Introductory Review Cellscience Reviews Vol.2 No.3 ISSN 1742-8130

Regulation and development of protein translation in neuronal processes

J. Brian McCarthy

Introduction

Local protein synthesis was first proposed as a mechanism to modify specific adult synapses in 1965 by David Bodian.¹ This initial finding of active subsynaptic ribosomes in dendrites¹ was soon followed by reports of ribosomes in axons.^2-4 Interest in local protein synthesis has grown since those early reports, becoming one of the most active areas of current neuroscience investigation.

It is now firmly established that local protein synthesis serves as a cellular mechanism by which neurons regulate physiological events in both dendrites and axons. The breadth and complexity of this capability has only been realized within the last few years. Many excellent reviews have been written about local protein synthesis in neuronal processes, and readers are encouraged to utilize these.^5-24 This review highlights some of the exciting progress and trends made within the last few years of local protein synthesis investigation.

Machinery in Dendrites and Axons

Neurons maintain the cellular machinery to generate new proteins within the complex architecture of their dendrites and axons. Over the past 25 years, investigations have paved the way by defining the existence of protein synthetic machinery in neuronal processes. Recent investigations have made particular progress in understanding how messenger ribonucleic acids (mRNAs) reach distal sites of translation.

RNA

To date, mRNAs encoding a wide variety of proteins have been identified in dendrites, including important cytoskeletal, signaling, and synaptic proteins.^5-24 Not limited to dendrites, a number of mRNAs and their respective proteins have also been identified in axons. Calmodulin mRNAs are found in both dendrites and axons²⁵, while cytoskeletal protein, heat shock protein, ER protein, injury-response and neurodegeneration protein encoding mRNAs are reported in injury-conditioned dorsal root ganglion axons.²⁶ In addition, mRNA encoding growth-associated protein 43 (GAP43) is associated with ribosomes and the RNA stabilizing protein (HuD) in developing growth cones.²⁷

Targeting signals within some RNAs are known to confer localization specificity. MAP2 (mammalian microtubule-associated protein 2) mRNA, postsynaptic scaffolding protein (Shank 1) mRNA, and vasopressin mRNA, for example, contain dendritic targeting elements (DTEs) within their 3’untranslated region (3’UTR) that act to direct localization into dendrites.^28,29 In contrast, the atypical protein kinase C isoform [Protein Kinase M zeta (PKMζ)] contains two DTEs; one close to its 5’UTR which directs export, and another within its 3’UTR for delivery to distal dendritic locations.³⁰ In some cases, trans-acting factors are involved in the regulation of dendritic localization: the hematopoietic zinc finger (Hzf) has been found to regulate dendritic localization of the type 1 inositol 1,4,5-triphosphate receptor (IP3RI).³¹ Recent evidence indicates that some RNAs, including MAP2 undergo translational initiation via internal ribosome entry sites³², a process that may increase the translational efficiency of RNAs in dendrites.³²

RNA Trafficking

In addition to a wide variety of mRNAs, recent reports show that neurons also maintain machinery for the complex activities of RNA targeting and processing in distal processes. Some RNAs enter dendrites as part of macromolecular structures named ‘RNA granules’.³³ These structures are densely packed motile units containing ribosomes and associated proteins. Staufen, one of the RNA granule proteins, serves a necessary role in mammalian dendritic RNA targeting,³⁴ and is also involved in Drosophila long-term memory³⁵ Additional proteins have been reported to associate with RNA granules in dendrites or dendritic spines, including the RNA-binding protein (SAM68), and the RNA splicing factor [translocated in liposarcoma (TLS/FUS)].^36-38 TLS in turn can bind mRNAs encoding actin-related proteins, including mRNA encoding the actin-stabilizing protein (Nd1-L).³⁹ This interaction, along with an abnormal dendritic spine morphology in TLS-null neurons, has lead to theories that TLS may be involved in actin reorganization in spines.³⁹ BC1 (a translational repressor RNA) and BC200 are also transported into dendrites as ribonucleoprotein particles were they bind the translational initiation regulator, PolyA-Binding Protein (PABP).⁴⁰ PABP is reported to bind DTEs within the 3’ UTR of some mRNAs.²⁰ Motor proteins involved in RNA transport have been found to associate with ribosomal domains and RNA granules in both axons and dendrites. Myosin and kinesin associate with ribosomal domains in myelinated axons, and kinesin actively transports RNA granules into dendrites.^41,42

Dendrites

Great progress has been made in understanding mRNA processing and translational regulation in dendrites (Figure 1; A, B). Recent evidence shows that precursor-mRNA (pre-RNA) splicing complexes are functional in dendrites, and that the capacity to splice RNA is maintained in isolated dendrites.⁴³ Translational initiation is reported to be under the regulation of the small-untranslated RNA (BC1). BC1 represses translation by interacting with the initiation factor (eIF4A) and PABP to inhibit the assembly of translational initiation complexes.^44,45

Figure 1. Model of RNA trafficking and translation within dendrites. A. Pseudocolor image of dendritic arbor of cerebellar Purkinje neuron. B. Image of dendritic spines showing morphology. C. Model of mRNA activity-dependent recruitment of RNA granules from the soma to the spine heads in response to synaptic (NMDA receptor) activation. Activity-dependent signals are retrograde and RNA granule transport is anterograde.

