Local Protein Synthesis In Axons Research Articles

Biological Roles of Local Protein Synthesis in Axons: A Journey of Discovery.

The remit of this review is to give an autobiographical account of our discovery of the role of local protein synthesis in axon guidance. The paper reporting our initial findings was published in 2001 (10). Here, I describe some of the work that led to this publication, the skepticism our findings initially received, and the subsequent exciting years of follow-up work that helped gradually to convince the neuroscience community of the existence and functional importance of local protein synthesis in multiple aspects of axon biology-guidance, branching, synaptogenesis, and maintenance. The journey has been an exhilarating one, taking me into a new field of RNA biology, with many unexpected twists and turns. In retelling it here, I have tried to recall the major influences on my thinking at the time rather than give a comprehensive review, and I apologize for any omissions due to my own ignorance during that era.

Ribosome-Associated Vesicles promote activity-dependent local translation.

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its activity-driven deployment to local translation sites remain unclear. Here, we employed cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes. Stimulation via chemically-induced long-term potentiation causes RAV accumulation in distal sites to drive local translation. We also demonstrate activity-driven changes in RAV generation and dynamics in vivo, identifying tubular ER shaping proteins in RAV biogenesis. Together, our work identifies a mechanism for ribosomal delivery to distal sites in neurons to promote activity-dependent local translation.

Rat Sciatic Nerve Axoplasm Proteome Is Enriched with Ribosomal Proteins during Regeneration Processes.

Axons are complex subcellular compartments that are extremely long in relation to cell bodies, especially in peripheral nerves. Many processes are required and regulated during axon injury, including anterograde and retrograde transport, glia-to-axon macromolecular transfer, and local axonal protein synthesis. Many in vitro omics approaches have been used to gain insight into these processes, but few have been applied in vivo. Here we adapted the osmotic ex vivo axoplasm isolation method and analyzed the adult rat sciatic-nerve-extruded axoplasm by label-free quantitative proteomics before and after injury. 2087 proteins groups were detected in the axoplasm, revealing translation machinery and microtubule-associated proteins as the most overrepresented biological processes. Ribosomal proteins (73) were detected in the uninjured axoplasm and increased their levels after injury but not within whole sciatic nerves. Meta-analysis showed that detected ribosomal proteins were present in in vitro axonal proteomes. Because local protein synthesis is important for protein localization, we were interested in detecting the most abundant newly synthesized axonal proteins in vivo. With an MS/MS-BONCAT approach, we detected 42 newly synthesized protein groups. Overall, our work indicates that proteomics profiling is useful for local axonal interrogation and suggests that ribosomal proteins may play an important role, especially during injury.

Age-related loss of axonal regeneration is reflected by the level of local translation

Regeneration capacity is reduced as CNS axons mature. Using laser-mediated axotomy, proteomics and puromycin-based tagging of newly-synthesized proteins in a human embryonic stem cell-derived neuron culture system that allows isolation of axons from cell bodies, we show here that efficient regeneration in younger axons (d45 in culture) is associated with local axonal protein synthesis (local translation). Enhanced regeneration, promoted by co-culture with human glial precursor cells, is associated with increased axonal synthesis of proteins, including those constituting the translation machinery itself. Reduced regeneration, as occurs with the maturation of these axons by d65 in culture, correlates with reduced levels of axonal proteins involved in translation and an inability to respond by increased translation of regeneration promoting axonal mRNAs released from stress granules. Together, our results provide evidence that, as in development and in the PNS, local translation contributes to CNS axon regeneration.

