Function and translational regulation of mRNA in developing axons

The capacity to synthesize proteins in axons is limited to early stages of neuronal development, while axons are undergoing elongation and pathfinding. Although the roles of local protein synthesis are not fully understood, it has been implicated in regulating the morphological plasticity of growth cones. Recent studies have identified specific mRNAs that are translated in growth cones in response to specific extracellular signals. In this review, we discuss the functional relevance of axonal protein translation for developing axons, the differences in translational capacity between developing and mature vertebrate axons, and possible pathways governing the specific translational activation of axonal mRNAs.     

RNA trafficking in axons

A substantial number of studies over a period of four decades have indicated that axons contain mRNAs and ribosomes, and are metabolically active in synthesizing proteins locally. For the most part, little attention has been paid to these findings until recently when the concept of targeting of specific mRNAs and translation in subcellular domains in polarized cells emerged to contribute to the likelihood and acceptance of mRNA targeting to axons as well. Trans-acting factor proteins bind to cis-acting sequences in the untranslated region of mRNAs integrated in ribonucleoprotein (RNPs) complexes determine its targeting in neurons. In vitro studies in immature axons have shown that molecular motors proteins (kinesins and myosins) associate to RNPs suggesting they would participate in its transport to growth cones. Tau and actin mRNAs are transported as RNPs, and targeted to axons as well as ribosomes. Periaxoplasmic ribosomal plaques (PARPs), which are systematically distributed discrete peripheral ribosome-containing, actin-rich formations in myelinated axons, also are enriched with actin and myosin Va mRNAs and additional regulatory proteins. The localization of mRNAs in PARPs probably means that PARPs are local centers of translational activity, and that these domains are the final destination in the axon compartment for targeted macromolecular traffic originating in the cell body. The role of glial cells as a potentially complementary source of axonal mRNAs and ribosomes is discussed in light of early reports and recent ultrastructural observations related to the possibility of glial-axon trans-endocytosis.     

Molecular biology of axons: "a turning point...".

Previous studies have shown that dendrites and axons contain both mRNAs and the machinery for local protein translation. While a number of studies in recent years have focused on the functional role of protein synthesis in dendrites, relatively less is know about the role of local translation in axons. Campbell and Holt (this issue of Neuron) show that local protein synthesis and degradation are required for proper chemotropic turning responses of isolated retinal growth cones.     

The evolving roles of axonally synthesized proteins in regeneration

Work emerging during the past decade has shown that axons, similar to dendrites, are capable of autonomously generating new proteins through translation of localized mRNAs. Even in mammals, neurons maintain the ability to target mRNAs and translational machinery into the axonal compartment well into adulthood. The biological functions of axonal protein synthesis in adult neurons are just now being revealed, and recent studies indicate that locally synthesized proteins facilitate regeneration. Local translation, in addition to protein degradation, is needed for growth cone formation after axotomy, for generating a retrogradely transported injury signal, and then to help structurally maintain the growing axon. Regulation of axonal protein synthesis by exogenous stimuli might provide a means to facilitate regeneration for neuronal populations that normally show poor regenerative capacity in the adult nervous system.     

Local translation and directional steering in axons

The assembly of functional neural circuits in the developing brain requires neurons to extend axons to the correct targets. This in turn requires the navigating tips of axons to respond appropriately to guidance cues present along the axonal pathway, despite being cellular 'outposts' far from the soma. Work over the past few years has demonstrated a critical role for local translation within the axon in this process in vitro, making axon guidance another process that requires spatially localized translation, among others such as synaptic plasticity, cell migration, and cell polarity. This article reviews recent findings in local axonal translation and discusses how new protein synthesis may function in growth cone guidance, with a comparative view toward models of local translation in other systems.     

Local protein synthesis in neuronal axons: why and how we study

Adaptive brain function and synaptic plasticity rely on dynamic regulation of local proteome. One way for the neuron to introduce new proteins to the axon terminal is to transport those from the cell body, which had long been thought as the only source of axonal proteins. Another way, which is the topic of this review, is synthesizing proteins on site by local mRNA translation. Recent evidence indicates that the axon stores a reservoir of translationally silent mRNAs and regulates their expression solely by translational control. Different stimuli to axons, such as guidance cues, growth factors, and nerve injury, promote translation of selective mRNAs, a process required for the axon’s ability to respond to these cues. One of the critical questions in the field of axonal protein synthesis is how mRNA-specific local translation is regulated by extracellular cues. Here, we review current experimental techniques that can be used to answer this question. Furthermore, we discuss how new technologies can help us understand what biological processes are regulated by axonal protein synthesis in vivo. [BMB Reports 2015; 48(3): 139-146]     

Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs

Subcellular regulation of protein synthesis requires the correct localization of messenger RNAs (mRNAs) within the cell. In this study, we investigate whether the axonal localization of neuronal mRNAs is regulated by extracellular stimuli. By profiling axonal levels of 50 mRNAs detected in regenerating adult sensory axons, we show that neurotrophins can increase and decrease levels of axonal mRNAs. Neurotrophins (nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3) regulate axonal mRNA levels and use distinct downstream signals to localize individual mRNAs. However, myelin-associated glycoprotein and semaphorin 3A regulate axonal levels of different mRNAs and elicit the opposite effect on axonal mRNA levels from those observed with neurotrophins. The axonal mRNAs accumulate at or are depleted from points of ligand stimulation along the axons. The translation product of a chimeric green fluorescent protein-beta-actin mRNA showed similar accumulation or depletion adjacent to stimuli that increase or decrease axonal levels of endogenous beta-actin mRNA. Thus, extracellular ligands can regulate protein generation within subcellular regions by specifically altering the localized levels of particular mRNAs.     

