Axonal mRNAs: Characterisation and role in the growth and regeneration of dorsal root ganglion axons and growth cones

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Abstract

We have developed a compartmentalised culture model for the purification of axonal mRNA from embryonic, neonatal and adult rat dorsal root ganglia. This mRNA was used un-amplified for RT-qPCR. We assayed for the presence of axonal mRNAs encoding molecules known to be involved in axon growth and guidance. mRNAs for β-actin, β-tubulin, and several molecules involved in the control of actin dynamics and signalling during axon growth were found, but mRNAs for microtubule-associated proteins, integrins and cell surface adhesion molecules were absent. Quantification of β-actin mRNA by means of qPCR showed that the transcript is present at the same level in embryonic, newborn and adult axons. Using the photoconvertible reporter Kaede we showed that there is local translation of β-actin in axons, the rate being increased by axotomy. Knock down of β-actin mRNA by RNAi inhibited the regeneration of new axon growth cones after in vitro axotomy, indicating that local translation of actin-related molecules is important for successful axon regeneration.

Introduction

Over the last two decades it has become clear that dendrites and also certain types of axons contain mRNA (Piper and Holt, 2004, Twiss and van Minnen, 2006, Giuditta et al., 2002, Kiebler and Bassell, 2006, Vogelaar and Fawcett, 2008a, Vogelaar and Fawcett, 2008b). This was first shown in invertebrate axons from squid and snails which contain numerous mRNAs, mainly encoding cytoskeletal and metabolic proteins, and proteins involved in local translation, as well as neuropeptides (VanMinnen et al., 1997, Gioio et al., 2001, Giuditta et al., 2002, Gioio et al., 2004, van Kesteren et al., 2006). There have been various studies on vertebrate neurites from developing neurons, axons from adult dorsal root ganglia (DRGs) that received a conditioning lesion, and also from cytoplasm squeezed out from adult nerves (axoplasm). These have identified localised cytoskeletal mRNAs, such as β-actin, β-tubulin, neurofilament, vimentin, mRNAs encoding small GTPases and CREB (Bassell et al., 1998, Eng et al., 1999, Lee and Hollenbeck, 2003, Sotelo-Silveira et al., 2006, Yao et al., 2006, Leung et al., 2006, Giuditta et al., 2002, Perlson et al., 2005, Wu et al., 2005, Cox et al., 2008). A study of pre-conditioned adult DRG neurons identified 27 mRNAs in these axons (Willis et al., 2005). While several mRNAs have been identified in immature mammalian axons, and in axons from developing Xenopus and goldfish, mRNAs appear to be present in only a few types of axons in the adult mammalian CNS. Studies on adult rat olfactory and hypothalamic axons demonstrated the presence of mRNA (Vassar et al., 1994, Wensley et al., 1995, Mohr and Richter, 2000, Nedelec et al., 2005). However, adult retinal axons do not contain detectable amounts of ribosomal protein, and in the spinal cord the only axons in which ribosomal protein is detectable are the central branches of DRG axons, suggesting that most adult CNS axons are not capable of local translation (Verma et al., 2005, Verma and Fawcett, unpublished results).

Several functions of local translation of axonal mRNA have been established. In Xenopus retinal axons it was shown that asymmetrical translation of β-actin mRNA is essential for growth cone turning (Yao et al., 2006, Leung et al., 2006). Knock-out mice for the RNA binding protein SMN1 show decreased axonal β-actin mRNA and protein in motor neuron axons in vitro, causing decreased axon growth and a reduction in growth cone size (Rossoll et al., 2003). siRNA directed against axonal rhoA abolished Sema3A-induced growth cone collapse of embryonic DRG axons in vitro (Wu et al., 2005), and local translation of CREB is involved in NGF signalling (Cox et al., 2008). Many types of axon are able to regenerate after axotomy, but their regenerative ability varies greatly, with PNS axons showing a strong regenerative response and many CNS axons showing little regeneration, even when presented with a permissive environment. The different regenerative ability of CNS and PNS axons can be modelled in vitro where DRG axons of all developmental stages are usually capable of regenerating a new growth cone after transection but adult retinal axons often fail to regenerate (Chierzi et al., 2005). The regenerative ability of DRG axons is much reduced by protein synthesis inhibitors and these axons contain ribosomal proteins and translation elongation factor at all developmental stages (Verma et al., 2005). However, the poorly regenerating adult retinal axons do not contain ribosomal protein, and their limited regeneration is not reduced by protein synthesis inhibitors (Verma et al., 2005). Local axonal translation of vimentin and importins play an important part in retrograde signalling from damaged sensory axons to the cell body (Hanz et al., 2003, Perlson et al., 2006).

PNS axotomy affects axonal transport, including probably that of mRNA (Willis et al., 2007). Currently, no localised mRNA identification data exist on axons from adult mammalian non-conditioned DRGs. In the present study we describe a new compartmented system for obtaining pure axonal material from DRG explants. We have used it to look for the presence of candidate mRNAs encoding proteins involved in the cytoskeleton, cytoskeletal control, signalling pathways and cell surface molecules. We have investigated axonal translation of β-actin mRNA and demonstrated its importance for successful axonal regeneration.

Section snippets

A new culture method for isolation of axon-only RNA

Obtaining sufficient axonal mRNA for quantitative studies, free of glial and neuronal cell body contamination, is challenging. We have developed a new compartmented culture system for extracting axonal material from adult, neonatal and embryonic rat DRG explants. In Fig. 1, we compare our method with the currently used compartmented culture systems. Because the compartment divider is not placed in the culture dish until robust axon growth has begun, the method overcomes the inability of some

Discussion

Although the presence of mRNA molecules in adult mammalian sensory axons is well established, only limited information is available as to their identity, and only in axons conditioned by a previous lesion (Vogelaar and Fawcett, 2008a, Vogelaar and Fawcett, 2008b). There is also a lack of data on comparisons between different developmental stages of mammalian axons. We have established a compartmentalised culture method which allows us to isolate rodent DRG axons of all developmental stages

Explant chambers

Dorsal root ganglia (DRGs) were dissected from embryonic (E16), neonatal (P0–P2), and adult (2 to 4-months-old) rats and nerve stumps were carefully cut off. Adult DRGs were cut into 2–3 smaller pieces. DRG explants (20–25 per dish) were plated in a row on top of scratches made with a Campenot pin rake (Tyler Research Corporation) in Nunclon dishes (NUNC) coated with PDL (20 μg/ml, Sigma) and laminin (1 μg/ml, Sigma) in a minimal amount of medium containing ITS+ (1:100, BD Biosciences),

Acknowledgments

This work was supported by grants from Action Medical Research, the Medical Research Council, the Wellcome Trust, the Henry Smith Charity and The Christopher and Dana Reeve Foundation.

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    1

    Present address: Molecular Neurobiology Laboratory, Department of Neurology, Heinrich-Heine University, Düsseldorf, Germany.

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    These authors contributed equally to this work.

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