Translating regeneration: Local protein synthesis in the neuronal injury response

Accepted Manuscript Title: Translating Regeneration: Local Protein Synthesis in the Neuronal Injury Response Authors: Sandip Koley, Meir Rozenbaum, Mike Fainzilber, Marco Terenzio PII: DOI: Reference:

S0168-0102(18)30580-7 https://doi.org/10.1016/j.neures.2018.10.003 NSR 4224

To appear in:

Neuroscience Research

Received date: Revised date: Accepted date:

13-7-2018 13-9-2018 2-10-2018

Please cite this article as: Koley S, Rozenbaum M, Fainzilber M, Terenzio M, Translating Regeneration: Local Protein Synthesis in the Neuronal Injury Response, Neuroscience Research (2018), https://doi.org/10.1016/j.neures.2018.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Translating Regeneration: Local Protein Synthesis in

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the Neuronal Injury Response

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Sandip Koley, Meir Rozenbaum, Mike Fainzilber, Marco Terenzio*

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Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel

*Corresponding author: [email protected]

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Tel: +972-8-934-4474

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Highlights for Koley et al., Translating Regeneration: Local Protein Synthesis in the Neuronal Injury Response

Local translation contributes to retrograde injury signaling and axon regeneration

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Numerous mRNAs localize to axons in different neuron types

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Diverse sources have been proposed for axonal ribosomes

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mTOR and other kinases drive local translation in axons

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Abstract

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Neurons need to overcome long distances in order to function in a mature mammal, for example motor neurons and sensory neurons project axons up to a meter long in humans.

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To this end, a sophisticated network of long-range signaling mechanisms enables communication between neuronal processes and somata. These mechanisms are

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activated during axonal injury and have essential roles both for sensing the injury and regulating subsequent regeneration. Here we survey the role of one such mechanism, axonal translation, which contributes to both retrograde injury signaling and as a source

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of proteins for regenerating axons. The nature of the axonal synthesis machinery has become progressively clearer over the past decade. A large number of axonally localized mRNAs have been identified, which cover a wide spectrum of protein families and axonal ribosomes have been detected, even though their origin is still subject to debate. Various kinase pathways, most prominently mTOR, have been implicated in driving local translation in axons. Finally, new technologies are becoming available to visualize axonal

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translation and enable proteomic analyses. These technological improvements offer new

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avenues towards comprehensive characterization of the axonal translational machinery.

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Keywords: Local translation; Ribosome; mTOR; Axonal mRNA; Nerve regeneration.

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Introduction Cells need to manage a plethora of different cellular processes in an energy efficient way and this is especially true for highly polarized cells such as neurons, where the cell body diameter can be orders of magnitude smaller than the length of the respective axon.

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One of the mechanisms by which neurons deal with metabolic and signaling requirements of their subcellular compartments is the localization of their translational machinery and specific mRNAs (Rangaraju et al., 2017), enabling local generation of proteins required for the physiology of these compartments. Localized protein synthesis gives neuronal processes autonomous control over their structure and function (Gomes et al., 2014). Since one copy of an mRNA can be used for multiple rounds of translation, local storage

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of mRNAs may enable rapid responses to extracellular stimuli or metabolic needs, possibly faster than what would be possible under limitations of anterograde transport

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rates of proteins synthesized in the soma. Furthermore, given the extensive area

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represented by axons and dendrites compared to the cell body, anterograde transport of

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mRNAs might also offer an energy-efficient alternative to protein transport. Axonal mRNA localization is achieved by anterograde transport of mRNAs bound to

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RNA-binding proteins (RBP). The sequences that drive mRNA axonal localization are

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often found in their 3’ untranslated regions (3’UTRs), though 5’UTR localization motifs have also been described (Andreassi and Riccio, 2009; Sahoo et al., 2018). The most

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known example of this mechanism is the “zip-code” axonal targeting sequence present within the 3’ untranslated region (UTR) of -actin (Costa and Willis, 2018; Sahoo et al.,

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2018). The presence of a zip-code determines binding to the RBP ZBP-1 (RNA-binding protein zip-code-binding protein 1), which facilitates axonal localization (Patel et al., 2012; Welshhans and Bassell, 2011; Willis et al., 2011). Although axonal localization

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elements have not been described to date in protein-coding sequences (CDS) of higher eukaryotes, CDS-linked localization elements have been described in yeast and may exist in mammals (Sahoo et al., 2018). Across the animal kingdom several organisms show significant regenerative potential, allowing re-growth of amputated or injured parts of their body. Such

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regenerative potential is most prevalent in simple metazoans, but has also been described for specific tissues and organs in vertebrates (Galliot et al., 2017). In most mammals, however, regenerative potential is progressively lost during development, with the exception of specific organs such as skin, liver, pancreas, heart and the peripheral nervous system (Iismaa et al., 2018). The unique morphology of neurons makes them

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vulnerable to axonal injury, which by definition represents a type of compartmentalized event that must be sensed locally and signaled to the neuronal cell body to initiate the appropriate somatic response. Thus, sensing long-range signaling is central to regeneration ability in neurons, and axonal mRNA translation appears to be a critical component of peripheral damage response mechanisms. Indeed, it has been shown that adult dorsal root ganglion (DRG) axons are capable of incorporating 3H-leucine upon

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axotomy, indicating translation of preexisting mRNAs (Verma et al., 2005). These mRNAs

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can act as mediators of the injury-related stress response and generate secondary signals that are relayed to the cell body. The functions of these signals have been detailed

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in several studies describing the axonal local translation of RNA transcripts such as

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importin 1, vimentin, RanBP1, STAT3 and PPAR (Lezana et al., 2016; Mahar and Cavalli, 2018; Rishal and Fainzilber, 2014). Furthermore, studies with transgenic mice

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(Donnelly et al., 2011).

