Axonal Protein Synthesis and Degradation Are Necessary for Efficient Growth Cone Regeneration

Abstract

Axonal regeneration can occur within hours of injury, the first step being the formation of a new growth cone. For sensory and retinal axons, regenerative ability in vivo correlates with the potential to form a new growth cone after axotomy in vitro. We show that this ability to regenerate a new growth cone depends on local protein synthesis and degradation within the axon. Axotomy in vitro leads to a fourfold to sixfold increase in 3 H-leucine incorporation in both neurones and axons, starting within 10 min and peaking 1 h after axotomy. Application of protein synthesis inhibitors (cycloheximide and anisomycin) to cut axons, including axons whose cell bodies were removed, or proteasome inhibitors (lactacystin and N-acetyl-Nor-Leu-Leu-Al) all result in a reduction in the proportion of transected axons able to reform growth cones. Similar inhibition of growth cone formation was observed on addition of target of rapamycin (TOR), p38 MAPK (mitogen-activated protein kinase), and caspase-3 inhibitors. Comparing retinal and sensory axons of different developmental stages, levels of ribosomal protein P0 and phosphorylated translation initiation factor are high in sensory axons, lower in embryonic axons, and absent in adult retinal axons. Conditioning lesions, which increase the regenerative ability of sensory axons, lead to increases in intra-axonal protein synthetic and degradative machinery both in vitro and in vivo. Collectively, these findings suggest that local protein synthesis and degradation, controlled by various TOR-, p38 MAPK-, and caspase-dependent pathways, underlie growth cone initiation after axotomy.

The RNA binding and transport proteins staufen and fragile X mental retardation protein are expressed by rat primary afferent neurons and localize to peripheral and central axons

“…An increasing body of evidence indicates that DRG axons contain mRNA and that these axons are translationally competent (Zheng et al, 2001;Verma et al, 2005;Willis et al, 2005). In this study, we have shown that DRG and TG neurons express the RNA binding and transport proteins stau and FMRP and that these proteins localize to DRG axons.…”

“…There is now compelling evidence that translation occurs in axons (Koenig, 1967;Nixon, 1980;Koenig, 1991;Eng et al, 1999;Brittis et al, 2002;Piper and Holt, 2004), as mRNA and translational machinery have been localized to CNS (Tiedge et al, 1993;Aronov et al, 2001;Brittis et al, 2002), sympathetic (Olink-Coux andHollenbeck, 1996;Eng et al, 1999) and sensory (Zheng et al, 2001;Li et al, 2004a;Verma et al, 2005;Willis et al, 2005) axons. Moreover, an emerging physiological role for local translation in axons has been described for growth cone guidance and collapse (Zheng et al, 2001;Li et al, 2004a;Verma et al, 2005;Wu et al, 2005).Primary sensory neurons of the dorsal root ganglion (DRG) and trigeminal ganglion (TG) have among the longest axons of the entire neuraxis (greater than one meter for some human DRG neurons). Given the relatively long time interval required for axonal transport of proteins to occur in these neurons following somal synthesis, it would be advantageous if regulable, ondemand protein synthesis would occur at distal sites (for review see: Alvarez et al, 2000;Alvarez, 2001).…”

“…Extra-somal translation in neurons requires the presence of specific substrates, including mRNA and translational machinery, as well as mechanisms for their transport to these distal sites of protein synthesis. To this end, various components of the translational machinery have been identified in the axons of DRG neurons (Koenig, 1991;Pannese and Ledda, 1991;Verma et al, 2005), and translation itself has been shown to occur in these axons (Koenig, 1991;Zheng et al, 2001;Verma et al, 2005;Willis et al, 2005). However, with the exception of zipcode binding protein 1 (ZBP1), which transports β-actin mRNA to growth cones (Zhang et al, 2001), RNA binding and transport molecules have not been shown to be present in axons, and none, to our knowledge, have been localized to the axons of DRG afferents.…”

Targeting Axonal Protein Synthesis in Neuroregeneration and Degeneration

“…During the development of the nervous system, guided axon growth requires intra-axonal mRNA translation, and it is thus not surprising that local protein synthesis is also crucial for axon regeneration. The formation of a new growth cone after axotomy of developing axons in vitro requires both local protein synthesis and degradation [30], and upon injury of mature axons, mRNAs and protein synthesis machinery are rapidly recruited into axons and intraaxonal translation is upregulated or re-activated within these mature axons [31][32][33][34]. Locally synthesized proteins are required for communication from the injured axons to their soma and likely participate in the formation of the growth bulb at the site of injury [35,36].…”

C. elegansas a genetic model to identify novel cellular and molecular mechanisms underlying nervous system regeneration

“…54,55 The application of the p38 inhibitor significantly eliminated the growth cone formation and axon regeneration in the cultured DRG neurons. 56 These observations indicate an evolutionarily conserved role of the MAP synaptic branch somehow controls the intrinsic growth state of injured neurons and has an inhibitory effect on distal axon regeneration.…”