Axonal Mrna Transport and Translation at a Glance Pabitra K

CELL SCIENCE AT A GLANCE Axonal mRNA transport and translation at a glance Pabitra K. Sahoo1, Deanna S. Smith1, Nora Perrone-Bizzozero2 and Jeffery L. Twiss1,*

ABSTRACT efforts are now uncovering new roles for locally synthesized proteins Localization and translation of mRNAs within different subcellular in neurological diseases and injury responses. In this Cell Science at domains provides an important mechanism to spatially and a Glance article and the accompanying poster, we provide an temporally introduce new proteins in polarized cells. Neurons make overview of how axonal mRNA transport and translation are use of this localized protein synthesis during initial growth, regulated, and discuss their emerging links to neurological regeneration and functional maintenance of their axons. Although disorders and neural repair. the first evidence for protein synthesis in axons dates back to 1960s, KEY WORDS: Axonal mRNA transport, Axonal mRNA translation, improved methodologies, including the ability to isolate axons to RNA granule, Post-transcriptional regulation, Protein synthesis, purity, highly sensitive RNA detection methods and imaging Ribonucleoprotein particle approaches, have shed new light on the complexity of the transcriptome of the axon and how it is regulated. Moreover, these Introduction Neurons are highly polarized cells with cytoplasmic extensions that 1Department of Biological Sciences, University of South Carolina, 715 Sumter St., can extend for millimeters, or even up to meters in large vertebrates. CLS 401, Columbia, SC 29208, USA. 2Department of Neurosciences, University of Axons and dendrites constitute the vast majority of the volume and New Mexico School of Medicine, 1 University of New Mexico, MSC08 4740, Albuquerque, NM 87131, USA. surface area of a neuron, and neurons use localized protein synthesis in these cytoplasmic extensions to spatially and temporally regulate *Author for correspondence ([email protected]) the protein content of these subcellular domains (Jung et al., 2014). D.S.S., 0000-0002-3014-641X; N.P., 0000-0002-7608-1332; J.L.T., 0000-0001- Axons provide long-range connections between neurons and their 7875-6682 targets that allow the brain, spinal cord and peripheral nerves to Journal of Cell Science

1 CELL SCIENCE AT A GLANCE Journal of Cell Science (2018) 131, jcs196808. doi:10.1242/jcs.196808 communicate. With the known transport rates of proteins, organelles 2013). Although mRNA protein-coding sequences (CDS) that and other macromolecules, the distal axon must respond to mediate axonal targeting have not yet been found for axonal environmental stimuli well before anything could be transported mRNAs, CDS motifs have been described in yeast (Kilchert and there from the cell body. Localized protein synthesis is one way to Spang, 2011), so it is likely that CDS motifs for axonal mRNA overcome this distance constraint, but the neuroscience community targeting will also be uncovered. In another commonality with largely overlooked the possibility of localized mRNA translation in dendrites and non-neuronal systems, RNA-binding proteins (RBPs) axons until recent years (Box 1). Generating proteins locally within binding to these motifs are necessary for axonal mRNA transport distal axons brings unique advantages for growth, survival and (Khalil et al., 2018; Korsak et al., 2016). Thus, the interaction of an function to these far reaches of the cytoplasm of a neuron, and recent mRNA with RBPs is sequence dependent, but consensus RNA unbiased analyses point to thousands of different mRNAs in axons. motifs unique to axonal mRNA targeting have yet to be found. In this Cell Science at a Glance article, we aim to summarize new Moreover, some axonal localization motifs can also target mRNAs advances in the field and point out where knowledge gaps exist. We into dendrites (Tiruchinapalli et al., 2003; Vuppalanchi et al., 2010), focus on the regulation of mRNA transport and translation and the which could reflect common uses for the encoded proteins in axons unique functions served from axonally synthesized proteins, and dendrites. highlighting how neuronal health is affected by these mechanisms The secondary structures of the RNA motifs are thought to where loss or gain of function result in disease or altered axon growth influence their interaction with RBPs (Gomes et al., 2014). The capacity. Obviously, it is not possible to cover the entire field in this ability to bioinformatically compare secondary structures across short article, so we refer the reader to several recent reviews for more RNA species is advancing, so common structural motifs may be detailed summaries (Batista and Hengst, 2016; Costa and Willis, discovered soon. Next-generation sequencing of RNAs from RBP 2017; Kar et al., 2018; Tasdemir-Yilmaz and Segal, 2016). immunoprecipitations also holds promise to uncover RBP- recognition motifs that are shared between axonal mRNAs, as How does the neuron know which mRNAs to localize into seen for recent work with motor axon transcriptomes (Rotem et al., axons? 