Master thesis:
Mechanisms of selective mRNA transport and translation
in neuronal axons
Remko Goossens - 3804097
Master program: Molecular and Cellular Life Sciences
Supervision: Professor Casper Hoogenraad, PhD.
13-05-2013
Contents
Summary ................................................................................................................................................. 3
Introduction............................................................................................................................................. 3
Axon growth ............................................................................................................................................ 4
Axoplasmic transport .............................................................................................................................. 5
Ribonucleoprotein particles .................................................................................................................... 7
Local mRNA translation during axonal development.............................................................................. 7
Growth cone stimulation and turning ................................................................................................... 10
Local mRNA translation in mature neurons .......................................................................................... 13
Local mRNA translation during injury and regeneration....................................................................... 13
The axonal mRNA transcriptome .......................................................................................................... 15
Retrograde signaling by axonally synthesized proteins ........................................................................ 16
Ribosome specificity in mRNA translation ............................................................................................ 17
Involvement of glial cells ....................................................................................................................... 18
Conclusion ............................................................................................................................................. 19
References ............................................................................................................................................. 20
Cover: Figure 801 as found in the 1918 edition of ‘Gray’s Anatomy’: “Diagram of the distribution of
the cutaneous branches of the posterior divisions of the spinal nerves”1.
2
Summary
Selectively targeting mRNA to subcellular locations for local translation allows the distal regions of
cells to rapidly respond to extracellular cues. In developing neurons, axons obtain a certain level of
autonomy by changing the local proteome in the growth cone during their navigation through the
developing nervous system. Studies of mature axons have shown that active local translation still
plays an important role in axon maintenance, with evidence suggesting that local translation of
mRNA is also indispensible for mounting a sufficient regenerative response upon damage of the
axon. Axons seem to gradually lose their local translation abilities as they become more mature,
meaning that a thorough understanding of how this ability can be restored could prove beneficial
for treating people suffering from damage to their central nervous system. This thesis aims to
review literature concerning the mechanisms involved in the selective localization of mRNAs by
axoplasmic transport, and will also cover the functionality and regulating factors which induce
local translation in the axon.
Introduction
Neurons are a highly specialized eukaryotic cell type capable of transmitting signals through their
ability to receive and send electric stimuli. These electric signals are generated by a change of the
electric potential of the neurons plasma membrane which spreads through the cell. In longer
neurons this electric signal is present as a rapid, self–propagating excitation called an action
potential2. The main cell body is referred to as the soma, from which neurons develop protrusions
called neurites (Figure 1), which will either develop into signal receiving dendrites, or a signal
transmitting axon. While a neuron can have several dendrites, it will always have only one long
axon2. Axons in the human body can reach lengths of up to 1 meter, an example of this are the
neurons stretching from the base of the spinal cord to the tip of the big toe in the sciatic nerve2.
Dendrites are highly branched, enabling them to receive signals from axons through the numerous
spines present on them3. The amount of input signals that a single neuron is able to receive from
other neurons can be as high as 100.000. Axons also branch out to form synaptic connections, but
this only occurs at their tips and not on the axonal shaft, with this high amount of branches and
synapses the axon is able to transmit its signal to many targets simultaneously2.
The structure of axons highly depends on the dynamics of the cytoskeleton. Axons contain bundles of
overlapping microtubule strands which are all oriented with their plus ends towards the axon
terminals2, 4. Experiments conducted by Witte et. al. showed that axons have increased microtubule
stability through acetylation in the distal regions of the axonal shaft, stabilizing the structure of the
axon5. The microtubule strands are also used to guide the transport of many cellular components
through vesicles carried by motor proteins. The cargo of these vesicles ranges from proteins to entire
organelles3. Anterograde transport away from the soma towards the axonal extremities over
microtubules is arranged by the kinesin-family of motor proteins3. The kinesin-family of motor
proteins are all plus end directed with different members of the kinesin superfamily being able to
carry specific subsets of organelles or protein containing vesicles. This diversity between the different
kinesin motor proteins enables the cell to specifically regulate and target anterograde transport
vesicles2, 3. Retrograde transport over microtubules is used to carry cargo towards the soma for
degradation and recycling of unwanted materials, but also to transport retrograde signaling factors.
3
This form of axonal transport is facilitated by vesicles which are carried by the dynein family of minus
end directed motor proteins2.
Figure 1: A vertebrate neuron with the direction of transmitted signals indicated by the arrows. Depicted are
the cell body (Soma), dendrites and the axon with its terminal branches. Adapted from Molecular Biology of
the Cell, 4th edition by Alberts et. al. 2
The extremities of the neurites are extended outwards during their development by means of a
growth cone. These structures contain high levels of actin which the growth cone depends on for
guidance and forming protrusions2, 4. The actin filaments are also used line the cortex of the axon, in
close proximity to the plasma membrane2, 4. Both the pre- and postsynaptic terminals found in
mature neurons contain high levels of actin, which is crucial for synapse formation3.
Local translation of mRNA has been shown to be of great importance in the function of the growth
cone, which for example needs active translation of β-actin mRNA to properly navigate through the
developing nervous system6. A range of extracellular guidance cues can affect the local translation of
different factors which ultimately lead to either collapse or attractive growth of the growth cone.
Also, mature neurons failing to translate mRNA in their axon seem to exhibit poor regenerative
capacities6. In this thesis, the currently known mechanisms and functions of selective mRNA
transport and translation in the neuronal axon will be discussed. Highlighting the importance of these
mechanisms in axonal maintenance should give an insight in the knowledge gathered thus far, and
the challenges which still lay ahead until their function is fully understood. A comprehensive
understanding of the mechanisms supporting the regenerative capacities of neurons through local
mRNA translation could prove useful for treatment of central nervous system (CNS) injuries.
Axon growth
To create connections between cells of the developing nervous system, axons need to be able to
navigate towards an appropriate target. The axons encounter a plethora of signals that affect axonal
elongation, branching, pathfinding, synaptogenesis and neuronal differentiation7, 8. This pathfinding
process is facilitated by the axonal growth cone, a highly motile structure rich in actin. Actin filaments
are also used line the cortex of the axon, in close proximity to the plasma membrane 2, 4. Of the three
actin isoforms, α, β and γ, only β-actin has been implied to be involved in directional movement9, 10.
4
The growth cone is able to produce filopodia and lamellipodia for pathfinding during its migration2.
Filopodia are protruding and stabilized from the growth cone in reaction to secreted proteins in the
direct environment and the reaction of the filopodia to these external cues is very sensitive2. By
repelling or attracting the growth cone with secreted soluble proteins, cells used as guideposts are
able to guide in which direction the growing axon advances through the extracellular matrix2. When a
filopodium comes across one of these guideposts during protrusion, it will form an adhesive contact.
