Peripheral axotomy induces long-term c-Jun amino-terminal kinase-1 activation and activator protein-1 binding activity by c-Jun and junD in adult rat dorsal root ganglia In vivo

Abstract

One of the earliest documented molecular events after sciatic nerve injury in adult rats is the rapid, long-term upregulation of the immediate early gene transcription factor c-Jun mRNA and protein in lumbar dorsal root ganglion (DRG) neurons, suggesting that c-Jun may regulate genes that are important both in the early post-injury period and during later peripheral axonal regeneration. However, neither the mechanism through which c-Jun protein is increased nor the level of its post-injury transcriptional activity in axotomized DRGs has been characterized. To determine whether transcriptional activation of c-Jun occurs in response to nerve injury in vivo and is associated with axonal regeneration, we have assayed axotomized adult rat DRGs for evidence of jun kinase activation, c-Jun phosphorylation, and activator protein-1 (AP-1) binding. We report that sciatic nerve transection resulted in chronic activation of c-Jun amino-terminal kinase-1 (JNK) in L4/L5 DRGs concomitant with c-Jun amino-terminal phosphorylation in neurons, and lasting AP-1 binding activity, with both c-Jun and JunD participating in DNA binding complexes. The timing of JNK activation was dependent on the distance of the axotomy site from the DRGs, suggesting the requirement for a retrograde transport-mediated signal. AP-1 binding and c-Jun protein returned to basal levels in DRGs as peripheral regeneration was completed but remained elevated in the case of chronic sprouting, indicating that c-Jun may regulate target genes that are involved in axonal outgrowth.

Fig. 1.

JNK is activated in vivo in dorsal root ganglia (DRG) after peripheral nerve transection, with timing dependent on distance of the lesion from the DRGs. JNK activity in L4/L5 DRGs was assessed using an immune complex kinase assay at times from 15 min to 30 d after proximal (a) or distal (b) nerve transection. Levels of JNK activation were determined as a ratio of the activity in the injured versus contralateral, uninjured DRGs. The graph shows the mean levels of activation in injured DRGs, with error bars indicating SEM. Thedotted line represents the average level of JNK activity in right versus left DRGs of three unoperated rats. For each time point, n = 3 animals. p values were calculated using the Student’s t test; *p < 0.05 or **p < 0.01 in comparison of injured versus control DRGs. Below each graph are representative autoradiographs from selected time points, showing GST-c-Jun fusion protein phosphorylated by immunoprecipitated JNK isolated from injured (I) and contralateral uninjured (C) DRGs. EachI/C represents DRGs from an individual rat.

Fig. 2.

JNK protein levels do not change after axotomy. Protein extracts prepared from injured and contralateral uninjured DRGs were immunoblotted for JNK at progressive times after proximal (•) or distal (▪) axotomy. The intensity of the JNK signal was measured by densitometric analysis of ECL autoradiographs, and a ratio between injured and contralateral DRGs was determined. Each data point is representative of the injured/contralateral JNK ratio in three different animals. Error bars represent SEM. There was no statistically significant difference in JNK levels between injured and contralateral, uninjured DRGs at any time, for either surgical procedure.

Fig. 3.

Phosphorylated c-Jun is localized to axotomized DRG neurons. Twenty-four hours after proximal nerve transection, axotomized (a) and contralateral untransected (b) DRGs were removed and processed as 35 mm free-floating sections for immunostaining with an antibody specific for serine-63 phosphorylated c-Jun. Intense nuclear staining was restricted to the nuclei of DRG neurons and was strongly evident in axotomized DRGs. Sections shown are representative of immunostaining in DRGs from three separately analyzed animals.

Fig. 4.

Specific AP-1 binding activity in axotomized DRGs. Protein extracts from injured (lanes 1, 3–9) and contralateral uninjured (lanes 2,10) DRGs were assayed for their ability to bind radiolabeled Hmt II_A AP-1 (lanes 1–8) or mutant (lanes 9, 10) sequences, at 72 hr after proximal axotomy. Incubation of injured DRG extracts with the indicated amounts of unlabeled AP-1 (lanes 3–5) reduced the amount of axotomy-induced binding to radiolabeled AP-1, whereas there was no reduction in specific AP-1 binding in the presence of unlabeled mutant AP-1 oligonucleotides (lanes 6–8). A band of more rapid mobility appearing in both injured and uninjured DRGs was competed by both AP-1 and mutant oligonucleotides, indicating that this protein complex does not specifically recognize the Hmt II_A AP-1 sequence, nor does it have high affinity for the mutant AP-1 (lanes 9, 10). We focused our studies on the more slowly migrating, injury-induced complex, indicated by <.

