The Glial Connectome: Identifying key cellular and molecular mediators of neuroinflammation in a murine model of prion disease

While neuroinflammation and glial activation are observed by immunohistochemistry early in prion disease, the precise dynamics of inter-cellular interactions remain unclear. Identifying the phenotype and the mode of activation of these cells throughout the disease may help elucidate this enigma.

Using a murine model of prion disease, we investigated neuroimmune crosstalk across five disease stages (70, 90, 127, 155, and 170 days post-intraperitoneal RML infection), spanning the preclinical to clinical transition. Using flow cytometry analysis of ex vivo brain cells, we identified proinflammatory phenotypic changes of microglia prior to pathology. Complementing this, multiplex MSD assays showed significant upregulation of proinflammatory cytokines and chemokines, at early time points compared to age matched uninfected controls. To identify the cellular origin of these mediators, we colocalised key cytokines with glial cells- microglia, astrocytes, oligodendrocytes and OPCs. This revealed multiple glial cell types contributing to early cytokine production, indicating a complex inter-glial communication network active well before clinical onset.

To explore the molecular mechanisms underlying this crosstalk we analyse activation of key inflammatory pathways (cGAS-STING, mTOR, MYD88, STAT1/3, and NF-κB), using primary glial and neuronal cultures alongside immunoblotting. Finally, we apply OLINK proximity extension-based proteomics to brain homogenates across the same timepoints to identify candidate biomarkers associated with early cellular activation and disease onset. This complements our targeted cytokine profiling and adds translational potential.

Together, our temporally resolved, multi-modal approach provides novel insights into the cellular and molecular mechanisms driving early neuroinflammation in prion disease.