Translating Axon Guidance Cues

Cell Adhesion & Migration, 2007

While initially thought to be essentially a developmental characteristic observed in artificial in vitro models, local protein synthesis in growth cones has been described in the adult, and more interestingly, during nerve regeneration. This emerging field is under intense investigation, revealing new functions of localized protein synthesis that include axon guidance, growth cone adaptation and sensitivity modulation at intermediate targets or axon regeneration. Here, we will review these functions and provide a short survey of the current knowledge on mechanisms of mRNA transport and regulation of localized protein synthesis. In addition, we will consider what lessons can be learned from localized protein synthesis in dendrites and what developments can be expected next in the field. This latter question relates to the crucial point of which technical strategy to adopt for an ideal and pertinent analysis of the phenomenon.

2013

Neurons have complex and often extensively elongated processes. This unique cell morphology raises the problem of how remote neuronal territories are replenished with proteins. For a long time, axonal and presynaptic proteins were thought to be exclusively synthesized in the cell body, which delivered them to peripheral sites by axoplasmic transport. Despite this early belief, protein has been shown to be synthesized in axons and nerve terminals, substantially alleviating the trophic burden of the perikaryon. This observation raised the question of the cellular origin of the peripheral RNAs involved in protein synthesis. The synthesis of these RNAs was initially attributed to the neuron soma almost by default. However, experimental data and theoretical considerations support the alternative view that axonal and presynaptic RNAs are also transcribed in the flanking glial cells and transferred to the axon domain of mature neurons. Altogether, these data suggest that axons and nerve terminals are served by a distinct gene expression system largely independent of the neuron cell body. Such a local system would allow the neuron periphery to respond promptly to environmental stimuli. This view has the theoretical merit of extending to axons and nerve terminals the marginalized concept of a glial supply of RNA (and protein) to the neuron cell body. Most long-term plastic changes requiring de novo gene expression occur in these domains, notably in presynaptic endings, despite their intrinsic lack of transcriptional capacity. This review enlightens novel perspectives on the biology and pathobiology of the neuron by critically reviewing these issues.

Journal of Neuroscience, 2009

Recent evidence suggests that growth cone responses to guidance cues require local protein synthesis. Using chick neurons, we investigated whether protein synthesis is required for growth cones of several types to respond to guidance cues. First, we found that global inhibition of protein synthesis stops axonal elongation after 2 h. When protein synthesis inhibitors were added 15 min before adding guidance cues, we found no changes in the typical responses of retinal, sensory, and sympathetic growth cones. In the presence of cycloheximide or anisomycin, ephrin-A2, slit-3, and semaphorin3A still induced growth cone collapse and loss of actin filaments, nerve growth factor (NGF) and neurotrophin-3 still induced growth cone protrusion and increased filamentous actin, and sensory growth cones turned toward an NGF source. In compartmented chambers that separated perikarya from axons, axons grew for 24-48 h in the presence of cycloheximide and responded to negative and positive cues. Our results indicate that protein synthesis is not strictly required in the mechanisms for growth cone responses to many guidance cues. Differences between our results and other studies may exist because of different cellular metabolic levels in in vitro conditions and a difference in when axonal functions become dependent on local protein synthesis.

J Comp Neurol, 1995

The effects of actinomycin D were studied in cultured grasshopper embryos at different stages of development by following the outgrowth patterns of identified neurones known as aCC, pCC, and Q1. When administered at stages occurring before 31% of embryonic development, actinomycin D (0.05-0.10 pM for 24-48 hours) prevented axon extension, whereas it did not affect the development of the nervous system in embryos older than 34% of development. At 31-3494) of development, actinomycin D perturbed pathfinding of aCC without blocking axon extension. Thus, only 22% of the aCCs (n = 271) in embryos treated with actinomycin D extended an axon along the intersegmental nerve as in control embryos. In the remaining embryos, aCC failed to turn into the intersegmental nerve root; its growth cone remained in the longitudinal connective, above or below the turning point. Neurones of the group caudal to the intersegmental nerve root could extend along either the anterior or posterior commissure of the next posterior segment. In contrast to the observations made with aCC, only 1.206 of pCC (n = 166) and 0.0% of Q1 (n = 45) in embryos treated with actinomycin D showed axon growth along aberrant pathways. The position of the growth cones of most pCCs and all Qls observed were in various points along their normal pathway. Both pCC and Q1, as a population, showed an extension rate significantly lower than that of their control counterparts. The effect of actinomycin D on aCC pathway choice was probably mediated by inhibition of RNA synthesis, because incorporation of uridine into RNA was reduced by 40%. The labelling of several monoclonal antibodies (lC10,3B11,7F7) that recognise surface glycoproteins (lachesin, fasciclin I, and REGA-1) involved in nervous system development of grasshopper embryos was suppressed. Our results suggest that the navigation of some axons along different pathways requires the synthesis of new mRNA. T: 19% wiley-~iss, Inc.

