Background: The complement factor C1q is released by microglia, localizes on weak synapses and acts as a tag for microglia-mediated synaptic pruning, a fundamental process for proper circuit refinement across early postnatal life, reactivated in neurodegeneration and Alzheimer’s disease. However, how C1q tags synapses at specific times remains elusive. We explored the possible involvement of extracellular vesicles (EVs) released by microglia in C1q delivery to synapses designated for removal.

Methods: We optimized a protocol to extract large and small EVs from the interstitial fluid of mouse brain tissue. By Western Blot, TRPS, single molecules arrays (Simoa) and confocal imaging, we measured the amount, cell source and complement cargo of EVs extracted from WT postnatal pups and adult mice brains, and from C9orf72KO adult brains displaying enhanced microglia-mediated pruning. Then, we performed in vitro and ex vivo studies to inquire microglial EVs involvement in C1q deposition to the synapse and synaptic engulfment, using approaches to increase or reduce microglial EVs production by EVs supplementation C9orf72 silencing or pharmacological inhibition (GW4869).

Results: We show that microglia release more EVs carrying C1q in C9orf72KO adult mice, providing a link between microglial EVs production and synaptic removal. Moreover, we report that production of C1q carrying microglial EVs peaks during the pruning period in the early postnatal hippocampus (postnatal day P17), confirming a positive correlation between EVs production and synaptic removal in a physiological setting. In neuron-microglia co-cultures, microglial EVs, labelled by the fluorescent dye mCLING, make preferential contacts with synapses, deliver C1q to pre-synapses that externalize phosphatidylserine and promote synaptic removal. Interestingly, C9orf72 KO microglia engulf more synaptic terminals and decreases synaptic density to a greater extent compared to WT microglia whereas inhibition of EVs release by GW4869 restores normal pre-synaptic density, providing mechanistic evidence linking EVs release to synaptic remodeling.

Conclusion: This study identifies microglial EVs as delivery vehicles for C1q to synapses targeted for removal and implicates abnormal EVs production from microglia in neurodegeneration and age-related disorders characterized by dysregulated synaptic pruning. Analysis of specific subpopulations of brain EVs opens new perspectives to the understanding of EVs roles in physiology and pathology.