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have shown that availability of ZBP-1 is a limiting factor in peripheral nerve regeneration

Electron microscopy (EM) images of polysomes at the base of dendritic spines in

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the mature hippocampus was the trigger for investigations of local translation in dendrites (Steward and Levy, 1982). Since similar findings could not be obtained in axons at that

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time, the possibility of a similar mechanism in axons has been a topic of intense debate (Sahoo et al., 2018). Early estimates suggested that 200-400 mRNAs are found in axons

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in invertebrates, while more recent studies have shown the presence of more than 6000 axonal mRNAs in mouse embryonic sensory neurons (Kar et al., 2017). The large pool of axonally localized mRNAs is now known to include proteins with diverse functions, including cytoskeletal, heat shock proteins, ER chaperones and even proteins associated with neurodegeneration (Willis et al., 2005). The diversity of proteins translated after injury suggests a central role of this mechanism in the neuronal response to peripheral insults.

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Two functions have been attributed to localized mRNA translation in response to injury: support of a survival response against the insult and initiation of long range axonal signaling to the cell body as part of the regenerative response. Both functions have been well documented in past decade (Mahar and Cavalli, 2018), but while much is known on the identity of axonally synthesized proteins and the means of their localization, initiation

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and regulation of axonal local translation is still poorly understood. Here we summarize studies on axonal local translation after injury, with a focus on how localized protein synthesis is initiated and controlled.

Translating injury signals

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During development, neurons extend their axons to make contact with their targets; however, this propensity for elongating growth is greatly reduced once such contact is

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made. Upon injury, neurons must switch to an “elongating growth state” that allows

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regeneration of axonal tracts. This reprogramming is dependent on the integration of fast

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and slow long-range signals coming from the injury site (Doron-Mandel et al., 2015). Fast signals are thought to be represented by a calcium influx in the axoplasm following injury,

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which gives rise to a depolarization wave propagated along the axon to the cell soma, while slow injury signaling relies on local translation and motor-based transport of locally

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activated signals (Rishal and Fainzilber, 2014). Neurons of the central nervous system (CNS) have a vastly reduced ability to regenerate compared to peripheral nervous system

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(PNS) neurons. This is in part because of the presence of an inhibitory microenvironment in the CNS, and in part due to differences in intrinsic regenerative mechanisms, which

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are discussed in detail in the following recent reviews (Doron-Mandel et al., 2015; Mahar and Cavalli, 2018, Glock et al., 2017). Interestingly, DRG neurons are characterized by a

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bifurcating axon with two branches, peripheral and central, which differ greatly in their regenerative potential. These differences are exemplified by contrasting responses to conditioning lesions, whereby lesioning a DRG peripheral branch induces an increase in growth ability of the injured neurons, while injury of the central branch does not (Neumann and Woolf, 1999; Richardson and Verge, 1987; Smith et al., 1997). This section will detail

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long-range signaling mechanisms connected to axonal local translation that have been described in the peripheral branch of DRG neurons. After axonal injury, calcium is released from intracellular stores and activates several signaling pathways, including the mitogen-activated protein kinase (MAPK) and

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Ca2+/calmodulin-dependent protein kinase (CaMK) pathways. CaMK is responsible for the phosphorylation of the cAMP response element binding protein (CREB) , which is a nuclear transcription factor and directly influences gene expression (Ghosh-Roy et al., 2010; Yan et al., 2016). Calcium signaling also activates proteases such as calpain, which is responsible for structural remodeling at the site of lesion by cleaving cytoskeletal actin and spectrin (Spira et al., 2001). The dismantling of these structural elements helps

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reduce membrane tension and accelerates membrane fusion, thus facilitating the closure of the membrane rupture caused by injury (Kamber et al., 2009). Calcium signaling was

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also reported to activate the dual-leucine zipper kinase 1 (DLK-1), which phosphorylates

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mitogen-activated protein kinase (MAPK) and stimulates retrograde signaling to activate

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expression of the injury responsive genes (Mahar and Cavalli, 2018; Tedeschi and Bradke, 2013). Interestingly, calcium has also been shown to activate a variety of

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signaling mediators capable of inducing the transformation of differentiated axonal segments into growth cones, an organelle with a central role in axonal regeneration (He

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and Jin, 2016). Most recently, calcium has been reported to stimulate translation of HIF1a, a transcription factor that plays a crucial role in axonal regeneration by mediating the

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activation of HIF responsive genes (Cho et al., 2015; Hui et al., 2006).

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The slow retrograde signaling response is mediated by the molecular motor dynein, which retrogradely transports locally translated and/or activated protein effectors from the site of lesion to the cell body. Early support for activation of local axonal signaling after

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nerve injury was provided by work on Aplysia, where injection of axoplasm from crushed nerves into the soma of uninjured sensory neurons triggered an injury-like response (Ambron et al., 1995). More recently it emerged that many of the locally activated injury signals are a consequence of local translation and is now clear that axonal local translation is essential for both retrograde injury signaling (Rishal and Fainzilber, 2014;

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Terenzio et al., 2017) and for regenerative axon outgrowth in sensory neurons (Sahoo et al., 2018). Initial studies were based on the observation that, after lesion, axons of cultured adult sensory neurons contain ribosomal proteins, translation initiation factors and

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ribosomal RNAs (Zheng et al., 2001) and that inhibition of protein synthesis in neurites leads to growth cone retraction and reduces the number of regenerating neurites (Chierzi et al., 2005; Zheng et al., 2001). In addition, genome-wide analyses of axonal preparations of embryonic and adult DRG neurons cultured on compartmentalized microporous membrane inserts identified large numbers of axonal mRNAs (Gumy et al., 2011; Minis et al., 2014; Willis et al., 2007). Transgenic overexpression of localization

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elements of axonal mRNAs affects neuronal outgrowth and leads to reduced regeneration, due to competitive displacement of endogenous mRNAs from the RBP

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HuD-ZBP1 (Donnelly et al., 2013, 2011; Yoo et al., 2013).