2017). However, multiple RBPs can bind to the same mRNA and Just as RNAs are localized into dendrites and the subcellular regions impart different fates to the mRNA. Interactions of mRNAs with of non-neuronal cells, mRNA transport into axons is driven by RBPs likely begin in the nucleus, either co-transcriptionally or sequences inherent to the RNAs. These sequence motifs have most shortly after transcription, and the fate of an mRNA with regard to often been found in 3′ untranslated regions (UTR) of the mRNAs its subcellular mRNA localization is conferred by the sequential (Andreassi and Riccio, 2009; Gomes et al., 2014), but 5′UTR- binding of multiple RBPs. Evidence for this is the interaction of localization motifs have also been uncovered (Merianda et al., β-actin mRNA (ACTB) with zip code binding protein 2 (ZBP2, also called KHSRP and FUBP2) and ZBP1 (also called IGF2BP1 and IMP1) (Pan et al., 2007). Box 1. History of axonal mRNA translation As new knowledge of localization motifs emerges, it will need to be Early electron microscopy (EM) analyses of rodent brain showed interpreted in the context of multiple protein interactors and different evidence for polysomes at the base of dendritic spines in mature neuron types, as well as for the protein–protein interactions that occur hippocampus (Steward and Levy, 1982), which spurred decades of at different sites within the soma and along the axon. For example, research focusing on the role of dendritically synthesized proteins in recent work shows that γ-actin mRNA localizes into motor axons synaptic plasticity (Namjoshi and Raab-Graham, 2017). Those same (Moradi et al., 2017), whereas it appears to be restricted to the soma of early EM studies shed doubt on the possibility that axons can synthesize sensory and cortical neurons (Bassell et al., 1998; Zheng et al., 2001). proteins, as no polysomes were found in the axons of the mature hippocampus (Steward and Levy, 1982). Further doubt that translation In addition, CREB mRNA localizes to embryonic sensory axons, but occurs in axons came from instances of mRNAs having been detected in not axons of sympathetic neurons (Andreassi et al., 2010; Cox et al., hypothalamic and olfactory axons, yet ribosomes appeared to be lacking 2008). Axonal mRNA populations can similarly change with growth (Denis-Donini et al., 1998; Mohr and Richter, 1992). Thus, it was states and as the neuron matures (Gumy et al., 2011; Shigeoka et al., suggested that the translational machinery and mRNAs are excluded 2016; Taylor et al., 2009). In-depth axonal RNA profiles of different from vertebrate axons (Steward, 1997), even though biochemical neuronal subtypes will undoubtedly uncover more differences in evidence already pointed to the possibility of intra-axonal protein synthesis in the 1960s (Koenig, 1965a,b; Koenig, 1967a,b) and, axonal transcriptomes, physiological states and pathological states, shortly thereafter, EM evidence for ribosomes in axons of the PNS and it is important to keep in mind that differential gene expression as was published (Bunge, 1973; Tennyson, 1970; Yamada et al., 1971). well as combinations of RBPs could drive these. A series of critical studies taking advantage of the size of the squid giant axon as a tractable model to test for intra-axonal protein synthesis Mechanism for the regulation of axonal mRNA transport culminated in clear evidence for intra-axonal protein synthesis in those Just as with proteins and organelles, mRNAs are actively invertebrate neurons (Giuditta et al., 1980, 1986, 1991). Subsequent transported into axons by molecular motors. Because of the studies in vertebrates, and then mammals, showed that axons synthesize proteins even in adults (Perry and Fainzilber, 2014; Twiss unified polarity of microtubules in axons, the plus-end-directed and van Minnen, 2006). Fueled by recent technical and experimental kinesin motor proteins are used for long-range anterograde transport advances, the field has now moved from phenomenon to understanding in axons on microtubules, whereas myosin motor proteins are used the functions served by axonally synthesized proteins in neuronal for short-range transport on microfilaments (see poster) (Kalinski development, injury responses and disease states. Recent studies are et al., 2015b). Dynein motor-dependent retrograde movements have starting to uncover how axonal mRNA transport and translation are been observed for some axonal RBPs, but it is not clear whether regulated, including the use of in vivo systems for analyses of axonal mRNA localization and protein synthesis in mature neurons (Kalinski mRNAs are bound to these retrogradely moving RBPs. This could et al., 2015a; Shigeoka et al., 2016). Recent work has also uncovered be a mechanism for relocating mRNAs within an axon, for instance how mRNA splice variants contribute to subcellular transcriptomes that in response to extracellular stimuli, or for delivering RBPs back to undoubtedly include axonal mRNAs (Taliaferro et al., 2016). the cell body for reuse, as has been shown for La protein (van

