A Neuron, Microglia, and Astrocyte Triple Co-culture Model to Study Alzheimer’s Disease

Abstract

Glial cells are essential to understand Alzheimer’s disease (AD) progression, given their role in neuroinflammation and neurodegeneration. There is a need for reliable and easy to manipulate models that allow studying the mechanisms behind neuron and glia communication. Currently available models such as co-cultures require complex methodologies and/or might not be affordable for all laboratories. With this in mind, we aimed to establish a straightforward in vitro setting with neurons and glial cells to study AD. We generated and optimized a 2D triple co-culture model with murine astrocytes, neurons and microglia, based on sequential seeding of each cell type. Immunofluorescence, western blot and ELISA techniques were used to characterize the effects of oligomeric Aβ (oAβ) in this model. We found that, in the triple co-culture, microglia increased the expression of anti-inflammatory marker Arginase I, and reduced pro-inflammatory iNOS and IL-1β, compared with microglia alone. Astrocytes reduced expression of pro-inflammatory A1 markers AMIGO2 and C3, and displayed a ramified morphology resembling physiological conditions. Anti-inflammatory marker TGF-β1 was also increased in the triple co-culture. Lastly, neurons increased post-synaptic markers, and developed more and longer branches than in individual primary cultures. Addition of oAβ in the triple co-culture reduced synaptic markers and increased CD11b in microglia, which are hallmarks of AD. Consequently, we developed a straightforward and reproducible triple co-cultured model, where cells resemble physiological conditions better than in individual primary cultures: microglia are less inflammatory, astrocytes are less reactive and neurons display a more mature morphology. Moreover, we are able to recapitulate Aβ-induced synaptic loss and CD11b increase. This model emerges as a powerful tool to study neurodegeneration and neuroinflammation in the context of AD and other neurodegenerative diseases.

Mutual interaction of neurons and astrocytes derived from iPSCs with APP V717L mutation developed the astrocytic phenotypes of Alzheimer’s disease

“…Luchena et al (2022) developed the triplet co-culture model of the murine rat neurons, astrocytes, and microglia. Similar to our study, the neurons in this triplet co-culture model developed longer neurite branches and expressed more post-synaptic markers, while the co-cultured astrocytes showed ramified morphology compared to the monocultured astrocytes [ 60 ]. Furthermore, microglia in the triplet co-culture model express less pro-inflammatory markers and more anti-inflammatory markers [ 60 ].…”

“…Similar to our study, the neurons in this triplet co-culture model developed longer neurite branches and expressed more post-synaptic markers, while the co-cultured astrocytes showed ramified morphology compared to the monocultured astrocytes [ 60 ]. Furthermore, microglia in the triplet co-culture model express less pro-inflammatory markers and more anti-inflammatory markers [ 60 ]. In addition, co-culture of iPSC-derived neurons and rat astrocytes accelerated the maturation of iPSC-derived neurons [ 61 ].…”

Regulation of LPS-Induced Inflammatory Responses in HMC3 Microglia by Nicotine and its Neuroprotective Effects

“…To investigate the indirect neuroprotective effect of nicotine, we established a co-culture system of human-derived microglia and neurons. Co-culture models are often used in the study of neurological diseases to more closely mimic the actual disease state, such as in the study of Alzheimer's disease, where Luchena Celia et al created a triple co-culture model consisting of mouse astrocytes, neurons, and microglia [49] . To investigate the effect of microglia on cortical neurons, Lopez-Lengowski Kara et al generated microglia from human induced pluripotent stem cells (iPSCs) and co-cultured them with cortical neurons [50] .…”

β-asarone inhibits autophagic neuron death by downregulating reactive astrocytes-derived SPARC expression in LPS-induced SH-SY5Y cells

“…Previous studies have shown that astrocytes are the most widely distributed neuroglia in the CNS. They exhibit complex interactions with neurons, and their activation mediates neuronal in ammation, which plays an important role in the development of neurodegeneration [23]. The astrocyte response to neuronal damage leads to the development of reactive astrocytes, a common feature of neurodegeneration disease, especially AD [24][25][26][27].…”