Upon sciatic nerve lesion, locally activated injury signals must be transported back

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to the soma and nucleus in order to elicit a transcriptional response. The nuclear access requirement suggested that retrograde injury signaling molecules might contain nuclear

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localization signals (Ambron et al., 1995; Schmied and Ambron, 1997). Indeed, the

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nuclear import factors importins  and 1 were found to be present in rodent sensory axons, and injection of nuclear localization signal (NLS) peptides at axonal injury sites

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reduced the capacity of sensory neurons to respond to injury (Hanz et al., 2003). Multiple importin  isoforms were found to be present in intact nerves, while importin 1 was

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shown to be locally translated upon axonal injury (Hanz et al., 2003). Local translation of importin 1 enables formation of functional importin complexes and the mounting of an NLS-mediated retrograde injury response (Hanz et al., 2003). Similarly to -actin and

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GAP-43, the axonal localizing element of importin 1 mRNA is encoded in its 3’UTR (Perry et al., 2012), and was narrowed down to a stem loop motif of 34 nucleotides, which interacts with the RNA-binding protein nucleolin (Perry et al., 2016). Notably, mice lacking the axon-localizing region of importin 1 3’-UTR reveal sequestration of this mRNA from axons and an attenuated regenerative response to sciatic nerve crush (Perry et al., 2012).

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In addition, Ras-related nuclear protein binding 1 (RanBP1) was also found to be locally translated in axons after sciatic nerve crush. RanBP1 triggers the hydrolysis of importinbound RanGTP, which results in its detachment from importin , freeing it to interact with newly translated importin 1 (Yudin et al., 2008). Finally, local synthesis of the

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cytoskeletal protein vimentin after sciatic nerve crush enables retrograde transport of phosphorylated ERK1/2 to neuronal cell bodies, since proteolytic cleavage products of vimentin bridge between pERK1/2 and importin 1 (Perlson et al., 2006, 2005).

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway is an important growth modulator and one of its members, the signal transducer and activator of transcription 3 (STAT3) was shown to be involved in cell

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survival and regeneration (Ben-Yaakov et al., 2012; Qiu et al., 2005; Schwaiger et al.,

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2000). STAT3 was indeed found to be locally translated in axons after sciatic nerve crush and subsequently retrogradely transported via its interaction with importin 5 to DRG

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nuclei, where it regulates survival after injury (Ben-Yaakov et al., 2012). Interestingly,

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STAT3 was described to associate with the nuclear import machinery through a noncanonical NLS sequence (Ben-Yaakov et al., 2012). Furthermore, DLK, which can be

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activated by disruption of the actin or microtubule cytoskeleton in response to injury

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(Valakh et al., 2015), was also suggested to be involved in retrograde transport of activated p-STAT3 after sciatic nerve lesion (Shin et al., 2012). Similarly, the ligand‐ activated nuclear receptor PPARγ was described to be involved in the neuronal injury

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response (Lezana et al., 2016). Its mRNA has been found in axons of sensory neurons, where is locally translated as a consequence of sciatic nerve crush (Lezana et al., 2016).

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PPARγ is then retrogradely transported in a dynein-dependent manner to the sensory

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neurons nuclei, where it promotes axonal regeneration (Lezana et al., 2016). In addition to the locally translated signals described above, there is a cohort of

retrograde injury signaling molecules that are activated but not translated locally, and may link to dynein motor complexes in an importin-independent manner. Examples include cJun N-terminal kinase (JNK) (Cavalli et al., 2005) and Sunday Driver (syd) (Abe et al., 2009). For further details on the roles and regulation of these molecules in retrograde injury signaling please see a recent review (Mahar and Cavalli, 2018). 9

The mRNAs that participate in the initial wave of local translation after injury must have been in the axon prior to the insult. It does not seem likely that neurons would evolve to invest significant energy and resources in “stockpiling” axonal mRNAs in preparation for an injury that might never occur, hence what is the role of this mRNA ensemble in normal neuronal biology? Interestingly, a recently published mechanism that allows

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neurons to sense their own length relies on the axonal mRNA axonal localization of known injury response elements and their translation at axon tips (Albus et al., 2013; Perry et al., 2016; Rishal et al., 2012). The suggested mechanism is based on frequency-based oscillatory signaling, which integrates nucleolin-mediated anterograde mRNA transport, peripheral local translation and dynein-mediated retrograde transport (Perry et al., 2016; Rishal et al., 2012), sharing many similarities with axonal retrograde injury signaling.

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These similarities suggest that a conserved cell size/axon length sensing mechanism can

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be utilized for injury responses if needed.