This subsequently leads to rapid assembly or disassembly of actin filaments in the growth cone,
causing the growth cone to turn towards the guidepost cell2. Interplay of negative and positive
stimulation by both soluble and insoluble factors are responsible for accurately guiding the growing
axon over long distances to its destination, at a rate of approximately 1 mm per day2. Simultaneously
with growth cone protrusion modulated by actin, microtubules grow into the growth cone and shrink
back by dynamic instability2. The microtubules are protected from disassembly upon signaling from
the adhesive contact site, stabilizing the established axonal region behind the growth cone2. When
the growth cone reaches its target destination, it will transform into a highly branched presynaptic
terminal to connect to its target, while becoming enriched with synaptic vesicles to mediate
signaling6.
While axons and dendrites have vastly different structures and organization of the cytoskeleton,
developing neurons are known to possess the ability to develop a new axon from an existing dendrite
upon loss of the existing axon4, 11. Upon axonal lesion close (<35 µm) to the soma of a mature
hippocampal neuron, one of the dendrites will undergo an identity change, causing it to restructure
its microtubule layout into a plus-end out exclusive fashion4, 11. Generally, the dendrite closest to the
severed axon will achieve the restructuring towards an axon, while the other dendrites keep their
original function and structure4. If the loss of the axon occurs in a more distal position (>35 µm), the
axon will regrow instead, resembling the situation of a young developing neuron11. This data
postulates that mature neurons are able to retain some degree of plasticity, even when integrated in
neuronal networks11. It is unclear though if this principle can be applied to regeneration of CNS
injuries in vivo11.
Axoplasmic transport
Axoplasmic transport of cellular components is crucial for maintenance of neuron structure and
function12. Many different cargoes are transported through the axon in order for them to arrive at
the synapse. Among these are: mRNAs, neurotransmitter receptors, ion channels, membrane
proteins, mitochondria and synaptic vesicle precursors, which to the greater extent are all
synthesized and preassembled in the soma3. Various studies concerning the trafficking of these
synaptic cargoes have revealed the existence of multiple mechanism by which the cell can specifically
target cargoes to the correct destination. For example, it is necessary for the cell to specifically target
vesicles containing presynaptic cargoes towards the axons, while vesicles containing components for
the postsynaptic structure like glutamate receptors should be routed specifically towards dendritic
branches3.
One mechanism that has been described for this sorting describes aspecific transport of synaptic
cargoes towards both the axon and dendrites, which are then selectively retained at the
compartment requiring this cargo3, 4. An example of this has been described by Bel et. al. who studied
neuronal targeting of Contactin-associated protein 2 (Caspr2). This neuronal transmembrane protein
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has been found to be mutated in disorders like autism, but its exact function is unknown13. It was
shown that Caspr2 targets both the dendritic and axonal surfaces, but that it is selectively
endocytosed in the somatodendendritic compartment in order to clear it from these
compartments13, which has also previously been reported by Fache et. al. for the Nav1.2 sodium
channels14. These proteins accumulate at the Axon Initial Segment (AIS) before being inserted into
the axonal tips, and are eliminated from the soma and dendrites by endocytosis14.
Other than compartment-specific endocytosis, it has been described that the Kv1.2 potassium
channels are regulated through polarized vesicular trafficking and does not depend on endocytic
internalization13, 15. Kv1.2 contains a targeting signal for direct delivery to axons, mediated by KIF3
kinesin13. Many of the models concerning trafficking in neurons proposed it to be facilitated to a
great extent by the microtubule plus-end directed motor protein kinesin3, 16, which was described
earlier.
Mitochondrial axoplasmic transport is used for dynamic distribution of mitochondria throughout the
axon12. The direction of mitochondrial transport relies on specific signaling stimuli and corresponds
with whether the axons are growing or retracting12. Long range axonal transport of mitochondria is
facilitated by kinesin and dynein motors along the microtubules12. Kinesin-1 (KIF5) is considered to be
the primary motor for anterograde transport of mitochondria, while retrograde transport is thought
to be mainly driven by cytoplasmic dynein12, 17. Kinesin-1 function might be mediated through Ca2+
control, with elevated Ca2+ levels being responsible for halting anterograde mitochondrial movement
in myelinated axons12, 18. Linking of kinesin-1 and mitochondria in turn has been proposed to be
managed through syntabulin17. The interaction between mitochondria and dynein has been
suggested to be facilitated through Dynein Intermediate Chain (DIC) and dynactin, but as direct
evidence is not available and no membrane receptor has been identified to date, this remains to be
further investigated12. Interestingly, mutations in kinesin-1 abrogate both retro- and anterograde
transport of mitochondria12. At the same time, dynein mutations only inhibit retrograde movement,
indicating that these motors are interdependent for mitochondrial transport12.
As axonal degradation of mitochondria through mechanisms like mitophagy has never been shown
before, it is assumed that damaged or excessive mitochondria are retrogradely transported for
processing in the cell body12. For the assembly of mitochondria, it has been revealed by microarrays
and qPCR that CNS derived axons can contain many of the mitochondrial mRNAs12, 19. Although
inhibitors of cytosolic translation reduce mitochondrial function, indicating that local translation of
mitochondrial mRNAs has an important function, it remains unclear what the exact role of this
mechanism is12. The high rate of axonal transport of mitochondria might be the main mechanism for
maintaining the mitochondria supply, while de novo biogenesis in axons is used for repair or as a
backup.
In dendrites, RNA-containing granules are transported by binding to the C-terminal tail of KIF5
kinesin16. These granules contain a large amount of PURα and PURβ, both ribonucleo-binding
proteins. PURα-containing granules colocalize selectively with mRNAs like the αCaMKII and ARC
mRNAs, but not with tubulin mRNA16.
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Ribonucleoprotein particles
In order for the neuron to be able to locally translate mRNA, it is of great importance to be able to
regulate RNA transport. First, the cell needs to process the required mRNA in the nucleus, after
which RBPs bind the cis-acting elements of the mRNA to mark it for subcellular localization20.
Subsequently, the mRNA bound RBPs assemble into granules together with ribosomal subunits and
translation initiation factors to form the RNP20. It is known that RNA is transported in granules which
can be as large as 1000S and are able to bind to the motor protein KIF5, as depicted in Figure 221. As a
few dozen different RNA-binding proteins and mRNA particles have been shown to take part into
forming these RNP complexes, making it unclear if the transport of specific mRNAs is regulated by
specific RBPs9, 21. Recent reports suggest the involvement of the α-subunit of the Golgi associated
coat protein I (COPI) in the formation of RNPs containing β-actin mRNA22. Also, Vg1RBP for example
is able to interact with the mRNA for both β-actin and cofilin9. These granules were seen to be
colocalizing in dendrites21, but a similar mechanism might exist for selective axonal transport.
