REPEATED INFLAMMATORY CHALLENGES INDUCE ASTROCYTE-ASSOCIATED INFLAMMATORY PRIMING AND ALZHEIMER’S DISEASE–RELATED MOLECULAR SIGNATURES IN HUMAN CORTICAL ORGANOIDS

Neurodegenerative diseases (NDs) represent a growing public health challenge with major socio-economic impact. Aging is the major risk factor for NDs such as Alzheimer’s disease (AD), yet the mechanisms linking aging to brain vulnerability remain elusive. Emerging evidence suggests that neuroinflammation is not merely a consequence of NDs but can also contribute to disease initiation and progression. Importantly, aging is associated with chronic low-grade inflammation. Astrocytes are key mediators of brain inflammatory responses and have been increasingly implicated in aging-associated dysfunctions and NDs. Our lab recently identified specific astrocytic dysfunctions associated with neuroinflammation in a human AD model. Accordingly, we hypothesize that repeated inflammatory challenges may induce persistent astrocytic dysfunctions promoting aging-associated inflammatory states and neurodegenerative vulnerability

I established a human cortical organoid (hCO) platform derived from multiple hiPSC lines, enabling long-term longitudinal analyses in a microglia-free 3D context designed to isolate neuron–astrocyte interactions. hCOs were subjected to a cytokine cocktail mimicking microglial inflammatory signaling, using paradigms of single or repeated exposures separated by resolution periods. Inflammatory outputs were assessed longitudinally using ELISA, qPCR, immunochemistry and transcriptomics.

hCOs mounted robust pro-inflammatory responses ( IL-6, IL-8, and CCL2 overexpression). Critically, repeated challenges resulted in a progressive amplification and prolongation of cytokine release, independent of organoid age,consistent with inflammatory priming or immune-like memory–like behavior. Parallel experiments in hiPSC-derived astrocyte monocultures recapitulated these effects, supporting astrocytes as major contributors to this phenomenon. Preliminary immunohistochemical analyses revealed changes in AD related molecular markers (AT8, 6E10) following repeated inflammatory stimulations.