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Translational modulation by kinases and phosphatases The previous section briefly surveyed a few examples of mRNAs which are locally

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translated in axons in response to injury, leading to the question how is axonal translation initiated and controlled? Peripheral injury activates multiple signaling pathways, several

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of which are directly or indirectly implicated in the regulation of axonal translation. mRNA translation is a complex, tightly regulated process, which consumes approximately 20%

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of total cellular energy (Roux and Topisirovic, 2012). The complexity and high energetic cost of translation suggests that it must be tightly regulated. In addition, translation can

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be activated or modulated by a variety of stress conditions, including environmental (heat shock, UV irradiation), extracellular (hormones, growth factors) or intracellular (nutrients,

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ATP) signaling (Ma and Blenis, 2009; Sonenberg and Hinnebusch, 2009; Wek et al., 2006). These diverse stimuli can control translation in subtle ways, ranging from global increases or decreases to selective activation for specific transcripts via the use of IRES (intra ribosomal entry site) (Truitt and Ruggero, 2016) or upstream ORF (uORF) control elements (Somers et al., 2013), which constitute non-canonical mechanisms of mRNA translation. The major pathways activated upon stress are calcium signaling, MAPKs,

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RSKs, MNK and mTOR pathways and cellular phosphatases, all of which may influence translation (Morrison, 2012). Calcium signaling was discussed above as one of the first signaling pathways to be activated after axonal injury. In this section we will focus on the role of kinases and phosphatases in axonal translation.

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Members of the MAPK family such as ERK, JNK, and p38 have been shown to regulate translation by controlling the phosphorylation state of their downstream effectors. ERK activation in human alveolar macrophages inhibits JNK activity, leading to the activation of protein phosphatase 1 and consequent dephosphorylation of eIF2A, which in turn regulates cell survival by activating translation (Monick et al., 2006). Similarly, ERK has been shown to be locally translated and phosphorylated upon axonal injury; activated

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ERK is subsequently transported back to the cell body in a vimentin dependent manner (Perlson et al., 2005). Although ERK’s role as a retrograde survival factor and promoter

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of axonal regeneration after injury is known, it is unclear whether or not ERK is able to

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influence peripheral axonal mRNA translation directly. P38 kinase has also been shown

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to regulate cellular translation by controlling the phosphorylation state of eIF4E in malignant cholangiocytes, bile duct epithelial cells (Yamagiwa et al., 2003). Activation of

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p38 in a coordinated manner with JNK was found to promote axonal regeneration after injury in C. elegans, although this is likely due to cell body activity, rather than a direct

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effect on local translation (Nix et al., 2011). MAPK interacting kinases (MNKs) were described to be important translational regulators in human cell lines. Two different MNKs

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have been implicated in these processes, with MNK1a and MNK2a being predominantly cytosolic, whereas MNK1b and MNK2b are present both in the nucleus and cytosol

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(O’Loghlen et al., 2007; Scheper et al., 2003). MNK1a and MNK2a are both activated by ERK1/2 and p38, which correlates to the phosphorylation at Ser-209 of eIF4E that

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reduces its affinity to 5’mG-cap (Fonseca et al., 2007; Whalen et al., 1996), a phenomena that leads to an increase of global cellular translation in a broad variety of cell types (Kaspar et al., 1990; Manzella et al., 1991). Indeed, phosphorylation of eIF4E has been shown to modulate translation of specific transcripts connected to inflammatory responses and the extracellular matrix (Min et al., 2017) and other mRNAs such as eIF2α, eEF2, and CCT2 (Amorim et al., 2018). In regards to neuronal physiology, MNK

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mediated phosphorylation and modulation of protein synthesis was described in nociceptive plasticity linked to pain conditions, through the action of inflammatory cytokines such as IL6, which mediates the activation of the eIF4F complex via the MNKeIF4E axis to increase translation (Melemedjian et al., 2010). Although this signaling axis has been reported to contribute to nociceptive plasticity by regulating calcium signaling in

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DRG neurons (Moy et al., 2017), there is no evidence to date for a possible role of this pathway in local mRNA translation during axonal regeneration.

A major signaling pathway that plays a key role in axonal local translation in injured axons of the peripheral nervous system is the mammalian target of rapamycin (mTOR) (Terenzio et al., 2018). mTOR is a serine threonine kinase that coordinates different

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molecular signaling pathways, acting as a metabolic regulator to integrate diverse cellular responses including stress, nutrient or energy sensing, nucleotide biosynthesis,

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autophagy and mRNA translation (Malik et al., 2013). Neuroscientists have long had a

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keen interest in mTOR involvement in neuronal plasticity, given its ability to regulate the

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rate of translation in response to extracellular stimuli (Switon et al., 2017). Indeed, a body of work has demonstrated key roles for mTOR in long-term potentiation (LTP), long-term

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depression (LTD), and learning and memory (Switon et al., 2017). In addition, mTOR was shown to be critical for neuronal survival, differentiation, and morphogenesis (Switon et

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al., 2017).