Transports of these granules occurs bidirectionally, suggesting that next to KIF5 there are also dynein
motors involved in a tug-of-war21. The speed of bidirectional transport of a β-actin mRNA/ZPB1
complex in developing neurites approaches speeds of 1µm/s23. This speed is similar to the rate of
transport observed for membrane bound vesicles and organelles transported by kinesin and dynein
motors23, giving another indication for their possible involvement in transport of such complexes.
Local mRNA translation during axonal development
It is clear that directed growth of the axonal growth cone relies on specific subcellular localization of
proteins in response to extracellular stimulation. The establishment of this local proteome could be
facilitated through active protein transport subsequent to translation, but it has been shown that
mRNAs are able to obtain specific subcellular localization in for example mammalian and Drosophila
cells 24, 25. Furthermore, an exclusive somatic derivation of axonal and presynaptic proteins could
cause problems due to the long distance that the proteins would need to travel to arrive at the
extremities of the axon causing a long delay in signaling, also considering the half-life of the
transported proteins26. Localization of mRNAs to specific regions in the cell seems to be a common
phenomenon, as a large scale in situ hybridization screen in Drosophila embryos showed25. 3370
genes were analyzed, with 71% of the expressed mRNAs showing subcellular distribution in various
patterns25. Screenings of the mRNAs present in the axon reveal that many transcripts are highly
enriched in the axon when compared to the soma, which indicates that accumulation of specific
mRNAs in the axons relies on active transport rather than diffusion27. Localization of the RNA plays a
role in the establishment and maintenance of polarity in a range of cell types24. As the amount of
data about subcellular targeting of mRNAs increases it becomes apparent that local synthesis of
proteins plays a role in many cellular events28. The local translation of mRNA in the axon provides
neurons with a high degree of autonomy for these regions by permitting rapid local responses to
extrinsic cues28.
Several hundreds of mRNAs have been identified to be present in axons. The mRNAs of for example
β-actin, β-tubulin, vimentin, NF-L, heat shock proteins, ER-proteins and more were detected in the
axon by RT-PCR analysis29. A study using a hybridized cDNA array with mRNA derived from pure
axonal preparations identified over 200 mRNAs that were present in injured Dorsal Root Ganglion
(DRG)29. GO-term analysis indicated that the most abundant mRNAs belonged to groups involved
7
with ribosomal and translational machinery, next to proteins with roles in mitochondria and
oxidative phosphorylation29. Interestingly, many of the mRNAs found in naïve and injured CNS
neurons are also present in in axons of the PNS29.
mRNAs have for example been found to accumulate in protruding pseudopodia of murine fibroblasts,
in which the RNA molecules anchor their 3’ untranslated region in granules24. Furthermore, the RNA
contained in the granules associates with Adenomatous Polyposis Coli (APC) and Fragile X Mental
Retardation Protein (FRMP)24. Kislauskis et. al. reported that the localization of β-actin mRNA in
chicken embryo fibroblasts is regulated by a highly conserved sequence of 54 nucleotides at the
3’UTR, called a zipcode30.
The first instances of selective RNA transport in neurons were recorded to occur by the delivery of
selected mRNA molecules towards postsynaptic sites in dendrites31, and the dendritic growth cone32.
Axonal transport of mRNA was thought to be rather rare, and to occur only in specialized neurons
like magnocellular hypothalamic cells and cells in the olfactory epithelium31. Furthermore, it was
deemed unlikely that protein synthesis occurred in mature mammalian axons, as they seemed to
contain no translational machinery6, 31.
After it was discovered in squid giant axons that the axoplasm also contains mRNAs, ribosomal RNA
and active polysomes, it was believed to be plausible that neurons are able to use local protein
translation to manage their local proteome6. Bassell et. al., for example, demonstrated that β-actin
mRNA is present in distinct granules in the axonal growth cones of cultured cerebrocortical
neurons23, 33. The presence of polysomes within these growth cones was also observed33, and axons
have been seen to contain endoplasmatic reticulum (ER), which exists in the form of connecting
tubules running parallel with the axon34. These tubules are able to extend into the synapse where the
ER often associates closely with a mitochondrion34. Formation of Ribonucleoprotein particles (RNP)
(Figure 2) consisting of a complex between the cis-acting element in the 3’UTR zipcode sequence of
β-actin mRNA and the Zipcode Binding Protein 1 (ZBP1) was observed in neurons by Zhang et. al23.
ZBP1 is a RNA-binding protein (RBP) of the VICKZ family, and keeps the β-actin mRNA in a
translationally silent state during transport from the nucleus due to its binding to the 3’UTR zipcode6,
35, 36
. Formation of this complex is necessary for dynamic trafficking of the mRNA in the neurites,
which is used to selectively increase the local concentration of β-actin through the translation of the
mRNA in the growth cones23. This transport of β-actin mRNA towards the growth cones of both
dendrites and axons is a response to neurotrophin stimulation23, and the supply of β-actin is
subsequently used to stimulate the forward movement of the growth cone23, 37. Upon arrival at the
target destination at the periphery of the cell, translation of the transported mRNA is promoted by
the tyrosine kinase Src which docks and subsequently phosphorylates ZBP1 at a residue contained
within a Src Homology 3 (SH3)-binding motif, which is crucial for its ability to bind RNA35, 36. Guidance
gradients induce an increased or decreased rate of protein synthesis by regulating the activity of the
Src kinase through its phosphorylation36. This mechanism of transcriptional silencing by bound ZBP1
allows the mRNA to be transported through the cytoplasm without being hindered by polysome
formation. Ca2+ signaling has an upstream role in the protein synthesis required for establishing
responses to guidance cues36. This response requires the asymmetric localization and translation of
β-actin mRNA in the growth cone for Ca2+-dependent turning of the growth cone as shown by Yao et.
al. by using a turning assay36. Calcium has a versatile range of roles in intracellular signals in almost
every cell type, including neurons, where Ca2+ signaling regulates synaptic plasticity, contributes to
8
dendritic action potential, release of neurotransmitters and gene transcription for example34, 38. As
different studies have shown that transcriptional repression of β-actin mRNA is lost upon
phosphorylation of ZBP1 by Src, it is likely that inhibition of Src may be involved in repulsive turning
responses. This block of mRNA release on the near side of signaling would result in decreased
motility36.
Figure 2: RNA-specific transport and translation
in the axon. Represented are several mechanisms
of extrinsic cues stimulating the active transport
of mRNA and subsequent translation as
described herein. For example: stimulation of the
growth cone with brain-derived neurotrophic
factor (BDNF) will activate Src-kinase through the
TrkB receptor. Src will phosphorylate Zipcode
Binding Protein 1 (ZBP1) leading to release of, up
until then, translationally silenced β-actin mRNA.