Fragile X mental retardation protein regulates accumulation of the active zone protein Munc18-1 in presynapses via local translation in axons during synaptogenesis

Fragile X mental retardation protein (FMRP), a causative gene (FMR1) product of Fragile X syndrome (FXS), is an RNA-binding protein to regulate local protein synthesis in dendrites for postsynaptic functions. However, involvement of FMRP in local protein synthesis in axons for presynaptic functions remains unclear. Here we investigated role of FMRP in local translation of the active zone protein Munc18-1 during presynapse formation. We found that leucine-rich repeat transmembrane neuronal 2 (LRRTM2)-conjugated beads, which promotes synchronized presynapse formation, induced simultaneous accumulation of FMRP and Munc18-1 in presynapses of axons of mouse cortical neurons in neuronal cell aggregate culture. The LRRTM2-induced accumulation of Munc18-1 in presynapses was observed in axons protein-synthesis-dependently, even physically separated from cell bodies. The accumulation of Munc18-1 was enhanced in Fmr1-knockout (KO) axons as compared to wild type (WT), suggesting FMRP-regulated suppression for local translation of Munc18-1 in axons during presynapse formation. Using naturally formed synapses of dissociated culture, structured illumination microscope revealed that accumulation of Munc18-1 puncta in Fmr1-KO neurons increased significantly at 19 days in vitro, as compared to WT. Our findings lead the possibility that excessive accumulation of Munc18-1 in presynapses at early stage of synaptic development in Fmr1-KO neurons may have a critical role in impaired presynaptic functions in FXS.

Fragile X granules are a family of axonal ribonucleoprotein particles with circuit-dependent protein composition and mRNA cargos.

Local axonal protein synthesis plays a crucial role in the formation and function of neuronal circuits. Understanding the role of this mechanism in specific circuits requires identifying the protein composition and mRNA cargos of the ribonucleoprotein particles (RNPs) that form the substrate for axonal translation. FXGs (Fragile X granules) are axonal RNPs present in a stereotyped subset of mature axons in the intact brain that contain one or more of the Fragile X related (FXR) proteins (FMRP, FXR2P, and FXR1P) along with mRNA and ribosomes. Here we performed a systematic survey of the FXR protein composition and mRNA association of FXGs in the brain. We have identified four FXG types that can be categorized based on their FXR protein complement. All FXGs contain FXR2P, with FMRP and/or FXR1P present in circuit-selective subsets. Individual neuronal cell types predominantly express a single FXG type, with FMRP-containing FXGs the most prevalent in forebrain neurons. All FXG types associate with ribosomes and mRNA, but the specific mRNA cargos are a function of FXG type, brain region and neuron class. Transcripts for β-catenin and its regulator APC associate with a subset of forebrain FXGs. Moreover, both these transcripts can colocalize within individual FXGs, suggesting that the axonal translation of functionally related proteins may be coordinately regulated with high spatiotemporal resolution. Cell type-dependent expression of specific RNP types with distinct mRNA cargos, such as FXGs, presents a potential mechanism for regulating local translation and its output in a circuit-dependent manner.

Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury.

Axons in the peripheral nervous system respond to injury by activating retrograde injury signaling (RIS) pathways, which promote local axonal protein synthesis (LPS) and neuronal regeneration. RIS is also initiated following injury of neurons in the central nervous system (CNS). However, regulation of the localization of axonal mRNA required for LPS is not well understood. We used a hippocampal explant system to probe the regulation of axonal levels of RIS-associated transcripts following axonal injury. Axonal levels of importin β1 and RanBP1 were elevated biphasically at 1 and 24 hrs after axotomy. Transcript levels for β-actin, a prototypic axonally synthesized protein, were similarly elevated. Our data suggest differential regulation of axonal transcripts. At 1 hr after injury, deployment of actinomycin revealed that RanBP1, but not importin β1, requires de novo mRNA synthesis. At 24 hrs after injury, use of importazole revealed that the second wave of increased axonal mRNA levels required importin β-mediated nuclear import. We also observed increased importin β1 axonal protein levels at 1 and 6 hrs after injury. RanBP1 levels and vimentin levels fluctuated but were unchanged at 3 and 6 hrs after injury. This study revealed temporally complex regulation of axonal transcript levels, and it has implications for understanding neuronal response to injury in the CNS.

Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors

Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca(2+), resulting from Ca(2+) influxes through calcium-permeable AMPA receptors, voltage-gated Ca(2+) channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca(2+) influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca(2+) and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain.

Identification of precursor microRNAs within distal axons of sensory neuron.

