Expression of Jun, Fos and ATF-2 proteins in axotomized explanted and cultured adult rat dorsal root ganglia

Abstract

The expression of c-Jun, JunB, JunD, c-Fos and ATF-2 transcription factors was studied in L4/L5 dorsal root ganglion neurons of adult rats, in order to determine the extent to know to which extend the expression of transcription factors in vitro parallels the pathophysiological expression in vivo. First, dorsal root ganglia were dissociated and cultured for up to 15 days in vitro (culture). Second, the dorsal root and the peripheral nerve fibres were transected close at the dorsal root ganglia, and the completely axotomized dorsal root ganglia were kept in artificial cerebrospinal fluid for up to 24 h. This procedure (explantation) preserves the intraganglionic morphology intact. Culture evoked a persistent expression of c-Jun and JunD in the majority of small neurons independent on neurite extension. In contrast, the number of large neurons with c-Jun decreased and with JunD increased with incubation time. JunB and c-Fos, which were also visible in the majority of neurons, strongly decreased with culture time in both small and large neurons. ATF-2 was visible in the vast majority of neurons and did not change during the observation period. Incubation with brain-derived neurotrophic factor for 15 days reduced JunB expression and raised c-Fos expression, but did not affect c-Jun or JunD labellings. Explantation of dorsal root ganglia evoked a dramatic and rapid induction of c-Jun in neurons located in the periphery of the ganglia, an area that showed prominent apoptosis as visualized by transferase dUTP nick end-labelling, followed by a delayed increase in neurons of the central parts of dorsal root ganglia. Expression of JunB showed a dramatic increase within 2 h in the whole ganglion, but disappeared within the following hours. JunD dropped from its basal levels within 4 h and was almost absent after 8 h. c-Fos did not appear until 6 h, when transferase dUTP nick end-labelling also became detectable, and remained visible in a rather small number of neurons. As with culture, incubation of explanted dorsal root ganglia with brain-derived neurotrophic factor prevented the initial rise in JunB, accelerated and enhanced c-Fos expression, but did not alter c-Jun and JunD expression. Immunoreactivity of ATF-2 declined or disappeared in those dorsal root ganglia compartments that showed a rise in c-Jun and transferase dUTP nick end-labelling. These findings demonstrate that inducible transcription factors such as Jun and Fos proteins are differentially expressed in adult neurons in vitro when compared to pathophysiological conditions in vivo such as nerve fibre transection (axotomy or rhizotomy). Moreover, the comparison between the explantation and culture experiments suggests that it is the complete axotomy of neurons that provokes those expression patterns found in neuronal cultures of adult neurons. The rapid and persisting expression of c-Jun during neurite extension and apoptosis points at the activation of a pivotal program that might be determined by the presence or absence of ATF-2 and that is involved in regeneration or degeneration.