Presynaptic release-regulating C5a receptors in the spinal cord of healthy mice and of animals suffering from experimental autoimmune encephalomyelitis: impact of the C5aR antagonist DF2593A.

Complement releases glutamate from the spinal cord glutamatergic synaptosomes through mechanisms involving amino-acid transporters (EAATs) and G protein coupled receptors (GPCRs), as suggested by the efficiency of the EAAT blocker dl-t-BOA and of entrapped Pertussis toxin (PTx) to half its releasing activity. The GPCR-mediated component involves C5aR1s since the C5aR1 non-competitive negative allosteric modulator DF2593A and the orthosteric antagonist W54011 concentration-dependently prevent the complement-evoked release of preloaded [3H]D-aspartate. Antagonism, however, emerged only after the prolonged exposure of synaptosomes to these ligands, suggesting a multiple-phase model of receptor antagonism. Consistent with the involvement of this receptor, spinal cord synaptosomal lysates showed C5aR1 immunostaining corresponding to a dimeric assembly of the protein. We next examined mice suffering from the experimental autoimmune encephalomyelitis (EAE), at the acute stage of the disease (21 ± 1 days post immunization). Spinal cord homogenates from these mice show decreased MOG immunopositivity, suggestive of diffuse demyelination, but increased GFAP, vimentin, CD11b and CD45 staining, predictive of ongoing astrocytosis, microgliosis and infiltration of lymphocytes. These signs were paralleled by the overaccumulation of C1q, C3 and C5 components and increased C5aR1 staining, but conserved GLT1 one, in synaptosomal lysates. Lastly, the complement-evoked [3H]D-Aspartate overflow from spinal cord synaptosomes was amplified when compared to controls, but still significantly reduced by DF2593A. These findings prove the existence of presynaptic release-regulating DF2593A-sensitive C5aR1s in spinal cord glutamatergic terminals, opening the road to therapeutic application of C5aR1 modulators to control symptoms associated with altered synaptic transmission during multiple sclerosis.