Activity-Dependent Regulation

Protein synthesis is reported to undergo activity-dependent regulation in dendrites. A number studies have not only observed protein translation in isolated dendrites and dendritic fragments^46-49, but have shown that neuronal depolarization stimulates translation in dendrites.^48-50 In addition to regulating protein synthesis, depolarization is reported to increase the number of ribosome granules in dendrites⁵¹, increase Staufen RNA complexes in dendrites⁵², initiate the reorganization of RNA granules into actively transcribing polyribosomes³³, and induce the translocation of the RNA-binding protein (Sam68) into dendrites (Figure 1, C).³⁶ Importantly, activation of NMDA ionotropic glutamate receptors was found to target mRNA encoding the activity-regulated cytoskeleton-associated protein (Arc) to synapses activated by the stimulus.⁵³ In addition, activation of the metabotropic glutamate receptor (mGluR5) causes a translocation of the RNA binding protein (TLS) to dendritic spines via microtubules and actin filaments, and serves to regulate dendritic spine morphology.⁵⁴

An interesting recent study indicates that activity-dependent mRNA processing and silencing may occur near synapses. An enzyme which enables cells to precisely dissect genetic material (Dicer) and a component of the RNA-induced silencing complex (eIF2c) are released from the post-synaptic density (PSD) by calpain following NMDA treatment.⁵⁵

Synaptic Plasticity

Local protein synthesis, since it was first discussed in 1965, has been proposed as a mechanism to modify selective synapses.¹ In fact, recent studies support a role for local protein synthesis in long-term synaptic plasticity. During long-term potentiation (LTP), elongation factor 1A (eEF1A) mRNA increases in dendrites⁵⁶, and polyribosomes accumulate in dendritic spines (Figure 1, C).⁵⁷ Importantly, local protein synthesis in dendrites is needed for the late-component of LTP.⁵⁸ Long-term depression has also been reported to be associated with the dendritic translation of eEF1A⁵⁹, and a general requirement for protein synthesis in dendrites.⁶⁰ The role of local protein synthesis in synaptic plasticity is further supported by experiments that disrupt calcium/calmodulin-dependent protein kinase II α (CaMKIIα) mRNA trafficking into dendrites. This disruption caused reduced CaMKII at synapses, reduced late-phase LTP, and impaired memory performance.⁶¹ CaMKII protein translation in dendrites is reported to be activity dependent, and requires an intact CaMKII 3’ UTR.⁶² Importantly, local protein synthesis near synapses has also been found to be required for long-term facilitation (LTF) in invertebrates^63,64, elongation factors (eEF1A) are transported to terminals after LTF⁶⁵, and the stable maintenance of LTF requires an mRNA polyadenylation element binding protein (CPEB).⁶⁶

Neurotrophins

Recent evidence indicates that the brain-derived neurotrophic factor (BDNF) regulates local protein synthesis in dendrites. BDNF stimulates synthesis of a reporter protein containing both the 3’ and 5’ UTRs of CaMKIIα in dendrites.⁶⁷ BDNF stimulates the association of CaMKII, an NMDA receptor subunit, and Homer2 with polyribosomes, and regulates homer2 translation in dendrites by the rapamycin-phosphatidylinositol 3-kinase-dependent pathway.⁶⁸ In addition, BDNF induces both the translocation of the eukaryotic initiation factor 4E (eIF4E) to mRNA granules and dendritic spines⁶⁹, and the dissociation of RNG105 (an RNA-binding protein that represses translation) from RNA granules.⁷⁰ Using fluorescence resonance energy transfer (FRET) analysis, a recent report indicated that eukaryotic initiation factor assembly and disassembly was enhanced by BDNF.⁷¹

Additionally, the BDNF-induced growth of dendritic filopodia requires dendritic localization of β-actin mRNA and its associated mRNA-binding protein zip code-binding protein-1 (ZBP1). Recent evidence indicates that knock-down of ZBP1 causes a reduction of β-actin mRNA in dendrites, and impairs BDNF-induced dendritic filopodia growth.⁷²

Additional Regulators

Protein synthetic machinery is regulated by additional stimuli in dendrites, including hormones, amines, and experience. Ribosome numbers increase in dendrites during estrogen-induced synaptogenesis in the hippocampus.⁷³ Contextual learning induces the association of HuD (an RNA-binding protein) with polyribosomes.⁷⁴ Insulin stimulates PSD95 translation in synaptic fractions via the PI3K-Akt-mTOR signaling pathway⁷⁵, and dopamine (D1/D5 receptor) activation induces the local protein synthesis of a GFP-based reporter in dendrites.⁷⁶

