FUNCTIONAL EFFECTS AND RECIPIENT CELLS FOLLOWING PRO-INFLAMMATORY SMALL EXTRACELLULAR VESICLES TRANSFER TO NAÏVE SPINAL CORD EXPLANTS

Small extracellular vesicles (sEVs) are lipid bilayer-enclosed nanoparticles carrying relevant biomolecules. Despite extensive investigation of vesicles’ role as intercellular messengers in the CNS, sEVs’ contribution to neuroinflammation - a process involving transcellular signaling to modulate neuron-glia functional states - remains elusive. To investigate sEVs’ role in neuroinflammation we exploited organotypic spinal cord cultures (OSCs, Thalhammer et al., 2022), which preserve native neuron-glia spatial organization and intra-segmental circuitry. Pro-inflammatory sEVs were separated from cytokine-challenged (sEVs_ck; Giacco et al., 2019) OSCs via differential centrifugation steps followed by ultracentrifugation or size-exclusion chromatography and characterized by electron microscopy and western blot. In a second set of experiments, sEVs_ck were transferred to naïve spinal explants. sEVs_ck induced a distinct temporal pattern of tissue reactivity in recipient OSCs: microglial amoeboid shift represented the earliest response (4 h), followed by increased astrocytic calcium event frequency and elevation of pro-inflammatory cytokines in the extracellular milieu (24 h). Whole-cell patch-clamp further confirmed the pro-inflammatory nature of sEVs_ck, revealing increased neuronal excitability at 24 h. To investigate whether sEVs_ck signaling exhibits cell-type directionality, donor OSCs were engineered to release sEVs_ck carrying Cre recombinase and delivered to Ai9 reporter OSCs, enabling genetic tracing of recipient cells. Overall, our findings not only identify sEVs as important physiopathology-defining vectors in neuroinflammation but also suggest that sEV dissemination may follow cell-specific routes, rather than non-specific diffusion, with broad implications for identifying key cellular actors in neuroinflammation propagation.