Abstract
Axonal regeneration is enhanced by the prior ;conditioning' of peripheral nerve lesions. Here we show that Xenopus dorsal root ganglia (DRG) with attached peripheral nerves (PN-DRG) can be conditioned in vitro, thereafter showing enhanced neurotrophin-induced axonal growth similar to preparations conditioned by axotomy in vivo. Actinomycin D inhibits axonal outgrowth from freshly dissected PN-DRG, but not from conditioned preparations. Synthesis of mRNAs that encode proteins necessary for axonal elongation might therefore occur during the conditioning period, a suggestion that was confirmed by oligonucleotide microarray analysis. Culturing PN-DRG in a compartmentalized system showed that inhibition of protein synthesis (but not RNA synthesis) in the distal nerve impaired the conditioning response, suggesting that changes in gene expression in cultured DRG depend on the synthesis and retrograde transport of protein(s) in peripheral nerves. The culture system was also used to demonstrate retrograde axonal transport of several proteins, including thioredoxin (Trx). Cyclopentenone prostaglandins, which react with Trx, blocked the in vitro conditioning effect, whereas inhibition of other signalling pathways thought to be involved in axonal regeneration did not. This suggests that Trx and/or other targets of these electrophilic prostaglandins regulate axonal regeneration. Consistent with this hypothesis, morpholino-induced suppression of Trx expression in dissociated DRG neurons was associated with reduced neurite outgrowth.
Highlights
We demonstrate that Xenopus dorsal root ganglia (DRG) with attached short peripheral nerves (PN-DRG) that were incubated for 3 days in serum-free medium show enhanced axonal growth in response to neurotrophins, similar to PN-DRG axotomized by sciatic nerve lesions, even in the presence of actinomycin D (ActD), indicating prior synthesis of mRNAs necessary to support axonal regeneration
Pharmacological inhibition of Trx function by using cyclopentenone prostaglandins blocked the in vitro conditioning effect, whereas inhibition of other signalling pathways thought to be involved in axonal regeneration did not, suggesting that Trx and/or other redox-sensitive factors regulate regenerationassociated genes (RAGs) expression and axonal regeneration
De novo transcription is required for axonal growth following conditioning in vitro Since neurite elongation rates of dissociated rat DRG neurons increase spontaneously during culture (Smith and Skene, 1997), which suggests the ‘conditioning lesion effect’ is mimicked in vitro, we investigated this possibility using Xenopus PN-DRG, which show excellent survival in culture (Tonge et al, 2004)
Summary
Axotomy causes extensive changes in gene expression (Costigan et al, 2002; Xiao et al, 2002; Tanabe et al, 2003; Cameron et al, 2003; Boeshore et al, 2004; Nilsson and Kanje, 2005; del Signore et al, 2006) and it is believed that induction of these regenerationassociated genes (RAGs) is important for axonal regeneration because it is faster in vivo and in vitro after prior ‘conditioning’ peripheral nerve lesions (Oblinger and Lasek, 1984; Lankford et al, 1998; Ekström et al, 2003 and references therein). In their phosphorylated form, the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) are retrogradely transported and involved in initiation of axonal regeneration (Perlson et al, 2005)
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