Niekerk et al., 2007). Journal of Cell Science

2 CELL SCIENCE AT A GLANCE Journal of Cell Science (2018) 131, jcs196808. doi:10.1242/jcs.196808

mRNAs are transported in protein complexes that have been referred to as ‘RNA transport granules’, which are Box 2. Axonal protein synthesis in neurological disease ribonucleoprotein complexes (RNPs) (Kar et al., 2017). Analyses Peripheral neuropathy of neural RNP contents show that there are many different RBPs, NGF regulates transcription and subsequent axonal transport of Bclw to variable components of translational machinery and proteins that are protect axons of developing PNS neurons from degeneration (Cosker needed for interaction with motor proteins, as well as many different et al., 2016). In mature neurons, the chemotherapeutic agent Paclitaxel decreases the amount of axonal SFPQ–BclW RNPs (Pease-Raissi et al., mRNAs that are components of them (Elvira et al., 2006; Kanai 2017). Peripheral neuropathy is a significant complication for some et al., 2004; Krichevsky and Kosik, 2001). Notably, these analyses chemotherapeutics, and this Paclitaxel-induced axon degeneration is represent pooled lysates, so the RNPs are those from dendrites, prevented by exogenous peptides targeting the interaction Bclw with the axons and soma. Subsequent work with dendritically localizing inositol-phosphate-3 receptor (Pease-Raissi et al., 2017). RBPs points to there being multiple dendritic RNPs with different Viral infection α contents (Fritzsche et al., 2013; Miller et al., 2009). It is highly Neuronal infection with -Herpes viruses requires retrograde transport of viral particles from distal axons to the nucleus for latent infection (Taylor likely that axons also contain a diversity of different RNPs, but more and Enquist, 2015), and intra-axonal protein synthesis is needed for this sensitive methods will be needed to systematically dissect the retrograde transport (Koyuncu et al., 2013). Although it is not clear which makeup of RNPs isolated directly from axons. axonal proteins are required, axonal translation of Kpnb1, Dctn1 and Although several RBPs have been shown to localize into axons Pafahb1 can modify retrograde transport. (see poster), the number is vastly smaller than the thousands of Motor neuron disease mRNAs that have been detected in axons (Kar et al., 2018). These Altered axonal mRNA translation is implicated in pathogenesis of motor neuron degeneration. SMA has an onset during childhood, with motor RBPs likely interact with many different mRNAs, and are analogous ‘ ’ neuron death resulting from loss of SMN. SMN has a well known role in to RNA regulons (Keene, 2007), but much is still to be learned RNP assembly (Khalil et al., 2018), but its axonal localization and the regarding the breadth and functions of axonal RBPs. Individual decreased in axonal β-actin mRNA seen upon depletion of SMN suggests axonal mRNAs can also interact with several different RBPs. For that it has other functions (Le et al., 2005; Rossoll et al., 2003; Zhang et al., example, although ZBP1 is required for axonal localization of β-actin 2006). Consistent with this, SMN depletion from cultured motor neurons mRNA, heterogeneous nuclear (hn)RNP R, spinal motor neuron markedly changes axonal mRNA levels, including mRNAs needed for (SMN) and HuD (also called ELAVL4) proteins also bind to axonal axon growth and synaptic function (Saal et al., 2014). The discovery that β mutations in the RBPs TDP-43 and Fus/TLS can be causative for ALS -actin mRNA (Glinka et al., 2010; Kim et al., 2015; Rossoll et al., pointed to possible post-transcriptional dysregulation in mature motor 2003; Tiruchinapalli et al., 2003). The interaction with hnRNP R and neurons. Motor neurons expressing ALS mutants of TDP-43 showed ZBP1 contributes to β-actin mRNA localization in motor axons, decreased mobility of axonal RNPs and reduced axonal transport of Nefl while ZBP1 or ZBP1 together with HuD may be sufficient for its (Alami et al., 2014). Recent RNA-seq work showed that ALS-causing axonal localization in other neurons (Glinka et al., 2010; Kim et al., TDP-43 mutations alter the axonal content of both mRNAs and miRNAs in 2015; Rossoll et al., 2002). In addition, both HuD and ZBP1 can bind cultured spinal motor neurons (Rotem et al., 2017). Alzheimer’sdisease to the AU-rich element (ARE) of Gap43, but it is not clear whether Work from the Hengst laboratory has surprisingly shown that activation of ZBP1 binds the RNA directly or in complex with HuD (Yoo et al., axonally synthesized proteins can also trigger neuronal degeneration. 