Retinal Ganglion Cell Axon Regeneration Requires Complement and Myeloid Cell Activity within the Optic Nerve

Abstract

Axon regenerative failure in the mature CNS contributes to functional deficits following many traumatic injuries, ischemic injuries, and neurodegenerative diseases. The complement cascade of the innate immune system responds to pathogen threat through inflammatory cell activation, pathogen opsonization, and pathogen lysis, and complement is also involved in CNS development, neuroplasticity, injury, and disease. Here, we investigated the involvement of the classical complement cascade and microglia/monocytes in CNS repair using the mouse optic nerve injury (ONI) model, in which axons arising from retinal ganglion cells (RGCs) are disrupted. We report that central complement C3 protein and mRNA, classical complement C1q protein and mRNA, and microglia/monocyte phagocytic complement receptor CR3 all increase in response to ONI, especially within the optic nerve itself. Importantly, genetic deletion ofC1q,C3, orCR3attenuates RGC axon regeneration induced by several distinct methods, with minimal effects on RGC survival. Local injections of C1q function-blocking antibody revealed that complement acts primarily within the optic nerve, not retina, to support regeneration. Moreover, C1q opsonizes and CR3+microglia/monocytes phagocytose growth-inhibitory myelin debris after ONI, a likely mechanism through which complement and myeloid cells support axon regeneration. Collectively, these results indicate that local optic nerve complement-myeloid phagocytic signaling is required for CNS axon regrowth, emphasizing the axonal compartment and highlighting a beneficial neuroimmune role for complement and microglia/monocytes in CNS repair.SIGNIFICANCE STATEMENTDespite the importance of achieving axon regeneration after CNS injury and the inevitability of inflammation after such injury, the contributions of complement and microglia to CNS axon regeneration are largely unknown. Whereas inflammation is commonly thought to exacerbate the effects of CNS injury, we find that complement proteins C1q and C3 and microglia/monocyte phagocytic complement receptor CR3 are each required for retinal ganglion cell axon regeneration through the injured mouse optic nerve. Also, whereas studies of optic nerve regeneration generally focus on the retina, we show that the regeneration-relevant role of complement and microglia/monocytes likely involves myelin phagocytosis within the optic nerve. Thus, our results point to the importance of the innate immune response for CNS repair.

The Onset of Whole-Body Regeneration in Botryllus schlosseri: Morphological and Molecular Characterization

“…Correct regeneration of lost organs in vertebrates necessitates a finely tuned interplay between inflammatory response, neovascularization and ECM remodeling to recruit stem/progenitor cells to the regenerative area and to organize the rebuilding tissues (reviewed in Mastellos et al, 2013 ). For instance, beyond its role as sentinels of immunity, C3 stimulate retina regeneration in chicken and mice ( Haynes et al, 2013 ; Peterson et al, 2021 ), while in mouse complement proteins regulate wound-healing and angiogenesis in a complex, not fully resolved manner (reviewed in Markiewski et al, 2020 ). In ascidians, C3 expression has been reported in various epithelial cells and hemocytes of several species ( Pinto et al, 2003 ; Raftos et al, 2004 ; Giacomelli et al, 2012 ).…”

Neuroinflammation, Microglia and Implications for Retinal Ganglion Cell Survival and Axon Regeneration in Traumatic Optic Neuropathy

“…It has been reported that complement protein C1q and C3 promoted axon regeneration and function recovery after SCI (81,82). Genetic ablation of C1q, C3 and CR3 receptors, completely abolished the growth-promoting effects induced by several manipulations known to promote axon regeneration in adult mice after ONC (80), including intraocular inflammation using zymosan treatment (83)(84)(85)(86), zinc chelator TPEN to block the accumulation of mobile zinc ions in injured RGCs (87), and deletion of phosphatase and tensin homolog (PTEN) with oncomodulin treatment (88,89). Resident microglia are known to express and produce both complement proteins C1q and C3 in the retina (90,91).…”

“…A recent study highlighted that complement activation in resident retinal microglia and infiltrating macrophages are pre-requisite for successful axon regeneration after ONC. Both complement proteins C1q and C3 as well as the complement receptor CR3, were markedly elevated in the optic nerve but not in the mouse retinae shortly after ONC (80). It has been reported that complement protein C1q and C3 promoted axon regeneration and function recovery after SCI (81,82).…”

“…Resident microglia are known to express and produce both complement proteins C1q and C3 in the retina (90,91). To address the roles of resident retinal microglia in the production of complement proteins and activation of complement signaling cascades, the authors attempted to deplete microglia using PLX5622 which allows the studies of RGC axon regeneration in the absence of microglia (80). PLX5622 and its structural homolog PLX3397 could eliminate virtually all microglia from the adult CNS through binding the colony-stimulating factor-1 receptor (CSF1R) (92)(93)(94).…”

The onset of whole-body regeneration inBotryllus schlosseri: morphological and molecular characterization

“…For instance, beyond its role as sentinels of immunity, C3 stimulate retina regeneration in chicken and mice (Haynes et al, 2013;Peterson et al, 2021), while in mouse complement proteins regulate woundhealing and angiogenesis in a complex, not fully resolved manner (reviewed in Markiewski et al, 2020). In ascidians, C3 expression has been reported in various epithelial cells and hemocytes of several species (Pinto et al, 2003;Raftos et al, 2004;Giacomelli et al, 2012).…”