Jun-Qi Zheng

Recent studies have begun to focus on the signals that regulate axonal protein synthesis and the functional significance of localized protein synthesis. However, identification of proteins that are synthesized in mammalian axons has been mainly based on predictions. Here, we used axons purified from cultures of injury-conditioned adult dorsal root ganglion (DRG) neurons and proteomics methodology to identify axonally synthesized proteins. Reverse transcription (RT)-PCR from axonal preparations was used to confirm that the mRNA for each identified protein extended into the DRG axons. Proteins and the encoding mRNAs for the cytoskeletal proteins beta-actin, peripherin, vimentin, gamma-tropomyosin 3, and cofilin 1 were present in the axonal preparations. In addition to the cytoskeletal elements, several heat shock proteins (HSP27, HSP60, HSP70, grp75, alphaB crystallin), resident endoplasmic reticulum (ER) proteins (calreticulin, grp78/BiP, ERp29), proteins associated with neurodegenerative diseases (ubiquitin C-terminal hydrolase L1, rat ortholog of human DJ-1/Park7, gamma-synuclein, superoxide dismutase 1), anti-oxidant proteins (peroxiredoxins 1 and 6), and metabolic proteins (e.g., phosphoglycerate kinase 1 (PGK 1), alpha enolase, aldolase C/Zebrin II) were included among the axonally synthesized proteins. Detection of the mRNAs encoding each of the axonally synthesized proteins identified by mass spectrometry in the axonal compartment indicates that the DRG axons have the potential to synthesize a complex population of proteins. Local treatment of the DRG axons with NGF or BDNF increased levels of cytoskeletal mRNAs into the axonal compartment by twofold to fivefold but had no effect on levels of the other axonal mRNAs studied. Neurotrophins selectively increased transport of beta-actin, peripherin, and vimentin mRNAs from the cell body into the axons rather than changing transcription or mRNA survival in the axonal compartment.

Although intradendritic protein synthesis has been documented in adult neurons, the question of whether axons actively synthesize proteins remains controversial. Adult sensory neurons that are conditioned by axonal crush can rapidly extend processes in vitro by regulating the translation of existing mRNAs (Twiss et al., 2000). These regenerating processes contain axonal but not dendritic proteins. Here we show that these axonal processes of adult sensory neurons cultured after conditioning injury contain ribosomal proteins, translational initiation factors, and rRNA. Pure preparations of regenerating axons separated from the DRG cell bodies can actively synthesize proteins in vitro and contain ribosome-bound beta-actin and neurofilament mRNAs. Blocking protein synthesis in these regenerating sensory axons causes a rapid retraction of their growth cones when communication with the cell body is blocked by axotomy or colchicine treatment. These findings indicate that axons of adult mammalian neurons can synthesize proteins and suggest that, under some circumstances, intra-axonal translation contributes to structural integrity of the growth cone in regenerating axons. By immunofluorescence, translation factors, ribosomal proteins, and rRNA were also detected in motor axons of ventral spinal roots analyzed after 7 d in vivo after a peripheral axonal crush injury. Thus, adult motor neurons are also likely capable of intra-axonal protein synthesis in vivo after axonal injury.

Recent advances in understanding the role of neurotrophins on activity-dependent plasticity have provided insight into how behavior can affect specific aspects of neuronal biology. We present evidence that voluntary exercise can prime adult dorsal root ganglion neurons for increased axonal regeneration through a neurotrophin-dependent mechanism. Dorsal root ganglion neurons showed an increase in neurite outgrowth when cultured from animals that had undergone 3 or 7 days of exercise compared with sedentary animals. Neurite length over 18-22 h in culture correlated directly with the distance that animals ran. The exercise-conditioned animals also showed enhanced regrowth of axons after an in vivo nerve crush injury. Sensory ganglia from the 3- and 7-day-exercised animals contained higher brain-derived neurotrophic factor, neurotrophin 3, synapsin I, and GAP43 mRNA levels than those from sedentary animals. Consistent with the rise in brain-derived neurotrophic factor and neurotrophin 3 during exercise, the increased growth potential of the exercise-conditioned animals required activation of the neurotrophin signaling in vivo during the exercise period but did not require new mRNA synthesis in culture.

PURPOSE: To establish an in vitro model of axonal regeneration from mammalian retinal ganglion cells and to evaluate the role of PKC isozymes in promoting such retinal axon regeneration. METHODS: Postnatal day-3 mice were subjected to optic nerve crush, and then retinal ganglion cells (RGCs) were used for culture 5 days later. RGCs were selected using anti-Thy 1.2-coated magnetic beads and plated onto a merosin substrate. Changes in axonal localization of PKC and axonal regeneration were examined in cultured RGCs by immunofluorescence. Changes in PKC isozyme mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). The role of PKC in RGC neurite outgrowth was examined by treatment with activators or pharmacological inhibitors of PKC activity. RESULTS: RGCs subjected to optic nerve crush injury demonstrated more rapid neurite outgrowth in vitro when compared with RGCs isolated from naïve retina. The neurites of these injury-conditioned RGCs showed both an increased rate of extension and enhanced PKC localization in culture. Injury-conditioned RGCs had elevated PKC isozyme mRNA levels, which probably contributed to the increased level of PKC protein in injury-conditioned RGC axons. Pharmacological activation of PKC enhanced neurite growth, whereas inhibition of PKC suppressed neurite growth in both the conditioned and naïve RGCs. CONCLUSIONS: RGCs actively respond to axonal injury by regulating expression of genes that promote neurite outgrowth. PKC-alpha and -beta isozymes are among the growth-associated proteins that are upregulated after injury. Results of pharmacological manipulation of PKC activity support the argument that increased PKC levels enhance neurite regrowth after axonal injury.