Transport of transcribed mRNA occurs most likely
through binding of RNA Binding Proteins (RBPs)
to the mRNAs 3’-untranslated regions (UTRs),
which proceed to form ribonucleoproteins
(RNPs). These RNPs are transported by motor
protein such as kinesin along the microtubules
towards their distal targets. The possibility for
ribosome mediated mRNA specificity is also
depicted. CPEB1: cytoplasmic polyadenylation
element binding protein, FAK: focal adhesion
kinase, GRB7: growth factor receptor-bound
protein 7, KOR1: κ-type opioid receptor, NT3:
neurotrophin 3, TRK: tyrosine receptor kinase.
Adapted from Jung et. al. Nat. Rev. Neurosci. 13,
308-324 (2012).6.
As pre-existing β-actin monomers are abundantly present in growth cones found in fibroblast
lamellipodia, as well as in chick motor neurons derived from the spinal cord39, it seems unclear what
the advantage of local translation could be over transporting β-actin monomers from the cell body6.
Calculations concerning the amount of actin needed for polymerization compared to the theoretical
rate of translation of the actin mRNA in migrating fibroblasts estimated that the amount of β-actin
synthesized locally could only supply about 7% of the β-actin required40. This would make it seem
unlikely that locally translated β-actin exerts its effects on the asymmetric nucleation of the actin
filaments by sheer numbers alone. However, these calculations do not take into account the
possibility that targeting and translation of the mRNA in a single confined location could be
substantially increase the contribution of the de novo synthesized actin to the polymerization of the
cytoskeleton40.
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It has been proposed that the primary role of this de novo synthesis of β-actin away from the cell
body could lie with different post-translational modification (PTM) of the molecules. The lack of
arginylation and glutathionylation on newly synthesized β-actin molecules could potentially induce a
stronger nucleation signal than of existing monomers containing these modifications, acting as an
attractive gradient6. Arginylation of β-actin has been described to regulate the properties of actin
filaments of murine firbroblasts in vivo41. Actin polymerization assays performed on cell extracts from
Ate-/- mice (Arg–transfer RNA (-tRNA) protein transferase) showed that a lack of active arginylation
increases the rate of actin filament aggregation, although in these cells, this results in exhibited
defects in spreading and lamella formation41. Another study found that deglutathionylation of actin
also leads to a highly increased rate of polymerization in A431 cells42. As the data concerning
arginylation and glutathionylation was gathered by using non-neuronal cells, it remains to be
determined if these PTMs play a role in distinguishing locally synthesized actin in the axon from
soma-derived actin monomers which were transported anterogradely.
Among the proteins synthesized de novo in the axon, there are proteins which promote local
mitochondrial function6, 43. Upon inhibition of axonal mRNA translation or mitochondrial protein
import by transfection of the HSP90 C-terminal domain into the axon, a decrease in mitochondrial
membrane potential of up to 65% was observed in cultured primary sympathetic neurons43. This loss
in membrane potential leads to dysfunction of mitochondria and eventually axon degeneration6. An
in vivo study of X. leavis retinal ganglion cells recently showed by utilizing axonal ribosome
immunoprecipitation that the mRNA for intermediate filament protein lamin B2 (LB2) is axonally
translated44. LB2 associates with the axonal mitochondria, and inhibition of LB2 leads to
mitochondrial dysfunction and degeneration of the axon44. These data support the idea that local
translation of mitochondrial components supports mitochondria function and is essential for
maintenance of healthy axons. Mutations in either lamin family members or mitochondrial proteins
respectively, can cause Charcot-Marie-Tooth (CMT) disease type 245, 46. This is a recessive autosomal
disease in which patients suffer from chronic peripheral neuropathy and axonal degeneration45.
Another disease causing intellectual disability and autism is the Fragile X syndrome (FXS), in which
the translational repressor Fragile X Mental Retardation Protein (FMRP) is silenced due to genetic
defects47. Immunohistochemistry analysis indicates that FMRP is localized to e.g. the sciatic nerve
and dorsal roots, at the same locations as transient receptor potential (TRP) channel type 148. FRMP
acts as a key regulator of mRNA translation in synaptic plasticity is known to be present in axons and
growth cones47, 48. It is therefore plausible that like in other neurodevelopmental disorders such as
autism spectrum disorder, dysregulation of axonal protein synthesis will be identified as an
underlying cause of diseases like FXS6.
Growth cone stimulation and turning
As stated earlier, the growth cone of an axon is guided during the development of the nervous
system in response to extracellular signals from the environment9, 36. Turning the growth cone relies
on the ability of the cone to create intracellular asymmetry in response to the gradients of guidance
cues presented by the extracellular environment9.
Chemotropic factors like semaphorin 3A (Sema3A), Slit2, brain-derived neurotrophic factor (BDNF)
and Netrin-1 are involved in guiding the growing axon9. These chemotropic factors can act attractive,
10
inducing turning and forward movement of the growth cone towards the factor, or repulsive, causing
collapse of the growth cone, which is a retraction of filopodia and lamellipodia39.
Sema3A is a repulsive chemotropic factor expressed in the embryonic Central Nervous System
(CNS)49. Upon stimulation of the receptor tyrosine kinase (RTK) neuropilin-1 (a receptor specific for
class-3 semaphorins) by Sema3A, protein synthesis in the isolated retinal growth cones is rapidly
induced through activation of translation initiation factors like eukaryotic translation initiation factor
4E (eIF-4E)49, 50. This activation occurs through a PI(3)K independent pathway, as PI(3)K inhibitors like
wortmannin and LY294002 do not inhibit responses of the growth cone to Sema3A49. Activation of
eIF-4E is achieved when its binding protein, the global translational regulator eIF-4E-binding protein 1
(BP1) gets phosphorylated by mammalian Target of Rapamycine (mTOR)6, 49, leading to release of eIF4E. Subsequently, eIF-4E binds to the cap of mRNA and activates translation of the molecule. In the
case of Sema3A stimulation this leads to local translation of RhoA mRNA9, 49, 51. RhoA is a small
guanosine triphosphatase (GTPase) which regulates the actin cytoskeleton51. This local translation of
RhoA induces collapse of the axonal growth cone through promoting actin depolymerization51.
In the brain of mice, Sema3E and its receptor PlexinD1 act as a repelling factor for the trajectory of
developing axons of corticofugal and striatonigral neurons52. However, in subiculo-mammillary
neurons, coexpression of Sema3E, PlexinD1 and Neuropilin-1 functions as an attractive cue52.