A set of specific precursor microRNAs (pre-miRNAs) are reported to localize into neuronal dendrites, where they could be processed locally to control synaptic protein synthesis and plasticity. However, it is not clear whether specific pre-miRNAs are also transported into distal axons to autonomously regulate intra-axonal protein synthesis. Here, we show that a subset of pre-miRNAs, whose mature miRNAs are enriched in axonal compartment of sympathetic neurons, are present in axons of neurons both in vivo and in vitro by quantitative PCR and by in situ hybridization. Some pre-miRNAs (let 7c-a and pre-miRs-16, 23a, 25, 125b-1, 433, and 541) showed elevated axonal levels, while others (pre-miRs-138-2, 185, and 221) were decreased in axonal levels following injury. Dicer and KSRP proteins are also present in distal axons, but Drosha is found restricted to the cell body. These findings suggest that specific pre-miRNAs are selected for localization into distal axons of sensory neurons and are presumably processed to mature miRNAs in response to extracellular stimuli. This study supports the notion that local miRNA biogenesis effectively provides another level of temporal control for local protein synthesis in axons.

Introduction to the special issue on local protein synthesis in axons

Introduction to the special issue on local protein synthesis in axons

Efficient Retrograde Transport of Pseudorabies Virus within Neurons Requires Local Protein Synthesis in Axons

After replicating in epithelial cells, alphaherpesviruses such as pseudorabies virus (PRV) invade axons of peripheral nervous system neurons and undergo retrograde transport toward the distant cell bodies. Although several viral proteins engage molecular motors to facilitate transport, the initial steps and neuronal responses to infection are poorly understood. Using compartmented neuron cultures to physically separate axon infection from cell bodies, we found that PRV infection induces local protein synthesis in axons, including proteins involved in cytoskeletal remodeling, intracellular trafficking, signaling, and metabolism. This rapid translation of axonal mRNAs is required for efficient PRV retrograde transport and infection of cell bodies. Furthermore, induction of axonal damage, which also induces local protein synthesis, prior to infection reduces virion trafficking, suggesting that host damage signals and virus particles compete for retrograde transport. Thus, similar to axonal damage, virus infection induces local protein translation in axons, and viruses likely exploit this response for invasion.

Morphological evidence for a transport of ribosomes from Schwann cells to regenerating axons

Recently, we showed that Schwann cells transfer ribosomes to injured axons. Here, we demonstrate that Schwann cells transfer ribosomes to regenerating axons in vivo. For this, we used lentiviral vector-mediated expression of ribosomal protein L4 and eGFP to label ribosomes in Schwann cells. Two approaches were followed. First, we transduced Schwann cells in vivo in the distal trunk of the sciatic nerve after a nerve crush. Seven days after the crush, 12% of regenerating axons contained fluorescent ribosomes. Second, we transduced Schwann cells in vitro that were subsequently injected into an acellular nerve graft that was inserted into the sciatic nerve. Fluorescent ribosomes were detected in regenerating axons up to 8 weeks after graft insertion. Together, these data indicate that regenerating axons receive ribosomes from Schwann cells and, furthermore, that Schwann cells may support local axonal protein synthesis by transferring protein synthetic machinery and mRNAs to these axons.

RNAi pathway is functional in peripheral nerve axons

Recent observations demonstrated that translation of mRNAs may occur in axonal processes at sites that are long distances away from the neuronal perikaria. While axonal protein synthesis has been documented in several studies, the mechanism of its regulation remains unclear. The aim of this study was to investigate whether RNA interference (RNAi) may be one of the pathways that control local protein synthesis in axons. Here we show that sciatic nerve contains Argonaute2 nuclease, fragile X mental retardation protein, p100 nuclease, and Gemin3 helicase-components of the RNA-induced silencing complex (RISC). Application of short-interfering RNAs against neuronal beta-tubulin to the sciatic nerve initiated RISC formation, causing a decrease in levels of neuronal beta-tubulin III mRNA and corresponding protein, as well as a significant reduction in retrograde labeling of lumbar motor neurons. Our observations indicate that RNAi is functional in peripheral mammalian axons and is independent from the neuronal cell body or Schwann cells. We introduce a concept of local regulation of axonal translation via RNAi.

