Axoplasmic proteins containing nuclear localization signals (NLS) signal retrogradely by an unknown mechanism in injured nerve. Here we demonstrate that the importin/karyopherin alpha and beta families underlie this process. We show that importins are found in axons at significant distances from the cell body and that importin beta protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity NLS binding complex that traffics retrogradely with the motor protein dynein. Trituration of synthetic NLS peptide at the injury site of axotomized dorsal root ganglion (DRG) neurons delays their regenerative outgrowth, and NLS introduction to sciatic nerve concomitantly with a crush injury suppresses the conditioning lesion induced transition from arborizing to elongating growth in L4/L5 DRG neurons. These data suggest a model whereby lesion-induced upregulation of axonal importin beta may enable retrograde transport of signals that modulate the regeneration of injured neurons.

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 ␤-actin, peripherin, vimentin, ␥-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, ␣B 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, ␥-synuclein, superoxide dismutase 1), anti-oxidant proteins (peroxiredoxins 1 and 6), and metabolic proteins (e.g., phosphoglycerate kinase 1 (PGK 1), ␣ 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 ␤-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 exerciseconditioned 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.neurotrophin ͉ gene expression ͉ plasticity ͉ neural activity ͉ experience A lterations in neuronal activity can lead to lasting changes in the ability of the nervous system to transduce information (1). Such synaptic plasticity is well documented in the developing nervous system where the levels of neuronal activity can influence the eventual organization of cortical circuits (2). More recent studies (3) indicate that activity-dependent plasticity is retained into adulthood. The morphological basis of lasting forms of activity-dependent synaptic plasticity is manifest as an overall change in the number and͞or area of synaptic contacts (1-3). In the developed organism, both of these processes require remodeling of synaptic structures, either through retraction of existing neuronal processes or by growth of new neuronal processes.Formation of synaptic contacts and growth is a dynamic process that is largely affected through interactions with the environment, such that experience can imprint the nervous system by regulating these events (3). Neurotrophins, originally described for their role on growth and differentiation of neurons, are becoming recognized as regulators of synaptic plasticity (4, 5). The levels of the neurotrophins and͞or their receptors can be altered by neuronal activity, thus providing a potential means to perpetuate changes in synaptic transmission (6, 7). Brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) are important for the regulation of sensorimotor function taking place at the muscle-dorsal root ganglion (DRG)-spinal cord interface (8-10). We previously showed that the expression of BDNF and NT-3 is increased in the spinal cord and skeletal muscle after voluntary exercise and this alters expression of synapsin I mRNA in motor neurons (11-13). Here, we have asked how voluntary exer...

The present study describes a new analytical approach for the detection and characterization of GSH-trapped reactive metabolites using multiple reaction monitoring (MRM) as the survey scan to trigger the acquisition of enhanced product ion (EPI) spectra on a triple quadrupole linear ion mass spectrometer. The MRM scan step was carried out following up to 114 MRM transitions from the protonated molecules of potential GSH adducts to their product ions derived from a neutral loss of 129 or 307 Da. MRM transition protocols were constructed on the basis of common bioactivation reactions predicted to occur in human liver microsomes (HLM). The effectiveness and reliability of the approach were evaluated using acetaminophen, diclofenac, and carbamazepine as model compounds. The total ion chromatograms of the MRM for the HLM incubations with these compounds and GSH clearly displayed a number of GSH adducts, including acetaminophen-GSH adducts and carbamazepine-GSH adducts that were not previously observed in HLM incubations. In addition, clomipramine and mefenamic acid that have the frame structures susceptible to P450-mediated bioactivation were investigated. As a result, the MRM-EPI analysis revealed multiple GSH adducts of clomipramine and mefenamic acid in HLM incubations possibly mediated by epoxide and/or quinone imine intermediates. Compared with the neutral loss (NL) and precursor ion (PI) scanning analysis, the MRM-based approach provided superior sensitivity and selectivity for GSH adducts. It also enabled the sensitive acquisition of EPI spectra with rich fragmentation in the same LC/MS run, which were useful for the rapid structure elucidation of GSH adducts and the elimination of false positives. The MRM-EPI experiment can be employed for high throughput screening of reactive metabolites and should be especially applicable to compounds of the same chemotype. Also, it can be applied in conjunction with the PI or NL scan as a comprehensive method for the analysis of reactive metabolites in a drug discovery setting.