Intra-axonal protein synthesis - a new target for neural repair?

Jeffery L. Twiss, Ashley L. Kalinski, Rahul Sachdeva, John D. Houle1 Department of Biological Sciences, University of South Carolina, Columbia, SC, USA Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA

INVITED REVIEW

Intra-axonal protein synthesis - a new target for neural repair?

Jeffery L. Twiss1,*, Ashley L. Kalinski1,?, Rahul Sachdeva2,?, John D. Houle2
1 Department of Biological Sciences, University of South Carolina, Columbia, SC, USA
2 Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA

How to cite this article: Twiss JL, Kalinski AL, Sachdeva R, Houle JD (2016) Intra-axonal protein synthesis - a new target for neural repair? Neural Regen Res 11(9):1365-1367.

Funding: Research in the authors’ laboratories that is related to the topic of this review has been supported by grants from the National Institutes of Health (R01-NS041596 and R01-NS089663 to JLT; P01-NS055976 to JDH), National Science Foundation (MCB-1020970 to JLT), Department of Defense/Congressionally Mandated Research Program (W81XWH-13-1-0308 to JLT), US-Israel Binational Science Foundation (2011329 to JLT), and Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (to JLT). Twiss is the incumbent South Carolina SmartState Chair in Childhood Neurotherapeutics at University of South Carolina.

Jeffery L. Twiss, M.D., Ph.D., twiss@mailbox.sc.edu.

? Current address for Ashley L. Kalinski is Department of Cell and Developmental Biology,

University of Michigan, Ann

Arbor, MI, USA.

? Current address for Rahul

Sachdeva is International

Collaboration on Repair

Discoveries (ICORD),

University of British Columbia, Vancouver, British Columbia, Canada.

orcid:

0000-0001-7875-6682

(Jeffery L. Twis)

Accepted: 2016-06-10

Although initially argued to be a feature of immature neurons with incomplete polarization, there is clear evidence that neurons in the peripheral nervous system retain the capacity for intra-axonal protein synthesis well into adulthood. This localized protein synthesis has been shown to contribute to injury signaling and axon regeneration in peripheral nerves. Recent works point to potential for protein synthesis in axons of the vertebrate central nervous system. mRNAs and protein synthesis machinery have now been documented in lamprey, mouse, and rat spinal cord axons. Intra-axonal protein synthesis appears to be activated in adult vertebrate spinal cord axons when they are regeneration-competent. Rat spinal cord axons regenerating into a peripheral nerve graft contain mRNAs and markers of activated translational machinery. Indeed, levels of some growth-associated mRNAs in these spinal cord axons are comparable to the regenerating sciatic nerve. Markers of active translation tend to decrease when these axons stop growing, but can be reactivated by a second axotomy. These emerging observations raise the possibility that mRNA transport into and translation within axons could be targeted to facilitate regeneration in both the peripheral and central nervous systems.

mRNA transport; translational control; RNA binding protein; axon regeneration; spinal cord injury; peripheral nerve injury

Post-transcriptional regulation provides a means to fine tune gene expression. Since a single messenger RNA (mRNA) can be translated into several copies of a protein, controlling the rate of translation for individual mRNAs can have major effects on the levels of a protein generated. The stability of an individual mRNA also directly impacts the amount of that mRNA available for translation. Proteins and small non-coding mRNAs (e.g., RNA binding proteins [RBP] and micro-RNAs [miRNA], respectively) interact with mRNAs and modulate their translation and stability. RBPs also play a role in subcellular localization of mRNAs. Polarized cells use mRNA localization to introduce new proteins in distinct subcellular domains. Neurons are highly polarized and they use mRNA transport and localized translational control in both their dendrites and axons. Protein synthesis in dendrites has largely been associated with synaptic efficacy. With the much greater distance that axons traverse, localized protein synthesis would be an appealing mechanism for the distal axon to locally regulate its protein levels. However, initial ultrastructural studies showing polyribosomes in dendrites failed to detect these in the axonal compartment, suggesting that ribosomes and other translational machinery are excluded from axons. With substantial advances in molecular tools and cellular techniques over the last two decades, many groups have now unequivocally shown that axons have the capacity to synthesize proteins (see Perry and Fainzilber, 2014 and references within).