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The mTOR pathway has also attracted keen interest in the axonal regeneration field due to its influence on regeneration of CNS neurons after axonal injury. A role for mTOR

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protein complexes in axonal regeneration was first suggested by the observation that deletion of mTOR inhibitors such as phosphatase and tensin homolog

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tuberous sclerosis complex (TSC) 1 in rodent retinal ganglion cells (RGCs) and peripheral

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sensory neurons stimulates axonal survival and regeneration (Belin et al., 2015; Christie et al., 2010; Park et al., 2008). The relative contribution of different downstream effectors of the mTOR pathway in the integration of injury stress signals is still not clear. Two mTOR effectors, S6 kinase (S6K) and 4EBP, were suggested to have distinct role in axonal regeneration after injury in RGC cells (Yang et al., 2014). Deletion of 4EBPs showed increased axonal survival but had very limited effect on axonal regeneration

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(Yang et al., 2014), while the effect of S6K seems to depend on the neuronal subtype in question. AAV mediated overexpression of S6K1 showed enhanced regeneration in RGC cells, while its knockdown or pharmacological inhibition caused increased neurite outgrowth in hippocampal neurons (Al-Ali et al., 2017; Yang et al., 2014). Perturbation of mTOR pathways affects regeneration in PNS neurons as well (Abe et al.,

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2010; Cho et al., 2014; Terenzio et al., 2018) and the complexity of the effects reported so far suggest that both mTORC1 and mTORC2 are involved. Genetic deletion of mTOR inhibitors like TSC2 enhances axonal regeneration and outgrowth in peripheral neurons (Abe et al., 2010), although it has also been reported that chemical inhibition of mTORC1 using rapamycin does not suppress axonal outgrowth in adult sensory DRG neurons (Christie et al., 2010). A recent study using torin-1, an mTOR complex inhibitor that blocks

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both mTORC1 and mTORC2, showed significant reduction of proprioceptive neuron

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outgrowth and survival upon injection of the inhibitor at the lesion site concomitantly with injury (Terenzio et al., 2018). Another recent study showed that chronic activation of the

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mTOR pathway by TSC2 genetic deletion in mouse peripheral sensory neurons, causes

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aberrant skin innervation by peptidergic nociceptors and consequent behavioral alterations (Carlin et al., 2018), indicating that activating mTOR as a possible therapy for

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axonal injury will require tight control of degree and duration of pathway activation.

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A number of reports suggested that mTOR effects in peripheral axon regeneration are mediated by promotion of axon growth regulators such as GAP43 (Abe et al., 2010)

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or syntaxin 13 (Cho et al., 2014). FRAP analysis in axons of hippocampal neurons in culture suggested that mTOR mRNA might undergo axonal synthesis, with modulation by

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miRNA-183 (Kye et al., 2014). Proof of in vivo axon localization and local translation of mTOR mRNA was recently shown in PNS axons (Terenzio et al., 2018). Axonal mTOR was shown to be activated upon injury and to promote a burst of local translation in the

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axon, including the translation of its own mRNA (Terenzio et al., 2018) (Figure 1). A proteomic analysis revealed a cohort of about 230 localized mRNAs under mTOR translational control, including canonical components of the retrograde injury signaling complex such as importin beta1, vimentin and STAT3 (Terenzio et al., 2018).

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In addition to kinases, phosphatases can also play important roles in the regulation of translation (Mahar and Cavalli, 2018). For example, PTEN is a lipid phosphatase that has been reported to negatively regulate mTOR-driven translation by activating the tuberous sclerosis complex (TSC) (Guertin and Sabatini, 2007; Luo et al., 2003). The study of mTOR local translation in axons also revealed an inverse correlation between

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PTEN and mTOR axonal levels, suggesting the existence of a mechanism in uninjured nerve by which mTOR activation might be kept in check by PTEN (Terenzio et al., 2018). However, the mechanism of PTEN downregulation in the axon upon injury is still unclear. Interestingly, another recent study reported that exosomes containing NADPH oxidase 2 (NOX2) complexes are translocated from macrophages to injured axons in peripheral nerve (Hervera et al., 2018). Retrograde transport of these complexes lead to the

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oxidation and subsequent inactivation of PTEN in neuronal cell bodies, resulting in the

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stimulation of the PI3K-Akt axis and regenerative outgrowth (Hervera et al., 2018). It remains to be seen if local release of NOX2 in axons might account for the local

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downregulation of PTEN after peripheral nerve lesion.

PTEN also acts as a negative regulator of axonal regeneration in adult retinal

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ganglionic cells, as shown by increased regeneration of CNS neurons upon genetic deletion of PTEN (Park et al., 2008; Sun et al., 2011). Serine/threonine phosphatases

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also play an important role in modulating mTOR function. Indeed protein phosphatase 2A (PP2A) mediates the dephosphorylation of AKT (Andjelković et al., 1996) as well as of

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S6K and mTOR itself (Ballou et al., 1988; Van Kanegan et al., 2005) thus modulating the activity of the mTOR pathway. In addition, inhibition of PP2A by okadaic acid activates

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S6K in hippocampal neurons, which in turn phosphorylates FMRP, a negative regulator of mRNA translation, facilitating protein synthesis-dependent synaptic plasticity

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(Narayanan et al., 2008). PP2A activation has also been shown to facilitate axonal growth and recovery after injury in the CNS (Cheng et al., 2015; Zhu et al., 2010). The myoinositol monophosphatase-1 (Impa1) has also been described to be axonally locally translated and promote axonal integrity in sympathetic neurons. Inhibition of local translation of Impa1 resulted in axonal degeneration mediated by impaired CREB activation in the nucleus (Andreassi et al., 2010).

