Abstract
Studies have found that molecular targets that regulate tissue development are also involved in regulating tissue regeneration. Erythropoietin-producing hepatocyte A4 (EphA4) not only plays a guiding role in neurite outgrowth during the development of the central nervous system (CNS) but also induces injured axon retraction and inhibits axon regeneration after spinal cord injury (SCI). EphA4 targets several ephrin ligands (including ephrin-A and ephrin-B) and is involved in cortical cell migration, axon guidance, synapse formation and astrocyte function. However, how EphA4 affects axon regeneration after SCI remains unclear. This study focuses on the effect and mechanism of EphA4-regulated astrocyte function in neuronal regeneration after SCI. Our research found that EphA4 expression increased significantly after SCI and peaked at 3 days post-injury; accordingly, we identified the cellular localization of EphA4 and ephrin-B ligands in neurons and astrocytes after SCI. EphA4 was mainly expressed on the surface of neurons, ephrin-B1 and ephrin-B3 were mainly localized on astrocytes, and ephrin-B2 was distributed on both neurons and astrocytes. To further elucidate the effect of EphA4 on astrocyte function after SCI, we detected the related cytokines secreted by astrocytes in vivo. We found that the levels of neurotrophic factors including nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) increased significantly after SCI (NGF peaked at 3 days and bFGF peaked at 7 days); the expression of laminin and fibronectin increased gradually after SCI; the expression of inflammatory factors [interleukin (IL)-1β and IL-6] increased significantly from 4 h to 7 days after SCI; and the levels of glial fibrillary acidic protein (GFAP), a marker of astrocyte activation, and chondroitin sulphate proteoglycan (CSPG), the main component of glial scars, both peaked at 7 days after SCI. Using a damaged astrocyte model in vitro, we similarly found that the levels of related cytokines increased after injury. Consequently, we observed the effect of damaged astrocytes on neurite outgrowth and regeneration, and the results showed that damaged astrocytes hindered neurite outgrowth and regeneration; however, the inhibitory effect of injured astrocytes on neurite regeneration was reduced following ephrin-B receptor knockdown or inflammatory inhibition at 24 h after astrocyte injury. Our results showed that EphA4 regulates the secretion of neurotrophic factors, adhesion molecules, inflammatory factors and glial scar formation by binding with the ligand ephrin-B located on the surface of astrocytes. EphA4 affects neurite outgrowth and regeneration after SCI by regulating astrocyte function.
Highlights
After spinal cord injury (SCI), Eph family members and their ligands accumulate in the proximal axon stump and reactive astrocytes and play an important role in the formation of glial scars and neurite regeneration (Bundesen et al 2003)
To determine the effect of Erythropoietin-producing hepatocyte A4 (EphA4) binding to ephrin-B ligands on neurite outgrowth and regeneration after SCI, we first detected the expression of EphA4 and examined the cell localization of EphA4 and ephrin-B ligands after SCI
The results showed that EphA4 expression increased significantly, peaked at 3 days, and maintained at a high level from 7 to 28 days after SCI (Fig. 1a and b)
Summary
After spinal cord injury (SCI), Eph family members and their ligands (ephrins) accumulate in the proximal axon stump and reactive astrocytes and play an important role in the formation of glial scars and neurite regeneration (Bundesen et al 2003). Some research found that the number of astrocytes and the area of glial scars were significantly reduced after lateral spinal cord hemisection in epha4−/− mice (Herrmann et al 2010); in contrast, epha gene deletion did not significantly affect the reactive proliferation of astrocytes or promote the formation of astrocyte fibrosis scar after spinal cord hemisection in mice (Goldshmit et al 2004) This discrepancy cannot be explained by different animal models, Turnley et al re-examined the proliferation of astrocytes after injury in epha null mice and found that the reactivity of astrocytes in the injured area did not decrease, and the degree of other phenotypes such as that of the spinal dorsal cord did not change. The results suggest that EphA4 expression in the injured area after SCI is involved in the regulation of astrocyte function, which affects the regeneration and functional reconstruction of neuronal processes after SCI
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