In the retinal growth cones of Xenopus laevis, signaling of Slit2 results in rapid activation of the
Mitogen-Activated protein kinases (MAPKs) p38 and ERK1/ERK2, which activate the MAP kinase
interacting kinase 1 (Mnk1)53. Mnk1 activates eIF-4E by phosphorylation of eIF-4EBP1, independent
from mTOR53. The signaling cascade induced by Slit2 stimulation finally gives rise to the local
translation of Cofilin mRNA9, 53. Cofilin is an actin depolymerizing protein, and will result in repulsion
of the growth cone by inducing its collapse53. Cofilin mRNA has been reported to immunoprecipitate
together with Vg1RBP, the X. leavis homolog of ZBP19, 53. There does not seem to be a known zipcode
sequence present in the cofilin mRNA though, so the mechanism by which this mRNA would be
selectively targeted to the growth cone is currently unknown36.
BDNF and Netrin-1 are chemotropic factors which facilitate an attractive cue for the growth cone,
inducing regional cytoskeletal assembly6. BDNF gradients cause an attractive cue for growth cones by
inducing an increase in observed β‑actin mRNA-ZBP1 positive puncta in growth cones36. An
asymmetric distribution of phosphorylated Src mediated through Ca2+ signaling is also induced36,
which will lead to an increase in the release of mRNA transported by ZBP136, 54. Simultaneous
stimulation of the growth cone with BDNF and the second messenger cyclic-AMP or addition of a
protein kinase A (PKA) inhibitor will induce a repulsive turn instead of an attractive turning response
in cultured X. laevis spinal neurons54. It is possible that other factors which normally act as attractive
cues, like Netrin-1, modulate cellular cAMP levels of neurons to induce different responses54. Cell
contact of the protruding axon with a guidepost cell in the developing nerve system might also be a
signal which raises cAMP levels, forcing the growth cone to migrate further along the path of
guidepost cells54.
Just like Sema3A, Netrin-1 is also expressed in the embryonic CNS49. Stimulation of growth cones
with netrin-1 and BDNF induces activation of MAPK55. This gradient of netrin-1 then leads to release
of active eIF-4E through mTOR activation in the same way described previously for Sema3A49. In this
context the spatial distribution of active eIF-4E induces a polarized increase of β‑actin translation at
11
the site where growth cone turning will occur9. Also, β-actin mRNA has been seen to bind to Vg1RBP
and Vg1RBP containing granules are seen to translocate rapidly into X. leavis filopodia upon netrin-1
stimulation, which increases the synthesis of β-actin9. Depending on the site of stimulation with a
gradient of netrin-1, the Vg1RBP-eGFP granules used in this study show asymmetric localization into
these filopodia9. Use of the inhibitor of eukaryotic translation cycloheximide (CHX) or antisense
morpholinos directed against β-actin blocked the observed attractive turning by netrin-19, giving
another indication of the importance of asymmetric β-actin distribution and translation for the
steering of axons through a gradient of positive cues. Yet another sign of the involvement of mTOR in
the netrin-1 signaling cascade is the observation that rapamycine, a specific inhibitor of mTOR, will
block netrin-1 mediated initiation of translation49.
Next to the attractive guidance cue of netrin-1, it is also possible for netrin-1 to act as a repulsive
factor56, 57. In the developing eye of X. laevis, netrin-1 excludes axon growth of retinal ganglion cells
(RGC) when the extracellular matrix also contains laminin-156, 57. This mechanism involves lowering
the levels of cytosolic cAMP57, a factor which blocks laminin-1 induced growth57. The involvement of
cAMP is further supported by the notion that Netrin-1 regulated cytoskeleton polymerization is
dependent on active Protein Kinase A (PKA), a kinase that is only active under higher cAMP levels57.
This laminin-1 based repulsion is for example particularly important to guide axons out of the eye at
the optic nerve head56, or to steer axons of the developing brain into the CNS57.
Stimulation of both embryonic and adult axons with neurotrophins, such as neurotrophin 3 (NT-3),
BDNF or nerve growth factor (NGF) will induce active transport of β-actin mRNA9, 58, as well as
localization of the β-actin protein to the growth cone23. The response observed when chick forebrain
neurons are stimulate with NT-3 is similar to the response observed when exposing neurons to
cAMP, indicating that also NT-3 might be involved in this signaling pathway58. This response also
includes stimulation of cAMP-dependent PKA activity which precedes microtubule dependent
localization of β-actin mRNA to the growth cone58. Possibly, this reaction is mediated through the
binding of NT-3 or BDNF to tyrosine kinase receptors Tropomyosin-related kinase C (TrkC) (NT-3
growth factor receptor) or TrkB (BDNF/NT-3 growth factors receptor) which are present within the
growth cone58, 59. Downstream of TrkB signaling is MAPK, inducing local translation through mTOR59,
60
. Also, TrkB mediates axon-dendrite polarization to skew in the direction of an axon if a
hippocampal neurite is exposed to extracellular BDNF through a TrkB dependent increase of cAMP’s
and PKA’s downstream targets59.
Experiments on cultured X. laevis spinal neurons showed that the growth cone exhibits the ability to
adapt when continuously stimulated with Netrin-1 or BDNF 50, 55. When continuously exposed to
either of these chemotropic factors, the ability of growth cones to detect and respond to gradients of
Netrin-1, BDNF and Sema3A undergoes consecutive phases of desensitization and resensitization
when the basal concentrations of said factors increases50, 55. Desensitization from e.g. Netrin-1 and
BDNF cues are accompanied by a reduction of Ca2+ signaling, and resensitization does not occur upon
inhibition of either MAPK or protein translation by the ribosomes55. Desensitization occurs at a level
which is upstream of BDNF and netrin-1’s signaling effectors55, and has been reported to be
dependent on rapid highly specific endocytosis of the receptors50. This is supported by the
observation that blocking endocytosis prevents desensitization of any of the membrane receptors50.
The speed at which the growth cones are able to recover their sensitivity vary between publications,
from several minutes to more than an hour50. This might be due to different experimental
12
procedures, and by the use of different types of neurons, e.g. young spinal neurons compared to
older retinal neurons50. This cycling between phases of sensitivity and insensitivity allows the growth
cone to migrate rapidly towards the source of the guidance cues by alternating attractive and
repulsive turning55. MAPK activated resensitization of the growth cone seems to either enhance
recruitment or synthesis in the components required for signaling of BDNF and netrin-155. Apart from
the possible involvement of MAPKs ability to initiate protein translation, treatment with translational
inhibitors has shown that constitutive protein synthesis is critical for axonal growth cone
resensitization55. Other theories favor the option that the cycling in sensitivity functions as an
adaptation mechanism for the changes in background levels of guidance cues50. This adaptation
would make the growth cones able to appropriately respond to the ever increasing levels of guidance
cues as they move on from attractive intermediate targets50.
