Endoh-Yamagami, S. | bioRxiv Science

bioRxiv2025

Human aged astrocytes induce neurotoxicity in response to inflammatory stimuli

Astrocytes play a critical role in neuroinflammation and the pathogenesis of neurodegenerative diseases. Here we found that human induced pluripotent stem cell (iPSC)-derived astrocytes responded differently to inflammatory triggers compared to rodent astrocytes, showing increased neurotoxicity when exposed to TNF- and IFN-{gamma}. Furthermore, astrocytes with senescent features showed even higher levels of neurotoxicity in the presence of TNF- and IFN-{gamma}, suggesting a potential link between aging and neurodegenerative diseases. It was also demonstrated that LPS-activated neuron/astrocyte/microglia tri-culture produced TNF-, leading to neurotoxicity in the tri-culture when IFN-{gamma} was present. Through compound screening, we identified Janus kinase inhibitors capable of preventing neurotoxicity in astrocytes induced by TNF- and IFN-{gamma}, demonstrating the potential use of neurotoxic astrocytes as a platform for drug screening. These results provide insight into the complex relationship between aging, inflammation, and neurodegenerative diseases, emphasizing the potential of targeting astrocytes as a novel therapeutic approach for addressing neurodegenerative diseases.

neuroscience↗

bioRxiv2025

The tri-culture system reveals an activation cascade from microglia through astrocytes to neurons during neuroinflammation

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. To elucidate these mechanisms, multicellular culture systems are required, although the availability of the culture systems using human cells is limited. 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. Previously, differentiation of astrocytes from iPSCs was commonly induced using serum. However, serum stimulation has been reported to cause irreversible activation of astrocytes. Therefore, we generated astrocytes using a serum-free method and established a tri-culture system. 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-{kappa}B revealed that LPS specifically activates microglia through the TLR4 receptor, which subsequently releases TNF-, 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.

neuroscience↗