CCAAT enhancer-binding protein (C/EBP) in focal cortical ischemia: Expression and functional evidence for its role in neurite formation

Stroke is the leading cause of disability in the adult population. Many studies indicate that axonal outgrowth and new synapse formation are key events in improving functional outcome after stroke. However, the molecular mechanisms by which neuritogenesis and synaptogenesis are regulated following ischemic stroke are not known. The C/EBP (CCAAT-enhancer binding protein) family has been identified as a key factor in phenotypic modulation and differentiation, although mainly in non-nervous tissue. A growing body of mostly indirect evidence suggests it may have comparable roles in the nervous system. We sought to investigate its role in neuroplastic responses. The objective of this project was twofold: to characterize the expression of C/EBP isoforms (C/EBP-α, -β, and -δ) in the cerebral cortex after stroke and to identify, in vitro, a potential role for these in promoting neurite outgrowth. The cell types and regions expressing C/EBP-α, -β, and -δ were identified by applying single- and double-immunohistochemical labeling techniques to the brain sections of animals subjected to cortical photothrombosis and recovered from a few hours to several days. C/EBP-α and -β were found in neurons while C/EBP δ appeared in astrocytes and in endothelial cells. The levels of these proteins increased after stroke with marked upregulation observed in C/EBPα- and C/EBP β-positive cells. C/EBP-α and -β were elevated in the surviving tissue that immediately surrounded the site of injury, and C/EBP β was additionally enhanced in the homotopic area of the contralateral cerebral cortex. To identify a potential role in enhancing neurite outgrowth in vitro, C/EBP-α and -β isoforms induced after stroke were first identified by Western analysis and then were overexpressed in PC12 cells pretreated with nerve growth factor (NGF) to induce neurite outgrowth and immunofluorescence microscopy was performed. A complex, but reproducible pattern emerged where wild type isoforms inhibited neurite extension while the C/EBP β isoforms that increased after stroke significantly enhanced synaptophysin-positive neurite extension.; These findings support the hypothesis that C/EBP α and C/EBP β are expressed in neurons after stroke and indicate a potential neurite modulating role for C/EBP.