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Ribosomes in the axon A major effort has been directed towards the characterization of the nature of the axonal translational machinery. In the 1980s Steward and Levy described the presence of the protein synthesis machinery in neuronal processes from the rodent hippocampus

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(Steward and Levy, 1982). Ultrastructural analysis detected ribosomes at the bases of dendritic spines, while mature CNS axons seemed devoid of them (Steward and Levy, 1982). This observation lead to the notion that axons of vertebrates, unlike dendrites, do not have the ability to synthesize proteins (Steward, 1997). However, other studies conducted in invertebrate axons suggested the presence of protein synthesis machinery, including ribosomes and RNAs (Koenig and Giuditta, 1999). The squid giant axon in

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particular provided a very useful early model due to its large size, which allowed for the

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retrieval of sufficient axoplasm to detect protein synthesis with the biochemical and molecular tools available at that time (Giuditta et al., 2002). Studies conducted using a

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polyclonal antibody against rat brain ribosomes and immuno-electron microscopy

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described the presence of ribosomes and polyribosomes in the squid axon (Sotelo et al., 1999), while polysome-bound mRNAs were subsequently described in the same model

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(Crispino et al., 1997). Protein synthetic capacity was then also reported in myelinated

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axons of vertebrates (Kun et al., 2007; Sotelo-Silveira et al., 2008), and β -actin mRNA and polyribosomes were identified in growth cones of chick cortical neurons (Bassell et al., 1998). In addition, mRNAs and rRNAs were shown to enter early neurites destined to

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become axons in cortical and hippocampal neurons (Kleiman et al., 1994). Interestingly, these early studies in vertebrate axons were conducted on CNS neurons, which might

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have been an unfortunate choice given the observation by Verma and colleagues that levels of axonal translational machinery correlate with neuronal regenerative capacity

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(Verma et al., 2005). Indeed, it is possible that the increased propensity of PNS neurons to regenerate reflects a higher inherent ability to synthesize axonal proteins. Traditional electron microscopy has been used to detect ribosomes in myelinated PNS axons (Zelená, 1970; Zelená, 1972) and ribosome-containing plaque-like domains were identified by concentrated electron spectroscopic imaging (ESI) (Koenig et al., 2000; Koenig and Martin, 1996). These domains were described to be enriched in RNA and 15

molecular motors such as Myosin Va and Kinesin (Sotelo-Silveira et al., 2004), and shown to contain -actin and its RNA binding protein ZBP1 (Sotelo-Silveira et al., 2008). Recently, ultrastructural analyses of ribosomes in motor axons performed by transmission electron microscopy on wild type mice and spinal muscular atrophy (SMA) mice revealed axonal ribosomes in both genotypes, though the SMA mice were characterized by a 27%

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decrease in axonal ribosomes (Bernabò et al., 2017). In addition, peripheral nerve segments grafted into the adult rat transected spinal cord were showed to create a permissive environment that allows for spinal cord neuron axons to grow (Kalinski et al., 2015). These axons contained mRNAs and members of the translational machinery such as 5S rRNA, phosphorylated S6 ribosomal protein, eIF2α, and 4EBP1 (Kalinski et al., 2015). Mature CNS axons in the adult rat brain were also found to contain part of the

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translational machinery, which include ribosomes, the translational regulator FMRP and

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a subset of FMRP mRNA targets, associated with Fragile X granules (Akins et al., 2017).

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Moreover, a recent study described an increase of phosphorylated S6 ribosomal protein in axons of the sciatic nerve after injury, which was connected with the activation of the

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mTOR pathway and local translation (Terenzio et al., 2018). Interestingly, hot spots of localized mRNA translation in axons of sensory neurons were shown to contain stalled

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axonal mitochondria and components of the translational machinery such as the

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ribosomal protein L10a and ribosomal 5.8S RNA (Spillane et al., 2013). The origin of axonal ribosomes has not yet been conclusively established (Sotelo

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et al., 2014; Twiss and Fainzilber, 2009). Different explanations might account for the presence of axonal ribosomes, one being transport from the soma and another the

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delivery to axons from an extra-neuronal source. In the 1970s it was proposed that ribosomes might be transferred from glial cells to the squid giant axon (Gainer et al., 1977; Lasek et al., 1977). Interestingly, axonal vesicles containing ribosomes that were

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decorated by an anti‐ribosome antibody were observed by electron microscopy analysis of axons from rat sciatic nerve (Kun et al., 2007). As the study was limited to static images, it was not possible to determine the origin of these vesicles. Strikingly, polysomal clusters packed in multimembrane vesicles were observed in injured peripheral nerve axons that were separated from the cell body using a ‘Wallerian degeneration slow’ (Wlds) mutant

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mice, which exhibits a slower kinetics of axonal degeneration (Court et al., 2008). The authors of that study described the transfer of fluorescently tagged Schwann cell ribosomes to axons (Court et al., 2008), which were further characterized by electron microscopy as enclosed by two sets of membranes, the outermost being probably axonal in nature and the other glial (Court et al., 2008). A subsequent study from the same group

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showed in vivo transfer of Schwan cell derived ribosomes to regenerating axons by labelling ribosomes in Schwann cells via lentiviral mediated expression of ribosomal protein L4 fused to eGFP (Court et al., 2011). Transected axons of rat and mouse sciatic nerves were found to accumulate in vivo newly-synthesized RNA in the absence of the neuronal cell bodies, which might indicate mRNA transfer from Schwann cells to axons (Sotelo et al., 2013). Interestingly, axonal ribosomal content was showed to increase in

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axons from a mouse model of multiple sclerosis (Shakhbazau et al., 2016).This increase

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was suggested to be at least partially due to glia to axon transfer of ribosomes, perhaps

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triggered by demyelination (Shakhbazau et al., 2016).