In the peripheral nervous system (PNS), intra-axonal protein contributes to axon regeneration. Studies from the Fainzilber group indicate that synthesis of Importin β1, RanBP1, vimentin, and Stat3α proteins in distal axons is used to signal injury to the cell body (Perry and Fainzilber, 2014). Importin β1 (KPNB1) is a member of the karyopherin protein family that imports proteins across the nuclearmembrane. The axonally-generated Importin β1 protein dimerizes with cell body-synthesized Importin α3 protein that arrives in axons through anterograde transport. Together with dynein motor protein, the Importin β1/α3 heterodimer transports signaling proteins to the nucleus to modulate an injury-induced transcriptional response (Perry et al., 2012). Dimerization of these two proteins is made possible by intra-axonal translation of Importin β1 that is activated by increases in axoplasmic Ca2+after injury. This increase in axoplasmic Ca2+also triggers translation of RanBP1 mRNA in axons, whose protein product regulates a RanGTPase that frees axonal Importin α3 for binding to the newly translated Importin β1 protein (Yudin and Fainzilber, 2009). On the other hand, axonally synthesized GAP43 and β-actin proteins are used locally for growth of sensory axons, and changes in the amount of GAP43 or β-actin mRNA targeted into axons contributes to the type of axon outgrowth (see (Gomes et al., 2014 and references within). Other axonally synthesized proteins contribute to axon growth in vitro, but it is not clear whether they play the same role in vivo or not. For example, intra-axonal translation of RhoA mRNA has been linked to chondroitin sulfate proteoglycan (CSPG)-mediated growth inhibition in cultured neurons (Walker et al., 2012a). Beyond growth, locally generated proteins have been implicated in maintenance of axons, cell survival, and mitochondrial respiration in cultured neurons and sometimes in vivo for developing neurons (see Gomes et al., 2014; Perry and Fainzilber, 2014 and references within).

Figure 1 Molecular targets for modulating intra-axonal protein synthesis.

The functions outlined above for proteins synthesized in cultured neurons and in vivo in the developing nervous system and adult PNS could conceivably be harnessed for neural repair strategies once more is known of the molecular mechanisms regulating mRNA transport and translation. Recent works from several groups point to the possibility that adult CNS neurons also have the capacity for synthesizing proteins in their axons. Akins et al. (2012) have shown that axons in adult mouse have granular profiles of the fragile X mental retardation (FMRP) and fragile X related (FXR1, FXR2) RBPs. These tend to concentrate in regions of the brain with relatively higher plasticity like the olfactory nerve/bulb (Akins et al., 2012). Walker et al. (2012b) reported intra-axonal translation in adult mouse spinal cord by delivering an exogenous mRNA directly into axons using SinB is viral particles. In cultured neurons, delivering adenylate cyclase mRNA into axons with this method allowed for axon growth in the face of growth-inhibitory CSPGs. Using a peripheral nerve graft (PNG) into the adult rat spinal cord, Kalinski et al. (2015) showed that ascending spinal cord axons contain mRNAs and translational machinery as they are regenerating in the PNG. Levels of the translational machinery seem to decline as the axons reached ends of the PNGs and stop growing, suggesting that the axon’s translational activity may reflect the growth supporting environment of the PNG (Sachdeva et al., 2016). Keeping with this notion, Selzer et al. (2016) recently showed that regenerating reticulospinal axons in the Lamprey spinal cord contain mRNAs and translational machinery. In contrast to higher vertebrates, some reticulospinal axons in the Lamprey can spontaneously regenerate after spinal cord injury. Together these studies indicate that, at least under some conditions, CNS axons have the mRNA transcripts and necessary translational machinery to generate new proteins.

It is tempting to speculate that mRNA translation in the spinal cord axons noted above is a reflection of regenerative growth. As noted the Lamprey reticulospinal axons can spontaneously regenerate, and the PNGs analyzed above similarly support regeneration compared to the non-permissive environment of the mammalian spinal cord (Kalinski et al., 2015; Selzer et al., 2016). So could the failure of early ultrastructural studies to visualize ribosomes in axons have resulted from investigators looking in the wrong place or under the wrong conditions? Recent work from the Hengst group is in support of this notion. Baleriola et al. (2014) showed that ATF4 mRNA is transported into adult hippocampal axons in vivo, where it is locally translated after application of exogenous amyloid-β peptide (Baleriola et al., 2014). While a provocative finding for the neurodegeneration field, this study emphasizes that these mammalian CNS neurons retain the capacity for intra-axonal protein synthesis into adulthood. Depending on the specific mRNA targets, activating mRNA transport into and translation within axons seems to be utilized for a neuron’s injury responses or increasing its axon’s intrinsic regeneration capacity.

