The transcription factor ATF-3 promotes neurite outgrowth

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Abstract

Dorsal root ganglion (DRG) neurons regenerate after a peripheral nerve injury but not after injury to their axons in the spinal cord. A key question is which transcription factors drive the changes in gene expression that increase the intrinsic growth state of peripherally injured sensory neurons? A prime candidate is activating transcription factor-3 (ATF-3), a transcription factor that we find is induced in all DRG neurons after peripheral, but not central axonal injury. Moreover, we show in adult DRG neurons that a preconditioning peripheral, but not central axonal injury, increases their growth, correlating closely with the pattern of ATF-3 induction. Using viral vectors, we delivered ATF-3 to cultured adult DRG neurons and find that ATF-3 enhances neurite outgrowth. Furthermore, ATF-3 promotes long sparsely branched neurites. ATF-3 overexpression did not increase c-Jun expression. ATF-3 may contribute, therefore, to neurite outgrowth by orchestrating the gene expression responses in injured neurons.

Introduction

Axons in the adult central nervous system fail to regenerate after injury, while those in the peripheral nervous system are capable of long distance growth and successful target reinnervation (Plunet et al., 2002, Snider et al., 2002). This difference results to a substantial extent from the non-permissive growth environment in the CNS which is due to the presence both of myelin-associated growth inhibitory proteins that cause axon growth cone collapse and of chondroitin sulfate proteoglycans that form a barrier for growth (Hunt et al., 2002, Filbin, 2003, Profyris et al., 2004, Schwab, 2004, Silver and Miller, 2004). Another major obstacle, however, is the absence or limited increase in the expression of growth-associated genes in injured CNS neurons in marked contrast to the robust increase found in neurons whose peripheral axons are injured (Plunet et al., 2002, Bareyre and Schwab, 2003, Mason et al., 2003).

DRG neurons with myelinated axons represent a particularly useful model for studying regeneration as they have a peripheral axonal branch that extends in the PNS and which regenerates after a nerve injury and a central axon that ascends in the dorsal columns of the spinal cord that does not regenerate after injury (Snider et al., 2002, Donnerer, 2003). Peripheral but not central axonal injury triggers substantial changes in gene expression in DRG neurons that may contribute to the regenerative capacity of the neurons after peripheral lesions (Schreyer and Skene, 1993, Chong et al., 1994, Costigan et al., 2002, Xiao et al., 2002). Richardson and colleagues found over 20 years ago that peripheral nerve lesions substantially increase the extent and rate of regeneration of injured dorsal column axons into a peripheral nerve graft (Richardson and Issa, 1984, Richardson and Verge, 1986, Oudega et al., 1994). Injuring the peripheral axons of DRG neurons before injuring their central axons enables, moreover, the central axons to regenerate into and beyond lesion sites in the spinal cord even in the absence of the permissive environment provided by a peripheral nerve graft (Neumann and Woolf, 1999). This is presumed to reflect a “conditioning” by the peripheral nerve lesion of the neurons into an active growth state, enabling the injured central axons to grow. A key question though is what drives the expression of those genes that increase intrinsic growth in DRG neurons after peripheral axonal injury?

Activating transcription factor-3 (ATF-3), a basic leucine zipper transcription factor, is rapidly upregulated in all injured DRG neurons after peripheral but not central axonal injury and is downregulated after reinnervation of peripheral targets (Tsujino et al., 2000, Bloechlinger et al., 2004). ATF-3 is typically induced by stress stimuli and cellular damage and has both apoptosis-promoting and survival functions in different tissues (Allen-Jennings et al., 2001, Hai and Hartman, 2001, Okamoto et al., 2001, Perez et al., 2001, Fan et al., 2002, Kawauchi et al., 2002, Nobori et al., 2002, Hartman et al., 2004). In the nervous system, ATF-3 appears to have a survival role (Herdegen et al., 1997). When delivered to the hippocampus, ATF-3 protects CA3 pyramidal neurons from kainic-acid-induced apoptosis (Francis et al., 2004) and rescues neonatal superior cervical ganglion neurons from apoptosis due to NGF withdrawal (Nakagomi et al., 2003). In order to study whether ATF-3 induction in DRG neurons after peripheral nerve injury contributes to the injury-induced increase in their intrinsic growth activity, we delivered the transcription factor to adult primary DRG cultured neurons using an HSV-based amplicon vector and demonstrate that ATF-3 promotes neurite outgrowth in these neurons.

Section snippets

Peripheral nerve injury induces ATF-3 expression and primes adult DRG neurons for growth

ATF-3 mRNA and protein levels were, as expected from earlier studies (Tsujino et al., 2000), induced in L4/L5 DRG neurons of adult rats after a peripheral nerve transection (Fig. 1). In situ hybridization and immunostaining revealed no expression of ATF-3 mRNA and protein in DRGs from naive non-injured rats (Figs. 1A and D respectively) but a strong upregulation in DRG neurons when measured 1 and 4 days after either transection of a sciatic (SNT) or spinal segmental nerve (spinal nerve lesion,

Discussion

Axonal regeneration involves actin stabilization along the growing axon shaft as well as its polymerization and microtubule assembly at the leading edge of the growth cone. New membrane is formed, and adhesion molecules, ion channels and multiple receptors are expressed (Heidemann, 1996, De Wit and Verhaagen, 2003, Tessier-Lavigne and Goodman, 1996, Yu and Bargmann, 2001). Protein synthesis both in the neuron cell body and locally in the axon enables cytoskeletal assembly (Steward, 2002,

Construction of HSV-based amplicon vectors

An HSV-based amplicon vector pHIAP (Seijffers and Woolf, 2004) that contains the enhanced green fluorescent protein (EGFP) reporter gene under control of the HSV immediate early (IE4/5) promoter and the human placental alkaline phosphatase (hPLAP) reporter gene fused to an IRES element under control of the CMV promoter was used as a backbone for construction of amplicons pHIAP43 and pHATF3. pHIAP43 contains rat GAP-43 coding sequence under control of the CMV promoter upstream of the IRES

Acknowledgments

We are grateful to Dr. Xandra O. Breakefield for providing the HSV amplicon and packaging system and to Dr. Miguel Sena-Esteves at the viral core. We thank Dr. Constance L. Cepko for providing the hPLAP gene, Dr. J. H. Pate Skene for the GAP-43 cDNA and Igor A. Bagayev at the confocal microscope for assistance. This work was supported by the NIH.

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