added. Live-cell imaging was initiated at the beginning of the treatments, where time-lapse images were acquired every 5 minutes for 4 hours.

Results, Conclusions, and Discussions: Our results show that FITC-Aβ particles were effectively cleared in the tri-culture configuration by the microglia engulfing FITC-Aβ particles (Fig 1A&B). Microglia size increased in response to Aβ treatment. In addition, we observed overall increase of proinflammatory cytokines (TNF-α, IL-1α, IL-1β, IL-6) in tri-culture but not in co-culture upon FITC-Aβ addition (Fig 1C). Furthermore, microglia motility was altered due Aβ particles in tri-culture (Fig 1E) exhibiting shorter displacements (Fig 1F) and directionality (Fig 1G), but not in mono-culture (Fig 1D), emphasizing the importance cellular crosstalk in the tri-culture. These results highlight the potential of the tri-culture model to investigate microglial clearance of Aβ particles, alterations in microglia motility, and neuronal-glial communication under Aβ exposure. We expect that the model will allow for further mechanistic studies of AD and assist in screening therapeutic modalities.

Acknowledgements (Optional): We gratefully acknowledge the support from the National Institutes of Health via NIH R03-NS118156, P30-ES023513, R01-EB034279.