RNA motifs help to regulate axonal responsiveness to molecular guidance cues

Navigating axons interact en route with intermediate targets, one well-characterized example of which is the CNS midline. Navigating spinal cord commissural axons are guided initially towards the midline, even though these axons will eventually grow onwards to their targets on the contralateral side. Thus, the midline serves as an important intermediate target. The growth cones are attracted to it at first, but can ignore or overcome these attractive signals to keep growing beyond the midline. One mechanism that could control varying responsiveness to midline cues in axons is local protein synthesis. However, until now its existence in axons has been debated. A recent paper by Brittis et al. now shows evidence for local protein synthesis in axons and demonstrates that this mechanism is used to regulate protein expression within commissural axons at the midline [ 1. Brittis P.A. et al. Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target. Cell. 2002; 110: 223-235 Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar ].

Influence of nerve cell body and neurolemma cell on local axonal protein synthesis following neurotomy

The influence of the nerve cell body and neurolemma cell on selected aspects of local axonal protein synthesis as a consequence of neurotomy was studied in the hypoglossal nerve. The onset of the phase of rapid local protein synthesis in axons of the proximal stump, occurring 12–15 hr after transection, was not affected by decentralization at the time of neurotomy. This indicated that the perikaryon was not required to induce the rapid local synthesis response. Moreover, other experiments demonstrated that this response could be induced in intact nerve in regions subjected to gentle blunt dissection of the nerve away from surrounding tissue. Incorporation of [ 3H] leucine into axonal protein in proximal stumps, 18 hr after transection, occurred without apparent delay and proceeded at a linear rate through 2 hr in vitro. In addition, incubation in vitro with colchicine produced no significant effect on local synthesis of axonal protein. These findings were consistent with an intra-axonal site of protein synthesis and a local induction of the phase of rapid axonal protein synthesis.

Axonal protein synthesizing activity during the early outgrowth period following neurotomy

Axonal protein synthesizing activity in the rabbit hypoglossal nerve was assayed in vitro 12–96 hr following unilateral nerve transection. Microscopic samples of myelin-free axons from the central stump region and a more proximal region were analyzed directly for total protein content and corresponding radioactivity using microchemical methods. Fifteen to 21 hr following transection, there was a doubling of protein content in axons from the proximal stump region, and a delayed increase in axons located more proximally. These findings were consistent with the reported damming of cytoplasmic debris within axons following nerve injury. At the same time intervals, there was a greater than 20-fold increase in protein synthesizing activity, normalized to axonal protein content of zero-hr control nerve. With [ 3H] Leucine incorporation into axonal protein, almost complete inhibition by cycloheximide but not by chloramphenicol occurred. The results show that there is a phase of rapid local axonal protein synthesis in the region of nerve injury which is induced after a latent period of 12–15 hr following transection, and which peaks at 18 hr. This phase is almost completed by 24 hr, and is followed by a phase of elevated protein synthesis which is about twice that of control and constant through 96 hr after transection.

The transport of proteins in Mauthner axon in fish as studied by autoradiography and interference microscopy

AbstractUsing 1 C‐Leucine the rate of transport of proteins in single nerve fiber was investigated by quantitative autoradiography using the desheathed, hand‐free isolated Mauthner axons of fish.After different times of incubation of living fish in the radioactive medium a non‐uniform labelling of the isolated axons along their length was observed, which showed a characteristic pattern. The distribution of the label indicates: (a) a continuous supply of labelled proteins from the cell body to the axon, (b) accumulation of labelled proteins near the terminal end of the axon, (c) two different rates of the somato‐axonal transport of proteins (30–40 mm 24hr; 3.5 mm/24 hr).After the transection of the spinal cord no incorporation into the segments of the axon below the lesion was detectable.No changes of the dry mass between the proximal and distal parts of the Mauthner axon were detectable in interference microscope.The alternative explanations for the observed pattern of axonal labelling, i.e. local protein synthesis in axons and somato‐axonal flow of proteins, are discussed.