2013). The binding of some axonal RBPs, such as nucleolin, FUS/ Stimulation of hippocampal neurons with Aβ1-42 peptide, a causative TLS, YB-1 (also known as YBX1), Hermes (also known as CD44), agent for Alzheimer’s disease, triggers translation of Atf4 mRNA in axons TDP-43 (also known as TARDBP) and FMRP, to individual mRNAs with subsequent retrograde transport of ATF4 causing cell death (Baleriola has been characterized, but not been tested for interactions with other et al., 2014). axonal mRNAs (Antar et al., 2006; Kar et al., 2017; Perry et al., 2016). These RBPs undoubtedly will have multiple mRNA targets among the axonal mRNAs, as was recently shown for SFPQ, which Regulation of mRNA dynamics within the axonal co-assembles with Bclw (also known as Bcl2l2) and lamin B2 (Lb2, compartment also known as Lmnb2) into RNA-transport granules (Cosker et al., mRNAs are thought to be in a translationally suppressed state during 2016). Other axonal RBPs, including TRF2-S (also known as their transport as RNPs (Wells, 2006). For example, ZPB1 undergoes TERF2), ZBP1 and HuD, have been shown to interact with multiple phosphorylation in distal axons, thereby decreasing its RNA-binding mRNAs that can localize to axons (Bolognani et al., 2010; affinity and releasing β-actin mRNA for translation (Hüttelmaier Jønson et al., 2007; Zhang et al., 2015). However, these et al., 2005). Phosphorylation of ZBP1 is regulated by extracellular interactions can only been inferred based on cross-referencing stimuli, as sonic hedgehog (SHH) has been shown to increase axonal co-immunoprecipitating mRNAs from brain or non-neuronal cell β-actin mRNA translation in growth cones of the developing spinal lysates to axonal transcriptomes, rather than having been validated as cord (Lepelletier et al., 2017). Thus, ZBP1 contributes to both true interactions within axons. transport and translational regulation of β-actin mRNA, and this Altered axonal RNA transport can impact axon growth, function function may extend to other mRNA targets of ZBP1. and survival, and several RBPs linked to neurological diseases have RBPs can also regulate the stability of their target mRNAs. HuD, been detected in axons. These include TDP-43 and FUS/TLS, which is needed for axonal localization of Gap43 (Yoo et al., 2013) which are mutated in amyotrophic lateral sclerosis (ALS), SMN, and has been well characterized to stabilize mRNAs (Beckel- whose deficiency causes spinal muscular atrophy (SMA), and Mitchener et al., 2002; Gomes et al., 2017), can also contribute to SFPQ, whose loss from axons contributes to peripheral neuropathy mRNA translational regulation (Fukao et al., 2009). By contrast, (Box 2). Analyses of cellular and animal models of ALS, SMA and KHSRP can also bind to the ARE of Gap43, but this interaction chemotherapy-induced neuropathy have shown alterations in axonal destabilizes the mRNA (Bird et al., 2013). This suggests that HuD mRNA localization (Alami et al., 2014; Pease-Raissi et al., 2017; and KHSRP can sometimes compete for binding to the same Rotem et al., 2017; Saal et al., 2014), which leads to the intriguing mRNAs, but with different outcomes (Gardiner et al., 2015). possibility that alterations in axonal protein synthesis can impact Interestingly, KHSRP binds to nuclear β-actin mRNA and facilitates disease pathogenesis or progression. its interaction with ZBP1 for axonal localization (Pan et al., 2007), Journal of Cell Science

3 CELL SCIENCE AT A GLANCE Journal of Cell Science (2018) 131, jcs196808. doi:10.1242/jcs.196808 emphasizing that RBPs can have different functions in different provide a means to control where proteins are synthesized in axons. subcellular compartments. The microtubule-binding adenomatous polyposis protein (APC) mRNAs can also compete for binding to the same RBP (see binds to subsets of localized mRNAs, including axonal mRNAs, poster). β-actin mRNA and Gap43 compete for binding to ZBP1 for and links these mRNAs to microtubules (Mili et al., 2008; Preitner their transport into axons (Donnelly et al., 2011; Yoo et al., 2013). et al., 2014; Villarin et al., 2016). This entails a mechanism to Furthermore, Gap43 and Nrn1 compete for binding to HuD in concentrate mRNAs near sites where their protein products are sensory neurons, but not in CNS neurons that express higher levels needed, but how this is coordinated at a molecular level is not of HuD (Gomes et al., 2017). Changes in transcription or stability, known. Beyond the cytoskeleton, work from the Flanagan which can change