Upregulation of activating transcription factor 3 (ATF3) by intrinsic CNS neurons regenerating axons into peripheral nerve grafts

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Abstract

The expression of the transcription factor ATF3 in the brain was examined by immunohistochemistry during axonal regeneration induced by the implantation of pieces of peripheral nerve into the thalamus of adult rats. After 3 days, ATF3 immunoreactivity was present in many cells within approximately 500 μm of the graft. In addition, ATF3-positive cell nuclei were found in the thalamic reticular nucleus (TRN) and medial geniculate nuclear complex (MGN), from which most regenerating axons originate. CNS cells with ATF3-positive nuclei were predominantly neurons and did not show signs of apoptosis. The number of ATF3-positive cells had declined by 7 days and further by 1 month after grafting when most ATF3-positive cells were found in the TRN and MGN. 14 days or more after grafting, some ATF3-positive nuclei were distorted and may have been apoptotic. In some experiments of 1 month duration, neurons which had regenerated axons to the distal ends of grafts were retrogradely labeled with DiAsp. ATF3-positive neurons in these animals were located in regions of the TRN and MGN containing retrogradely labeled neurons and the great majority were also labeled with DiAsp. SCG10 and c-Jun were found in neurons in the same regions as retrogradely labeled and ATF3-positive cells. Thus, ATF3 is transiently upregulated by injured CNS neurons, but prolonged expression is part of the pattern of gene expression associated with axonal regeneration. The co-expression of ATF3 with c-jun suggests that interactions between these transcription factors may be important for controlling the program of gene expression necessary for regeneration.

Introduction

Axons in injured peripheral nerves regenerate vigorously following injury, but most axons in the CNS show only an initial sprouting response, which is soon aborted. The failure of axonal regeneration in the CNS is believed to result, in part, from an inadequate cell body response to axotomy. When motor or primary sensory (DRG) neurons are axotomized, they undergo dramatic changes in morphology and gene expression (e.g., Chong et al., 1992, Lieberman, 1971, Mason et al., 2002) but some CNS neurons show virtually no response to axotomy (Kloss et al., 1999, Mason et al., 2003a, Mason et al., 2003b, Miller et al., 1989). Some populations of CNS neurons can regenerate axons over long distances through nerve grafts implanted into the brain or spinal cord (Morrow et al., 1993, Richardson et al., 1980), and this ability can be correlated with the extent of the cell body response to axotomy (Anderson et al., 1998). The two thalamic nuclei with the greatest capacity for axonal regeneration are the thalamic reticular nucleus (TRN) and subregions of the medial geniculate nucleus (MGN) (Benfey et al., 1985, Morrow et al., 1993). Following nerve graft implantation, neurons in these nuclei show similar changes in growth-related gene expression to those seen in regenerating peripheral neurons (Mason et al., 2003a, Mason et al., 2003b). The key molecules for orchestrating the programs of gene expression necessary for regenerating an axon are transcription factors. The transcription factor whose expression has been most closely associated with axonal regeneration is c-jun, which is upregulated by DRG and motor neurons following peripheral nerve injury (Jenkins and Hunt, 1991) and by intrinsic CNS neurons when regenerating axons into peripheral nerve grafts (Vaudano et al., 1998). More recently, it has been recognized that another transcription factor, ATF3, is also upregulated strongly and selectively by injured sensory and motor neurons and following peripheral nerve injury (Tsujino et al., 2000, Tsuzuki et al., 2001, Tsuzuki et al., 2002). Its expression is correlated with the duration of axonal regeneration (Tsujino et al., 2000). ATF-3 is one member of a large family of bZip leucine zipper transcription factors that bind to promoters responsive to cAMP and phorbol ester at the related cAMP (CRE) and phorbol ester response elements and AP-1 sites (reviewed in Hai and Hartman, 2001). ATF3 is particularly interesting in the context of axonal regeneration because it can form heterodimers with c-Jun (see review by Hai and Hartman, 2001) and, in contrast to c-Jun, is undetectable in the great majority of undamaged neurons. Heterodimers of ATF3 and c-Jun bind to different sites and presumably have different effects on transcription than homodimers. ATF3 is a stress-induced factor and is also upregulated by injured CNS neurons (Takeda et al., 2000) but whether such neurons express ATF3 when regenerating axons is not clear. We have therefore examined the pattern of ATF3 expression in intrinsic CNS neurons when regenerating axons into peripheral nerve grafts in the thalamus. We have also correlated ATF3 expression with that of c-jun and SCG10, a protein which is strongly upregulated by most regenerating neurons (Mason et al., 2002) and therefore acts as a good marker for such cells.

Section snippets

Surgery

All surgical procedures were approved by the UCL ethical committee and licensed by the Home Office. Twenty-three adult female Sprague–Dawley rats, weighing 180–250 g at the time of implantation, received peripheral (tibial) nerve grafts to the thalamus. The rats were anesthetized with Halothane, nitrous oxide, and oxygen mixture. Segments approximately 1.5 cm long of the left tibial nerve were removed and one end implanted through a craniotomy into the left thalamus, using coordinates taken

3 and 7 days after grafting

Large numbers of cells with ATF3 immunopositive nuclei were present in the cerebral cortex, hippocampus, and thalamus surrounding the graft (Fig. 1, Fig. 4, inset). The labeling was restricted to nuclei within the size range of approximately 12 μm to 20 μm in diameter and, therefore, probably in neurons (Fig. 2). The nuclei were oval or round and appeared normal in size and shape (Fig. 2). There were a few, considerably smaller, immunopositive nuclei, 5–8 μm in diameter, but it was not possible

Discussion

We have shown that ATF3 is rapidly and strongly upregulated by many neurons around peripheral nerve grafts in the thalamus of adult rats. By 1 month after grafting, the number of ATF3-positive neurons had declined dramatically and most were found in thalamic nuclei known to contain neurons with a high regenerative capacity. SCG10 and c-Jun were expressed by similar numbers of neurons in the same locations as ATF3-positive cells. Retrograde labeling showed that most of the neurons that retained

Acknowledgment

This work was supported by BBSRC.

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