Dendritic Protein Synthesis and Disease

Important recent progress has implicated local protein synthesis in the pathology of Fragile X mental retardation syndrome, a disease characterized by an absence of the RNA-binding protein [Fragile X mental retardation protein (FMRP)]. FMRP, which is involved in mRNA transport and translational control, is required for metabotropic glutamate receptor (mGluR)-induced local protein synthesis in dendrites.⁷⁷ FMRP mRNA granules display activity-dependent trafficking to filopodia, dendritic spines, and growth cones.⁷⁸ The continuing elucidation of proteins that associate with FMRP will aid in understanding the pathology of Fragile X syndrome.^79,80

In addition, epileptogenic stimuli induce BDNF mRNA targeting to dendrites⁸¹, and BC1 RNA (a translation repressor) which regulates postsynaptic protein synthesis is itself down-regulated following epileptiform activity.⁸² Although the implications of these findings for epilepsy are not fully understood, local protein synthesis is theorized to be upregulated following epileptiform activity.

AXONS

Recent reports show that local protein synthesis in axons plays an important role in axon guidance, pathfinding, and in growth cone collapse and regeneration (Figure 2, A).

Figure 2. Growth cone regulation in vitro A. Developing growth cone in cell culture showing principal structural elements. B. Growth cone under higher magnification with competing guidance factors, one attractive (EN-2) and one repulsive (Sem3a).

Axon Guidance

Response to axon guidance cues involves the upregulation of proteins within axons. Recent evidence shows that proteins that respond to guidance cues are translated locally within axonal subregions, and that this upregulation requires intact 3’ UTRs.⁸³ A number of factors that regulate axon guidance and local proteins synthesis in axons have been identified. In one case, axon guidance has been shown to involve the transcription factor engrailed-2 (EN-2). EN-2 enters growth cones, stimulates the phosphorylation of proteins involved in translational initiation, triggers local protein synthesis in growth cones, and aids in axon guidance (Figure 2, B).⁸⁴ Another factor, semaphorin 3A (Sem3A) is a secreted guidance cue that repels axons from inappropriate targets and induces cytoskeletal rearrangements to initiate growth cone collapse (Figure 2, B). Recent reports show that Sem3A is involved in the regulation of initiation factor (eIF-4E) phosphorylation in growth cones.⁸⁵ Sem3A also induces local translation of the small GTPase (RhoA) in axons.⁸⁶ RhoA mRNA is targeted to developing axons and growth cones by a 3’ UTR targeting element. The local translation of RhoA in axons, which functions to regulate the actin cytoskeleton, is sufficient for Sem3A-regulated growth cone collapse.⁸⁶

Actin-binding proteins, including β-thymosin, are known to regulate axonal development by binding monomeric actin and preventing actin polymerization. Recent investigations in invertebrates show that β-thymosin mRNA accumulates at turning points and growth cones.⁸⁷ A knockdown of β-thymosin mRNA in isolated neurites resulted in abnormal outgrowth, supporting a role for local β-thymosin translation in the regulation of neurite outgrowth.⁸⁷

Growth Cone Regeneration

Following injury, the generation of new growth cones requires local protein synthesis in axons.⁸⁸ This local protein synthesis-dependent growth cone regeneration is under the regulation of target of rapamycin (TOR), p38 mitogen-activated protein kinase (MAPK), and caspase-dependent signaling pathways in axons.⁸⁸ Thus, axonal regeneration requires local protein translation for growth cone integrity, and coincides with the appearance of β-actin and neurofilament mRNAs in regenerating axons.⁸⁹ In invertebrates, axon regeneration is accompanied by the appearance of aplysia β-thymosin (apβT) mRNA and local translation of soluble apβT in axons.⁹⁰ Additionally, nuclear-encoded mitochondrial proteins are synthesized in synaptic terminals of the squid giant axon.⁹¹ This is theorized to provide a mechanism for regulating the synapse’s local energy generating system. In invertebrates, injected G-protein coupled receptor (GPCR) mRNA is actively translated in axons.⁹²

Neurotrophins

Neurotrophins also play a role in the regulation of local protein synthesis in axons. BDNF, which serves in axon pathfinding, increases cytoskeletal mRNAs in DRG axons²⁶, and induces a form of synaptic potentiation that requires local protein synthesis in developing axons.⁹³

Concluding Remarks

Progress in understanding local protein synthesis within neuronal processes has advanced at an exponential pace in recent years. Local RNA trafficking and protein translation are known to contribute to cellular processes as diverse as synaptogenesis, axon outgrowth / regeneration, growth cone collapse, synaptic remodeling and plasticity. In the upcoming years, new studies will uncover the roles played by local protein translation in the pathology of disease, and provide new therapeutic targets for drug development.

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