When axons from cultured retinal ganglion cells are severed from the soma, they remain able to
respond to local translation dependent guidance cues from Sema3A and netrin-149. Growth cone
turning was abolished by inhibition of protein translation49, 55.
Local mRNA translation in mature neurons
For a long time, it was thought to be unlikely that mature axons contained mRNA, as in situ
hybridization studies concerning developing cortical axons showed mRNA levels which gradually
became undetectable as the neuron matured61. The identification of mRNA transcripts in the axon
was mainly impaired due to the techniques available which limited the detection threshold19. For
example, recent work with a combination of microfluidic chambers for harvesting axons with
subsequent microarrays did allow the identification of transcripts in cultured naïve matured cortical
axons19. These microfluidic chambers make it possible to isolate axonal material without the risk of
non-neuronal or soma contamination62. This data was supported by utilizing Fluorescence in situ
hybridization (FISH)19. Unlike the mRNA translation required for adaptive responses in developing
neurons, it is also possible for local mRNA translation to occur in mature neurons upon events which
require a plastic response, such as regeneration6.
Local mRNA translation during injury and regeneration
Injury of the axon has severe consequences for the fate of the cell and nerve it belongs to, including
disconnection from the distal target. Possible responses to injury include induction of pathways
leading to either sprouting, axonal degeneration or cell death, depending on the cellular context and
extent of the damage endured63. The severed axon will undergo Wallerian degeneration, while the
part connected to the soma will be able to reseal the damaged tip and attempt to regenerate29.
Neurons from the mammalian CNS possess little regenerative capabilities in vivo, but it has been
shown that when these cells are exposed to the right molecular context, they do have the ability to
survive longer and extend new axons to a certain degree63. PNS axons on the other hand, have
greater success rates in regenerating correctly, regrowing long distances to reconnect to their
target29. The ability of axons to regenerate after injury is in general reduced as the neuron matures,
and coincides with reduced levels of protein synthesis. If local protein synthesis is unable to assist in
forming a proper new growth cone, the first step in regeneration, it is unlikely that the axon will be
able to navigate back to its destination29. It is not entirely clear what causes the PNS neurons to have
13
a better regenerating potential, but possibly the environment of the CNS contains too many
inhibitory factors, like Sema3A, for the axon to be able to regenerate29.
Another 3’UTR localization motif was discovered in the mRNA encoding for the RAs Related (Ran)
GTPase RanBP1 by Yudin et. al64. Ran GTPase is considered to be a regulator of nuclear transport,
localizing to axons in its GTP-bound state6. There it prevents retrograde transport of cargo by
inhibiting binding to dynein through importin-α6. Importins are part of the karyopherin group of
proteins and bind to the specific Nuclear Localization Signal (NLS) present in the amino acid sequence
of certain proteins65. Upon injury of the nerve, the Ran GTPase activator protein RanBP1 is locally
translated in order to stimulate dissociation of RanGTP from importin-α by converting RanGTP to the
RanGDP form, that is unable to bind importins64. Local synthesis of importin-β is also induced by
RanBP1, which subsequently binds to the liberated importin-α. This importin-α and importin-β
complex binds to dynein, carrying NLS containing proteins like locally synthesized vimentin with
them6, 64, 66. This binding of dynein to importins indicates a Ran mediated mechanism of retrograde
signaling to occur in neurons through local mRNA translation upon damage of the axon64.
The 3’ UTR zipcodes present on mRNAs contain sequence elements that allow for specific translation
responses upon different input signals. Cox et. al. experimentally showed that stimulation of axons
with Nerve Growth Factor (NGF) leads to the induction of local translation of CREB, but not RhoA
mRNA, the latter being regulated by Sema3A7.
Local translation of mRNA has a few potential advantages compared to transporting translated
proteins. As a single molecule of mRNA can be used for protein translation numerous times, it
reduces the energy required for transporting a vast amount of proteins25. This process might also be
important to prevent proteins from coming into regions of the cell where their activity would be
detrimental25.
In axons it would also be faster to translate proteins locally as is demanded for the situation. It would
take a great amount of time to respond to the extracellular cues in the growth cone if the messenger
would first have to be transported back to the soma. Here the messenger would need to induce
transcription of required proteins, which in turn need to be packaged into vesicles for axonal
transport along the microtubules. By keeping translation machinery and a set of mRNA molecules in
the growth cone, the growing axon is able to respond faster to cues which require it to alter its local
proteome. Indeed, this has been confirmed by research performed on severed cultured dorsal root
ganglion neurons29. Some of these in vitro cells were able to reseal their severed axon and start the
formation of a growth cone within 20 minutes of axotomy, much faster than a signaling cascade to
the soma can be transported to induce a response29, 67. In vivo regeneration in Sprague Dawley rats
can be promoted by increasing the activity of mTOR at the site of injury by applying short interfering
RNA (siRNA) against phosphatase and tensin homologue (PTEN) or the specific inhibitor of PTEN
bisperoxovanadium68. PTEN is an inhibitor of PI(3)K-PKB signaling, which is upstream of mTOR6, 68.
The regenerative capabilities of the axon are ablated upon treatment with inhibitors of protein
synthesis like CHX and anisomycin67. Also, inhibitors of mTOR and p38 MAPK induced a similar
inhibition of growth cone formation67. The levels of phosphorylated initiation factor eIF4-4E have
been reported to decrease with developmental age of the neuron by the use of quantitive
immunofluorescence assays on cultured DRG and RGCs29. This also showed that eIF4-4E levels are
much lower in cells of the CNS than the PNS29, 67. Thus, regenerative capabilities of neurons seem to
14
be directly in line with their ability to synthesize proteins locally in the axon, with a critical role for
activating pathways such as mTOR activity.
Subcellular localization of mRNA is known to happen in animal cells through contribution of at least
three different mechanisms28. These include: localized protection of mRNA from degradation, active
localized transport by motor complexes utilizing the cytoskeleton, and localized anchorage of mRNA
molecules28.
After axotomy of cortical axons, localization of transcripts changes depending on the governed
function. mRNA molecules related to intracellular transport, mitochondria and cytoskeleton show
decreased localization to the regenerating axon, while mRNAs involved in axonal targeting and
synaptic function show increased localization19. The shift in observed localization of transcripts is
likely to govern an increased capacity for axonal outgrowth and targeting, with subsequent synapse
formation after injury of the axon19. This study on CNS axons also demonstrated that the axons of
these cells are still able to synthesize proteins locally to maintain autonomy, just like Peripheral
Nervous System (PNS) neurons19.