We posit that the proteins and non-coding RNAs responsible for regulating the transport and translation of mRNAs are rising as prime candidates for neural repair strategies. For example, the levels of zip code binding protein 1 (ZBP1) in adult sensory neurons limits how much β-actin and GAP43 mRNAs can localize into axons (see Gomes et al., 2014 and references within). So increasing ZBP1 expression could effectively increase transport of the mRNAs encoding growth-promoting proteins. However, ZBP1 binds to many different mRNAs and there is a pressing need to determine which mRNAs it transports into axons and if CNS and PNS neurons differ in their use or need for ZBP1. Likewise, many more RBPs are undoubtedly used for transport and translation of axonal mRNAs; there is a similar need to identify the axonal RBPs and the cohorts of mRNAs they interact with. miRNAs have also been detected in axons in culture and PNS axons in vivo. These non-coding RNAs provide a platform for modifying the translation and stability of mRNAs within the axons. Interestingly, the Yoo group recently showed that miRNA precursors (pre-miRNAs) localize to axons of the sciatic nerve (Kim et al., 2015). This raises the possibility for another step of regulatory control for intra-axonal protein synthesis that could be a target for future neural repair strategies. However, more work is needed to define the mRNA targets for the axonal miRNAs as well as the signaling mechanisms that control pre-miRNA-to-miRNA maturation in axons.

In summary, localized protein synthesis clearly can occur in both PNS and CNS axons. Although there are increasingly new functions identified for axonally synthesized proteins, growth of the axon remains a key mechanism for the protein products of axonal mRNAs. The molecular events briefly outlined above that contribute to regulation of axonal mRNA transport and translation (Figure 1) could indeed bring new strategies to facilitate axon regeneration. Evidence has been mounting for this possibility in the PNS, and studies over the past two years suggest that protein synthesis can be activated in at least some axons in the brain and spinal cord. While RBPs and miRNAs offer potential targets for facilitating axon regeneration, more work is needed to understand the molecular mechanisms underlying their mRNA interactions and activities as well as the breadth of axonal mRNAs that they affect.

Author contributions: JLT and JDH wrote the paper. ALK and RS edited initial draft and provided insight into research. Conflicts of interest: The authors declare no financial conflicts of interest.

References

Akins MR, Leblanc HF, Stackpole EE, Chyung E, Fallon JR (2012) Systematic mapping of fragile X granules in the mouse brain reveals a potential role for presynaptic FMRP in sensorimotor functions. J Comp Neurol 520:3687-3706.

Baleriola J, Walker CA, Jean YY, Crary JF, Troy CM, Nagy PL, Hengst U (2014) Axonally synthesized ATF4 transmits a neurodegenerative signal across brain regions. Cell 158:1159-1172.

Gomes C, Merianda TT, Lee SJ, Yoo S, Twiss JL (2014) Molecular determinants of the axonal mRNA transcriptome. Dev Neurobiol 74:218-232.

Kalinski AL, Sachdeva R, Gomes C, Lee SJ, Shah Z, Houle JD, Twiss JL (2015) mRNAs and protein synthetic machinery localize into regenerating spinal cord axons when they are provided a substrate that supports growth. J Neurosci 35:10357-10370.

Kim HH, Kim P, Phay M, Yoo S (2015) Identification of precursor microRNAs within distal axons of sensory neuron. J Neurochem 134:193-199.

Perry RB, Fainzilber M (2014) Local translation in neuronal processes--in vivo tests of a “heretical hypothesis”. Dev Neurobiol 74:210-217.

Perry RBT, Doron E, Iavnilovitch E, Rishal I, Dagan S, Tsoory M, Copolla G, Gomes C, McDonald M, Geschwind D, Twiss J, Yaron A, Fainzilber M (2012) Subcellular knockout of importin β1 perturbs axonal retrograde signaling. Neuron 75:294-305.

Sachdeva R, Farrell K, McMullen MK, Twiss JL, Houle JD (2016) Dynamic changes in local protein synthetic machinery in regenerating central nervous system axons after spinal cord injury. Neural Plasticity 2016:4087254.

Selzer M, Jin LQ, Pennise C, Rodemer W, Jahn K (2016) Protein Synthetic Machinery and mRNA in Regenerating Tips of Spinal Cord Axons in Lamprey. J Comp Neurol doi:10.1002/cne.24020.

Walker BA, Ji SJ, Jaffrey SR (2012a) Intra-axonal translation of RhoA promotes axon growth inhibition by CSPG. J Neurosci 32:14442-14447.

Walker BA, Hengst U, Kim HJ, Jeon NL, Schmidt EF, Heintz N, Milner TA, Jaffrey SR (2012b) Reprogramming axonal behavior by axon-specific viral transduction. Gene Ther 19:947-955.

Yudin D, Fainzilber M (2009) Ran on tracks - cytoplasmic roles for a nuclear regulator. J Cell Sci 122:587-593.

10.4103/1673-5374.191193

*Correspondence to: