Elsevier

Experimental Neurology

Volume 239, January 2013, Pages 148-157
Experimental Neurology

Lack of the transcription factor C/EBPδ impairs the intrinsic capacity of peripheral neurons for regeneration

https://doi.org/10.1016/j.expneurol.2012.10.012Get rights and content

Abstract

Adult neurons of the peripheral nervous system (PNS), in contrast to those of the central nervous system, have a remarkable capacity to repair themselves after injury, yet the mechanisms underlying this regenerative propensity of peripheral neurons are far from completely understood. Here we show that the transcription factor CCAAT enhancer binding protein delta (C/EBPδ) is necessary for the efficient axonal regeneration of dorsal root ganglia (DRG) neurons after sciatic nerve crush injury. Loss of C/EBPδ substantially impairs axonal growth in dissociated cultured DRG neurons. In addition, lack of C/EPBδ causes a major reduction in the regenerative response of DRG neurons to a conditioning lesion, which is a well known paradigm of injury that enhances axonal growth due to a transcription-dependent cell body response. C/EBPδ is required for the induction of selected regeneration-associated genes. For example, the expression of SPRR1A (small proline-rich repeat protein 1A) is greatly reduced in DRG neurons of C/EBPδ knockout mice during axonal regeneration compared to those in wild-type mice, while the expression of GAP-43 (growth associated protein-43) and galanin is not affected. Nevertheless, the expected prompt recovery of sciatic nerve function after injury is severely impaired in C/EBPδ knockout mice, having a delay time of approximately 1 month for reaching the full function of recovering wild-type mice, suggesting that a transcription mechanism mediated by C/EBPδ is required for efficient axonal regeneration. Taken together, our results identify C/EBPδ as a crucial component of the transcriptional regulatory machinery which underlies the intrinsic capacity of peripheral neurons for axonal regeneration.

Highlights

► The transcription factor C/EBPδ is associated with the neuronal response to injury. ► Lack of C/EBPδ impairs the early phase of axonal regeneration in peripheral nerves. ► C/EBPδ affects the regulation of selective genetic regenerative pathways. ► C/EBPδ is required for efficient functional recovery after peripheral nerve injury.

Introduction

Peripheral axons, unlike the axons of the central nervous system (CNS), regenerate successfully after injury. The mechanisms of peripheral nervous system (PNS) repair have been extensively investigated in sensory neurons of the dorsal root ganglia (DRG), which have peripheral and central axonal branches with respective different regenerative responses to injury (reviewed in Chen et al., 2007). Inducible events in the cell body of DRG neurons have been proposed to underlie the key mechanisms which are responsible for axonal regeneration in the sciatic nerve (Liu et al., 2011). The prominent paradigm of conditioning, in which an initial lesion to the peripheral axons of DRG neurons accelerates regeneration of their central axons after crush injury (Richardson and Verge, 1987), has revealed that axonal regeneration is governed by transcriptional mechanisms (Smith and Skene, 1997). In this context, transcription factors responsive to injury signals, and capable of regulating the expression of regeneration-associated genes (RAGs), are a focus for identifying the key intrinsic determinants of the neuronal regeneration program (Raivich, 2011). Consequently, such transcription factors are potential molecular targets in therapeutic strategies aimed at enhancing the intrinsic regeneration capacity of neurons in neurodegenerative conditions of the CNS (Yang and Yang, 2012). So far, several widely known transcription factor families have been implicated in axonal regeneration, including members of the cAMP responsive element binding protein, CREB (Gao et al., 2004, Seijffers et al., 2007), signal transducer and activator of transcription, STAT (Bareyre et al., 2011), activator protein 1, AP-1 (Raivich et al., 2004), SRY-box containing factor, SOX (Jankowski et al., 2009), and the kruppel-like factor, KLF, family (Moore et al., 2009), indicating to the existence of multitude regulatory pathways of regeneration in response to injury. Therefore, it is apparent that the prior identification of the complete milieu of the transcriptional pathways regulating axonal regeneration is of fundamental importance for characterizing the key regenerative mechanisms required for successful neuronal repair.

Here we hypothesized that an additional transcription factor family, the CAAT/enhancer binding protein (C/EBP) family, is involved in peripheral nerve repair. The C/EBP family, comprising six members, C/EBPα (alpha), β (beta), γ (gamma), δ (delta), ε (epsilon), and ζ (zeta), is well known for regulating the expression of a diverse array of genes in biological processes, such as cellular differentiation and proliferation, metabolism and inflammation, in non-neuronal systems (Ramji and Foka, 2002). Our hypothesis is supported by studies showing that C/EBPs are associated with neuronal processes related to nerve repair. For example, C/EBPβ has been associated with the transcriptional response to neuronal injury (Nadeau et al., 2005), C/EBPδ is widely expressed in spinal cord motor neurons and DRG (Sterneck et al., 1998), C/EBPδ and C/EBPβ are responsive to regenerative cAMP signals in neurons (McCauslin et al., 2006, Yukawa et al., 1998), and recently C/EBPs were found to be involved in axonal outgrowth in vitro (Macgillavry et al., 2011). In this study, based on the above hypothesis, we have identified C/EBPδ as focal member of the C/EBP family involved in axonal regeneration, and we have addressed its function in peripheral nerve repair in vivo.

Section snippets

Animals and surgery

Adult animals, 3–6 months old, of either sex were used in all the experiments, with the exception in behavioural experiments where only female animals were grouped for testing. C/EBPδ knockout (KO) mice were previously generated in the C56BL/6 strain (Sterneck et al., 1998). Heterozygous KO animals were bred on the same genetic background of the C56BL/6 strain (Charles River), and KO homozygous (C/EBPδ null) and wild-type (WT) offspring were used. The genotype of the mice was diagnosed by a

Expression of C/EBPδ is associated with axonal regeneration

In order to investigate the potential involvement of the C/EBP gene family in axonal regeneration, the mRNA expression of five C/EBP family members, C/EBPα, -β, -γ, -δ, and -ε, was examined in DRG after sciatic crush nerve injury. L4-L5 DRG from the injured (ipsilateral) and non-injured (contralateral) side were assayed for C/EBP gene expression by quantitative RT-PCR, at different time points after injury. The first time point chosen was 4 h after injury, representing early events in the

Discussion

In this study we have identified the transcription factor C/EBPδ as a regulator of neuronal growth in the repairing PNS in response to injury. We found that C/EBPδ is the only member of the C/EBP family whose expression parallels the early regenerative phase of DRG neurons after sciatic nerve crush injury, albeit the expression of two other members, C/EBPβ and -γ, is induced for shorter periods during the early response to injury. This pattern of C/EBPδ expression is in accordance with

Acknowledgments

We are grateful to Dr. Esta Sterneck from the National Cancer Institute, Frederick, for providing the C/EBPδ knockout mice and to Prof. Stephen Strittmatter from Yale University School of Medicine for providing the SPRR1A antibody. We thank Mr. Graham McPhail, from the Celllular Pathology Division of Barts and the London, for his expertise assistance in processing nerve tissue, and Prof. Adina Michael-Titus for critical reading our manuscript. LLH was supported by a PhD studentship from Barts

References (35)

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