Figure 3: Functional
classification of 444 mRNAs
with a known function
detected in X. laevis growth
cones by microarray
analysis. Adapted from
Zivraj et. al. J. Neurosci. 30,
15464-15478 (2010) 72.
The axonal mRNA transcriptome
Different studies have attempted to resolve the axonal transcriptome, the repertoire of mRNAs that
is present in embryonic and adult axons. Microarray analysis have been performed on neurons from
X. laevis, mice and rats, either embryonic or adult19, 62, 69, just as serial analysis of gene expression
(SAGE) in rat perinatal sympathetic neurons27. These studies identified thousands of mRNAs to be
present in axons, with great overlap between the different axons analyzed. As seen in Table 1, many
of the shared mRNAs encode for proteins involved in protein synthesis, mitochondrial function and
cytoskeleton formation6. Another distribution of the functional categories of growth cone mRNAs
found in X. laevis is shown in Figure 369, it should be noted that these percentages can vary
depending on the specific neuron examined. The axonal repertoire of mRNAs dynamically changes
over time as the neuron matures, with for example Tubulin-beta3 (Tubb3) mRNA being present in
embryonic but not adult DRG neurons62. Next to the overlapping mRNAs, there were also mRNAs
which were specifically enriched in certain types of axons6. Examples of this are the apparent PNS
exclusive distribution of CREB and Impa1 mRNA6. Bioinformatics analysis showed that many of the
transcripts found in the SAGE screen were enriched in the axon compared to the soma27. This
15
indicates that active transport but not diffusion is regulating the accumulation of mRNA in the axon27.
After mRNA translation has been completed, mRNAs can either re-associate with transport RNPs, be
incorporated into stress granules which sequester unneeded mRNA in the event of cellular stress, or
taken up by processing bodies (P bodies) that are used for either the storage or degradation of
mRNA70, 71.
Retrograde signaling by axonally synthesized proteins
Locally translated proteins commonly function at the site where they were synthesized, such as βactin28. But in contrast, the mRNA for cAMP response element binding protein (CREB) can be
synthesized in distal axons to undergo retrograde transport towards the soma’s nucleus. CREB is a
transcription factor which is translated within axons in response to stimulation of the NGF receptor
TrkA and promotes cell survival in DRG neurons7, 28. The way CREB is phosphorylated is different
depending on the site of translation, and a nuclear accumulation of pCREB and subsequent CREmediated transcription do not occur upon specific axonal knockdown of CREB mRNA7. Also, TrkA
kinase activity is required in the cell body for phosphorylation of CREB, indicating that CREB might be
phosphorylated by TrkA effectors, such as Erk57, 72. In rat sympathetic neurons, nuclear CREB
activation requires active myo-Inositol Monophosphatase-1 (IMPA1), which is also actively translated
in the axon upon NGF stimulation27. IMPA1 mRNA contains a specific localization element within its
3’UTR to target the molecule specifically towards the axon27.
Retrograde transport of proteins in the axoplasm such as CREB, is most likely mediated by the
molecular motor protein dynein7. Retrograde transport by dynein was shown for both the importin α
and importin β proteins65, 73, which are translated locally from axonal mRNA upon lesion of the nerve
in DRG neurons65, 73. This system might enable retrograde transport of signals modulating
regeneration of injured neurons by upregulation of axonal importins65, 73.
Species
Neuron type
Rat
Embryonic
SCG
Embryonic
DRG
Adult DRG
Rat
Rat
Subcellular
compartment
Axons
Protein
synthesis
Yes
Mitochondrial
Cytoskeletal
Ref
Yes
Cell-Cell
signaling
No
Yes
Axons
Yes
Yes
Yes
No
66
Young growth
cones
Mature axons
Yes
Yes
Yes
Yes
66
65
64
Neonatal
Yes
Yes
Yes
No
cerebral
cortical
74
X. laevis Embryonic
Young growth Yes
Yes
Yes
No
RGC
cones
74
X. laevis Embryonic
Old
growth Yes
Yes
Yes
Yes
RGC
cones
74
Mouse
Embryonic
Growth cones
Yes
Yes
Yes
No
RGC
Table 1: Selected functional groups of mRNAs identified to be enriched in the neurons of several species.
Dorsal Root Ganglion (DRG), Retinal Ganglion Cells (RGC), Superior cervical ganglion (SCG). Adapted from Jung
et. al. Nat. Rev. Neurosci. 13, 308-324 (2012).6.
Rat
16
Another transcription factor which has recently been identified to be involved in anterograde injury
signaling in the sciatic nerve and DRG is Signal Transducer and Activator of Transcription 3 (STAT3)73.
Upon damage of the axon STAT3 is locally translated and subsequently phosphorylated, bringing it
into an active state73. It is then transported retrogradely by binding to dynein and importin α573.
STAT3 has been described to act downstream of effectors ultimately mediating outgrowth and
survival responses in neurons73. The activity of STAT3 is indispensible for viability of lesioned adult
neurons and seems to work mainly in an apoptotic inhibiting mechanism73.
As mentioned earlier, upon nerve injury a retrograde signaling cascade mediated by axonally
translated Ran GTPase is activated, with vimentin as one of the cargoes which is transported through
an importin-dynein mediated mechanism6, 64, 66. Vimentin is also locally synthesized in the axoplasm
of sciatic nerves at the lesion site together with importin-β upon injury66. This injury also induces the
activation of MAPK ERK1/ERK2 by phosphorylation, allowing vimentin to bind to the phosphorylated
ERKs (pERKs)66. The ability of vimentin to bind both importin-β and pERK at the same time allows for
retrograde transport of pERK through dynein motors66. The importance of vimentin was confirmed
by co-immunoprecipitation experiments using axoplasm from injured mouse nerves, as this showed
the pERKs are coprecipitated with dynein in wildtype but not vimentin−/− mice66. Erks are able to
modulate gene expression by direct phosphorylation of transcription factors such as Elk166. Elk1 is
part of the E-twenty-six (ETS) family, and is known to be induced upon injury of the nerve66. Both the
activation of Elk1 and neural regeneration in general are inhibited or delayed upon defects in the
vimentin and pERK binding mechanism66, indicating the importance of retrograde transport mediated
by axonally synthesized proteins. This study also showed that the interaction between pERK2 and
vimentin was calcium dependent in vitro, while binding of nonphosphorylated ERK to vimentin was
actually reduced at higher calcium levels66. As it is known that intracellular calcium levels in axons are
significantly increased upon injury63, these findings again suggest a correlation between local protein
translation and functionality of these proteins upon nerve damage.