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Recent studies have used mouse models with genetically tagged ribosomes to examine the source of axonal ribosomes, with conflicting results. Two groups used the

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Ribotag mouse, which expresses an HA-tagged version of the ribosomal protein RPL22 in a tissue-specific manner (Sanz et al., 2009), and observed ribosomes of neuronal origin

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in growth cones of DRG neurons in culture (Perry et al., 2016) (Fig. 2A) and axons of retinal ganglionic cells in vivo (Shigeoka et al., 2018, 2016). Another group used the

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RiboTracker mouse strain, which expresses a fluorescent protein fusion of the L4 ribosomal protein (Müller et al., 2018). Comparison of the presence of tagged axonal L4

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in the SN using neuronal and glial Cre drivers of the RiboTracker allele revealed low levels of tagged L4 in axons of neuronal Cre mice, and higher levels using glial Cre (Müller et al., 2018). This apparent dichotomy could be resolved by assuming both neuronal and

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glial origins for axonal ribosomes. However, both models lack a systematic analysis of motor interaction or transport capabilities of the genetically tagged ribosomes. Furthermore, one should also note that bulky or misplaced tags may disrupt ribosome complex superstructures (Viero et al., 2015) or ribosome functions. Thus, more careful investigation of the precise composition of axonal ribosomes, including proof of the

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integration of the tagged protein in polysomes in vivo, is still needed to unambiguously establish their origin.

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Energetics of mRNA local translation A common argument in the literature is that local translation is cost-effective for the axon since it allows generation of multiple copies of a protein from a single transported mRNA. The implicit assumption is that axonal localization of the entire translational machinery is energy-efficient versus anterograde transport of the entire axonal proteome. However, we are not aware of any rigorous quantifications that would support this

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assumption. Moreover, local translation in the axon will require a localized mechanism in

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place to prevent unintended translational activity, which might also be energetically costly. Indeed, RNA axonal localization and local translation require considerable energetic

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investments by neurons, further compounded by the distance these mRNAs must cover

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to reach their destination. mRNA translation is one of the most energy consuming processes per se, accounting for ~25-30% of the total energy expenditure in mammalian

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cells (Buttgereit and Brand, 1995; Rolfe and Brown, 1997). Moreover, the energy

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requirement for translation varies for different mRNAs and cell types (Lynch and Marinov, 2015). The translational machinery derives its energy primarily from ATP consumption and cellular ATP is mainly produced by oxidative phosphorylation in mitochondria (Pontes

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et al., 2015). Although mitochondrial presence and movement in axons is well documented, their actual contribution to axonal ATP levels is still under debate

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(Magistretti, 2014; Rangaraju et al., 2014; Smith and Gallo, 2018; Zhu et al., 2012). Approximately 90% of the ATP present at the synapse is thought to be supplied by

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mitochondria, with the rest being a product of glycolysis (Harris et al., 2012; Sheng, 2017). This fraction may increase in times of metabolic stress, which may increase the requirement for glycolysis as an energy source at the synapse (Jang et al., 2016). Indeed, glycolytic enzymes have been found in presynaptic compartments and are essential for synaptic vesicle protein clustering (Jang et al., 2016).

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The relative contribution of mitochondria versus glycolysis to the axonal energy milieu has been revisited in recent years. Long-range molecular motor-driven transport was believed to derive its energy from axonal mitochondria (Sheng, 2014), but recent studies have highlighted important roles for glycolysis in axonal transport. Axonally transported organelles were associated with components of the glycolytic machinery such glyceraldehyde-3-phosphate

dehydrogenase

(GAPDH),

providing

on-board

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as

production of ATP in the transport complex (Zala et al., 2013). The extent to which these alternative energy sources can fuel transport of RNA granules and axonal local translation has not been addressed thus far (Hinckelmann et al., 2016; Schiavo and Fainzilber, 2013; Zala et al., 2013), apart from reports of mitochondrial co-localization at translational foci

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at branch points in axons (Spillane et al., 2013; Wong et al., 2017).

Mitochondria have been shown to play a major role in axonal regeneration.

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Mitochondrial density increases at the sites of axonal injury to promote regeneration,

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while failure to accumulate mitochondria in injured nerves decreases their regeneration

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capacity (Han et al., 2016). Factors like DLK-1 and the Armadillo repeat-containing Xlinked protein 1 (Armcx1) were shown to mobilize mitochondria to axonal injury sites

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(Cartoni et al., 2016). It therefore seems plausible that the energy required for axonal local translation would be primarily supplied by mitochondria and there are reports of co-

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localization between mitochondria and the translation machinery in yeast (Gold et al., 2017; Kellems et al., 1975). As noted above, NGF stimulation induces the formation of

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local mitochondrial hotspots at the axonal branching sites of mouse sensory neurons, which constitute active zones of localized mRNA translation of the actin cytoskeleton

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(Spillane et al., 2013). Along these lines, a recent study correlated the spatiotemporal dynamics of RNA granules with mitochondrial localization in retinal axons (Wong et al., 2017). RNA granules dock at branching sites similarly to mitochondria, which are hotspots

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for β-actin de novo synthesis (Wong et al., 2017). While these studies support a role for mitochondria in supplying energy for axonal local translation, more quantitative and comparative analyses of the relative contributions of mitochondria versus glycolysis machine will be require for definitive understanding.