Progress in Neurobiology, 2000

This article focuses on local protein synthesis as a basis for maintaining axoplasmic mass, and expression of plasticity in axons and terminals. Recent evidence of discrete ribosomal domains, subjacent to the axolemma, which are distributed at intermittent intervals along axons, are described. Studies of locally synthesized proteins, and proteins encoded by RNA transcripts in axons indicate that the latter comprise constituents of the so-called slow transport rate groups. A comprehensive review and analysis of published data on synaptosomes and identi®ed presynaptic terminals warrants the conclusion that a cytoribosomal machinery is present, and that protein synthesis could play a role in long-term changes of modi®able synapses. The concept that all axonal proteins are supplied by slow transport after synthesis in the perikaryon is challenged because the underlying assumptions of the model are discordant with known metabolic principles. The¯awed slow transport model is supplanted by a metabolic model that is supported by evidence of local synthesis and turnover of proteins in axons. A comparison of the relative strengths of the two models shows that, unlike the local synthesis model, the slow transport model fails as a credible theoretical construct to account for axons and terminals as we know them. Evidence for a dynamic anatomy of axons is presented. It is proposed that a distributed``sprouting program,'' which governs local plasticity of axons, is regulated by environmental cues, and ultimately depends on local synthesis. In this respect, nerve regeneration is treated as a special case of the sprouting program. The term merotrophism is proposed to denote a class of phenomena, in which regional phenotype changes are regulated locally without speci®c involvement of the neuronal nucleus. 7

Nature Cell Biology, 2009

During development, axon growth rates are precisely regulated to provide temporal control over pathfinding1 , 2. The precise temporal regulation of axonal growth is a key step in the formation of functional synapses and the proper patterning of the nervous system. The rate of axonal elongation is increased by factors such as netrin-1 and NGF, which stimulate axon outgrowth by incompletely defined pathways. To clarify the mechanism of netrin-1 and NGF-stimulated axon growth, we explored the role of local translation. We find that intraaxonal protein translation is required for stimulated but not basal axon outgrowth. To identify the mechanism of translation-dependent outgrowth, we examined the PAR complex, a cytoskeleton regulator3. We find that the PAR complex, like local translation, is required only for stimulated but not basal growth. PAR3 mRNA is localized to developing axons, and NGF and netrin-1 trigger its local translation. Selective ablation of PAR3 mRNA from axons abolishes the outgrowth-promoting effects of NGF. These results identify a novel role for local translation and the PAR complex in axonal outgrowth.

1999

The proteins needed for growth and maintenance of the axon are generally believed to be synthesized in the cell bodies and delivered to the axons by anterograde transport. However, recent reports suggest that some proteins can also be synthesized within axons. We used [ 35 S]methionine metabolic labeling to investigate axonal protein synthesis in compartmented cultures of sympathetic neurons from newborn rats. Incubation of distal axons for 4 hr with [ 35 S]methionine resulted in a highly specific pattern of labeled axonal proteins on SDS-PAGE, with 4 prominent bands in the 43-55 kDa range. The labeled proteins in axons were not synthesized in the cell bodies, because they were also produced by axons after the cell bodies had been removed. Two of the proteins were identified by immunoprecipitation as actin and ␤-tubulin. Axons synthesized Ͻ1% of the actin and tubulin synthesized in the cell bodies and transported into the axons, and 75-85% inhibition of axonal protein synthesis by cycloheximide and puromycin failed to inhibit axonal elongation. Nonetheless, the specific production by axons of the major proteins of the axonal cytoskeleton suggests that axonal protein synthesis arises from specific mechanisms and likely has biological significance. One hypothetical scenario involves neurons with long axons in vivo in which losses from turnover during axonal transport may limit the availability of cell body synthesized proteins to the distal axons. In this case, a significant fraction of axonal proteins might be supplied by axonal synthesis, which could, therefore, play important roles in axonal maintenance, regeneration, and sprouting.

Annual Review of Neuroscience, 1989

European Journal of Biochemistry, 1989

Secretion of proteins from the growth cone has been implicated in axon growth and synapse formation and might be involved in the transmission of a variety of axon-derived regulatory signals during neurogenesis. In order to identify axonally secreted proteins, dorsal-root-ganglia neurons from chicken embryos were cultured in a compartmentalized cell culture system that allows separate access to neuronal cell somas and axons. The proteins synthesized by the neurons were metabolically labeled by addition of [35S]methionine to the compartment containing the cell somas; the proteins released from the axons were harvested from the culture medium of the axonal compartment. Two-dimensional gel electrophoresis revealed two axonally secreted proteins with apparent molecular mass of 132-140 kDa and 54-60 kDa; they were termed axonin-I and axonin-2, respectively. Both axonins were found to be secreted from a variety of neuronal cell cultures, but not from any of the nonneuronal cultures investigated, and hence might be neuron-specific. Virtual absence of these proteins from the axonal protein pattern suggests constitutive secretion. The information acquired on coordinates and spot morphology of these proteins in two-dimensional gel electrophoresis provides a useful assay for their purification.

Current Opinion in Neurobiology, 2008

An increasing body of evidence indicates that local axonal translation is required for growing axons to respond appropriately to guidance cues and other stimuli. Recent studies suggest that asymmetrical synthesis of cytoskeletal proteins mediates growth cone turning and that local translation and retrograde transport of transcription factors mediate neuronal survival. Axonal translation is regulated partly by selective axonal localization of mRNAs and by translation initiation factors and RNA-binding proteins. We discuss possible rationales for local axonal translation, including distinct properties of nascent proteins, precise localization, and axonal autonomy.