Complement Protein C3 Modulates Neurite Outgrowth of Primary Neurons in vitro

After a spinal cord injury there is a failure of axons to regenerate due to injury induced processes that include the disruption of the blood spinal barrier, formation of a glial scar, myelin associated inhibition, and inflammation.  The inflammatory response after a spinal cord injury has been shown to have both beneficial and detrimental effects, affecting injury dynamics and behavioral recovery after injury.  An often understudied branch of the inflammatory response after spinal cord injury is the complement system; complement proteins have been shown to increase after spinal cord injury and localize with neurons, oligodendrocytes, and disrupted myelin.  However, the role of complement in the spinal cord is not well understood.  Traditionally, the complement system is involved in cell lysis, opsonization, and recruitment of inflammatory cells.  There is a small set of literature describing alternative functions of complement protein C3, which include neuroprotection, neural progenitor cell differentiation and development.  However, the role of complement proteins on neurite outgrowth of primary neurons has not been previously studied.  We set out to investigate the role of the complement protein C3 on myelin mediated inhibition of neurite outgrowth.  Dissociated primary neuron growth assay experiments containing an inhibitory myelin substrate and C3 were conducted.  For neurons grown in the presence of myelin, C3 was found to exacerbate neurite inhibition.  The known functions of C3 are always mediated through the C3 cleavage products C3a and C3b, which are responsible for the recruitment of inflammatory cells and cell opsonization.  C3a is further cleaved into the immunologically inactive form, C3a desArg.  Interestingly, myelin has previously been shown to contain serine proteases, which are able to cleave C3.  Thus an ELISA for C3a was performed, when C3 was in the presence of myelin C3a production increased considerably.  Therefore, we hypothesized that one of the cleavage products of C3 (C3a, C3b, or C3a desArg) would be sufficient to inhibit neurite outgrowth of primary neurons in the absence of myelin.  However, when primary neurons were exposed to individual purified C3 cleavage products in media, neither C3a, C3b, nor C3a desArg was sufficient to inhibit neurite outgrowth in the absence of myelin.  Our results may indicate that an interaction between myelin and C3 is required to exacerbate the inhibition of neurite growth, particularly that one or more of the C3 cleavage products may directly augment the myelin-mediated inhibitory pathways. Support: NIH Grant NS43428-01, NIH Grant GM-69337