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Future perspectives A more detailed picture is emerging regarding the regulation of axonal local translation, particularly regarding the local response to axonal injury. As discussed above, mTOR is likely a core regulator of the axonal translational response, however further

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investigation that includes the exploration of non-canonical translational mechanisms is needed in order to reach a thorough understanding. The mTOR pathway partly regulates the translation initiation step during which the eukaryotic small 40S ribosomal subunit is recruited to the mRNA’s 5’-terminal cap structure (Nandagopal and Roux, 2015). However, mTOR also drives the translation of mRNAs characterized by the presence of a 5’ terminal oligopyrimidine tract (TOP mRNAs) in a manner that is dependent upon

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stress and amino acid availability (Nandagopal and Roux, 2015). Notably, TOP mRNAs

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represent some of the most abundant transcripts in axons and dendrites, where they are transported and locally translated (Gobert et al., 2008; Moccia et al., 2003; Poon et al.,

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2006; Tsokas et al., 2007) and proteins encoded by TOP mRNAs such as eEF1A, eEF2

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as well as the ribosomal protein S6 have been reported to be translated as a consequence of synaptic activity (Carroll et al., 2006, 2004; Giustetto et al., 2003; Huang et al., 2005;

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Tsokas et al., 2007, 2005). This body of evidence suggests that mTORC1 might

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participate in synaptic plasticity by locally increasing the availability of translation factors and other components of the translational apparatus (Nandagopal and Roux, 2015). The mechanisms that mediate mTOR control of TOP mRNA translation are still unclear, and

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further investigation will be required for detailed understanding of the translational control

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underlying axonal injury responses and neuronal regeneration. Furthermore, other non-canonical mechanisms were found to contribute to mRNA

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translation, most notably IRES mediated translation (Chappell et al., 2006), which was the first to be described and found essential during brain development (Audigier et al., 2008), synaptic activation (Pinkstaff et al., 2001) and axonal extension and growth cone steering (Choi et al., 2018). IRES-mediated localized translation of the ER chaperone GRP78, for example, was reported in axons, but its possible contribution to axonal homeostasis and/or regeneration still needs to be elucidated (Pacheco and Twiss, 2012).

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Non-canonical translation regulatory processes might offer a diversity of options for finely tuned regulation of protein synthesis, enabling tissue or cell specific or even gene specific regulation of mRNA translation upon need. In-depth analyses of the mechanistic regulation of axonal translation will likely reveal additional layers of regulation controlling

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this essential mechanism in neurons. Future progress in the field will undoubtedly benefit from different proteomic tools that are becoming available for the identification of newly synthesized polypeptides. The antibiotic puromycin can be used to label nascent polypeptides (Schmidt et al., 2009; Smith et al., 2005) (Fig. 2B) and retrieve translated proteins for mass spectrometry analysis (Forester et al., 2018). Non-canonical amino acids have also been used to

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selectively label and purify newly synthesized proteins (Dieterich et al., 2010, 2006). Both technologies can be modified to allow for cell selectivity and tissue specificity (Barrett et

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al., 2016; Buhr et al., 2015; Ngo et al., 2009). Puromycin or non-canonical amino acid

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labelling can also be used in combination with the proximity ligation assay (PLA) to enable

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the visualization of newly synthesized proteins of interest (Dieck et al., 2015; Sambandan et al., 2017; Terenzio et al., 2018). Ribosomal profiling methodologies are also becoming

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widely used, including TRAP (translating ribosome affinity purification) (Heiman et al., 2008) and Ribotag (Sanz et al., 2009), which allow for cell type-specific identification of

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ribosome-associated mRNAs. Finally, several tools have been recently developed for live imaging strategies to enable monitoring of the location and dynamics of single mRNA

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translation in vivo (Chekulaeva and Landthaler, 2016). For a more detailed summary of technical innovations that will drive future developments of the field please see these

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reviews (Chekulaeva and Landthaler, 2016; Glock et al., 2017; Kim and Jung, 2015).

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Acknowledgements We thank Dr. Indrek Koppel for his insightful comments on the manuscript. Our work in this area was supported by a Koshland Senior Postdoctoral Award to M.T., the European Research Council (Advanced Grant Neurogrowth), the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, and the Israel Science Foundation (1284/13).

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M.F. is the incumbent of the Chaya Professorial Chair in Molecular Neuroscience at the Weizmann Institute of Science.

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Figure Legends

Figure 1: Mechanisms of axonal mRNA localization, translation, and retrograde transport after injury. mRNAs, including mTOR mRNA, are anterogradely transported

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from the soma to and within axons along microtubules in complex with their respective RNA-binding proteins (RBP). Local translation of the axonal mRNAs is induced by mTOR after injury. mTOR activation results in the increase of de novo synthesized mTOR protein, which promotes further translation. De novo synthesized proteins, including transcription factors (TF), undergo retrograde transport by dynein via binding to adaptor proteins such as the importins and regulate transcriptional responses in the soma.

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Figure 2: Axonal translational machinery and protein synthesis. A) DRG neurons in

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culture from Islet-Cre RiboTag mice immunostained for the tagged ribosome epitope (HA) and ribosomal RNA (Y10B). These mice express a tagged version of the Rpl22 submit of

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the ribosome under the control of a neuronal promoter, thus allowing for cell type-specific measurement of translating mRNAs. A representative neuron is shown. Scale bar, 50 μm.

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A magnified panel of a growth cone is presented below (marked with an *) to better visualize the colocalization between axonal ribosomes and ribosomal RNA in axons.

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Scale bar, 5 μm. B) Visualization of axonal protein synthesis in the SN after injury using the antibiotic puromycin, which can be used to label nascent polypeptides, where it

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incorporates. Newly synthesized protein incorporating puromycin are then visualized using an antibody against it. Specifically, SN segments were incubated in DMEM medium with puromycin (100 g/ml) for 2h at 37oC or pre-incubated with cycloheximide (40 g/ml)

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for 30 minutes and subsequently subjected to Puromycin (100 g/ml) & Cicloheximide (40 g/ml) for 2h. A control incubated only in DMEM for 2h is also shown. The SN segments were then fixed in PFA and stained for puromycin (red) and the axonal marker NFH (green).

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