Fig. 5.

AP-1 binding activity by c-Jun and JunD in L4/L5 DRGs after proximal sciatic nerve transection. At the indicated times after proximal peripheral nerve transection, DRG protein extracts from injured (I) and contralateral uninjured (C) DRGs were assayed for activity toward an Hmt II_A AP-1 sequence, in the presence of antibodies against various jun family members. Shown are autoradiographs representative of DRGs from three separately analyzed rats at each time point. All animals were included in quantitative densitometric analysis discussed in the text (see Results). The level of AP-1 binding activity in each sample was determined as a ratio of AP-1 binding activity in injured versus contralateral, uninjured samples. In the case of antibody supershift experiments, a ratio was drawn between AP-1 binding activity in injured DRG samples with antibody versus without antibody. Antibodies used are indicated above each lane. A diffuse signal strongly present in extracts from injured DRGs (←AP-1) was not affected by incubation with antibodies to junB. Antibodies to c-Jun blocked formation of this complex, because these antibodies interfere with the DNA-binding domain of c-Jun. JunD antibodies diminished formation of the injury-induced complex and caused a portion to be retained near the top of the gel. The extent to which junD antibodies diminished formation of the specific AP-1 complex was quantified by densitometric analysis of autoradiographs, as described above and in Results. ← NS indicates a nonspecific, noninduced complex.

Fig. 6.

AP-1 binding and c-Jun protein levels in affected DRGs after distal ligation with cuff or sciatic nerve crush. At the indicated times after mid-thigh sciatic nerve transection with cuff (a, b) or crush (c, d), injured (I) or contralateral uninjured (C) DRGs were analyzed by EMSA with Hmt II_A AP-1 oligonucleotides (a, c) or enhanced chemiluminescent Western blotting for c-Jun protein (b, d). Shown are autoradiographs representative of samples analyzed from three rats at each time point, for each surgery. All animals were included in quantitative densitometric analysis described in Results. The level of injury-induced AP-1 binding activity in each sample was determined as a ratio of AP-1 binding activity in injured versus contralateral, uninjured samples. The participation of c-Jun in AP-1 binding activity was determined by the ability of an antibody against the c-Jun DNA binding domain to inhibit formation of AP-1 binding complexes. Samples treated with the c-Jun antibody are indicated above the lane (*). The specific AP-1 binding complex signal is shown by <. Samples analyzed by EMSA were also immunoblotted for c-Jun, under previously optimized experimental conditions that have been established to quantify changes in c-jun protein levels in axotomized DRGs using chemiluminescent detection (Kenney and Kocsis, 1997, 1998), also described in Materials and Methods. The c-Jun antibody used recognizes four major species: 80, 60, 46, and 30 kDa. Only the doublet appearing at 46 kDa is regulated by axonal transection. When extracts from UV-irradiated NIH 3T3 cells were immunoblotted for c-Jun using this antibody, the same hybridization pattern was observed, and the 46 kDa doublet was the only species induced by UV light. Therefore, this species was understood to represent c-Jun and was quantified by densitometric analysis of ECL autoradiographs. A ratio of c-jun immunoreactivity in injured versus the contralateral uninjured DRGs was then established to determine axotomy-induced changes in c-jun protein levels.

Fig. 7.

Hind footprints of animals at progressive times after right-side sciatic nerve crush. Regeneration of the sciatic nerve axons after crush was assessed just before animals were killed by obtaining hind footprints after the animals were anesthetized, and then during the dissection procedure by measuring the anatomical site of the axonal regeneration front (see Results). Footprints are shown for selected times after right-side nerve crush. n = 3 animals for each time point. DRGs removed from these animals were analyzed by EMSA and c-Jun immunoblotting (see Results and Fig.6).