the levels of either target mRNAs or RBPs, will laboratory has shown that ribosome-subunit-bound mRNAs can thus obviously affect the competition between mRNA and RBPs. form a complex with the cytoplasmic domain of deleted in colon Taken together, these observations provide evidence for the cancer (DCC), a component of the netrin receptor (Tcherkezian dynamic nature and composition of axonal RNPs. et al., 2010), thus providing an appealing mechanism to link Translation of mRNAs in axons obviously requires ribosomes, extracellular stimuli, such as netrin, to the translation of receptor- tRNAs and translation factors. Stimuli that regulate axonal bound mRNA subsets. It will be of high interest to determine translation can modify the activity of this translational whether this docking mechanism extends to other cell surface machinery in axons (Jung et al., 2012). A unique single- receptors and which mRNAs are docked. molecule imaging approach recently showed that there was an increase in axonal β-actin mRNA translation within 20 s of ligand Axonally synthesized proteins support axon growth stimulation (Ströhl et al., 2017), demonstrating that axon terminals Given the large distances that can separate the distal axon from its can rapidly activate translation in response to extracellular stimuli. soma, the axon must be able to rapidly respond to extracellular This also implies that prior studies that used standard imaging stimuli. This autonomy is perhaps best illustrated by the use of approaches and suggested translation within 5–20 min may have axonally synthesized proteins for growth of developing axons. underestimated how quickly axonal translation can occur, Axons sense attractant and repellant cues in their environment to including work from our laboratory (Pacheco and Twiss, 2012; navigate their way to the appropriate targets. β-actin mRNA and Vuppalanchi et al., 2012). ribosomes have been detected in growing axons of cultured neurons, A critical, yet unanswered, question is how translational specificity and axonal levels of β-actin mRNA increase in response to is driven at the level of individual axonal mRNAs beyond their neurotrophin-3 (NT-3, also known as NTF3) (Bassell et al., 1998; interactions with RBPs. There is evidence that intracellular Ca2+ Zhang et al., 1999). Attractant cues have also been shown to activate levels can impact the specificity for axonal mRNA translation (see translation in axons, which is needed for growth cone turning (see poster). Increases in axoplasmic Ca2+ after severing nerve injury (i.e. poster) (Campbell and Holt, 2001). Moreover, attractant stimuli can axotomy) have been implicated in the translation of Kpnb1, RanBP1, trigger movement of mRNAs and the translational machinery to Vim (encoding vimentin) and Stat3 (Ben-Yaakov et al., 2012; Hanz coordinate where proteins are generated within a growth cone et al., 2003; Perlson et al., 2005; Yudin et al., 2008). Additionally, (Leung et al., 2006; Yao et al., 2006). Repellant stimuli can also release of Ca2+ from endoplasmic reticulum (ER) stores increases modify mRNA transport and translation in growth cones (Piper translation of axonal Calr and Grp78 (also known as BiP and Hspa5), et al., 2006, 2005; Walker et al., 2012; Wu et al., 2005). The but not β-actin mRNA, through phosphorylation of eIF2α opposite outcomes with regard to growth direction, which are driven (Vuppalanchi et al., 2012). ER Ca2+ release can activate the by attractant and repellant cues, are determined in part by which unfolded protein response (UPR), and UPR-induced translation of proteins are synthesized. For example, translation of RhoA and Luman/CREB3 in peripheral nervous system (PNS) axons occurs cofilin 1 (Cfl1) is increased by repellant cues and that of β-actin after axotomy (Ying et al., 2014, 2015). Thus, axotomy-induced mRNA increased by attractant cues (Leung et al., 2006; Piper et al., elevated Ca2+ can drive translation of specific axonal mRNAs. 2006; Wu et al., 2005). Although many of these observations are Translation of mRNAs that are not part of this axotomy response is from cultured neurons, there is now ample evidence that translation likely to be regulated by other means, as indicated above for β-actin occurs in developing axons in vivo, and that there are specific mRNA. Translation of axonal mRNAs that encode components of the changes in axonally synthesized protein populations during axonal translational machinery brings the potential to impact subsequent pathfinding, such as changes in RhoA, β-actin and ErbA2 translation. For instance, translation of eIF2B2 and eIF4G2 in translation (Brittis et al., 2002; Shigeoka et al., 2016; Walker sympathetic axons contributes to axon growth (Kar et al., 2013). et al., 2012; Zivraj et al., 2010). A more generalized promotion of Axonal levels of