# Tri‐Culture System Reveals an Activation Cascade From Microglia Through Astrocytes to Neurons During Neuroinflammation

## TL;DR

A tri-culture system using human stem cells reveals how microglia, astrocytes, and neurons interact during neuroinflammation, a process linked to neurodegenerative diseases.

## Contribution

The study introduces a tri-culture system derived from human iPSCs to model and reveal a signaling cascade in neuroinflammation involving microglia, astrocytes, and neurons.

## Key findings

- LPS activates microglia via TLR4 and NF-κB, leading to TNF-α release.
- TNF-α activates astrocytes, which in turn increase neuronal excitability.
- The tri-culture system demonstrates an amplification of inflammatory signals through intercellular communication.

## Abstract

Neuroinflammation is involved in various neurodegenerative diseases, with glial cells playing crucial roles. It is known that neuroinflammation is initiated by microglia, which interact with astrocytes and neurons. However, the detailed molecular mechanisms underlying intercellular interactions during neuroinflammation are not fully understood. In this study, we developed a tri‐culture system of neurons, astrocytes, and microglia derived from human induced pluripotent stem cells (iPSCs) to evaluate their relationships in neuroinflammation. Microglia cocultured with the astrocytes and neurons exhibited a morphology with branched processes compared to the monoculture system, suggesting a homeostatic state. By applying lipopolysaccharide (LPS) stimulation to induce inflammation, the microglial morphology shifted to an amoeboid shape, accompanied by an increase in the expression of pro‐inflammatory cytokines. Additionally, nuclear translocation of NF‐κB revealed that LPS specifically activates microglia through the TLR4 receptor, which subsequently releases TNF‐α, leading to the activation of astrocytes. Furthermore, activated astrocytes were shown to enhance neuronal excitability. Using the tri‐culture system, we elucidated a part of the cascade involving microglia, astrocytes, and neurons during neuroinflammation and demonstrated the amplification of inflammatory signals through cell communication. This culture system will be valuable for conducting detailed investigations into the interactions between glia and neurons, advancing research on neurodegenerative diseases associated with neuroinflammation.

Neuroinflammatory signaling was examined using a tri‐culture of neurons, astrocytes, and microglia, derived from human induced pluripotent stem cells. Lipopolysaccharide (LPS) stimulated microglia via Toll‐like receptor 4 (TLR4) and nuclear factor kappa B (NF‐κB) activation, inducing tumor necrosis factor alpha (TNF‐α) release. TNF‐α subsequently activated astrocytes, resulting in increased neuronal excitability. This human tri‐culture system describes an intercellular cascade that amplifies neuroinflammatory signals relevant to neurodegenerative disease pathology.