Localized IRES-Dependent Translation of ER Chaperone Protein mRNA in Sensory Axons

Transport of neuronal mRNAs into distal nerve terminals and growth cones allows axonal processes to generate proteins autonomous from the cell body. While the mechanisms for targeting mRNAs for transport into axons has received much attention, how specificity is provided to the localized translational apparatus remains largely unknown. In other cellular systems, protein synthesis can be regulated by both cap-dependent and cap-independent mechanisms. The possibility that these mechanisms are used by axons has not been tested. Here, we have used expression constructs encoding axonally targeted bicistronic reporter mRNAs to determine if sensory axons can translate mRNAs through cap-independent mechanisms. Our data show that the well-defined IRES element of encephalomyocarditis virus (EMCV) can drive internal translational initiation of a bicistronic reporter mRNA in distal DRG axons. To test the potential for cap-independent translation of cellular mRNAs, we asked if calreticulin or grp78/BiP mRNA 5'UTRs might have IRES activity in axons. Only grp78/BiP mRNA 5'UTR showed clear IRES activity in axons when placed between the open reading frames of diffusion limited fluorescent reporters. Indeed, calreticulin's 5'UTR provided an excellent control for potential read through by ribosomes, since there was no evidence of internal initiation when this UTR was placed between reporter ORFs in a bicistronic mRNA. This study shows that axons have the capacity to translate through internal ribosome entry sites, but a simple binary choice between cap-dependent and cap-independent translation cannot explain the specificity for translation of individual mRNAs in distal axons.     

Neuronal ribonucleoprotein granules: Dynamic sensors of localized signals

Membrane‐less organelles, because of their capacity to dynamically, selectively and reversibly concentrate molecules, are very well adapted for local information processing and rapid response to environmental fluctuations. These features are particularly important in the context of neuronal cells, where synapse‐specific activation, or localized extracellular cues, induce signaling events restricted to specialized axonal or dendritic subcompartments. Neuronal ribonucleoprotein (RNP) particles, or granules, are nonmembrane bound macromolecular condensates that concentrate specific sets of mRNAs and regulatory proteins, promoting their long‐distance transport to axons or dendrites. Neuronal RNP granules also have a dual function in regulating the translation of associated mRNAs: while preventing mRNA translation at rest, they fuel local protein synthesis upon activation. As revealed by recent work, rapid and reversible switches between these two functional modes are triggered by modifications of the networks of interactions underlying RNP granule assembly. Such flexible properties also come with a cost, as neuronal RNP granules are prone to transition into pathological aggregates in response to mutations, aging, or cellular stresses, further emphasizing the need to better understand the mechanistic principles governing their dynamic assembly and regulation in living systems.     

Limited availability of ZBP1 restricts axonal mRNA localization and nerve regeneration capacity

Subcellular localization of mRNAs is regulated by RNA-protein interactions. Here, we show that introduction of a reporter mRNA with the 3'UTR of β-actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of β-actin mRNA, and introducing GFP with the 3'UTR of β-actin mRNA depletes axons of endogenous β-actin and GAP-43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of β-actin and GAP-43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.     

Molecular motors and mechanisms of directional transport in neurons

Intracellular transport is fundamental for neuronal morphogenesis, function and survival. Many proteins are selectively transported to either axons or dendrites. In addition, some specific mRNAs are transported to dendrites for local translation. Proteins of the kinesin superfamily participate in selective transport by using adaptor or scaffolding proteins to recognize and bind cargoes. The molecular components of RNA-transporting granules have been identified, and it is becoming clear how cargoes are directed to axons and dendrites by kinesin superfamily proteins. Here we discuss the molecular mechanisms of directional axonal and dendritic transport with specific emphasis on the role of motor proteins and their mechanisms of cargo recognition.     

Aβ1–42 triggers the generation of a retrograde signaling complex from sentinel mRNAs in axons

Neurons frequently encounter neurodegenerative signals first in their periphery. For example, exposure of axons to oligomeric Aβ_1‐42 is sufficient to induce changes in the neuronal cell body that ultimately lead to degeneration. Currently, it is unclear how the information about the neurodegenerative insult is transmitted to the soma. Here, we find that the translation of pre‐localized but normally silenced sentinel mRNAs in axons is induced within minutes of Aβ_1–42 addition in a Ca²⁺‐dependent manner. This immediate protein synthesis following Aβ_1–42 exposure generates a retrograde signaling complex including vimentin. Inhibition of the immediate protein synthesis, knock‐down of axonal vimentin synthesis, or inhibition of dynein‐dependent transport to the soma prevented the normal cell body response to Aβ_1–42. These results establish that CNS axons react to neurodegenerative insults via the local translation of sentinel mRNAs encoding components of a retrograde signaling complex that transmit the information about the event to the neuronal soma.