these two mRNAs are regulated by microRNAs axon growth can also be driven by localized synthesis, as (miRNAs; designated by the prefix miR), which in turn impacts exemplified by translation of Par3 (also known as Pard3), TC10 translation of other axonal mRNAs (Kar et al., 2013). miR-182 has (also known as Rhoq) and Gap43 in developing sensory neurons also been shown to attenuate axonal synthesis of cofilin 1, with the (Donnelly et al., 2013; Gracias et al., 2014; Hengst et al., 2009). guidance cue Slit2 attenuating effects of miR-182 (Bellon et al., As noted above, mRNAs of the different actin isoforms show a 2017). mRNAs encoding other translation factors, ribosomal proteins differential localization in axons of motor neurons compared with and RBPs feature prominently in motor, sensory and retinal ganglion other neuron types. β-actin synthesis in sensory and RGC axons has cell (RGC) axon transcriptomes (Briese et al., 2016; Gumy et al., been shown to trigger axon branching (Donnelly et al., 2013; Wong 2011; Minis et al., 2014; Zivraj et al., 2010), and some miRNAs have et al., 2017). However, local synthesis of axonal γ-actin rather than been found to be enriched in axons (Natera-Naranjo et al., 2010; Phay β-actin in motor axons is needed for branching, and the authors et al., 2015; Rotem et al., 2017). Effects of miRNAs, together with hypothesized that this difference reflects the higher degree of local generation of translational machinery, bring the potential to branching that spinal motor neurons undergo (Moradi et al., 2017). broadly impact translation of many axonal mRNAs. For the RGC axons, this branching occurs in vivo as the axons are Ribosomes and mRNAs have long been known to associate with approaching their targets to, presumably, facilitate target the cytoskeleton (Bassell and Singer, 2001), which could also innervation (Wong et al., 2017). Although these effects are Journal of Cell Science

4 CELL SCIENCE AT A GLANCE Journal of Cell Science (2018) 131, jcs196808. doi:10.1242/jcs.196808 limited to β-actin, it is appealing to speculate that other axonally et al., 2008). Adult PNS neurons show translation and retrograde translated proteins may impart different functions to different transport of the transcription factors Stat3α and PPARγ after axotomy neuronal populations. (Ben-Yaakov et al., 2012; Lezana et al., 2016). The chromatin- interacting protein HMGN5 is synthesized in hippocampal axons, Axonally synthesized proteins support survival and function and the protein product is transported to the nucleus to modulate gene of axons expression (Moretti et al., 2015). Axonal synthesis of ATF4 is Soma-to-axon signaling has been shown to support survival and stimulated by Aβ1-42 peptide and retrogradely transported ATF4 can function of axons through the delivery of mRNAs and localized stimulate neuronal apoptosis (Box 2) (Baleriola et al., 2014). A key translation of new proteins. Developing sensory neurons require unsolved issue for these retrogradely transported transcription factors nerve growth factor (NGF) for survival, and NGF triggers and chromatin modulators is how the proteins are distinguished from transcription of the Bclw gene with the subsequent delivery of Bclw those that are synthesized in and reside in the soma. mRNA into axons (Cosker et al., 2013). Withdrawal of NGF or Translation in axons also provides a means to control retrograde preventing axonal translation of Bclw results in axon degeneration transport. Axonal injury activates translation of importin-β1 mRNA (Cosker et al., 2013), and loss of axonal Bclw mRNA has recently (Kpnb1) in axons (Hanz et al., 2003). By forming a heterodimer been linked to neuropathy (Box 2) (Pease-Raissi et al., 2017). NGF- with an anterogradely transported importin-α protein, importin-β1 dependent survival of sympathetic axons has been shown to require protein links cargo proteins, as the transcription factors mentioned intra-axonal translation of myo-inositol monophosphatase-1 (Impa1), above, to the minus-end-directed microtubule motor protein dynein and a decrease in Impa1 translation attenuates CREB signaling in (Hanz and Fainzilber, 2006). This provides a route for axon-to- these neurons (Andreassi et al., 2010). Translation of Lb2 in RGC nuclear transport, and this axonal synthesis of importin-β1 is needed axons supports axon survival by maintaining mitochondrial function for subsequent axotomy-induced changes in gene expression that (Yoon et al., 2012). Further data link intra-axonal translation of other support nerve regeneration (Perry et al., 2012). Interestingly, axonal nuclear-encoded mitochondrial proteins to axon function. synthesis of importin-β1 also provides a cell-size-sensing Specifically, translation of CoxIV and Atp5g1 in sympathetic axons mechanism during development (Perry et