Nervy is a transcription factor of the myeloid translocation gene family of A kinase anchoring
proteins (AKAPs) which localizes to Drosophila’s axons75. Nervy has a non-nuclear function there, and
acts as an adaptor protein for linking PKA and the Plexin A receptor upon Sema1A stimulation7, 75. In
this case, formation of the aforementioned complex leads to axonal repulsion, and Nervy thus
functions as a mediating element for Sema1A guidance cues75.
Ribosome specificity in mRNA translation
Ribosomes consist of different RNA molecules and proteins, the ribosomal subunits. It has been
shown that genes encoding ribosomal proteins can be tissue specifically altered76, and that these
changes can perturb the translation of a specific subset of mRNAs, acting as a regulating factor for
protein expression76. This principle was displayed to exist in mice with mutations in the ribosomal
protein L38 (Rpl38) gene, which had specific defects in translation of Hox mRNAs76. RPL38 acts to
facilitate the formation of the 80S ribosome complex during translation initiation76. As many variable
compositions of ribosomes may exist, there might be many different types of ribosomes favoring
translation of specific mRNAs6. Axons contain many mRNAs encoding ribosomal proteins19, and these
mRNAs are again regulated by mTOR-S6K signaling through the 5’TOP-sequences77. Taken together,
this could mean that axons are able to utilize locally synthesized ribosomal components to generate
mRNA selective ribosomes to further increase the control of the axonal proteome6.
17
Involvement of glial cells
Another way that protein synthesis might be activated was is a mechanism in which ribosomes are
delivered from glial cells to the axon upon injury. This transfer of polyribosomes from Schwann cells
to desomatized sciatic nerve axons of mice was detected by labeling ribosomal proteins in Schwann
cells with eGFP utilizing a lentiviral vector, and subsequently tracking the translocalization78, 79. As
polyribosomes consist of multiple ribosomes in the process of simultaneously translating the same
mRNA molecule, it is postulated that Schwann cells are also able to transfer mRNA in addition to the
observed ribosomal transferring78, as depicted in figure 4B80. One possible mechanism that has been
described to facilitate this transfer are exosomes shuttling from glial cells, like oligodendrocytes, to
the neuron while carrying protein and RNA cargo81. It has been shown that oligodendrocytes in the
brain excrete exosomes in a Ca2+ dependent fashion82. Isolation of these exosomes by sucrose
density gradient centrifugation and subsequent proteomic analysis revealed that these vesicles can
contain proteins which are believed to play a role in the relief of cellular stress82. Nerve injury is
known to increase Ca2+ levels in the axon, perhaps it is therefore possible that glial cells play an
important role in the quick early response of the axon to this stress inducing event, but this has yet
to be elucidated. For the squid giant axon it has been described that it is able to receive RNAs
synthesized in the glial cells upon axonal depolarization26.
Figure 4: Schematic models of possible
originas of mRNA in neuronal axons.
Panel A represents the classic that
axonal mRNAs originate from the soma
(blue arrows). Panel B represents the
theory that glial cells may supply the
axon with RNA (red arrows), a pathway
which would be coexisting with the
somatic derived mRNA (dotted blue
arrows)80. Adapted from Giuditta et. al.
Physiol Rev 88: 515–555, 2008 80.
These mRNA transferring systems would allow the periphery of the neuron to rapidly respond to
stimuli in the environment by changing the proteome through mRNA transcription in the glial cells
instead of the soma80. While experimental data exists to support this theory, it is for example not
clear yet whether or not the targeting of glial RNA is specific towards the axon80. As oligodendrocytes
and Schwann cells perform the same functions, like forming myelin sheets, to some extent in the CNS
and PNS respectively, it is possible that they share a common mechanism of glial-neuron signaling.
The importance of such a glial-neuron interaction system is more likely to play an important role in
neurons with longer axons, as inherently the amount of cytoplasm contained within the axon can be
18
much greater than in the soma, perhaps making the trophic demands too high for the soma to
maintain on its own83.
The expression of these ribosomal proteins in the axon is regulated by the phosphatidylinositol 3kinase (PI(3)K) – Protein Kinase Beta (PKB, also known as Akt) – mTOR axis of signaling through
phosphorylation of mTORs target protein: ribosomal protein S6 kinase (S6K)77. The activated S6K in
turn, will promote the translation of mRNAs with 5’-TOP sequences found in their sequences77. This
mTOR regulated mechanism would ensure that the energy intensive translational machinery is not
synthesized at times when the cell lacks a supply of nutrients, like amino acids or energy, a situation
in which mTOR signaling will be inhibited by upstream signaling.
The RBP La binds the 5’-terminal oligopyrimidine tract (TOP) sequence present in the 5’UTR of
mRNAs and promotes their translation77. This 5’-TOP sequence is a unique feature of mRNAs
encoding for components of translational machinery, allowing them to be selectively targeted and
regulated77. Unmodified La mediates anterograde transport of RNPs in axons by interacting with
kinesin, but upon sumoylation of lysine 41, La will interact with dynein for retrograde transport84.
Conclusion
Neurons utilize the local translation of mRNA in their axons for a wide range of goals throughout
their life cycle. Among these roles are the herein described processes of axonal growth in the
embryonic nervous system and regeneration upon injury of the axon. These processes require rapid
responses to extrinsic stimuli, which have been observed to occur too fast for the soma to be able to
respond to a retrograde signal with axoplasmic transport. Local mRNA translation is also of
importance to retain homeostasis in the cell, like its role in mitochondrial maintenance. It is currently
unclear though how these responses are coordinated in the axonal tip to maintain the right balance
of all the effector proteins used, thus further research will have to elucidate this.
Also, although differences have been found in the translated mRNAs in the axons of different types
of neurons, no clear transcriptome has been established yet for each type. Further research could
give insight in the different proteomes, which might also help to explain the differences in
regenerative behavior observed upon e.g. injury. High throughput methods utilizing microarrays or
RNA-immunoprecipitations followed by deep sequencing could be useful for this purpose.
The establishment of the axonal mRNA repertoire also requires the enriched mRNAs to be selectively
transported towards the axon. Multiple cis-acting elements have been identified in for example the
3’-UTR of some mRNAs, but how other mRNAs lacking these sequences are selectively transported
remains unclear. The repertoire of mRNA molecules could be different in the axon shaft, tip, growth
cone and presynaptic terminals, but the amount of data available on this subject is limited.
The ability of axons to regenerate upon injury seems to be closely related to their local translation
capabilities. As these capabilities decrease as the axon matures together with a change in their mRNA
repertoire, it becomes harder for older neurons to start a plastic response when injured.
Advancements in the knowledge of how neurons regulate local translation might prove to be
beneficial in the future for treatment of humans that have sustained injuries to the CNS, by
modulating the regenerative capacities of the neurons.
19
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