al., 2016), emphasizing is needed to maintain mitochondrial ATP production in axons, which that different functions can be imparted by the same axonally in turn is required for axon function and survival (Aschrafi et al., synthesized protein under different physiological conditions. It will 2010, 2008; Kar et al., 2014). ATP is needed for translation, and be interesting to see if this results from a qualitative difference in translation-dependent branching of sensory axons by neurotrophins transported cargo or a quantitative difference in how much importin- requires local mitochondrial respiration at incipient branch points β1–importin-α3 is delivered under these two different settings. where translation occurs (Spillane et al., 2013). The transcriptomes of Recent work also indicates that the dynein-mediated retrograde axons for different neuronal subtypes contain many mRNAs of transport of vesicles can be modified by intra-axonal translation of nuclear-encoded mitochondrial proteins (Kar et al., 2018), so the dynein regulators. Local translation of mRNAs encoding p150Glued support of mitochondrial function might be a common role for and Lis1 (Dctn1 and Pafah1b1, respectively) is required for NGF- axonally synthesized proteins. induced retrograde transport of signaling endosomes, while only Dendritically synthesized proteins have a prominent role in locally synthesized Lis1 is needed for NGF-induced retrograde synaptic plasticity (Kosik, 2016), and there is emerging evidence that transport of large vesicles (Villarin et al., 2016). Both Lis1 and axonally synthesized proteins can support synaptic development and p150Glued have stimulatory effects on dynein (Baumbach et al., function. Changes in the translated mRNA populations were 2017; Gutierrez et al., 2017; King and Schroer, 2000; Pandey and observed in developing RGC axons as they reach their targets and Smith, 2011) and are involved in the initiation of transport from establish synaptic connections (Shigeoka et al., 2016). Axons of microtubule plus-ends (Jha et al., 2017; Moughamian and cultured cortical neurons also show shifts in translation in response to Holzbaur, 2012). Thus, the local synthesis of dynein regulators in glutamate, indicating that neurotransmitters can stimulate axonal distal axons constitutes an ‘on demand’ system to alter retrograde protein synthesis, as has been shown for dendritic translation (Hsu transport. Deletions or mutations of the human Lis1 (PAFAH1B1) et al., 2015). Although it is not clear which proteins are generated in gene cause lissencephaly, a brain malformation with severe axons in response to glutamate, intra-axonal synthesis of disruption of cortical development originating from altered synaptosomal-associated protein 25 (SNAP25), which is needed migration of neuronal precursors (Bertipaglia et al., 2017). for synaptic vesicle release, is rapidly increased during formation of However, Lis1 is also needed in mature neurons, since depletion presynaptic terminals in hippocampal neurons (Batista et al., 2017). or knockout of Lis1 in adult sensory neurons disrupts retrograde Translation in PNS sensory nerve endings has also been linked to the axonal transport (Hines et al., 2018; Pandey and Smith, 2011), and it development of neuropathic pain, and cytokine signaling in distal is intriguing to speculate that loss of axonally synthesized Lis1 may axons contributes to this (Khoutorsky and Price, 2017). Taken contribute to this effect in adult neurons. together, studies clearly indicate that axonally synthesized proteins can impact presynaptic function and neural activity. Future perspectives As we outline above, our knowledge of the axonal transcriptome and Axonally synthesized proteins provide a platform for its regulation has remarkably advanced our understanding of axonal retrograde signaling protein synthesis. Axonally synthesized proteins contribute to Axonally generated proteins provide a platform for the delivery of growth, function and survival responses within the axon, but also signals to the soma (see poster). Work in several different types provide an axon-to-soma signaling platform to coordinate neuronal of neurons and under different settings has shown that translation of responses culminating in growth, regeneration, survival, or cell death transcriptional modulators in axons can affect gene expression in the depending on the stimulus. These could have profound effects on nucleus. NGF-dependent synthesis of CREB in axons promotes the development and function of the CNS and PNS. Although we have survival of developing sensory neurons, where the retrogradely learned much about the axonal transcriptome and its regulation, there transported CREB functions as a nuclear transcription factor (Cox is still a lot to discover, and several pressing issues need to be Journal of Cell Science

5 CELL SCIENCE AT A GLANCE Journal of Cell Science (2018) 131, jcs196808. doi:10.1242/jcs.196808 addressed in future years. First, are there unique needs for axonally Batista, A. F. R., Martınez,́ J. C. and Hengst, U. (2017). Intra-axonal Synthesis of Cell Rep. synthesized proteins in different neuronal types, as noted for axon SNAP25 Is Required for the Formation of Presynaptic Terminals. 20, γ β 3085-3098. branching supported by -actin in motor and -actin in RGC and Baumbach, J., Murthy, A., McClintock, M. A., Dix, C. I., Zalyte, R., Hoang, H. T. sensory neurons? Second, many axonally synthesized proteins, such and Bullock, S. L. (2017). Lissencephaly-1 is a context-dependent regulator of as β-actin and Gap43, are also made in the soma and then transported the human dynein complex. eLife 6, e21768. Beckel-Mitchener, A. C., Miera, A., Keller, R. and Perrone-Bizzozero, N. I. into axons, and it is not clear which, if any, unique functions the (2002). Poly(A) tail length-dependent stabilization of GAP-43 mRNA by the RNA- axonally synthesized protein imparts and how. Third, RNA–protein binding protein HuD. J. Biol. Chem. 277, 27996-28002. interactions have a critical role in the delivery of mRNAs into axons Bellon, A., Iyer, A., Bridi, S., Lee, F. C. Y., Ovando-Vázquez, C., Corradi, E., and translation of mRNAs in axons, but we have only identified a few Longhi, S., Roccuzzo, M., Strohbuecker, S., Naik, S. et al. (2017). miR-182 regulates Slit2-mediated axon guidance by modulating the local translation of a axonal RBPs and a better understanding of the dynamics of axonal specific mRNA. Cell Rep. 18, 1171-1186. RNPs is needed. Finally, the question of how specificity is achieved Ben-Yaakov, K., Dagan, S. Y., Segal-Ruder, Y., Shalem, O., Vuppalanchi, D., in the translation of some mRNAs and not others in response to Willis, D. E., Yudin, D., Rishal, I., Rother, F., Bader, M. et al. (2012). Axonal EMBO J. stimuli needs to be addressed. Reversible RNA methylation, which transcription factors signal retrogradely in lesioned peripheral nerve. 31, 1350-1363. has recently been implicated in regulating axonal mRNA translation Bertipaglia, C., Goncalves, J. C. and Vallee, R. B. (2017). Nuclear migration in and regeneration (Yu et al., 2017), could bring transcript specificity mammalian brain development. Semin. Cell Dev. Biol. (in press). for translation. With continuingly emerging evidence for the Bird, C. W., Gardiner, A. S., Bolognani, F., Tanner, D. C., Chen, C.-Y., Lin, W.-J., Yoo, S., Twiss, J. L. and Perrone-Bizzozero, N. (2013). KSRP modulation of contribution of axonal protein synthesis to neurological disease and GAP-43 mRNA stability restricts axonal outgrowth in embryonic hippocampal regeneration, answering the issues above has the potential to open up neurons. PLoS ONE 8, e79255. new strategies for treatments. Bolognani, F., Contente-Cuomo, T. and Perrone-Bizzozero, N. I. (2010). Novel recognition motifs and biological functions of the RNA-binding protein HuD Nucleic Acids Res. Acknowledgements revealed by genome-wide identification of its targets. 38, 117-130. The authors thank Amar N. Kar, Seung Joon Lee, Terika Smith and Priyanka Patel Briese, M., Saal, L., Appenzeller, S., Moradi, M., Baluapuri, A. and Sendtner, M. for suggestions and discussions on the topic. (2016). Whole transcriptome profiling reveals the RNA content of motor axons. Nucleic Acids Res. 44, e33. Funding Brittis, P. A., Lu, Q. and Flanagan, J. G. (2002). Axonal protein synthesis provides Our work in this area has been supported by grants from the National Institutes of a mechanism for localized regulation at an intermediate target. Cell 110, 223-235. Health (R01-NS041596 and P01-NS055976 to J.L.T.; R01-NS089663 to N.P.-B. Bunge, M. B. (1973). Fine structure of nerve fibers and growth cones of isolated and J.L.T.; R01-NS056314 to D.S.S.), National Science Foundation (MCB- sympathetic neurons in culture. J. Cell Biol. 56, 713-735. 1020970), U.S. Department of Defense (W81XWH-13-1-0308), and the Dr Miriam Campbell, D. S. and Holt, C. E. (2001). Chemotropic responses of retinal growth and Sheldon G. Adelson Medical Research Foundation. J.L.T. is the SmartState cones mediated by rapid local protein synthesis and degradation. Neuron 32, Chair in Childhood Neurotherapeutics at the University of South Carolina. Deposited 1013-1016. in PMC for release after 12 months. Cosker, K. E., Pazyra-Murphy, M. F., Fenstermacher, S. J. and Segal, R. A. (2013). Target-derived neurotrophins coordinate transcription and transport of J. Neurosci. Cell science at a glance bclw to prevent axonal degeneration. 33, 5195-5207. A high-resolution version of the poster and individual poster panels are available for Cosker, K. E., Fenstermacher, S. 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